Key Specifications Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|H, M, R||IHC, IP, WB||M||Purified||Monoclonal Antibody|
|Safety Information according to GHS|
|Material Size||250 µg|
References | 105 Available | See All References
|Reference overview||Application||Species||Pub Med ID|
|Lichen Secondary Metabolite, Physciosporin, Inhibits Lung Cancer Cell Motility. |
Yang, Y; Park, SY; Nguyen, TT; Yu, YH; Nguyen, TV; Sun, EG; Udeni, J; Jeong, MH; Pereira, I; Moon, C; Ha, HH; Kim, KK; Hur, JS; Kim, H
PloS one 10 e0137889 2015
Lichens produce various unique chemicals that can be used for pharmaceutical purposes. To screen for novel lichen secondary metabolites showing inhibitory activity against lung cancer cell motility, we tested acetone extracts of 13 lichen samples collected in Chile. Physciosporin, isolated from Pseudocyphellaria coriacea (Hook f. & Taylor) D.J. Galloway & P. James, was identified as an effective compound and showed significant inhibitory activity in migration and invasion assays against human lung cancer cells. Physciosporin treatment reduced both protein and mRNA levels of N-cadherin with concomitant decreases in the levels of epithelial-mesenchymal transition markers such as snail and twist. Physciosporin also suppressed KITENIN (KAI1 C-terminal interacting tetraspanin)-mediated AP-1 activity in both the absence and presence of epidermal growth factor stimulation. Quantitative real-time PCR analysis showed that the expression of the metastasis suppressor gene, KAI1, was increased while that of the metastasis enhancer gene, KITENIN, was dramatically decreased by physciosporin. Particularly, the activity of 3'-untranslated region of KITENIN was decreased by physciosporin. Moreover, Cdc42 and Rac1 activities were decreased by physciosporin. These results demonstrated that the lichen secondary metabolite, physciosporin, inhibits lung cancer cell motility through novel mechanisms of action.
|Spatial mapping of juxtacrine axo-glial interactions identifies novel molecules in peripheral myelination. |
Poitelon, Y; Bogni, S; Matafora, V; Della-Flora Nunes, G; Hurley, E; Ghidinelli, M; Katzenellenbogen, BS; Taveggia, C; Silvestri, N; Bachi, A; Sannino, A; Wrabetz, L; Feltri, ML
Nature communications 6 8303 2015
Cell-cell interactions promote juxtacrine signals in specific subcellular domains, which are difficult to capture in the complexity of the nervous system. For example, contact between axons and Schwann cells triggers signals required for radial sorting and myelination. Failure in this interaction causes dysmyelination and axonal degeneration. Despite its importance, few molecules at the axo-glial surface are known. To identify novel molecules in axo-glial interactions, we modified the 'pseudopodia' sub-fractionation system and isolated the projections that glia extend when they receive juxtacrine signals from axons. By proteomics we identified the signalling networks present at the glial-leading edge, and novel proteins, including members of the Prohibitin family. Glial-specific deletion of Prohibitin-2 in mice impairs axo-glial interactions and myelination. We thus validate a novel method to model morphogenesis and juxtacrine signalling, provide insights into the molecular organization of the axo-glial contact, and identify a novel class of molecules in myelination.
|The proto-oncogene c-Src and its downstream signaling pathways are inhibited by the metastasis suppressor, NDRG1. |
Liu, W; Yue, F; Zheng, M; Merlot, A; Bae, DH; Huang, M; Lane, D; Jansson, P; Lui, GY; Richardson, V; Sahni, S; Kalinowski, D; Kovacevic, Z; Richardson, DR
Oncotarget 6 8851-74 2015
N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor that plays a key role in regulating signaling pathways involved in mediating cancer cell invasion and migration, including those derived from prostate, colon, etc. However, the mechanisms and molecular targets through which NDRG1 reduces cancer cell invasion and migration, leading to inhibition of cancer metastasis, are not fully elucidated. In this investigation, using NDRG1 over-expression models in three tumor cell-types (namely, DU145, PC3MM and HT29) and also NDRG1 silencing in DU145 and HT29 cells, we reveal that NDRG1 decreases phosphorylation of a key proto-oncogene, cellular Src (c-Src), at a well-characterized activating site (Tyr416). NDRG1-mediated down-regulation of EGFR expression and activation were responsible for the decreased phosphorylation of c-Src (Tyr416). Indeed, NDRG1 prevented recruitment of c-Src to EGFR and c-Src activation. Moreover, NDRG1 suppressed Rac1 activity by modulating phosphorylation of a c-Src downstream effector, p130Cas, and its association with CrkII, which acts as a "molecular switch" to activate Rac1. NDRG1 also affected another signaling molecule involved in modulating Rac1 signaling, c-Abl, which then inhibited CrkII phosphorylation. Silencing NDRG1 increased cell migration relative to the control and inhibition of c-Src signaling using siRNA, or a pharmacological inhibitor (SU6656), prevented this increase. Hence, the role of NDRG1 in decreasing cell migration is, in part, due to its inhibition of c-Src activation. In addition, novel pharmacological agents, which induce NDRG1 expression and are currently under development as anti-metastatic agents, markedly increase NDRG1 and decrease c-Src activation. This study leads to important insights into the mechanism involved in inhibiting metastasis by NDRG1 and how to target these pathways with novel therapeutics.
|Immunohistochemical analysis of the natural killer cell cytotoxicity pathway in human abdominal aortic aneurysms. |
Hinterseher, I; Schworer, CM; Lillvis, JH; Stahl, E; Erdman, R; Gatalica, Z; Tromp, G; Kuivaniemi, H
International journal of molecular sciences 16 11196-212 2015
Our previous analysis using genome-wide microarray expression data revealed extreme overrepresentation of immune related genes belonging the Natural Killer (NK) Cell Mediated Cytotoxicity pathway (hsa04650) in human abdominal aortic aneurysm (AAA). We followed up the microarray studies by immunohistochemical analyses using antibodies against nine members of the NK pathway (VAV1, VAV3, PLCG1, PLCG2, HCST, TYROBP, PTK2B, TNFA, and GZMB) and aortic tissue samples from AAA repair operations (n = 6) and control aortae (n = 8) from age-, sex- and ethnicity-matched donors from autopsies. The results confirmed the microarray results. Two different members of the NK pathway, HCST and GRZB, which act at different steps in the NK-pathway, were actively transcribed and translated into proteins in the same cells in the AAA tissue demonstrated by double staining. Furthermore, double staining with antibodies against CD68 or CD8 together with HCST, TYROBP, PTK2B or PLCG2 revealed that CD68 and CD8 positive cells expressed proteins of the NK-pathway but were not the only inflammatory cells involved in the NK-pathway in the AAA tissue. The results provide strong evidence that the NK Cell Mediated Cytotoxicity Pathway is activated in human AAA and valuable insight for future studies to dissect the pathogenesis of human AAA.
|ROCK1 deficiency enhances protective effects of antioxidants against apoptosis and cell detachment. |
Surma, M; Handy, C; Chang, J; Kapur, R; Wei, L; Shi, J
PloS one 9 e90758 2014
We have recently reported that the homologous Rho kinases, ROCK1 and ROCK2, play different roles in regulating stress-induced stress fiber disassembly and cell detachment, and the ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has remarkable anti-apoptotic, anti-detachment and pro-survival effects against doxorubicin, a chemotherapeutic drug. This study investigated the roles of ROCK isoforms in doxorubicin-induced reactive oxygen species (ROS) generation which is believed to be the major mechanism underlying its cytotoxicity to normal cells, and especially to cardiomyocytes. Different antioxidants have been shown to provide a protective role reported in numerous experimental studies, but clinical trials of antioxidant therapy showed insufficient benefit against the cardiac side effect. We found that both ROCK1-/- and ROCK2-/- MEFs exhibited reduced ROS production in response to doxorubicin treatment. Interestingly, only ROCK1 deficiency, but not ROCK2 deficiency, significantly enhanced the protective effects of antioxidants against doxorubicin-induced cytotoxicity. First, ROCK1 deficiency and N-acetylcysteine (an anti-oxidant) treatment synergistically reduced ROS levels, caspase activation and cell detachment. In addition, the reduction of ROS generation in ROCK1-/- MEFs in response to doxorubicin treatment was in part through inhibiting NADPH oxidase activity. Furthermore, ROCK1 deficiency enhanced the inhibitory effects of diphenyleneiodonium (an inhibitor of NADPH oxidase) on ROS generation and caspase 3 activation induced by doxorubicin. Finally, ROCK1 deficiency had greater protective effects than antioxidant treatment, especially on reducing actin cytoskeleton remodeling. ROCK1 deficiency not only reduced actomyosin contraction but also preserved central stress fiber stability, whereas antioxidant treatment only reduced actomyosin contraction without preserving central stress fibers. These results reveal a novel strategy to enhance the protective effect of antioxidant therapy by targeting the ROCK1 pathway to stabilize the actin cytoskeleton and boost the inhibitory effects on ROS production, apoptosis and cell detachment.
|Interactome analysis of AMP-activated protein kinase (AMPK)-α1 and -β1 in INS-1 pancreatic beta-cells by affinity purification-mass spectrometry. |
Moon, S; Han, D; Kim, Y; Jin, J; Ho, WK; Kim, Y
Scientific reports 4 4376 2014
The heterotrimeric enzyme AMP-activated protein kinase (AMPK) is a major metabolic factor that regulates the homeostasis of cellular energy. In particular, AMPK mediates the insulin resistance that is associated with type 2 diabetes. Generally, cellular processes require tight regulation of protein kinases, which is effected through their formation of complex with other proteins and substrates. Despite their critical function in regulation and pathogenesis, there are limited data on the interaction of protein kinases. To identify proteins that interact with AMPK, we performed large-scale affinity purification (AP)-mass spectrometry (MS) of the AMPK-α1 and -β1 subunits. Through a comprehensive analysis, using a combination of immunoprecipitaion and ion trap mass spectrometry, we identified 381 unique proteins in the AMPKα/β interactomes: 325 partners of AMPK-α1 and 243 for AMPK-β1. Further, we identified 196 novel protein-protein interactions with AMPK-α1 and AMPK-β1. Notably, in our bioinformatics analysis, the novel interaction partners mediated functions that are related to the regulation of actin organization. Specifically, several such proteins were linked to pancreatic beta-cell functions, including glucose-stimulated insulin secretion, beta-cell development, beta-cell differentiation, and cell-cell communication.
|Downregulation of microRNA-100 enhances the ICMT-Rac1 signaling and promotes metastasis of hepatocellular carcinoma cells. |
Zhou, HC; Fang, JH; Luo, X; Zhang, L; Yang, J; Zhang, C; Zhuang, SM
Oncotarget 5 12177-88 2014
Metastasis is responsible for rapid recurrence of hepatocellular carcinoma (HCC) and poor survival of HCC patients. Here we showed that miR-100 downregulation in HCC tissues was significantly associated with venous invasion, advanced TNM stage, tumor nodule without complete capsule, poorer cell differentiation, and shorter recurrence-free survival. Both gain- and loss-of-function studies showed that miR-100 dramatically suppressed the ability of HCC cells to migrate and to invade through Matrigel in vitro. Analyses using mouse orthotopic xenograft model further revealed that xenografts of miR-100-stable-expressing HCC cells displayed a significant reduction in pulmonary metastasis, compared with control group. Subsequent investigations revealed that miR-100 directly inhibited the expression of isoprenylcysteine carboxyl methyltransferase (ICMT) and ras-related C3 botulinum toxin substrate 1 (Rac1) by binding to their 3'-UTRs, and in turn suppressed lamellipodia formation and matrix metallopeptidase 2 (MMP2) activation. Furthermore, knockdown of ICMT and Rac1 phenocopied the anti-metastasis effect of miR-100, whereas overexpression of the constitutively active Rac1 (Q61L) antagonized the function of miR-100. Taken together, miR-100 represses metastasis of HCC cells by abrogating the ICMT-Rac1 signaling. Downregulation of miR-100 contributes to HCC metastasis and the restoration of miR-100 is a potential strategy for cancer therapy.
|Wnt7a stimulates myogenic stem cell motility and engraftment resulting in improved muscle strength. |
Bentzinger, CF; von Maltzahn, J; Dumont, NA; Stark, DA; Wang, YX; Nhan, K; Frenette, J; Cornelison, DD; Rudnicki, MA
The Journal of cell biology 205 97-111 2014
Wnt7a/Fzd7 signaling stimulates skeletal muscle growth and repair by inducing the symmetric expansion of satellite stem cells through the planar cell polarity pathway and by activating the Akt/mTOR growth pathway in muscle fibers. Here we describe a third level of activity where Wnt7a/Fzd7 increases the polarity and directional migration of mouse satellite cells and human myogenic progenitors through activation of Dvl2 and the small GTPase Rac1. Importantly, these effects can be exploited to potentiate the outcome of myogenic cell transplantation into dystrophic muscles. We observed that a short Wnt7a treatment markedly stimulated tissue dispersal and engraftment, leading to significantly improved muscle function. Moreover, myofibers at distal sites that fused with Wnt7a-treated cells were hypertrophic, suggesting that the transplanted cells deliver activated Wnt7a/Fzd7 signaling complexes to recipient myofibers. Taken together, we describe a viable and effective ex vivo cell modulation process that profoundly enhances the efficacy of stem cell therapy for skeletal muscle.
|Novel mechanism of JNK pathway activation by adenoviral E1A. |
Romanov, VS; Brichkina, AI; Morrison, H; Pospelova, TV; Pospelov, VA; Herrlich, P
Oncotarget 5 2176-86 2014
The adenoviral oncoprotein E1A influences cellular regulation by interacting with a number of cellular proteins. In collaboration with complementary oncogenes, E1A fully transforms primary cells. As part of this action, E1A inhibits transcription of c-Jun:Fos target genes while promoting that of c-Jun:ATF2-dependent genes including jun. Both c-Jun and ATF2 are hyperphosphorylated in response to E1A. In the current study, E1A was fused with the ligand binding domain of the estrogen receptor (E1A-ER) to monitor the immediate effect of E1A activation. With this approach we now show that E1A activates c-Jun N-terminal kinase (JNK), the upstream kinases MKK4 and MKK7, as well as the small GTPase Rac1. Activation of the JNK pathway requires the N-terminal domain of E1A, and, importantly, is independent of transcription. In addition, it requires the presence of ERM proteins. Downregulation of signaling components upstream of JNK inhibits E1A-dependent JNK/c-Jun activation. Taking these findings together, we show that E1A activates the JNK/c-Jun signaling pathway upstream of Rac1 in a transcription-independent manner, demonstrating a novel mechanism of E1A action.
|EZH2 regulates cofilin activity and colon cancer cell migration by targeting ITGA2 gene. |
Ferraro, A; Boni, T; Pintzas, A
PloS one 9 e115276 2014
Reorganization of cytoskeleton via actin remodeling is a basic step of cell locomotion. Although cell migration of normal and cancer cells can be stimulated by a variety of intra- and extra-cellular factors, all paths ultimate on the regulation of cofilin activity. Cofilin is a small actin-binding protein able to bind both forms of actin, globular and filament, and is regulated by phosphorylation at Serine 3. Following phosphorylation at serine 3 cofilin is inactive, therefore cannot bind actin molecules and cytoskeleton remodeling is impaired. The histone methyltransferase EZH2 is frequently over expressed in many tumour types including colorectal cancer (CRC). EZH2 over activity, which results in epigenetic gene-silencing, has been associated with many tumour properties including invasion, angiogenesis and metastasis but little is known about the underneath molecular mechanisms. Herein, we report that EZH2 is able to control cofilin activity and consequently cell locomotion of CRC cell lines through a non-conventional novel axis that involves integrin signaling. Indeed, we show how genetic and pharmacological inhibition (DZNep and GSK343) of EZH2 function produces hyper phosphorylation of cofilin and reduces cell migration. We previously demonstrated by chromatin immuno-precipitation that Integrin alpha 2 (ITGα2) expression is regulated by EZH2. In the present study we provide evidence that in EZH2-silenced cells the signaling activity of the de-repressed ITGα2 is able to increase cofilin phosphorylation, which in turn reduces cell migration. This study also proposes novel mechanisms that might provide new anti-metastatic strategies for CRC treatment based on the inhibition of the epigenetic factor EZH2 and/or its target gene.
|VAV3 mediates resistance to breast cancer endocrine therapy. |
Aguilar, H; Urruticoechea, A; Halonen, P; Kiyotani, K; Mushiroda, T; Barril, X; Serra-Musach, J; Islam, A; Caizzi, L; Di Croce, L; Nevedomskaya, E; Zwart, W; Bostner, J; Karlsson, E; Pérez Tenorio, G; Fornander, T; Sgroi, DC; Garcia-Mata, R; Jansen, MP; García, N; Bonifaci, N; Climent, F; Soler, MT; Rodríguez-Vida, A; Gil, M; Brunet, J; Martrat, G; Gómez-Baldó, L; Extremera, AI; Figueras, A; Balart, J; Clarke, R; Burnstein, KL; Carlson, KE; Katzenellenbogen, JA; Vizoso, M; Esteller, M; Villanueva, A; Rodríguez-Peña, AB; Bustelo, XR; Nakamura, Y; Zembutsu, H; Stål, O; Beijersbergen, RL; Pujana, MA
Breast cancer research : BCR 16 R53 2014
Endocrine therapies targeting cell proliferation and survival mediated by estrogen receptor α (ERα) are among the most effective systemic treatments for ERα-positive breast cancer. However, most tumors initially responsive to these therapies acquire resistance through mechanisms that involve ERα transcriptional regulatory plasticity. Herein we identify VAV3 as a critical component in this process.A cell-based chemical compound screen was carried out to identify therapeutic strategies against resistance to endocrine therapy. Binding to ERα was evaluated by molecular docking analyses, an agonist fluoligand assay and short hairpin (sh)RNA-mediated protein depletion. Microarray analyses were performed to identify altered gene expression. Western blot analysis of signaling and proliferation markers, and shRNA-mediated protein depletion in viability and clonogenic assays, were performed to delineate the role of VAV3. Genetic variation in VAV3 was assessed for association with the response to tamoxifen. Immunohistochemical analyses of VAV3 were carried out to determine its association with therapeutic response and different tumor markers. An analysis of gene expression association with drug sensitivity was carried out to identify a potential therapeutic approach based on differential VAV3 expression.The compound YC-1 was found to comparatively reduce the viability of cell models of acquired resistance. This effect was probably not due to activation of its canonical target (soluble guanylyl cyclase), but instead was likely a result of binding to ERα. VAV3 was selectively reduced upon exposure to YC-1 or ERα depletion, and, accordingly, VAV3 depletion comparatively reduced the viability of cell models of acquired resistance. In the clinical scenario, germline variation in VAV3 was associated with the response to tamoxifen in Japanese breast cancer patients (rs10494071 combined P value = 8.4 × 10-4). The allele association combined with gene expression analyses indicated that low VAV3 expression predicts better clinical outcome. Conversely, high nuclear VAV3 expression in tumor cells was associated with poorer endocrine therapy response. Based on VAV3 expression levels and the response to erlotinib in cancer cell lines, targeting EGFR signaling may be a promising therapeutic strategy.This study proposes VAV3 as a biomarker and a rationale for its use as a signaling target to prevent and/or overcome resistance to endocrine therapy in breast cancer.
|Hax-1 is required for Rac1-Cortactin interaction and ovarian carcinoma cell migration. |
Gomathinayagam, R; Muralidharan, J; Ha, JH; Varadarajalu, L; Dhanasekaran, DN
Genes & cancer 5 84-99 2014
Hax-1 is a multifunctional protein, which is involved in diverse cellular signaling pathways including tumor cell survival and migration. We have shown previously that cell migration stimulated by the oncogenic G protein, G13, requires Hax-1 for the formation of a functional complex involving Gα13, Rac1, and cortactin. However, the role of Hax-1 in cancer cell migration or its role in Rac1-cortactin complex formation, which is known to be required for such migration remains to be characterized. Results focused on resolving the role of Hax-1 in ovarian cancer pathophysiology indicate that Hax-1 is overexpressed in ovarian cancer cells and the silencing of Hax-1 inhibits lysophosphatidic acid (LPA)- or fetal bovine serum-stimulated migration of these cells. In addition, silencing of Hax-1 greatly reduces Rac1-cortactin interaction and their colocalization in SKOV3 cells. Mapping the structural domains of Hax-1 indicates that it interacts with cortactin via domains spanning amino acids 1 to 56 (Hax-D1) and amino acids 113 to 168 (Hax-D3). Much weaker interaction with cortactin was also observed with the region of Hax-1 spanning amino acids 169 - 224 (Hax-D4). Similar mapping of Hax-1 domains involved in Rac1 interaction indicates that it associates with Rac1 via two primary domains spanning amino acids 57 to 112 (Hax-D2) and 169 to 224 (Hax-D4). Furthermore, expression of either of these domains inhibits LPA-mediated migration of SKOV3 cells, possibly through their ability to exert competitive inhibition on endogenous Hax-1-Rac1 and/or Hax-1-cortactin interaction. More significantly, expression of Hax-D4 drastically reduces Rac1-cortactin colocalization in SKOV3 cells along with an attenuation of LPA-stimulated migration. Thus our results presented here describe for the first time that Hax-1 interaction is required for the association between Rac1 and cortactin and that these multiple interactions are required for the LPA-stimulated migration of SKOV3 ovarian cancer cells.
|Impaired cell death and mammary gland involution in the absence of Dock1 and Rac1 signaling. |
Bagci, H; Laurin, M; Huber, J; Muller, WJ; Côté, JF
Cell death & disease 5 e1375 2014
Throughout life, the tight equilibrium between cell death and the prompt clearance of dead corpses is required to maintain a proper tissue homeostasis and prevent inflammation. Following lactation, mammary gland involution is triggered and results in the death of excessive epithelial cells that are rapidly cleared by phagocytes to ensure that the gland returns to its prepregnant state. Orthologs of Dock1 (dedicator of cytokinesis 1), Elmo and Rac1 (ras-related C3 botulinum toxin substrate 1) in Caenorhabditis elegans are part of a signaling module in phagocytes that is linking apoptotic cell recognition to cytoskeletal reorganization required for engulfment. In mammals, Elmo1 was shown to interact with the phosphatidylserine receptor Bai1 and relay signals to promote phagocytosis of apoptotic cells. Still, the role of the RacGEF Dock1 in the clearance of dying cells in mammals was never directly addressed. We generated two mouse models with conditional inactivation of Dock1 and Rac1 and revealed that the expression of these genes is not essential in the mammary gland during puberty, pregnancy and lactation. We induced mammary gland involution in these mice to investigate the role of Dock1/Rac1 signaling in the engulfment of cell corpses. Unpredictably, activation of Stat3 (signal transducer and activator of transcription 3), a key regulator of mammary gland involution, was impaired in the absence of Rac1 and Dock1 expression. Likewise, failure to activate properly Stat3 was coinciding with a significant delay in the initiation and progression of mammary gland involution in mutant animals. By using an in vitro phagocytosis assay, we observed that Dock1 and Rac1 are essential to mediate engulfment in epithelial phagocytes. In vivo, cell corpses accumulated at late time points of involution in Dock1 and Rac1 mutant mammary glands. Overall, our study demonstrated an unsuspected role for Dock1/Rac1 signaling in the initiation of mammary gland involution, and also suggested a role for this pathway in the clearance of dead cells by epithelial phagocytes.
|Epithelial splicing regulatory proteins 1 (ESRP1) and 2 (ESRP2) suppress cancer cell motility via different mechanisms. |
Ishii, H; Saitoh, M; Sakamoto, K; Kondo, T; Katoh, R; Tanaka, S; Motizuki, M; Masuyama, K; Miyazawa, K
The Journal of biological chemistry 289 27386-99 2014
ESRP1 (epithelial splicing regulatory protein 1) and ESRP2 regulate alternative splicing events associated with epithelial phenotypes of cells, and both are down-regulated during the epithelial-mesenchymal transition. However, little is known about their expression and functions during carcinogenesis. In this study, we found that expression of both ESRP1 and ESRP2 is plastic: during oral squamous cell carcinogenesis, these proteins are up-regulated relative to their levels in normal epithelium but down-regulated in invasive fronts. Importantly, ESRP1 and ESRP2 are re-expressed in the lymph nodes, where carcinoma cells metastasize and colonize. In head and neck carcinoma cell lines, ESRP1 and ESRP2 suppress cancer cell motility through distinct mechanisms: knockdown of ESRP1 affects the dynamics of the actin cytoskeleton through induction of Rac1b, whereas knockdown of ESRP2 attenuates cell-cell adhesion through increased expression of epithelial-mesenchymal transition-associated transcription factors. Down-regulation of ESRP1 and ESRP2 is thus closely associated with a motile phenotype of cancer cells.
|Rictor/mTORC2 regulates blood-testis barrier dynamics via its effects on gap junction communications and actin filament network. |
Mok, KW; Mruk, DD; Lee, WM; Cheng, CY
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 27 1137-52 2013
In the mammalian testis, coexisting tight junctions (TJs), basal ectoplasmic specializations, and gap junctions (GJs), together with desmosomes near the basement membrane, constitute the blood-testis barrier (BTB). The most notable feature of the BTB, however, is the extensive network of actin filament bundles, which makes it one of the tightest blood-tissue barriers. The BTB undergoes restructuring to facilitate the transit of preleptotene spermatocytes at stage VIII-IX of the epithelial cycle. Thus, the F-actin network at the BTB undergoes cyclic reorganization via a yet-to-be explored mechanism. Rictor, the key component of mTORC2 that is known to regulate actin cytoskeleton, was shown to express stage-specifically at the BTB in the seminiferous epithelium. Its expression was down-regulated at the BTB in stage VIII-IX tubules, coinciding with BTB restructuring at these stages. Using an in vivo model, a down-regulation of rictor at the BTB was also detected during adjudin-induced BTB disruption, illustrating rictor expression is positively correlated with the status of the BTB integrity. Indeed, the knockdown of rictor by RNAi was found to perturb the Sertoli cell TJ-barrier function in vitro and the BTB integrity in vivo. This loss of barrier function was accompanied by changes in F-actin organization at the Sertoli cell BTB in vitro and in vivo, associated with a loss of interaction between actin and α-catenin or ZO-1. Rictor knockdown by RNAi was also found to impede Sertoli cell-cell GJ communication, disrupting protein distribution (e.g., occludin, ZO-1) at the BTB, illustrating that rictor is a crucial BTB regulator.
|The anti-migratory effects of FKBPL and its peptide derivative, AD-01: regulation of CD44 and the cytoskeletal pathway. |
Yakkundi, A; McCallum, L; O'Kane, A; Dyer, H; Worthington, J; McKeen, HD; McClements, L; Elliott, C; McCarthy, HO; Hirst, DG; Robson, T
PloS one 8 e55075 2013
FK506 binding protein-like (FKBPL) and its peptide derivatives exert potent anti-angiogenic activity in vitro and in vivo and control tumour growth in xenograft models, when administered exogenously. However, the role of endogenous FKBPL in angiogenesis is not well characterised. Here we investigated the molecular effects of the endogenous protein and its peptide derivative, AD-01, leading to their anti-migratory activity. Inhibition of secreted FKBPL using a blocking antibody or siRNA-mediated knockdown of FKBPL accelerated the migration of human microvascular endothelial cells (HMEC-1). Furthermore, MDA-MB-231 tumour cells stably overexpressing FKBPL inhibited tumour vascular development in vivo suggesting that FKBPL secreted from tumour cells could inhibit angiogenesis. Whilst FKBPL and AD-01 target CD44, the nature of this interaction is not known and here we have further interrogated this aspect. We have demonstrated that FKBPL and AD-01 bind to the CD44 receptor and inhibit tumour cell migration in a CD44 dependant manner; CD44 knockdown abrogated AD-01 binding as well as its anti-migratory activity. Interestingly, FKBPL overexpression and knockdown or treatment with AD-01, regulated CD44 expression, suggesting a co-regulatory pathway for these two proteins. Downstream of CD44, alterations in the actin cytoskeleton, indicated by intense cortical actin staining and a lack of cell spreading and communication were observed following treatment with AD-01, explaining the anti-migratory phenotype. Concomitantly, AD-01 inhibited Rac-1 activity, up-regulated RhoA and the actin binding proteins, profilin and vinculin. Thus the anti-angiogenic protein, FKBPL, and AD-01, offer a promising and alternative approach for targeting both CD44 positive tumours and vasculature networks.
|Changes in the GEF-H1 pathways after traumatic brain injury. |
Sabirzhanova, I; Liu, C; Zhao, J; Bramlett, H; Dietrich, WD; Hu, B
Journal of neurotrauma 30 1449-56 2013
Brains undergo significant remodeling after traumatic brain injury (TBI). The Rho guanine triphosphate (GTP)ase pathways control brain remodeling during development and under pathological conditions. How the Rho GTPase pathways are regulated in the brain after TBI remains largely unknown, however. This study used the rat fluid percussion injury model to investigate changes in the Rho GTPase pathways after TBI. The results showed that TBI leads to activation and translocation of RhoA and Rac1 proteins from cytosolic fraction to the membrane fraction after injury. Consistently, the Rho guanine nucleotide exchange factors GEF-H1 and Cool-2/αPix are significantly activated by dephosphorylation and accumulation in the cytosolic fractions during the post-TBI phase. Because the Rho GTPase pathways are key regulators of brain remodeling, these results depict regulatory mechanisms of the Rho GTPase pathways after TBI, and pave the way for the study of therapeutic agents targeting the Rho GTPase pathways for functional recovery after TBI.
|The Rac1 splice form Rac1b promotes K-ras-induced lung tumorigenesis. |
Zhou, C; Licciulli, S; Avila, JL; Cho, M; Troutman, S; Jiang, P; Kossenkov, AV; Showe, LC; Liu, Q; Vachani, A; Albelda, SM; Kissil, JL
Oncogene 32 903-9 2013
Rac1b, an alternative splice form of Rac1, has been previously shown to be upregulated in colon and breast cancer cells, suggesting an oncogenic role for Rac1b in these cancers. Our analysis of NSCLC tumor and matched normal tissue samples indicates Rac1b is upregulated in a significant fraction of lung tumors in correlation with mutational status of K-ras. To directly assess the oncogenic potential of Rac1b in vivo, we employed a mouse model of lung adenocarcinoma, in which the expression of Rac1b can be conditionally activated specifically in the lung. Although expression of Rac1b alone is insufficient to drive tumor initiation, the expression of Rac1b synergizes with an oncogenic allele of K-ras resulting in increased cellular proliferation and accelerated tumor growth. Finally, we show that in contrast to our previous findings demonstrating a requirement for Rac1 in K-ras-driven cell proliferation, Rac1b is not required in this context. Given the partially overlapping spectrum of downstream effectors regulated by Rac1 and Rac1b, our findings further delineate the signaling pathways downstream of Rac1 that are required for K-ras driven tumorigenesis.
|Targeting GGTase-I activates RHOA, increases macrophage reverse cholesterol transport, and reduces atherosclerosis in mice. |
Khan, OM; Akula, MK; Skålen, K; Karlsson, C; Ståhlman, M; Young, SG; Borén, J; Bergo, MO
Circulation 127 782-90 2013
Statins have antiinflammatory and antiatherogenic effects that have been attributed to inhibition of RHO protein geranylgeranylation in inflammatory cells. The activity of protein geranylgeranyltransferase type I (GGTase-I) is widely believed to promote membrane association and activation of RHO family proteins. However, we recently showed that knockout of GGTase-I in macrophages activates RHO proteins and proinflammatory signaling pathways, leading to increased cytokine production and rheumatoid arthritis. In this study, we asked whether the increased inflammatory signaling of GGTase-I-deficient macrophages would influence the development of atherosclerosis in low-density lipoprotein receptor-deficient mice.Aortic lesions in mice lacking GGTase-I in macrophages (Pggt1b▵/▵) contained significantly more T lymphocytes than the lesions in controls. Surprisingly, however, mean atherosclerotic lesion area in Pggt1b▵/▵ mice was reduced by ≈60%. GGTase-I deficiency reduced the accumulation of cholesterol esters and phospholipids in macrophages incubated with minimally modified and acetylated low-density lipoprotein. Analyses of GGTase-I-deficient macrophages revealed upregulation of the cyclooxygenase 2-peroxisome proliferator-activated-γ pathway and increased scavenger receptor class B type I- and CD36-mediated basal and high-density lipoprotein-stimulated cholesterol efflux. Lentivirus-mediated knockdown of RHOA, but not RAC1 or CDC42, normalized cholesterol efflux. The increased cholesterol efflux in cultured cells was accompanied by high levels of macrophage reverse cholesterol transport and slightly reduced plasma lipid levels in vivo.Targeting GGTase-I activates RHOA and leads to increased macrophage reverse cholesterol transport and reduced atherosclerosis development despite a significant increase in inflammation.
|p16 Stimulates CDC42-dependent migration of hepatocellular carcinoma cells. |
Chen, YW; Chu, HC; Ze-Shiang Lin, ; Shiah, WJ; Chou, CP; Klimstra, DS; Lewis, BC
PloS one 8 e69389 2013
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. Tumor dissemination to the extra-hepatic region of the portal vein, lymph nodes, lungs or bones contributes to the high mortality seen in HCC; yet, the molecular mechanisms responsible for HCC metastasis remain unclear. Prior studies have suggested a potential link between accumulated cytoplasm-localized p16 and tumor progression. Here we report that p16 enhances metastasis-associated phenotypes in HCC cells - ectopic p16 expression increased cell migration in vitro, and lung colonization after intravenous injection, whereas knockdown of endogenous p16 reduced cell migration. Interestingly, analysis of p16 mutants indicated that the Cdk4 interaction domain is required for stimulation of HCC cell migration; however, knockdown of Cdk4 and Cdk6 showed that these proteins are dispensable for this phenomenon. Intriguingly, we found that in p16-positive HCC samples, p16 protein is predominantly localized in the cytoplasm. In addition, we identified a potential role for nuclear-cytoplasmic shuttling in p16-stimulated migration, consistent with the predominantly cytoplasmic localization of p16 in IHC-positive HCC samples. Finally, we determined that p16-stimulated cell migration requires the Cdc42 GTPase. Our results demonstrate for the first time a pro-migratory role for p16, and suggest a potential mechanism for the observed association between cytoplasmic p16 and tumor progression in diverse tumor types.
|Spatial regulation of VEGF receptor endocytosis in angiogenesis. |
Nakayama, M; Nakayama, A; van Lessen, M; Yamamoto, H; Hoffmann, S; Drexler, HC; Itoh, N; Hirose, T; Breier, G; Vestweber, D; Cooper, JA; Ohno, S; Kaibuchi, K; Adams, RH
Nature cell biology 15 249-60 2013
Activities as diverse as migration, proliferation and patterning occur simultaneously and in a coordinated fashion during tissue morphogenesis. In the growing vasculature, the formation of motile, invasive and filopodia-carrying endothelial sprouts is balanced with the stabilization of blood-transporting vessels. Here, we show that sprouting endothelial cells in the retina have high rates of VEGF uptake, VEGF receptor endocytosis and turnover. These internalization processes are opposed by atypical protein kinase C activity in more stable and mature vessels. aPKC phosphorylates Dab2, a clathrin-associated sorting protein that, together with the transmembrane protein ephrin-B2 and the cell polarity regulator PAR-3, enables VEGF receptor endocytosis and downstream signal transduction. Accordingly, VEGF receptor internalization and the angiogenic growth of vascular beds are defective in loss-of-function mice lacking key components of this regulatory pathway. Our work uncovers how vessel growth is dynamically controlled by local VEGF receptor endocytosis and the activity of cell polarity proteins.
|RasGRF2 Rac-GEF activity couples NMDA receptor calcium flux to enhanced synaptic transmission. |
Schwechter, B; Rosenmund, C; Tolias, KF
Proceedings of the National Academy of Sciences of the United States of America 110 14462-7 2013
Dendritic spines are the primary sites of excitatory synaptic transmission in the vertebrate brain, and the morphology of these actin-rich structures correlates with synaptic function. Here we demonstrate a unique method for inducing spine enlargement and synaptic potentiation in dispersed hippocampal neurons, and use this technique to identify a coordinator of these processes; Ras-specific guanine nucleotide releasing factor 2 (RasGRF2). RasGRF2 is a dual Ras/Rac guanine nucleotide exchange factor (GEF) that is known to be necessary for long-term potentiation in situ. Contrary to the prevailing assumption, we find RasGRF2's Rac-GEF activity to be essential for synaptic potentiation by using a molecular replacement strategy designed to dissociate Rac- from Ras-GEF activities. Furthermore, we demonstrate that Rac1 activity itself is sufficient to rapidly modulate postsynaptic strength by using a photoactivatable derivative of this small GTPase. Because Rac1 is a major actin regulator, our results support a model where the initial phase of long-term potentiation is driven by the cytoskeleton.
|srGAP1 regulates lamellipodial dynamics and cell migratory behavior by modulating Rac1 activity. |
Yamazaki, D; Itoh, T; Miki, H; Takenawa, T
Molecular biology of the cell 24 3393-405 2013
The distinct levels of Rac activity differentially regulate the pattern of intrinsic cell migration. However, it remains unknown how Rac activity is modulated and how the level of Rac activity controls cell migratory behavior. Here we show that Slit-Robo GAP 1 (srGAP1) is a modulator of Rac activity in locomotive cells. srGAP1 possesses a GAP activity specific to Rac1 and is recruited to lamellipodia in a Rac1-dependent manner. srGAP1 limits Rac1 activity and allows concomitant activation of Rac1 and RhoA, which are mutually inhibitory. When both GTPases are activated, the protrusive structures caused by Rac1-dependent actin reorganization are spatially restricted and periodically destabilized, causing ruffling by RhoA-induced actomyosin contractility. Depletion of srGAP1 overactivates Rac1 and inactivates RhoA, resulting in continuous spatiotemporal spreading of lamellipodia and a modal shift of intrinsic cell motility from random to directionally persistent. Thus srGAP1 is a key determinant of lamellipodial dynamics and cell migratory behavior.
|The human minor histocompatibility antigen 1 is a RhoGAP. |
de Kreuk, BJ; Schaefer, A; Anthony, EC; Tol, S; Fernandez-Borja, M; Geerts, D; Pool, J; Hambach, L; Goulmy, E; Hordijk, PL
PloS one 8 e73962 2013
The human minor Histocompatibility Antigen HMHA-1 is a major target of immune responses after allogeneic stem cell transplantation applied for the treatment of leukemia and solid tumors. The restriction of its expression to hematopoietic cells and many solid tumors raised questions regarding its cellular functions. Sequence analysis of the HMHA-1 encoding HMHA1 protein revealed the presence of a possible C-terminal RhoGTPase Activating Protein (GAP) domain and an N-terminal BAR domain. Rho-family GTPases, including Rac1, Cdc42, and RhoA are key regulators of the actin cytoskeleton and control cell spreading and migration. RhoGTPase activity is under tight control as aberrant signaling can lead to pathology, including inflammation and cancer. Whereas Guanine nucleotide Exchange Factors (GEFs) mediate the exchange of GDP for GTP resulting in RhoGTPase activation, GAPs catalyze the low intrinsic GTPase activity of active RhoGTPases, resulting in inactivation. Here we identify the HMHA1 protein as a novel RhoGAP. We show that HMHA1 constructs, lacking the N-terminal region, negatively regulate the actin cytoskeleton as well as cell spreading. Furthermore, we show that HMHA1 regulates RhoGTPase activity in vitro and in vivo. Finally, we demonstrate that the HMHA1 N-terminal BAR domain is auto-inhibitory as HMHA1 mutants lacking this region, but not full-length HMHA1, showed GAP activity towards RhoGTPases. In conclusion, this study shows that HMHA1 acts as a RhoGAP to regulate GTPase activity, cytoskeletal remodeling and cell spreading, which are crucial functions in normal hematopoietic and cancer cells.
|Specific β-containing integrins exert differential control on proliferation and two-dimensional collective cell migration in mammary epithelial cells. |
Jeanes, AI; Wang, P; Moreno-Layseca, P; Paul, N; Cheung, J; Tsang, R; Akhtar, N; Foster, FM; Brennan, K; Streuli, CH
The Journal of biological chemistry 287 24103-12 2012
Understanding how cell cycle is regulated in normal mammary epithelia is essential for deciphering defects of breast cancer and therefore for developing new therapies. Signals provided by both the extracellular matrix and growth factors are essential for epithelial cell proliferation. However, the mechanisms by which adhesion controls cell cycle in normal epithelia are poorly established. In this study, we describe the consequences of removing the β1-integrin gene from primary cultures of mammary epithelial cells in situ, using CreER. Upon β1-integrin gene deletion, the cells were unable to progress efficiently through S-phase, but were still able to undergo collective two-dimensional migration. These responses are explained by the presence of β3-integrin in β1-integrin-null cells, indicating that integrins containing different β-subunits exert differential control on mammary epithelial proliferation and migration. β1-Integrin deletion did not inhibit growth factor signaling to Erk or prevent the recruitment of core adhesome components to focal adhesions. Instead the S-phase arrest resulted from defective Rac activation and Erk translocation to the nucleus. Rac inhibition prevented Erk translocation and blocked proliferation. Activated Rac1 rescued the proliferation defect in β1-integrin-depleted cells, indicating that this GTPase is essential in propagating proliferative β1-integrin signals. These results show that β1-integrins promote cell cycle in mammary epithelial cells, whereas β3-integrins are involved in migration.
|Exome sequencing identifies recurrent somatic RAC1 mutations in melanoma. |
Krauthammer, M; Kong, Y; Ha, BH; Evans, P; Bacchiocchi, A; McCusker, JP; Cheng, E; Davis, MJ; Goh, G; Choi, M; Ariyan, S; Narayan, D; Dutton-Regester, K; Capatana, A; Holman, EC; Bosenberg, M; Sznol, M; Kluger, HM; Brash, DE; Stern, DF; Materin, MA; Lo, RS; Mane, S; Ma, S; Kidd, KK; Hayward, NK; Lifton, RP; Schlessinger, J; Boggon, TJ; Halaban, R
Nature genetics 44 1006-14 2012
We characterized the mutational landscape of melanoma, the form of skin cancer with the highest mortality rate, by sequencing the exomes of 147 melanomas. Sun-exposed melanomas had markedly more ultraviolet (UV)-like Cgreater than T somatic mutations compared to sun-shielded acral, mucosal and uveal melanomas. Among the newly identified cancer genes was PPP6C, encoding a serine/threonine phosphatase, which harbored mutations that clustered in the active site in 12% of sun-exposed melanomas, exclusively in tumors with mutations in BRAF or NRAS. Notably, we identified a recurrent UV-signature, an activating mutation in RAC1 in 9.2% of sun-exposed melanomas. This activating mutation, the third most frequent in our cohort of sun-exposed melanoma after those of BRAF and NRAS, changes Pro29 to serine (RAC1(P29S)) in the highly conserved switch I domain. Crystal structures, and biochemical and functional studies of RAC1(P29S) showed that the alteration releases the conformational restraint conferred by the conserved proline, causes an increased binding of the protein to downstream effectors, and promotes melanocyte proliferation and migration. These findings raise the possibility that pharmacological inhibition of downstream effectors of RAC1 signaling could be of therapeutic benefit.
|Phenotype-based high-content chemical library screening identifies statins as inhibitors of in vivo lymphangiogenesis. |
Schulz, MM; Reisen, F; Zgraggen, S; Fischer, S; Yuen, D; Kang, GJ; Chen, L; Schneider, G; Detmar, M
Proceedings of the National Academy of Sciences of the United States of America 109 E2665-74 2012
Lymphangiogenesis plays an important role in promoting cancer metastasis to sentinel lymph nodes and beyond and also promotes organ transplant rejection. We used human lymphatic endothelial cells to establish a reliable three-dimensional lymphangiogenic sprouting assay with automated image acquisition and analysis for inhibitor screening. This high-content phenotype-based assay quantifies sprouts by automated fluorescence microscopy and newly developed analysis software. We identified signaling pathways involved in lymphangiogenic sprouting by screening the Library of Pharmacologically Active Compounds (LOPAC)(1280) collection of pharmacologically relevant compounds. Hit characterization revealed that mitogen-activated protein kinase kinase (MEK) 1/2 inhibitors substantially block lymphangiogenesis in vitro and in vivo. Importantly, the drug class of statins, for the first time, emerged as potent inhibitors of lymphangiogenic sprouting in vitro and of corneal and cutaneous lymphangiogenesis in vivo. This effect was mediated by inhibition of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and subsequently the isoprenylation of Rac1. Supplementation with the enzymatic products of HMG-CoA reductase functionally rescued lymphangiogenic sprouting and the recruitment of Rac1 to the plasma membrane.
|Traumatic noise activates Rho-family GTPases through transient cellular energy depletion. |
Chen, FQ; Zheng, HW; Hill, K; Sha, SH
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 12421-30 2012
Small GTPases mediate transmembrane signaling and regulate the actin cytoskeleton in eukaryotic cells. Here, we characterize the auditory pathology of adult male CBA/J mice exposed to traumatic noise (2-20 kHz; 106 dB; 2 h). Loss of outer hair cells was evident 1 h after noise exposure in the basal region of the cochlea and spread apically with time, leading to permanent threshold shifts of 35, 60, and 65 dB at 8, 16, and 32 kHz. Several biochemical and molecular changes correlated temporally with the loss of cells. Immediately after exposure, the concentration of ATP decreased in cochlear tissue and reached a minimum after 1 h while the immunofluorescent signal for p-AMPKα significantly increased in sensory hair cells at that time. Levels of active Rac1 increased, whereas those of active RhoA decreased significantly 1 h after noise attaining a plateau at 1-3 h; the formation of a RhoA-p140mDia complex was consistent with an activation of Rho GTPase pathways. Also at 1-3 h after exposure, the caspase-independent cell death marker, Endo G, translocated to the nuclei of outer hair cells. Finally, experiments with the inner ear HEI-OC1 cell line demonstrated that the energy-depleting agent oligomycin enhanced both Rac1 activity and cell death. The sum of the results suggests that traumatic noise induces transient cellular ATP depletion and activates Rho GTPase pathways, leading to death of outer hair cells in the cochlea.
|EGFR inhibition in glioma cells modulates Rho signaling to inhibit cell motility and invasion and cooperates with temozolomide to reduce cell growth. |
Ramis, G; Thomàs-Moyà, E; Fernández de Mattos, S; Rodríguez, J; Villalonga, P
PloS one 7 e38770 2012
Enforced EGFR activation upon gene amplification and/or mutation is a common hallmark of malignant glioma. Small molecule EGFR tyrosine kinase inhibitors, such as erlotinib (Tarceva), have shown some activity in a subset of glioma patients in recent trials, although the reported data on the cellular basis of glioma cell responsiveness to these compounds have been contradictory. Here we have used a panel of human glioma cell lines, including cells with amplified or mutant EGFR, to further characterize the cellular effects of EGFR inhibition with erlotinib. Dose-response and cellular growth assays indicate that erlotinib reduces cell proliferation in all tested cell lines without inducing cytotoxic effects. Flow cytometric analyses confirm that EGFR inhibition does not induce apoptosis in glioma cells, leading to cell cycle arrest in G(1). Interestingly, erlotinib also prevents spontaneous multicellular tumour spheroid growth in U87MG cells and cooperates with sub-optimal doses of temozolomide (TMZ) to reduce multicellular tumour spheroid growth. This cooperation appears to be schedule-dependent, since pre-treatment with erlotinib protects against TMZ-induced cytotoxicity whereas concomitant treatment results in a cooperative effect. Cell cycle arrest in erlotinib-treated cells is associated with an inhibition of ERK and Akt signaling, resulting in cyclin D1 downregulation, an increase in p27(kip1) levels and pRB hypophosphorylation. Interestingly, EGFR inhibition also perturbs Rho GTPase signaling and cellular morphology, leading to Rho/ROCK-dependent formation of actin stress fibres and the inhibition of glioma cell motility and invasion.
|GEP100/Arf6 is required for epidermal growth factor-induced ERK/Rac1 signaling and cell migration in human hepatoma HepG2 cells. |
Hu, Z; Du, J; Yang, L; Zhu, Y; Yang, Y; Zheng, D; Someya, A; Gu, L; Lu, X
PloS one 7 e38777 2012
Epidermal growth factor (EGF) signaling is implicated in the invasion and metastasis of hepatoma cells. However, the signaling pathways for EGF-induced motility of hepatoma cells remain undefined.We found that EGF dose-dependently stimulated the migration of human hepatoma cells HepG2, with the maximal effect at 10 ng/mL. Additionally, EGF increased Arf6 activity, and ectopic expression of Arf6 T27N, a dominant negative Arf6 mutant, largely abolish EGF-induced cell migration. Blocking GEP100 with GEP100 siRNA or GEP100-△PH, a pleckstrin homology (PH) domain deletion mutant of GEP100, blocked EGF-induced Arf6 activity and cell migration. EGF also increased ERK and Rac1 activity. Ectopic expression GEP100 siRNA, GEP100-△PH, or Arf6-T27N suppressed EGF-induced ERK and Rac1 activity. Furthermore, blocking ERK signaling with its inhibitor U0126 remarkably inhibited both EGF-induced Rac1 activation as well as cell migration, and ectopic expression of inactive mutant form of Rac1 (Rac1-T17N) also largely abolished EGF-induced cell migration.Taken together, this study highlights the function of the PH domain of GEP100 and its regulated Arf6/ERK/Rac1 signaling cascade in EGF-induced hepatoma cell migration. These findings could provide a rationale for designing new therapy based on inhibition of hepatoma metastasis.
|R-Ras is required for murine dendritic cell maturation and CD4+ T-cell priming. |
Singh, G; Hashimoto, D; Yan, X; Helft, J; Park, PJ; Ma, G; Qiao, RF; Kennedy, CR; Chen, SH; Merad, M; Chan, AM
Blood 119 1693-701 2012
R-Ras is a member of the RAS superfamily of small GTP-binding proteins. The physiologic function of R-Ras has not been fully elucidated. We found that R-Ras is expressed by lymphoid and nonlymphoid tissues and drastically up-regulated when bone marrow progenitors are induced to differentiate into dendritic cells (DCs). To address the role of R-Ras in DC functions, we generated a R-Ras-deficient mouse strain. We found that tumors induced in Rras(-/-) mice formed with shorter latency and attained greater tumor volumes. This finding has prompted the investigation of a role for R-Ras in the immune system. Indeed, Rras(-/-) mice were impaired in their ability to prime allogeneic and antigen-specific T-cell responses. Rras(-/-) DCs expressed lower levels of surface MHC class II and CD86 in response to lipopolysaccharide compared with wild-type DCs. This was correlated with a reduced phosphorylation of p38 and Akt. Consistently, R-Ras-GTP level was increased within 10 minutes of lipopolysaccharide stimulation. Furthermore, Rras(-/-) DCs have attenuated capacity to spread on fibronectin and form stable immunologic synapses with T cells. Altogether, these findings provide the first demonstration of a role for R-Ras in cell-mediated immunity and further expand on the complexity of small G-protein signaling in DCs.
|Interplay between β1-integrin and Rho signaling regulates differential scattering and motility of pancreatic cancer cells by snail and Slug proteins. |
Shields, MA; Krantz, SB; Bentrem, DJ; Dangi-Garimella, S; Munshi, HG
The Journal of biological chemistry 287 6218-29 2012
The Snail family of transcription factors has been implicated in pancreatic cancer progression. We recently showed that Snail (Snai1) promotes membrane-type 1 matrix metalloproteinase (MT1-MMP)- and ERK1/2-dependent scattering of pancreatic cancer cells in three-dimensional type I collagen. In this study, we examine the role of Slug (Snai2) in regulating pancreatic cancer cell scattering in three-dimensional type I collagen. Although Slug increased MT1-MMP expression and ERK1/2 activity, Slug-expressing cells failed to scatter in three-dimensional collagen. Moreover, in contrast to Snail-expressing cells, Slug-expressing cells did not demonstrate increased collagen I binding, collagen I-driven motility, or α2β1-integrin expression. Significantly, inhibiting β1-integrin function decreased migration and scattering of Snail-expressing cells in three-dimensional collagen. As Rho GTPases have been implicated in invasion and migration, we also analyzed the contribution of Rac1 and Rho signaling to the differential migration and scattering of pancreatic cancer cells. Snail-induced migration and scattering were attenuated by Rac1 inhibition. In contrast, inhibiting Rho-associated kinase ROCK1/2 increased migration and scattering of Slug-expressing cells in three-dimensional collagen and thus phenocopied the effects of Snail in pancreatic cancer cells. Additionally, the increased migration and scattering in three-dimensional collagen of Slug-expressing cells following ROCK1/2 inhibition was dependent on β1-integrin function. Overall, these results demonstrate differential effects of Snail and Slug in pancreatic cancer and identify the interplay between Rho signaling and β1-integrin that functions to regulate the differential scattering and migration of Snail- and Slug-expressing pancreatic cancer cells.
|The guanine-nucleotide-exchange factor P-Rex1 is activated by protein phosphatase 1α. |
Barber, MA; Hendrickx, A; Beullens, M; Ceulemans, H; Oxley, D; Thelen, S; Thelen, M; Bollen, M; Welch, HC
The Biochemical journal 443 173-83 2012
P-Rex1 is a GEF (guanine-nucleotide-exchange factor) for the small G-protein Rac that is activated by PIP3 (phosphatidylinositol 3,4,5-trisphosphate) and Gβγ subunits and inhibited by PKA (protein kinase A). In the present study we show that PP1α (protein phosphatase 1α) binds P-Rex1 through an RVxF-type docking motif. PP1α activates P-Rex1 directly in vitro, both independently of and additively to PIP3 and Gβγ. PP1α also substantially activates P-Rex1 in vivo, both in basal and PDGF (platelet-derived growth factor)- or LPA (lysophosphatidic acid)-stimulated cells. The phosphatase activity of PP1α is required for P-Rex1 activation. PP1β, a close homologue of PP1α, is also able to activate P-Rex1, but less effectively. PP1α stimulates P-Rex1-mediated Rac-dependent changes in endothelial cell morphology. MS analysis of wild-type P-Rex1 and a PP1α-binding-deficient mutant revealed that endogenous PP1α dephosphorylates P-Rex1 on at least three residues, Ser834, Ser1001 and Ser1165. Site-directed mutagenesis of Ser1165 to alanine caused activation of P-Rex1 to a similar degree as did PP1α, confirming Ser1165 as a dephosphorylation site important in regulating P-Rex1 Rac-GEF activity. In summary, we have identified a novel mechanism for direct activation of P-Rex1 through PP1α-dependent dephosphorylation.
|Unconjugated Bilirubin Restricts Oligodendrocyte Differentiation and Axonal Myelination. |
Barateiro, Andreia, et al.
Mol. Neurobiol., (2012) 2012
High levels of serum unconjugated bilirubin (UCB) in newborns are associated with axonal damage and glial reactivity that may contribute to subsequent neurologic injury and encephalopathy (kernicterus). Impairments in myelination and white matter damage were observed at autopsy in kernicteric infants. We have recently reported that UCB reduces oligodendrocyte progenitor cell (OPC) survival in a pure OPC in vitro proliferative culture. Here, we hypothesized that neonatal hyperbilirubinemia may also impair oligodendrocyte (OL) maturation and myelination. We used an experimental model of hyperbilirubinemia that has been shown to mimic the pathophysiological conditions leading to brain dysfunction by unbound (free) UCB. Using primary cultures of OL, we demonstrated that UCB delays cell differentiation by increasing the OPC number and reducing the number of mature OL. This finding was combined with a downregulation of Olig1 mRNA levels and upregulation of Olig2 mRNA levels. Addition of UCB, prior to or during differentiation, impaired OL morphological maturation, extension of processes and cell diameter. Both conditions reduced active guanosine triphosphate (GTP)-bound Rac1 fraction. In myelinating co-cultures of dorsal root ganglia neurons and OL, UCB treatment prior to the onset of myelination decreased oligodendroglial differentiation and the number of myelinating OL, also observed when UCB was added after the onset of myelination. In both circumstances, UCB decreased the number of myelin internodes per OL, as well as the myelin internode length. Our studies demonstrate that increased concentrations of UCB compromise myelinogenesis, thereby elucidating a potential deleterious consequence of elevated UCB.
|Constitutive overexpression of the oncogene Rac1 in the airway of recurrent respiratory papillomatosis patients is a targetable host-susceptibility factor. |
Lucs, AV; Wu, R; Mullooly, V; Abramson, AL; Steinberg, BM
Molecular medicine (Cambridge, Mass.) 18 244-9 2012
Recurrent respiratory papillomatosis (RRP) is caused by human papillomaviruses (HPVs), primarily types 6 and 11. The disease is characterized by multiple recurrences of airway papillomas, resulting in high levels of morbidity and significant mortality. The prevalence of latent HPV in the larynx of the general population is much greater than the prevalence of RRP, suggesting a host-susceptibility factor for disease. We report that the oncogene Rac1 and its downstream product cyclooxygenase-2 (COX-2) are both constitutively expressed at high levels throughout the airway of these patients, independent of active HPV infection. Use of the COX-2 inhibitor celecoxib in primary papilloma cell culture resulted in the downregulation of HPV transcription. Furthermore, a proof-of-principle study treating three patients with severe RRP with celecoxib resulted in remission of disease in all cases. Therefore, we have identified the first pharmacologically targetable host-susceptibility pathway that contributes to RRP recurrence.
|Rnd3/RhoE Is down-regulated in hepatocellular carcinoma and controls cellular invasion. |
Grise, Florence, et al.
Hepatology, 55: 1766-75 (2012) 2012
We performed a review of public microarray data that revealed a significant down-regulation of Rnd3 expression in hepatocellular carcinoma (HCC), as compared to nontumor liver. Rnd3/RhoE is an atypical RhoGTPase family member because it is always under its active GTP-bound conformation and not sensitive to classical regulators. Rnd3 down-regulation was validated by quantitative real-time polymerase chain reaction in 120 independent tumors. Moreover, Rnd3 down-expression was confirmed using immunohistochemistry on tumor sections and western blotting on human tumor and cell-line extracts. Rnd3 expression was significantly lower in invasive tumors with satellite nodules. Overexpression and silencing of Rnd3 in Hep3B cells led to decreased and increased three-dimensional cell motility, respectively. The short interfering RNA-mediated down-regulation of Rnd3 expression induced a loss of E-cadherin at cell-cell junctions that was linked to epithelial-mesenchymal transition through the up-regulation of the zinc finger E-box binding homeobox protein, ZEB2, and the down-regulation of miR-200b and miR-200c. Rnd3 knockdown mediated tumor hepatocyte invasion in a matrix-metalloproteinase-independent, and Rac1-dependent manner. CONCLUSION: Rnd3 down-regulation provides an invasive advantage to tumor hepatocytes, suggesting that RND3 might represent a metastasis suppressor gene in HCC.
|The metastasis gene NEDD9 product acts through integrin β3 and Src to promote mesenchymal motility and inhibit amoeboid motility. |
Ahn, J; Sanz-Moreno, V; Marshall, CJ
Journal of cell science 125 1814-26 2012
Neural precursor expressed, developmentally down-regulated 9 (NEDD9), a member of the Cas family of signal transduction molecules, is amplified at the genetic level in melanoma, and elevated expression levels have been shown to correlate with melanoma progression and metastasis. NEDD9 interacts with the guanine nucleotide exchange factor DOCK3 to promote Rac activation and the elongated, mesenchymal-type of tumour cell invasion, but the molecular mechanisms through which NEDD9 promotes melanoma metastasis are not fully understood. We show that signalling through increased NEDD9 levels requires integrin β3 signalling, which leads to elevated phosphorylation of integrin β3. This results in increased Src and FAK but decreased ROCK signalling to drive elongated, mesenchymal-type invasion in environments that contain vitronectin. NEDD9 overexpression does not affect ROCK signalling through activation of RhoA but decreases ROCKII signalling through Src-dependent phosphorylation of a negative regulatory site Tyr722. In NEDD9-overexpressing melanoma cells, inhibition of Src with dasatinib results in a switch from Rac-driven elongated, mesenchymal-type invasion to ROCK-dependent rounded, amoeboid invasion. These findings brings into question whether dasatinib would work as a therapeutic agent to block melanoma invasion and metastasis. On the basis of the in vitro data presented here, a combination treatment of dasatinib and a ROCK inhibitor might be a better alternative in order to inhibit both elongated, mesenchymal-type and rounded, amoeboid motility.
|Comparative study of human and mouse postsynaptic proteomes finds high compositional conservation and abundance differences for key synaptic proteins. |
Bayés, A; Collins, MO; Croning, MD; van de Lagemaat, LN; Choudhary, JS; Grant, SG
PloS one 7 e46683 2012
Direct comparison of protein components from human and mouse excitatory synapses is important for determining the suitability of mice as models of human brain disease and to understand the evolution of the mammalian brain. The postsynaptic density is a highly complex set of proteins organized into molecular networks that play a central role in behavior and disease. We report the first direct comparison of the proteome of triplicate isolates of mouse and human cortical postsynaptic densities. The mouse postsynaptic density comprised 1556 proteins and the human one 1461. A large compositional overlap was observed; more than 70% of human postsynaptic density proteins were also observed in the mouse postsynaptic density. Quantitative analysis of postsynaptic density components in both species indicates a broadly similar profile of abundance but also shows that there is higher abundance variation between species than within species. Well known components of this synaptic structure are generally more abundant in the mouse postsynaptic density. Significant inter-species abundance differences exist in some families of key postsynaptic density proteins including glutamatergic neurotransmitter receptors and adaptor proteins. Furthermore, we have identified a closely interacting set of molecules enriched in the human postsynaptic density that could be involved in dendrite and spine structural plasticity. Understanding synapse proteome diversity within and between species will be important to further our understanding of brain complexity and disease.
|BRAF and RAS oncogenes regulate Rho GTPase pathways to mediate migration and invasion properties in human colon cancer cells: a comparative study. |
Makrodouli, E; Oikonomou, E; Koc, M; Andera, L; Sasazuki, T; Shirasawa, S; Pintzas, A
Molecular cancer 10 118 2011
Colorectal cancer is a common disease that involves genetic alterations, such as inactivation of tumour suppressor genes and activation of oncogenes. Among them are RAS and BRAF mutations, which rarely coexist in the same tumour. Individual members of the Rho (Ras homology) GTPases contribute with distinct roles in tumour cell morphology, invasion and metastasis. The aim of this study is to dissect cell migration and invasion pathways that are utilised by BRAFV600E as compared to KRASG12V and HRASG12V oncoproteins. In particular, the role of RhoA (Ras homolog gene family, member A), Rac1 (Ras-related C3 botulinum toxin substrate 1) and Cdc42 (cell division cycle 42) in cancer progression induced by each of the three oncogenes is described.Colon adenocarcinoma cells with endogenous as well as ectopically expressed or silenced oncogenic mutations of BRAFV600E, KRASG12V and HRASG12V were employed. Signalling pathways and Rho GTPases were inhibited with specific kinase inhibitors and siRNAs. Cell motility and invasion properties were correlated with cytoskeletal properties and Rho GTPase activities.Evidence presented here indicate that BRAFV600E significantly induces cell migration and invasion properties in vitro in colon cancer cells, at least in part through activation of RhoA GTPase. The relationship established between BRAFV600E and RhoA activation is mediated by the MEK-ERK pathway. In parallel, KRASG12V enhances the ability of colon adenocarcinoma cells Caco-2 to migrate and invade through filopodia formation and PI3K-dependent Cdc42 activation. Ultimately increased cell migration and invasion, mediated by Rac1, along with the mesenchymal morphology obtained through the Epithelial-Mesenchymal Transition (EMT) were the main characteristics rendered by HRASG12V in Caco-2 cells. Moreover, BRAF and KRAS oncogenes are shown to cooperate with the TGFβ-1 pathway to provide cells with additional transforming properties.This study discriminates oncogene-specific cell migration and invasion pathways mediated by Rho GTPases in colon cancer cells and reveals potential new oncogene-specific characteristics for targeted therapeutics.Full Text Article
|Geranylgeranyltransferase type I (GGTase-I) deficiency hyperactivates macrophages and induces erosive arthritis in mice. |
Khan, OM; Ibrahim, MX; Jonsson, IM; Karlsson, C; Liu, M; Sjogren, AK; Olofsson, FJ; Brisslert, M; Andersson, S; Ohlsson, C; Hultén, LM; Bokarewa, M; Bergo, MO
The Journal of clinical investigation 121 628-39 2011
RHO family proteins are important for the function of inflammatory cells. They are modified with a 20-carbon geranylgeranyl lipid in a process catalyzed by protein geranylgeranyltransferase type I (GGTase-I). Geranylgeranylation is viewed as essential for the membrane targeting and activity of RHO proteins. Consequently, inhibiting GGTase-I to interfere with RHO protein activity has been proposed as a strategy to treat inflammatory disorders. However, here we show that mice lacking GGTase-I in macrophages develop severe joint inflammation resembling erosive rheumatoid arthritis. The disease was initiated by the GGTase-I-deficient macrophages and was transplantable and reversible in bone marrow transplantation experiments. The cells accumulated high levels of active GTP-bound RAC1, CDC42, and RHOA, and RAC1 remained associated with the plasma membrane. Moreover, GGTase-I deficiency activated p38 and NF-κB and increased the production of proinflammatory cytokines. The results challenge the view that geranylgeranylation is essential for the activity and localization of RHO family proteins and suggest that reduced geranylgeranylation in macrophages can initiate erosive arthritis.
|The F-BAR domain protein PACSIN2 associates with Rac1 and regulates cell spreading and migration. |
de Kreuk, BJ; Nethe, M; Fernandez-Borja, M; Anthony, EC; Hensbergen, PJ; Deelder, AM; Plomann, M; Hordijk, PL
Journal of cell science 124 2375-88 2011
The Rac1 GTPase controls cytoskeletal dynamics and is a key regulator of cell spreading and migration mediated by signaling through effector proteins, such as the PAK kinases and the Scar and WAVE proteins. We previously identified a series of regulatory proteins that associate with Rac1 through its hypervariable C-terminal domain, including the Rac1 activator β-Pix (also known as Rho guanine-nucleotide-exchange factor 7) and the membrane adapter caveolin-1. Here, we show that Rac1 associates, through its C-terminus, with the F-BAR domain protein PACSIN2, an inducer of membrane tubulation and a regulator of endocytosis. We show that Rac1 localizes with PACSIN2 at intracellular tubular structures and on early endosomes. Active Rac1 induces a loss of PACSIN2-positive tubular structures. By contrast, Rac1 inhibition results in an accumulation of PACSIN2-positive tubules. In addition, PACSIN2 appears to regulate Rac1 signaling; siRNA-mediated loss of PACSIN2 increases the levels of Rac1-GTP and promotes cell spreading and migration in a wound healing assay. Moreover, ectopic expression of PACSIN2 reduces Rac1-GTP levels in a fashion that is dependent on the PACSIN2-Rac1 interaction, on the membrane-tubulating capacity of PACSIN2 and on dynamin. These data identify the BAR-domain protein PACSIN2 as a Rac1 interactor that regulates Rac1-mediated cell spreading and migration.
|Dual function of protein kinase C (PKC) in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced manganese superoxide dismutase (MnSOD) expression: activation of CREB and FOXO3a by PKC-alpha phosphorylation and by PKC-mediated inactivation of Akt, respectively. |
Chung, YW; Kim, HK; Kim, IY; Yim, MB; Chock, PB
The Journal of biological chemistry 286 29681-90 2011
12-O-tetradecanoylphorbol-13-acetate (TPA) has been shown to induce transcriptional activation of human manganese superoxide dismutase (MnSOD) mRNA in human lung carcinoma cells, A549, mediated by a protein kinase C (PKC)-dependent activation of cAMP-responsive element-binding protein (CREB)-1/ATF-1-like factors. In this study, we showed that MnSOD protein expression was elevated in response to TPA or TNF-α, but not to hydrogen peroxide treatment. TPA-induced generation of reactive oxygen species (ROS) was blocked by pretreatment of the PKC inhibitor BIM and NADPH oxidase inhibitor DPI. Small interfering RNA (siRNA) experiments indicated that knocking down the NADPH oxidase components e.g. Rac1, p22(phox), p67(phox), and NOXO1 in A549 cells impaired TPA-induced MnSOD expression. To identify the PKC isozyme involved, we used a sod2 gene response reporter plasmid, pSODLUC-3340-I2E-C, capable of sensing the effect of TNF-α and TPA, to monitor the effects of PKC isozyme-specific inhibitors and siRNA-induced knockdown of specific PKC isozyme. Our data indicate that TPA-induced MnSOD expression was independent of p53 and most likely mediated by PKC-α-, and -ε-dependent signaling pathways. Furthermore, siRNA-induced knock-down of CREB and Forkhead box class O (FOXO) 3a led to a reduction in TPA-induced MnSOD gene expression. Together, our results revealed that TPA up-regulates, in part, two PKC-dependent transcriptional pathways to induce MnSOD expression. One pathway involves PKC-α catalyzed phosphorylation of CREB and the other involves a PKC-mediated the PP2A catalyzed dephosphorylation of Akt at Ser(473) which in turn leads to FOXO3a Ser(253) dephosphorylation and its activation.
|P-Rex1 and Vav1 cooperate in the regulation of formyl-methionyl-leucyl-phenylalanine-dependent neutrophil responses. |
Lawson, CD; Donald, S; Anderson, KE; Patton, DT; Welch, HC
Journal of immunology (Baltimore, Md. : 1950) 186 1467-76 2011
G protein-coupled receptor (GPCR) activation elicits neutrophil responses such as chemotaxis and reactive oxygen species (ROS) formation, which depend on the small G protein Rac and are essential for host defense. P-Rex and Vav are two families of guanine-nucleotide exchange factors (GEFs) for Rac, which are activated through distinct mechanisms but can both control GPCR-dependent neutrophil responses. It is currently unknown whether they play specific roles or whether they can compensate for each other in controlling these responses. In this study, we have assessed the function of neutrophils from mice deficient in P-Rex and/or Vav family GEFs. We found that both the P-Rex and the Vav family are important for LPS priming of ROS formation, whereas particle-induced ROS responses and cell spreading are controlled by the Vav family alone. Surprisingly, fMLF-stimulated ROS formation, adhesion, and chemotaxis were synergistically controlled by P-Rex1 and Vav1. These responses were more severely impaired in neutrophils lacking both P-Rex1 and Vav1 than those lacking the entire P-Rex family, the entire Vav family, or both P-Rex1 and Vav3. P-Rex1/Vav1 (P1V1) double-deficient cells also showed the strongest reduction in fMLF-stimulated activation of Rac1 and Rac2. This reduction in Rac activity may be sufficient to cause the defects observed in fMLF-stimulated P1V1 neutrophil responses. Additionally, Mac-1 surface expression was reduced in P1V1 cells, which might contribute further to defects in responses involving integrins, such as GPCR-stimulated adhesion and chemotaxis. We conclude that P-Rex1 and Vav1 together are the major fMLFR-dependent Dbl family Rac-GEFs in neutrophils and cooperate in the control of fMLF-stimulated neutrophil responses.
|RhoA and RhoC have distinct roles in migration and invasion by acting through different targets. |
Vega, FM; Fruhwirth, G; Ng, T; Ridley, AJ
The Journal of cell biology 193 655-65 2011
Several studies suggest that RhoA and RhoC, despite their sequence similarity, have different roles in cell migration and invasion, but the molecular basis for this is not known. Using RNAi, we show that RhoA-depleted cells became elongated and extended multiple Rac1-driven narrow protrusions in 2D and 3D environments, leading to increased invasion. These phenotypes were caused by combined but distinct effects of the Rho-regulated kinases ROCK1 and ROCK2. Depletion of ROCK2 induced multiple delocalized protrusions and reduced migratory polarity, whereas ROCK1 depletion selectively led to cell elongation and defective tail retraction. In contrast, RhoC depletion increased cell spreading and induced Rac1 activation around the periphery in broad lamellipodia, thereby inhibiting directed migration and invasion. These effects of RhoC depletion are mediated by the formin FMNL3, which we identify as a new target of RhoC but not RhoA. We propose that RhoA contributes to migratory cell polarity through ROCK2-mediated suppression of Rac1 activity in lamellipodia, whereas RhoC promotes polarized migration through FMNL3 by restricting lamellipodial broadening.
|AP-1 (Fra-1/c-Jun)-mediated induction of expression of matrix metalloproteinase-2 is required for 15S-hydroxyeicosatetraenoic acid-induced angiogenesis. |
Singh, NK; Quyen, DV; Kundumani-Sridharan, V; Brooks, PC; Rao, GN
The Journal of biological chemistry 285 16830-43 2010
To understand the involvement of matrix metalloproteinases (MMPs) in 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE)-induced angiogenesis, we have studied the role of MMP-2. 15(S)-HETE induced MMP-2 expression and activity in a time-dependent manner in human dermal microvascular endothelial cells (HDMVECs). Inhibition of MMP-2 activity or depletion of its levels attenuated 15(S)-HETE-induced HDMVEC migration, tube formation, and Matrigel plug angiogenesis. 15(S)-HETE also induced Fra-1 and c-Jun expression in a Rac1-MEK1-JNK1-dependent manner. In addition, 15(S)-HETE-induced MMP-2 expression and activity were mediated by Rac1-MEK1-JNK1-dependent activation of AP-1 (Fra-1/c-Jun). Cloning and site-directed mutagenesis of MMP-2 promoter revealed that AP-1 site proximal to the transcriptional start site is required for 15(S)-HETE-induced MMP-2 expression, and Fra-1 and c-Jun are the essential components of AP-1 that bind to MMP-2 promoter in response to 15(S)-HETE. Hind limb ischemia led to an increase in MEK1 and JNK1 activation and Fra-1, c-Jun, and MMP-2 expression resulting in enhanced neovascularization and recovery of blood perfusion in wild-type mice as compared with 12/15-Lox(-/-) mice. Together, these results provide the first direct evidence for a role of 12/15-Lox-12/15(S)-HETE axis in the regulation of ischemia-induced angiogenesis.Full Text Article
|Clostridial glucosylating toxins enter cells via clathrin-mediated endocytosis. |
Papatheodorou, P; Zamboglou, C; Genisyuerek, S; Guttenberg, G; Aktories, K
PloS one 5 e10673 2010
Clostridium difficile toxin A (TcdA) and toxin B (TcdB), C. sordellii lethal toxin (TcsL) and C. novyi alpha-toxin (TcnA) are important pathogenicity factors, which represent the family of the clostridial glucosylating toxins (CGTs). Toxin A and B are associated with antibiotic-associated diarrhea and pseudomembraneous colitis. Lethal toxin is involved in toxic shock syndrome after abortion and alpha-toxin in gas gangrene development. CGTs enter cells via receptor-mediated endocytosis and require an acidified endosome for translocation of the catalytic domain into the cytosol. Here we studied the endocytic processes that mediate cell internalization of the CGTs. Intoxication of cells was monitored by analyzing cell morphology, status of Rac glucosylation in cell lysates and transepithelial resistance of cell monolayers. We found that the intoxication of cultured cells by CGTs was strongly delayed when cells were preincubated with dynasore, a cell-permeable inhibitor of dynamin, or chlorpromazine, an inhibitor of the clathrin-dependent endocytic pathway. Additional evidence about the role of clathrin in the uptake of the prototypical CGT family member toxin B was achieved by expression of a dominant-negative inhibitor of the clathrin-mediated endocytosis (Eps15 DN) or by siRNA against the clathrin heavy chain. Accordingly, cells that expressed dominant-negative caveolin-1 were not protected from toxin B-induced cell rounding. In addition, lipid rafts impairment by exogenous depletion of sphingomyelin did not decelerate intoxication of HeLa cells by CGTs. Taken together, our data indicate that the endocytic uptake of the CGTs involves a dynamin-dependent process that is mainly governed by clathrin.
|Breast cancer-specific mutations in CK1epsilon inhibit Wnt/beta-catenin and activate the Wnt/Rac1/JNK and NFAT pathways to decrease cell adhesion and promote cell migration. |
Foldynová-Trantírková, S; Sekyrová, P; Tmejová, K; Brumovská, E; Bernatík, O; Blankenfeldt, W; Krejcí, P; Kozubík, A; Dolezal, T; Trantírek, L; Bryja, V
Breast cancer research : BCR 12 R30 2010
Breast cancer is one of the most common types of cancer in women. One of the genes that were found mutated in breast cancer is casein kinase 1 epsilon (CK1epsilon). Because CK1epsilon is a crucial regulator of the Wnt signaling cascades, we determined how these CK1epsilon mutations interfere with the Wnt pathway and affect the behavior of epithelial breast cancer cell lines.We performed in silico modeling of various mutations and analyzed the kinase activity of the CK1epsilon mutants both in vitro and in vivo. Furthermore, we used reporter and small GTPase assays to identify how mutation of CK1epsilon affects different branches of the Wnt signaling pathway. Based on these results, we employed cell adhesion and cell migration assays in MCF7 cells to demonstrate a crucial role for CK1epsilon in these processes.In silico modeling and in vivo data showed that autophosphorylation at Thr 44, a site adjacent to the breast cancer point mutations in the N-terminal lobe of human CK1epsilon, is involved in positive regulation of the CK1epsilon activity. Our data further demonstrate that, in mammalian cells, mutated forms of CK1epsilon failed to affect the intracellular localization and phosphorylation of Dvl2; we were able to demonstrate that CK1epsilon mutants were unable to enhance Dvl-induced TCF/LEF-mediated transcription, that CK1epsilon mutants acted as loss-of-function in the Wnt/beta-catenin pathway, and that CK1epsilon mutants activated the noncanonical Wnt/Rac-1 and NFAT pathways, similar to pharmacological inhibitors of CK1. In line with these findings, inhibition of CK1 promoted cell migration as well as decreased cell adhesion and E-cadherin expression in the breast cancer-derived cell line MCF7.In summary, these data suggest that the mutations of CK1epsilon found in breast cancer can suppress Wnt/beta-catenin as well as promote the Wnt/Rac-1/JNK and Wnt/NFAT pathways, thus contributing to breast cancer development via effects on cell adhesion and migration. In terms of molecular mechanism, our data indicate that the breast cancer point mutations in the N-terminal lobe of CK1epsilon, which are correlated with decreased phosphorylation activities of mutated forms of CK1epsilon both in vitro and in vivo, interfere with positive autophosphorylation at Thr 44.
|p120ctn and P-cadherin but not E-cadherin regulate cell motility and invasion of DU145 prostate cancer cells. |
Kümper, S; Ridley, AJ
PloS one 5 e11801 2010
Adherens junctions consist of transmembrane cadherins, which interact intracellularly with p120ctn, beta-catenin and alpha-catenin. p120ctn is known to regulate cell-cell adhesion by increasing cadherin stability, but the effects of other adherens junction components on cell-cell adhesion have not been compared with that of p120ctn.We show that depletion of p120ctn by small interfering RNA (siRNA) in DU145 prostate cancer and MCF10A breast epithelial cells reduces the expression levels of the adherens junction proteins, E-cadherin, P-cadherin, beta-catenin and alpha-catenin, and induces loss of cell-cell adhesion. p120ctn-depleted cells also have increased migration speed and invasion, which correlates with increased Rap1 but not Rac1 or RhoA activity. Downregulation of P-cadherin, beta-catenin and alpha-catenin but not E-cadherin induces a loss of cell-cell adhesion, increased migration and enhanced invasion similar to p120ctn depletion. However, only p120ctn depletion leads to a decrease in the levels of other adherens junction proteins.Our data indicate that P-cadherin but not E-cadherin is important for maintaining adherens junctions in DU145 and MCF10A cells, and that depletion of any of the cadherin-associated proteins, p120ctn, beta-catenin or alpha-catenin, is sufficient to disrupt adherens junctions in DU145 cells and increase migration and cancer cell invasion.Full Text Article
|Role of Nogo-A in neuronal survival in the reperfused ischemic brain. |
Kilic, E; ElAli, A; Kilic, U; Guo, Z; Ugur, M; Uslu, U; Bassetti, CL; Schwab, ME; Hermann, DM
Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 30 969-84 2010
Nogo-A is an oligodendroglial neurite outgrowth inhibitor, the deactivation of which enhances brain plasticity and functional recovery in animal models of stroke. Nogo-A's role in the reperfused brain tissue was still unknown. By using Nogo-A(-/-) mice and mice in which Nogo-A was blocked with a neutralizing antibody (11C7) that was infused into the lateral ventricle or striatum, we show that Nogo-A inhibition goes along with decreased neuronal survival and more protracted neurologic recovery, when deactivation is constitutive or induced 24 h before, but not after focal cerebral ischemia. We show that in the presence of Nogo-A, RhoA is activated and Rac1 and RhoB are deactivated, maintaining stress kinases p38/MAPK, SAPK/JNK1/2 and phosphatase-and-tensin homolog (PTEN) activities low. Nogo-A blockade leads to RhoA deactivation, thus overactivating Rac1 and RhoB, the former of which activates p38/MAPK and SAPK/JNK1/2 via direct interaction. RhoA and its effector Rho-associated coiled-coil protein kinase2 deactivation in turn stimulates PTEN, thus inhibiting Akt and ERK1/2, and initiating p53-dependent cell death. Our data suggest a novel role of Nogo-A in promoting neuronal survival by controlling Rac1/RhoA balance. Clinical trials should be aware of injurious effects of axonal growth-promoting therapies. Thus, Nogo-A antibodies should not be used in the very acute stroke phase.
|Cardiac Rac1 overexpression in mice creates a substrate for atrial arrhythmias characterized by structural remodelling. |
Reil, JC; Hohl, M; Oberhofer, M; Kazakov, A; Kaestner, L; Mueller, P; Adam, O; Maack, C; Lipp, P; Mewis, C; Allessie, M; Laufs, U; Böhm, M; Neuberger, HR
Cardiovascular research 87 485-93 2010
The small GTPase Rac1 seems to play a role in the pathogenesis of atrial fibrillation (AF). The aim of the present study was to characterize the effects of Rac1 overexpression on atrial electrophysiology.In mice with cardiac overexpression of constitutively active Rac1 (RacET), statin-treated RacET, and wild-type controls (age 6 months), conduction in the right and left atrium (RA and LA) was mapped epicardially. The atrial effective refractory period (AERP) was determined and inducibility of atrial arrhythmias was tested. Action potentials were recorded in isolated cells. Left ventricular function was measured by pressure-volume analysis. Five of 11 RacET hearts showed spontaneous or inducible atrial tachyarrhythmias vs. 0 of 9 controls (P less than 0.05). In RacET, the P-wave duration was significantly longer (26.8 +/- 2.1 vs. 16.7 +/- 1.1 ms, P = 0.001) as was total atrial activation time (RA: 13.6 +/- 4.4 vs. 3.2 +/- 0.5 ms; LA: 7.1 +/- 1.2 vs. 2.2 +/- 0.3 ms, P less than 0.01). Prolonged local conduction times occurred more often in RacET (RA: 24.4 +/- 3.8 vs. 2.7 +/- 2.1%; LA: 19.1 +/- 6.3 vs. 1.2 +/- 0.7%, P less than 0.01). The AERP and action potential duration did not differ significantly between both groups. RacET demonstrated significant atrial fibrosis but only moderate systolic heart failure. RacET and statin-treated RacET were not significantly different regarding atrial electrophysiology.The substrate for atrial arrhythmias in mice with Rac1 overexpression is characterized by conduction disturbances and atrial fibrosis. Electrical remodelling (i.e. a shortening of AERP) does not play a role. Statin treatment cannot prevent the structural and electrophysiological effects of pronounced Rac1 overexpression in this model.
|Molecular dissection of integrin signalling proteins in the control of mammary epithelial development and differentiation. |
Akhtar, N; Marlow, R; Lambert, E; Schatzmann, F; Lowe, ET; Cheung, J; Katz, E; Li, W; Wu, C; Dedhar, S; Naylor, MJ; Streuli, CH
Development (Cambridge, England) 136 1019-27 2009
Cell-matrix adhesion is essential for the development and tissue-specific functions of epithelia. For example, in the mammary gland, beta1-integrin is necessary for the normal development of alveoli and for the activation of endocrine signalling pathways that determine cellular differentiation. However, the adhesion complex proteins linking integrins with downstream effectors of hormonal signalling pathways are not known. To understand the mechanisms involved in connecting adhesion with this aspect of cell phenotype, we examined the involvement of two proximal beta1-integrin signalling intermediates, integrin-linked kinase (ILK) and focal adhesion kinase (FAK). By employing genetic analysis using the Cre-LoxP system, we provide evidence that ILK, but not FAK, has a key role in lactogenesis in vivo and in the differentiation of cultured luminal epithelial cells. Conditional deletion of ILK both in vivo and in primary cell cultures resulted in defective differentiation, by preventing phosphorylation and nuclear translocation of STAT5, a transcription factor required for lactation. Expression of an activated RAC (RAS-related C3 botulinum substrate) in ILK-null acini restored the lactation defect, indicating that RAC1 provides a mechanistic link between the integrin/ILK adhesion complex and the differentiation pathway. Thus, we have determined that ILK is an essential downstream component of integrin signalling involved in differentiation, and have identified a high degree of specificity within the integrin-based adhesome that links cell-matrix interactions with the tissue-specific function of epithelia.Full Text Article
|A role for Gab1/SHP2 in thrombin activation of PAK1: gene transfer of kinase-dead PAK1 inhibits injury-induced restenosis. |
Wang, D; Paria, BC; Zhang, Q; Karpurapu, M; Li, Q; Gerthoffer, WT; Nakaoka, Y; Rao, GN
Circulation research 104 1066-75 2009
To understand the role of epidermal growth factor receptor (EGFR) transactivation in G protein-coupled receptor (GPCR) agonist-induced signaling events, we have studied the capacity of thrombin in the activation of Gab1-SHP2 in vascular smooth muscle cells (VSMCs). Thrombin activated both Gab1 and SHP2 in EGFR-dependent manner. Similarly, thrombin induced Rac1 and Cdc42 activation, and these responses were suppressed when either Gab1 or SHP2 stimulation is blocked. Thrombin also induced PAK1 activation in a time- and EGFR-Gab1-SHP2-Rac1/Cdc42-dependent manner. Inhibition of activation of EGFR, Gab1, SHP2, Rac1, Cdc42, or PAK1 by pharmacological or genetic approaches attenuated thrombin-induced VSMC stress fiber formation and motility. Thrombin activated RhoA in a time-dependent manner in VSMCs. LARG, a RhoA-specific GEF (guanine nucleotide exchange factor), was found to be associated with Gab1 and siRNA-mediated depletion of its levels suppressed RhoA, Rac1 and PAK1 activation. Dominant negative mutant-mediated interference of RhoA activation inhibited thrombin-induced Rac1 and PAK1 stimulation in VSMCs and their stress fiber formation and migration. Balloon injury induced PAK1 activity and interference with its activation led to attenuation of SMC migration from media to intima, resulting in reduced neointima formation and increased lumen size. Inhibition of thrombin signaling by recombinant hirudin also blocked balloon injury-induced EGFR tyrosine phosphorylation and PAK1 activity. These results show that thrombin-mediated PAK1 activation plays a crucial role in vascular wall remodeling and it could be a potential target for drug development against these vascular lesions.
|Cytoskeletal changes underlie estrogen's acute effects on synaptic transmission and plasticity. |
Kramár, EA; Chen, LY; Brandon, NJ; Rex, CS; Liu, F; Gall, CM; Lynch, G
The Journal of neuroscience : the official journal of the Society for Neuroscience 29 12982-93 2009
Estrogen, in addition to its genomic effects in brain, causes rapid and reversible changes to synaptic operations. We report here that these acute actions are due to selective activation of an actin-signaling cascade normally used in the production of long-term potentiation (LTP). Estrogen, or a selective agonist of the steroid's beta-receptor, caused a modest increase in fast glutamatergic transmission and a pronounced facilitation of LTP in adult hippocampal slices; both effects were completely eliminated by latrunculin, a toxin that prevents actin filament assembly. Estrogen also increased spine concentrations of filamentous actin and strongly enhanced its polymerization in association with LTP. A search for the origins of these effects showed that estrogen activates the small GTPase RhoA and phosphorylates (inactivates) the actin severing protein cofilin, a downstream target of RhoA. Moreover, an antagonist of RhoA kinase (ROCK) blocked estrogen's synaptic effects. Estrogen thus emerges as a positive modulator of a RhoAgreater than ROCKgreater than LIM kinasegreater than cofilin pathway that regulates the subsynaptic cytoskeleton. It does not, however, strongly affect a second LTP-related pathway, involving the GTPases Rac and Cdc42 and their effector p21-activated kinase, which may explain why its acute effects are reversible. Finally, ovariectomy depressed RhoA activity, spine cytoskeletal plasticity, and LTP, whereas brief infusions of estrogen rescued plasticity, suggesting that the deficits in plasticity arise from acute, as well as genomic, consequences of hormone loss.
|A novel role for activating transcription factor-2 in 15(S)-hydroxyeicosatetraenoic acid-induced angiogenesis. |
Zhao, T; Wang, D; Cheranov, SY; Karpurapu, M; Chava, KR; Kundumani-Sridharan, V; Johnson, DA; Penn, JS; Rao, GN
Journal of lipid research 50 521-33 2009
To investigate the mechanisms underlying 15(S)-HETE-induced angiogenesis, we have studied the role of the small GTPase, Rac1. We find that 15(S)-HETE activated Rac1 in human retinal microvascular endothelial cells (HRMVEC) in a time-dependent manner. Blockade of Rac1 by adenovirus-mediated expression of its dominant negative mutant suppressed HRMVEC migration as well as tube formation and Matrigel plug angiogenesis. 15(S)-HETE stimulated Src in HRMVEC in a time-dependent manner and blockade of its activation inhibited 15(S)-HETE-induced Rac1 stimulation in HRMVEC and the migration and tube formation of these cells as well as Matrigel plug angiogenesis. 15(S)-HETE stimulated JNK1 in Src-Rac1-dependent manner in HRMVEC and adenovirus-mediated expression of its dominant negative mutant suppressed the migration and tube formation of these cells and Matrigel plug angiogenesis. 15(S)-HETE activated ATF-2 in HRMVEC in Src-Rac1-JNK1-dependent manner and interference with its activation via adenovirus-mediated expression of its dominant negative mutant abrogated migration and tube formation of HRMVEC and Matrigel plug angiogenesis. In addition, 15(S)-HETE-induced MEK1 stimulation was found to be dependent on Src-Rac1 activation. Blockade of MEK1 activation inhibited 15(S)-HETE-induced JNK1 activity and ATF-2 phosphorylation. Together, these findings show that 15(S)-HETE activates ATF-2 via the Src-Rac1-MEK1-JNK1 signaling axis in HRMVEC leading to their angiogenic differentiation.Full Text Article
|NADPH oxidase expression and production of superoxide by human corneal stromal cells. |
O'Brien WJ, Heimann T, Rizvi F
Mol Vis 15 2535-43. 2009
PURPOSE: Superoxide (O(2) (.-)) may function as a second messenger or regulator of signal transduction when produced at low concentrations in the proper locations within cells. The purpose of these studies was to determine whether human corneal stromal (HCS) fibroblasts are capable of producing O(2) (.-) via nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, a family of protein complexes believed to be responsible for the localized and limited production of O(2) (.-) with regulatory activity.Full Text Article
|Specific roles of Rac1 and Rac2 in motile functions of HT1080 fibrosarcoma cells. |
Verena Niggli, Dominique Schlicht, Sarah Affentranger, Verena Niggli, Dominique Schlicht, Sarah Affentranger
Biochemical and biophysical research communications 386 688-92 2009
Rho family proteins are constitutively activated in the highly invasive human fibrosarcoma HT1080 cells. We now investigated the specific roles of Rac1 and Rac2 in regulating morphology, F-actin organization, adhesion, migration, and chemotaxis of HT1080 cells. Downregulation of Rac1 using specific siRNA probes resulted in cell rounding, markedly decreased spreading, adhesion, and chemotaxis of HT1080 cells. 2D migration on laminin-coated surfaces in contrast was not markedly affected. Selective Rac2 depletion did not affect cell morphology, cell adhesion, and 2D migration, but significantly reduced chemotaxis. Downregulation of both Rac1 and Rac2 resulted in an even more marked reduction, but not complete abolishment, of chemotaxis indicating distinct as well as overlapping roles of both proteins in chemotaxis. Rac1 thus is selectively required for HT1080 cell spreading and adhesion whereas Rac1 and Rac2 are both required for efficient chemotaxis.
|Rosuvastatin ameliorates the development of pulmonary arterial hypertension in the transgenic (mRen2)27 rat. |
DeMarco, VG; Habibi, J; Whaley-Connell, AT; Schneider, RI; Sowers, JR; Andresen, BT; Gutweiler, AA; Ma, L; Johnson, MS; Ferrario, CM; Dellsperger, KC
American journal of physiology. Heart and circulatory physiology 297 H1128-39 2009
We have recently reported that transgenic (mRen2)27 rats (Ren2 rats) exhibit pulmonary arterial hypertension (PAH), which is, in part, mediated by oxidative stress. Since 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) exhibit beneficial vascular effects independent of cholesterol synthesis, we hypothesized that rosuvastatin (RSV) treatment ameliorates PAH and pulmonary vascular remodeling in Ren2 rats, in part, by reducing oxidative stress. Six-week-old male Ren2 and Sprague-Dawley rats received RSV (10 mg x kg(-1) x day(-)1 ip) or vehicle for 3 wk. After treatment, right ventricular systolic pressure (RVSP) and mean arterial pressure (MAP) were measured. To evaluate treatment effects on pulmonary arteriole remodeling, morphometric analyses were performed to quantitate medial thickening and cell proliferation, whereas whole lung samples were used to quantitate the levels of 3-nitrotyrosine, superoxide, stable nitric oxide (NO) metabolites [nitrates and nitrites (NO(x))], and expression of NO synthase isoforms. In the Ren2 rat, RVSP is normal at 5 wk of age, PAH develops between 5 and 7 wk of age, and the elevated pressure is maintained with little variation through 13 wk. At 8 wk of age, left ventricular function and blood gases were normal in the Ren2 rat. Ren2 rats exhibited elevations in medial hypertrophy due to smooth muscle cell proliferation, 3-nitrotyrosine, NO(x), NADPH oxidase activity, and endothelial NO synthase expression compared with Sprague-Dawley rats. RSV significantly blunted the increase in RVSP but did not reduce MAP in the Ren2 rat; additionally, RSV significantly attenuated the elevated parameters examined in the Ren2 rat. These data suggest that statins may be a clinically viable adjunct treatment of PAH through reducing peroxynitrite formation.Full Text Article
|HSP90 beta regulates rapsyn turnover and subsequent AChR cluster formation and maintenance. |
Luo, S; Zhang, B; Dong, XP; Tao, Y; Ting, A; Zhou, Z; Meixiong, J; Luo, J; Chiu, FC; Xiong, WC; Mei, L
Neuron 60 97-110 2008
Rapsyn, an acetylcholine receptor (AChR)-interacting protein, is essential for synapse formation at the neuromuscular junction (NMJ). Like many synaptic proteins, rapsyn turns over rapidly at synapses. However, little is known about molecular mechanisms that govern rapsyn stability. Using a differential mass-spectrometry approach, we identified heat-shock protein 90beta (HSP90beta) as a component in surface AChR clusters. The HSP90beta-AChR interaction required rapsyn and was stimulated by agrin. Inhibition of HSP90beta activity or expression, or disruption of its interaction with rapsyn attenuated agrin-induced formation of AChR clusters in vitro and impaired the development and maintenance of the NMJ in vivo. Finally, we showed that HSP90beta was necessary for rapsyn stabilization and regulated its proteasome-dependent degradation. Together, these results indicate a role of HSP90beta in NMJ development by regulating rapsyn turnover and subsequent AChR cluster formation and maintenance.
|Distinct roles of class IA PI3K isoforms in primary and immortalised macrophages. |
Papakonstanti, EA; Zwaenepoel, O; Bilancio, A; Burns, E; Nock, GE; Houseman, B; Shokat, K; Ridley, AJ; Vanhaesebroeck, B
Journal of cell science 121 4124-33 2008
The class IA isoforms of phosphoinositide 3-kinase (p110alpha, p110beta and p110delta) often have non-redundant functions in a given cell type. However, for reasons that are unclear, the role of a specific PI3K isoform can vary between cell types. Here, we compare the relative contributions of PI3K isoforms in primary and immortalised macrophages. In primary macrophages stimulated with the tyrosine kinase ligand colony-stimulating factor 1 (CSF1), all class IA PI3K isoforms participate in the regulation of Rac1, whereas p110delta selectively controls the activities of Akt, RhoA and PTEN, in addition to controlling proliferation and chemotaxis. The prominent role of p110delta in these cells correlates with it being the main PI3K isoform that is recruited to the activated CSF1 receptor (CSF1R). In immortalised BAC1.2F5 macrophages, however, the CSF1R also engages p110alpha, which takes up a more prominent role in CSF1R signalling, in processes including Akt phosphorylation and regulation of DNA synthesis. Cell migration, however, remains dependent mainly on p110delta. In other immortalised macrophage cell lines, such as IC-21 and J774.2, p110alpha also becomes more prominently involved in CSF1-induced Akt phosphorylation, at the expense of p110delta.These data show that PI3K isoforms can be differentially regulated in distinct cellular contexts, with the dominant role of the p110delta isoform in Akt phosphorylation and proliferation being lost upon cell immortalisation. These findings suggest that p110delta-selective PI3K inhibitors may be more effective in inflammation than in cancer.
|Delta-catenin-induced dendritic morphogenesis. An essential role of p190RhoGEF interaction through Akt1-mediated phosphorylation. |
Kim, H; Han, JR; Park, J; Oh, M; James, SE; Chang, S; Lu, Q; Lee, KY; Ki, H; Song, WJ; Kim, K
The Journal of biological chemistry 283 977-87 2008
Delta-catenin was first identified through its interaction with Presenilin-1 and has been implicated in the regulation of dendrogenesis and cognitive function. However, the molecular mechanisms by which delta-catenin promotes dendritic morphogenesis were unclear. In this study, we demonstrated delta-catenin interaction with p190RhoGEF, and the importance of Akt1-mediated phosphorylation at Thr-454 residue of delta-catenin in this interaction. We have also found that delta-catenin overexpression decreased the binding between p190RhoGEF and RhoA, and significantly lowered the levels of GTP-RhoA but not those of GTP-Rac1 and -Cdc42. Delta-catenin T454A, a defective form in p190RhoGEF binding, did not decrease the binding between p190RhoGEF and RhoA. Delta-catenin T454A also did not lower GTP-RhoA levels and failed to induce dendrite-like process formation in NIH 3T3 fibroblasts. Furthermore, delta-catenin T454A significantly reduced the length and number of mature mushroom shaped spines in primary hippocampal neurons. These results highlight signaling events in the regulation of delta-catenin-induced dendrogenesis and spine morphogenesis.
|G alpha o mediates WNT-JNK signaling through dishevelled 1 and 3, RhoA family members, and MEKK 1 and 4 in mammalian cells. |
Bikkavilli, RK; Feigin, ME; Malbon, CC
Journal of cell science 121 234-45 2008
In Drosophila, activation of Jun N-terminal Kinase (JNK) mediated by Frizzled and Dishevelled leads to signaling linked to planar cell polarity. A biochemical delineation of WNT-JNK planar cell polarity was sought in mammalian cells, making use of totipotent mouse F9 teratocarcinoma cells that respond to WNT3a via Frizzled-1. The canonical WNT-beta-catenin signaling pathway requires both G alpha o and G alpha q heterotrimeric G-proteins, whereas we show that WNT-JNK signaling requires only G alpha o protein. G alpha o propagates the signal downstream through all three Dishevelled isoforms, as determined by epistasis experiments using the Dishevelled antagonist Dapper1 (DACT1). Suppression of either Dishevelled-1 or Dishevelled-3, but not Dishevelled-2, abolishes WNT3a activation of JNK. Activation of the small GTPases RhoA, Rac1 and Cdc42 operates downstream of Dishevelled, linking to the MEKK 1/MEKK 4-dependent cascade, and on to JNK activation. Chemical inhibitors of JNK (SP600125), but not p38 (SB203580), block WNT3a activation of JNK, whereas both the inhibitors attenuate the WNT3a-beta-catenin pathway. These data reveal both common and unique signaling elements in WNT3a-sensitive pathways, highlighting crosstalk from WNT3a-JNK to WNT3a-beta-catenin signaling.
|Simvastatin inhibits Staphylococcus aureus host cell invasion through modulation of isoprenoid intermediates. |
Horn, MP; Knecht, SM; Rushing, FL; Birdsong, J; Siddall, CP; Johnson, CM; Abraham, TN; Brown, A; Volk, CB; Gammon, K; Bishop, DL; McKillip, JL; McDowell, SA
The Journal of pharmacology and experimental therapeutics 326 135-43 2008
Patients on a statin regimen have a decreased risk of death due to bacterial sepsis. We have found that protection by simvastatin includes the inhibition of host cell invasion by Staphylococcus aureus, the most common etiologic agent of sepsis. Inhibition was due in part to depletion of isoprenoid intermediates within the cholesterol biosynthesis pathway and led to the cytosolic accumulation of the small GTPases CDC42, Rac, and RhoB. Actin stress fiber disassembly required for host invasion was attenuated by simvastatin and by the inhibition of phosphoinositide 3-kinase (PI3K) activity. PI3K relies on coupling to prenylated proteins, such as this subset of small GTPases, for access to membrane-bound phosphoinositide to mediate stress fiber disassembly. Therefore, we examined whether simvastatin restricts PI3K cellular localization. In response to simvastatin, the PI3K isoform p85, coupled to these small-GTPases, was sequestered within the cytosol. From these findings, we propose a mechanism whereby simvastatin restricts p85 localization, inhibiting the actin dynamics required for bacterial endocytosis. This approach may provide the basis for protection at the level of the host in invasive infections by S. aureus.
|X-linked chronic granulomatous disease secondary to skewed X chromosome inactivation in a female with a novel CYBB mutation and late presentation. |
Lewis, EM; Singla, M; Sergeant, S; Koty, PP; McPhail, LC
Clinical immunology (Orlando, Fla.) 129 372-80 2008
Chronic granulomatous disease (CGD) is characterized by defects in the superoxide producing enzyme NADPH oxidase causing phagocytes to improperly clear invading pathogens. Here we report findings of a late presenting 16-year-old female with X-linked CGD. The patient presented with community-acquired pneumonia, but symptoms persisted for 2 weeks during triple antimicrobial coverage. Cultures revealed Aspergillus fumigatus which was resolved through aggressive voriconazole treatment. Neutrophil studies revealed NADPH oxidase activity and flavocytochrome b(558) levels that were 4-8% of controls and suggested carrier status of the mother. We found a null mutation in the CYBB gene (c.252insAG) predicting an aberrant gp91(phox) protein (p.Cys85fsX23) in the heterozygous state. Methylation analysis demonstrated extremely skewed X chromosome inactivation favoring the maternally inherited defective gene. In conclusion, a novel mutation in the CYBB gene and an extremely skewed X-inactivation event resulted in the rare expression of the CGD phenotype in a carrier female.
|Phosphatidylinositol 3-kinase-dependent membrane recruitment of Rac-1 and p47phox is critical for alpha-platelet-derived growth factor receptor-induced production of reactive oxygen species. |
Bäumer, AT; Ten Freyhaus, H; Sauer, H; Wartenberg, M; Kappert, K; Schnabel, P; Konkol, C; Hescheler, J; Vantler, M; Rosenkranz, S
The Journal of biological chemistry 283 7864-76 2008
Platelet-derived growth factor (PDGF) plays a critical role in the pathogenesis of proliferative diseases. NAD(P)H oxidase (Nox)-derived reactive oxygen species (ROS) are essential for signal transduction by growth factor receptors. Here we investigated the dependence of PDGF-AA-induced ROS production on the cytosolic Nox subunits Rac-1 and p47(phox), and we systematically evaluated the signal relay mechanisms by which the alphaPDGF receptor (alphaPDGFR) induces ROS liberation. Stimulation of the alphaPDGFR led to a time-dependent increase of intracellular ROS levels in fibroblasts. Pharmacological inhibitor experiments and enzyme activity assays disclosed Nox as the source of ROS. alphaPDGFR activation is rapidly followed by the translocation of p47(phox) and Rac-1 from the cytosol to the cell membrane. Experiments performed in p47(phox)(-/-) cells and inhibition of Rac-1 or overexpression of dominant-negative Rac revealed that these Nox subunits are required for PDGF-dependent Nox activation and ROS liberation. To evaluate the signaling pathway mediating PDGF-AA-dependent ROS production, we investigated Ph cells expressing mutant alphaPDGFRs that lack specific binding sites for alphaPDGFR-associated signaling molecules (Src, phosphatidylinositol 3-kinase (PI3K), phospholipase Cgamma, and SHP-2). Lack of PI3K signaling (but not Src, phospholipase Cgamma, or SHP-2) completely abolished PDGF-dependent p47(phox) and Rac-1 translocation, increase of Nox activity, and ROS production. Conversely, a mutant alphaPDGFR able to activate only PI3K was sufficient to mediate these subcellular events. Furthermore, the catalytic PI3K subunit p110alpha (but not p110beta) was identified as the crucial isoform that elicits alphaPDGFR-mediated production of ROS. Finally, bromodeoxyuridine incorporation and chemotaxis assays revealed that the lack of ROS liberation blunted PDGF-AA-dependent chemotaxis but not cell cycle progression. We conclude that PI3K/p110alpha mediates growth factor-dependent ROS production by recruiting p47(phox) and Rac-1 to the cell membrane, thereby assembling the active Nox complex. ROS are required for PDGF-AA-dependent chemotaxis but not proliferation.
|Rac-dependent cyclin D1 gene expression regulated by cadherin- and integrin-mediated adhesion. |
Fournier, AK; Campbell, LE; Castagnino, P; Liu, WF; Chung, BM; Weaver, VM; Chen, CS; Assoian, RK
Journal of cell science 121 226-33 2008
Integrin-mediated adhesion to substratum is required for cyclin D1 induction in mesenchymal cells, but we show here that the induction of cyclin D1 persists despite blockade of ECM-integrin signaling in MCF10A mammary epithelial cells. E-cadherin-mediated cell-cell adhesion also supports cyclin D1 induction in these cells, and the combined inhibition of both E-cadherin and integrin adhesion is required to prevent the expression of cyclin D1 mRNA and protein. Our previous studies described a pro-proliferative effect of E-cadherin in MCF10A cells, mediated by Rac, and we now show that Rac is required for cyclin D1 mRNA induction by both E-cadherin and integrin engagement. The levels of p21Cip1 and p27Kip1, Cdk inhibitors that are also targets of integrin signaling, are not affected by E-cadherin-mediated cell-cell adhesion. Finally, we show that the increased expression of cyclin D1 mRNA associated with E-cadherin-dependent cell-cell adhesion is causally linked to an increased entry into S phase. Our results identify Rac signaling to cyclin D1 as a crucial pro-proliferative effect of E-cadherin-mediated cell-cell adhesion.Full Text Article
|SOD1 mutations disrupt redox-sensitive Rac regulation of NADPH oxidase in a familial ALS model. |
Harraz, Maged M, et al.
J. Clin. Invest., 118: 659-70 (2008) 2008
Neurodegeneration in familial amyotrophic lateral sclerosis (ALS) is associated with enhanced redox stress caused by dominant mutations in superoxide dismutase-1 (SOD1). SOD1 is a cytosolic enzyme that facilitates the conversion of superoxide (O(2)(*-)) to H(2)O(2). Here we demonstrate that SOD1 is not just a catabolic enzyme, but can also directly regulate NADPH oxidase-dependent (Nox-dependent) O(2)(*-) production by binding Rac1 and inhibiting its GTPase activity. Oxidation of Rac1 by H(2)O(2) uncoupled SOD1 binding in a reversible fashion, producing a self-regulating redox sensor for Nox-derived O(2)(*-) production. This process of redox-sensitive uncoupling of SOD1 from Rac1 was defective in SOD1 ALS mutants, leading to enhanced Rac1/Nox activation in transgenic mouse tissues and cell lines expressing ALS SOD1 mutants. Glial cell toxicity associated with expression of SOD1 mutants in culture was significantly attenuated by treatment with the Nox inhibitor apocynin. Treatment of ALS mice with apocynin also significantly increased their average life span. This redox sensor mechanism may explain the gain-of-function seen with certain SOD1 mutations associated with ALS and defines new therapeutic targets.
|Prostaglandin E2-mediated migration of human trophoblast requires RAC1 and CDC42. |
Nicola, C; Lala, PK; Chakraborty, C
Biology of reproduction 78 976-82 2008
The invasion of maternal decidua and uterine spiral arteries by a trophoblast subpopulation called extravillous trophoblast (EVT) is essential for the establishment of a normal placenta and an adequate blood flow toward the fetus. Derangements in these processes underlie pregnancy-related diseases like preeclampsia and intrauterine growth restriction. Many growth factors, growth factor binding proteins, and extracellular matrix components can positively or negatively regulate the proliferation, migration, and/or invasiveness of these EVT cells. RHO GTPases, including RHOA, RAC1, and CDC42, are ubiquitous proteins that control cytoskeletal changes by forming stress fibers and projecting lamellipodia and filopodia during cellular migration. We had previously shown that prostaglandin (PG) E(2) produced in abundance by the decidua promotes the migration of first-trimester human EVTs by increasing the intracellular concentration of calcium and activating calpain. Using our well-characterized immortalized EVT cell line, HTR-8/SVneo, as well as villus explants from first-trimester placentae, this study examined the role of RHO GTPases RAC1 and CDC42 in PGE(2)-mediated migratory responses of these cells. Though a RAC1 inhibitor, NSC23766 as well as RAC1 knockdown by siRNA decreased the migration of HTR-8/SVneo cells in a Transwell migration assay, this inhibition could not be restored by PGE(2) or 17-phenyl trinor PGE(2) (PGE receptor PTGER1 agonist) or PGE(1) Alcohol (PGE receptor PTGER4 agonist). Similar results were noted for EVT cell spreading in villus explants. Furthermore, CDC42 silencing using siRNA inhibited PGE(2)-induced migration of HTR-8/SVneo cells. Finally, the treatment of EVT cells with PGE(2), PTGER1 agonist, or PTGER4 agonist activated RAC1 and CDC42 at 10 min, suggesting that RAC1 and CDC42 play an essential role in PGE(2)-mediated migration of human EVTs.
|Regulation of microglial phagocytosis and inflammatory gene expression by Gas6 acting on the Axl/Mer family of tyrosine kinases. |
Christian Grommes, C Y Daniel Lee, Brandy L Wilkinson, Qingguang Jiang, Jessica L Koenigsknecht-Talboo, Brian Varnum, Gary E Landreth
Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology 3 130-40 2008
Removal of apoptotic cells is an essential process for normal development and tissue maintenance. Importantly, apoptotic cells stimulate their phagocytosis by macrophages while actively suppressing inflammatory responses. Growth arrest specific gene 6 (Gas6) is involved in this process, bridging phosphatidylserine residues on the surface of apoptotic cells to the Axl/Mer family of tyrosine kinases which stimulate phagocytosis. Animals with mutations or loss of these receptors exhibit phenotypes reflective of impaired phagocytosis and a hyperactive immune response. We report that Gas6 induces phagocytosis in microglia through a novel non-classical phagocytic mechanism. Gas6 stimulates a type-II-related phagocytic response, but requires Vav phosphorylation and Rac activation, distinguishing it from the classical type II mechanism. Importantly, Gas6 suppressed lipopolysaccharide-induced expression of the inflammatory molecules IL-1beta and iNOS. Gas6 inhibited iNOS expression through suppression of promoter activity. The present data provide direct evidence for the role of Gas6 receptors in mediating an anti-inflammatory response to ligands found on apoptotic cells with the simultaneous stimulation of phagocytosis. These data provide a mechanistic explanation for the phenotype observed in animals lacking Axl/Mer receptors.
|The involvement of the tyrosine kinase c-Src in the regulation of reactive oxygen species generation mediated by NADPH oxidase-1. |
Gianni, D; Bohl, B; Courtneidge, SA; Bokoch, GM
Molecular biology of the cell 19 2984-94 2008
NADPH oxidase (Nox) family enzymes are one of the main sources of cellular reactive oxygen species (ROS), which have been shown to function as second messenger molecules. To date, seven members of this family have been reported, including Nox1-5 and Duox1 and -2. With the exception of Nox2, the regulation of the Nox enzymes is still poorly understood. Nox1 is highly expressed in the colon, and it requires two cytosolic regulators, NoxO1 and NoxA1, as well as the binding of Rac1 GTPase, for its activity. In this study, we investigate the role of the tyrosine kinase c-Src in the regulation of ROS formation by Nox1. We show that c-Src induces Nox1-mediated ROS generation in the HT29 human colon carcinoma cell line through a Rac-dependent mechanism. Treatment of HT29 cells with the Src inhibitor PP2, expression of a kinase-inactive form of c-Src, and c-Src depletion by small interfering RNA (siRNA) reduce both ROS generation and the levels of active Rac1. This is associated with decreased Src-mediated phosphorylation and activation of the Rac1-guanine nucleotide exchange factor Vav2. Consistent with this, Vav2 siRNA that specifically reduces endogenous Vav2 protein is able to dramatically decrease Nox1-dependent ROS generation and abolish c-Src-induced Nox1 activity. Together, these results establish c-Src as an important regulator of Nox1 activity, and they may provide insight into the mechanisms of tumor formation in colon cancers.Full Text Article
|Novel functions of Ect2 in polar lamellipodia formation and polarity maintenance during "contractile ring-independent" cytokinesis in adherent cells. |
Kanada, M; Nagasaki, A; Uyeda, TQ
Molecular biology of the cell 19 8-16 2008
Some mammalian cells are able to divide via both the classic contractile ring-dependent method (cytokinesis A) and a contractile ring-independent, adhesion-dependent method (cytokinesis B). Cytokinesis A is triggered by RhoA, which, in HeLa cells, is activated by the guanine nucleotide-exchange factor Ect2 localized at the central spindle and equatorial cortex. Here, we show that in HT1080 cells undergoing cytokinesis A, Ect2 does not localize in the equatorial cortex, though RhoA accumulates there. Moreover, Ect2 depletion resulted in only modest multinucleation of HT1080 cells, enabling us to establish cell lines in which Ect2 was constitutively depleted. Thus, RhoA is activated via an Ect2-independent pathway during cytokinesis A in HT1080 cells. During cytokinesis B, Ect2-depleted cells showed narrower accumulation of RhoA at the equatorial cortex, accompanied by compromised pole-to-equator polarity, formation of ectopic lamellipodia in regions where RhoA normally would be distributed, and delayed formation of polar lamellipodia. Furthermore, C3 exoenzyme inhibited equatorial RhoA activation and polar lamellipodia formation. Conversely, expression of dominant active Ect2 in interphase HT1080 cells enhanced RhoA activity and suppressed lamellipodia formation. These results suggest that equatorial Ect2 locally suppresses lamellipodia formation via RhoA activation, which indirectly contributes to restricting lamellipodia formation to polar regions during cytokinesis B.Full Text Article
|The Rac effector p67phox regulates phagocyte NADPH oxidase by stimulating Vav1 guanine nucleotide exchange activity. |
Ming, W; Li, S; Billadeau, DD; Quilliam, LA; Dinauer, MC
Molecular and cellular biology 27 312-23 2007
The phagocyte NADPH oxidase catalyzes the reduction of molecular oxygen to superoxide and is essential for microbial defense. Electron transport through the oxidase flavocytochrome is activated by the Rac effector p67(phox). Previous studies suggest that Vav1 regulates NADPH oxidase activity elicited by the chemoattractant formyl-Met-Leu-Phe (fMLP). We show that Vav1 associates with p67(phox) and Rac2, but not Rac1, in fMLP-stimulated human neutrophils, correlating with superoxide production. The interaction of p67(phox) with Vav1 is direct and activates nucleotide exchange on Rac, which enhances the interaction between p67(phox) and Vav1. This provides new molecular insights into regulation of the neutrophil NADPH oxidase, suggesting that chemoattractant-stimulated superoxide production can be amplified by a positive feedback loop in which p67(phox) targets Vav1-mediated Rac activation.Full Text Article
|Modulation of COX-2 expression by statins in human monocytic cells. |
Habib, A; Shamseddeen, I; Nasrallah, MS; Antoun, TA; Nemer, G; Bertoglio, J; Badreddine, R; Badr, KF
FASEB journal : official publication of the Federation of American Societies for Experimental Biology 21 1665-74 2007
Macrophage cyclooxygenase-2 (COX-2) plays an important role in prostaglandin E2 and thromboxane A2 production. Statins are inhibitors of HMG CoA (3-Hydroxy-3-methylglutaryl coenzyme A) reductases and cholesterol synthesis, which block the expression of several inflammatory proteins independent of their capacity to lower endogenous cholesterol. In the present study, we investigated the effect of simvastatin and mevastatin on COX-2 induction in human monocytic cell line U937 and analyzed the underlying mechanisms. Pretreatment of U937 cells with simvastatin or mevastatin for 24 h resulted in a significant reduction in the lipopolysaccharide (LPS)-dependent induction of prostaglandin E2, thromboxane A2 synthesis, and COX-2 expression. Mevalonate, the direct metabolite of HMG CoA reductase, and farnesyl pyrophosphate and geranylgeranyl-pyrophosphate, intermediates of the mevalonate pathway, significantly reversed the inhibitory effect of statins on COX-2. An inhibitor of geranylgeranyl transferases, GGTI-286 mimicked the effect of statins on COX-2 expression. Cytonecrotic factor-1 increased LPS-dependent expression of COX-2. Treatment of cells with NSC 23766, an inhibitor of Rac, which we demonstrated to block Rac 2 activation, resulted in an inhibition of the LPS-dependent expression of COX-2. Whereas no effect was obtained with RhoA/C blocker, C3 exoenzyme. Gel retardation experiments and NFkappaB-p65 transcription factor assay showed that simvastatin and NSC 23766 decrease significantly NF-kappaB complex formation. In macrophages, the antiinflammatory effects of statins are mediated in part through the inhibition of COX-2 and prostanoids. Rac GTPase protein is identified as one of the targets of statins in this regulation.
|Activation of NADPH oxidase by transforming growth factor-beta in hepatocytes mediates up-regulation of epidermal growth factor receptor ligands through a nuclear factor-kappaB-dependent mechanism. |
Miguel M Murillo, Irene Carmona-Cuenca, Gaelle Del Castillo, Conrad Ortiz, César Roncero, Aránzazu Sánchez, Margarita Fernández, Isabel Fabregat
The Biochemical journal 405 251-9 2007
The TGF-beta (transforming growth factor-beta) induces survival signals in foetal rat hepatocytes through transactivation of EGFR (epidermal growth factor receptor). The molecular mechanism is not completely understood, but both activation of the TACE (tumour necrosis factor alpha-converting enzyme)/ADAM17 (a disintegrin and metalloproteinase 17; one of the metalloproteases involved in shedding of the EGFR ligands) and up-regulation of TGF-alpha and HB-EGF (heparin-binding epidermal growth factor-like growth factor) appear to be involved. In the present study, we have analysed the molecular mechanisms that mediate up-regulation of the EGFR ligands by TGF-beta in foetal rat hepatocytes. The potential involvement of ROS (reactive oxygen species), an early signal induced by TGF-beta, and the existence of an amplification loop triggered by initial activation of the EGFR, have been studied. Results indicate that DPI (diphenyleneiodonium) and apocynin, two NOX (NADPH oxidase) inhibitors, and SB431542, an inhibitor of the TbetaR-I (TGF-beta receptor I), block up-regulation of EGFR ligands and Akt activation. Different members of the NOX family of genes are expressed in hepatocytes, included nox1, nox2 and nox4. TGF-beta up-regulates nox4 and increases the levels of Rac1 protein, a known regulator of both Nox1 and Nox2, in a TbetaR-I-dependent manner. TGF-beta mediates activation of the nuclear factor-kappaB pathway, which is inhibited by DPI and is required for up-regulation of TGF-alpha and HB-EGF. In contrast, EGFR activation is not required for TGF-beta-induced up-regulation of those ligands. Considering previous work that has established the role of ROS in apoptosis induced by TGF-beta in hepatocytes, the results of the present study indicate that ROS might mediate both pro- and anti-apoptotic signals in TGF-beta-treated cells.Full Text Article
|Edaravone mimics sphingosine-1-phosphate-induced endothelial barrier enhancement in human microvascular endothelial cells. |
Omori, Kazuyoshi, et al.
Am. J. Physiol., Cell Physiol., 293: C1523-31 (2007) 2007
|Negative feedback regulation of Rac in leukocytes from mice expressing a constitutively active phosphatidylinositol 3-kinase gamma. |
Costa, C; Barberis, L; Ambrogio, C; Manazza, AD; Patrucco, E; Azzolino, O; Neilsen, PO; Ciraolo, E; Altruda, F; Prestwich, GD; Chiarle, R; Wymann, M; Ridley, A; Hirsch, E
Proceedings of the National Academy of Sciences of the United States of America 104 14354-9 2007
Polarization of chemotaxing cells depends on positive feedback loops that amplify shallow gradients of chemoattractants into sharp intracellular responses. In particular, reciprocal activation of phosphatidylinositol 3-kinases (PI3Ks) and small GTPases like Rac leads to accumulation, at the leading edge, of the PI3K product phosphatidylinositol 3,4,5-trisphosphate (PIP3). Mice carrying a "knockin" allele of the G protein-coupled receptor (GPCR)-activated PI3Kgamma, encoding a plasma membrane-targeted protein appeared normal, but their leukocytes showed GPCR-uncoupled PIP3 accumulation. In vivo, the mutation increased proliferation and decreased apoptosis, leading to leukocytosis and delayed resolution of inflammation in wound healing. Mutant leukocytes showed significantly impaired directional cell migration in response to chemoattractants. Stimulated mutant macrophages did not polarize PIP3 and showed a shortened Rac activation because of enhanced PI3K-dependent activation of RacGAPs. Together with the finding that chemoattractants stimulate a PIP3-dependent GAP activation in wild-type macrophages, these results identify a molecular mechanism involving PI3K- and RacGAP-dependent negative control of Rac that limits and fine-tunes feedback loops promoting cell polarization and directional motility.Full Text Article
|Expression of NADPH oxidase in rabbit corneal epithelial and stromal cells in culture. |
O'Brien, WJ; Krema, C; Heimann, T; Zhao, H
Investigative ophthalmology & visual science 47 853-63 2006
Reactive oxygen- and nitrogen-containing molecules produced in high concentrations are mediators of tissue damage caused by inflammation. The free radical molecules superoxide (O2-*) and nitric oxide (NO*), when produced at low concentrations, may function as second messengers or regulators of signal transduction. The purpose of these studies was to determine whether corneal epithelial and stromal cells are capable of producing O2-* via an NADPH oxidase complex.Rabbit corneal epithelial and stromal cells, grown as primary cultures and low-passage isolates, were used as the sources of RNA for RT-PCR with primers specific for mRNAs encoding the proteins that comprise an NADPH oxidase complex. The RT-PCR products were sequenced to confirm their identities. The production of proteins composing the oxidase complex was confirmed, and the proteins were identified by Western blot analysis. The production of superoxide in cell-free preparations was assessed by measurement of NADPH-dependent superoxide dismutase (SOD)-inhibitable cytochrome c reduction and by electron paramagnetic resonance (EPR) with a superoxide specific spin trap.Cell-free extracts of corneal epithelial and stromal cells produced superoxide in an NADPH-dependent manner, and this production was inhibited by SOD. EPR confirmed the identity of the reaction product as superoxide anion. Both rabbit corneal epithelial and stromal cells constitutively produced mRNAs encoding five proteins known to comprise a classic neutrophil-like NADPH oxidase complex. Production of NOX4, p22phox, p47phox, p67phox, and p40phox was confirmed by Western blot. Both epithelial and stromal cells expressed isoforms of Rac, a putative regulator of the activity of the complex.A constitutively expressed NADPH oxidase complex that includes NOX4 is a source of O2-* produced by rabbit corneal epithelial and stromal cells. Superoxide produced by the oxidation of NADPH via the NADPH oxidase complex is a potential contributor to signal transduction pathways as well as a potential participant in processes that occur during inflammation.
|Effects of constitutively active GTPases on fibroblast behavior. |
Zhang, ZG; Lambert, CA; Servotte, S; Chometon, G; Eckes, B; Krieg, T; Lapière, CM; Nusgens, BV; Aumailley, M
Cellular and molecular life sciences : CMLS 63 82-91 2006
The GTP-binding proteins RhoA, Cdc42 and Rac1 regulate the organization and turnover of the cytoskeleton and cell-matrix adhesions, structures bridging cells to their support, and translating forces, external or generated within the cell. To investigate the specific requirements of Rho GTPases for biomechanical activities of clonal cell populations, we compared side-by-side stable lines of human fibroblasts expressing constitutively active (CA) RhoA, Cdc42 or Rac1. There was no marked effect of any CA GTPase on cell adhesion to different extracellular matrix proteins. Cell spreading was CA Rho GTPase specific and independent of the extracellular matrix proteins allowing adhesion. Mechanical properties were dramatically restricted by CA RhoA on bi- and in tri-dimensional surroundings, were boosted by CA Rac1 on bi-dimensional surroundings only, and were not or marginally affected by CA Cdc42. In conclusion, the action of Rho GTPases appears to depend on the task cells are performing.
|Lipid rafts serve as a signaling platform for nicotinic acetylcholine receptor clustering. |
Zhu, D; Xiong, WC; Mei, L
The Journal of neuroscience : the official journal of the Society for Neuroscience 26 4841-51 2006
Agrin, a motoneuron-derived factor, and the muscle-specific receptor tyrosine kinase (MuSK) are essential for the acetylcholine receptor (AChR) clustering at the postjunctional membrane. However, the underlying signaling mechanisms remain poorly defined. We show that agrin stimulates a dynamic translocation of the AChR into lipid rafts-cholesterol and sphingolipid-rich microdomains in the plasma membrane. This follows MuSK partition into lipid rafts and requires its activation. Disruption of lipid rafts inhibits MuSK activation and downstream signaling and AChR clustering in response to agrin. Rapsyn, an intracellular protein necessary for AChR clustering, is located constitutively in lipid rafts, but its interaction with the AChR is inhibited when lipid rafts are perturbed. These results reveal that lipid rafts may regulate AChR clustering by facilitating the agrin/MuSK signaling and the interaction between the receptor and rapsyn, both necessary for AChR clustering and maintenance. These results provide insight into mechanisms of AChR cluster formation.
|Association of RhoGDIalpha with Rac1 GTPase mediates free radical production during myocardial hypertrophy. |
Custodis, F; Eberl, M; Kilter, H; Böhm, M; Laufs, U
Cardiovascular research 71 342-51 2006
Reactive oxygen species (ROS) contribute to the pathogenesis of myocardial hypertrophy. NADPH oxidase is a major source of ROS production. The small GTPase Rac1 mediates the activation of NADPH oxidase; however, the mechanism of Rac1 activation is incompletely understood.Transaortic constriction (TAC, C57/Bl6 mice, 360 microm, 21 days) increased the ratio of heart to body weight from [ per thousand] SHAM 4.16+/-0.09 to TAC 7.1+/-0.37, pless than 0.01. Treatment with rosuvastatin prevented pressure-induced cardiac hypertrophy (5.5+/-0.18, pless than 0.05). TAC induced a 4-fold up-regulation of myocardial NADPH oxidase activity as well as Rac1 activity; both effects were absent in statin-treated animals. In cultured rat cardiomyocytes, treatment with angiotensin II (AngII) increased translocation of Rac1 to cell membranes and Rac1 activity. AngII altered neither expression nor tyrosine phosphorylation of GTPase activating protein GAP-p190 and the guanine nucleotide exchange factors Vav and Tiam. Transaortic constriction as well as AngII increased the binding of Rho guanine nucleotide dissociation inhibitor (RhoGDIalpha) to Rac1. The association of RhoGDIalpha with Rac1 was mediated by phosphatidylinositol 3-kinase and depended on geranylgeranylation. Statin treatment inhibited RhoGDIalpha-Rac1 binding both in cultured cardiomyocytes and during myocardial hypertrophy in vivo. Transfection with RhoGDIalpha siRNA constructs potently reduced RhoGDIalpha protein expression, decreased AngII-induced superoxide production and lipid peroxidation, and inhibited AngII-induced leucine incorporation.Myocardial hypertrophy is characterized by activation of Rac1 and NADPH oxidase. The association of the regulatory protein RhoGDIalpha with Rac1 represents a necessary step in the Rac1-dependent release of ROS. Rac1-RhoGDIalpha binding may represent a target for anti-hypertrophic pharmacologic interventions, potentially by statin treatment.
|Phosphorylation of RhoGDI by Src regulates Rho GTPase binding and cytosol-membrane cycling. |
DerMardirossian, C; Rocklin, G; Seo, JY; Bokoch, GM
Molecular biology of the cell 17 4760-8 2006
Rho GTPases (Rac, Rho, and Cdc42) play important roles in regulating cell function through their ability to coordinate the actin cytoskeleton, modulate the formation of signaling reactive oxidant species, and control gene transcription. Activation of Rho GTPase signaling pathways requires the regulated release of Rho GTPases from RhoGDI complexes, followed by their reuptake after membrane cycling. We show here that Src kinase binds and phosphorylates RhoGDI both in vitro and in vivo at Tyr156. Analysis of Rho GTPase-RhoGDI complexes using in vitro assays of complexation and in vivo by coimmunoprecipitation analysis indicates that Src-mediated phosphorylation of Tyr156 causes a dramatic decrease in the ability of RhoGDI to form a complex with RhoA, Rac1, or Cdc42. Phosphomimetic mutation of Tyr156--greater than Glu results in the constitutive association of RhoGDI(Y156E) with the plasma membrane and/or associated cortical actin. Substantial cortical localization of tyrosine-phosphorylated RhoGDI is also observed in fibroblasts expressing active Src, where it is most evident in podosomes and regions of membrane ruffling. Expression of membrane-localized RhoGDI(Y156E) mutant is associated with enhanced cell spreading and membrane ruffling. These results suggest that Src-mediated RhoGDI phosphorylation is a novel physiological mechanism for regulating Rho GTPase cytosol membrane-cycling and activity.Full Text Article
|Lgl mediates apical domain disassembly by suppressing the PAR-3-aPKC-PAR-6 complex to orient apical membrane polarity. |
Yamanaka, T; Horikoshi, Y; Izumi, N; Suzuki, A; Mizuno, K; Ohno, S
Journal of cell science 119 2107-18 2006
The basolateral tumor suppressor protein Lgl is important for the regulation of epithelial cell polarity and tissue morphology. Recent studies have shown a physical and functional interaction of Lgl with another polarity-regulating protein machinery, the apical PAR-3-aPKC-PAR-6 complex, in epithelial cells. However, the mechanism of Lgl-mediated regulation of epithelial cell polarity remains obscure. By an siRNA method, we here show that endogenous Lgl is required for the disassembly of apical membrane domains in depolarizing MDCK cells induced by Ca2+ depletion. Importantly, this Lgl function is mediated by the suppression of the apical PAR-3-aPKC-PAR-6 complex activity. Analysis using 2D- or 3D-cultured cells in collagen gel suggests the importance of this suppressive regulation of Lgl on the collagen-mediated re-establishment of apical membrane domains and lumen formation. These results indicate that basolateral Lgl plays a crucial role in the disassembly of apical membrane domains to induce the orientation of apical membrane polarity, which is mediated by the suppression of apical PAR-3-aPKC-PAR-6 complex activity.
|3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors attenuate beta-amyloid-induced microglial inflammatory responses. |
Cordle, A; Landreth, G
The Journal of neuroscience : the official journal of the Society for Neuroscience 25 299-307 2005
Alzheimer's disease (AD) is characterized by extracellular deposits of fibrillar beta-amyloid (Abeta) in the brain, a fulminant microglial-mediated inflammatory reaction, and neuronal death. The use of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) is associated with a reduced risk of AD, which has been attributed to the cholesterol-lowering actions of these drugs. Statins have been reported recently to have anti-inflammatory actions in addition to their classic lipid-lowering effects. We report that statins robustly inhibited the Abeta-stimulated expression of interleukin-1beta and inducible nitric oxide synthase and the production of nitric oxide by microglia and monocytes. Statin treatment also blocked the rac1-dependent activation of NADPH oxidase and superoxide production. The anti-inflammatory actions of the statins were attributable to their ability to reduce the levels of isoprenyl intermediates in the cholesterol biosynthetic pathway. The effect of statins could not be reversed by exogenous cholesterol supplementation, indicating that the anti-inflammatory actions are distinct from their cholesterol-lowering actions. The addition of the isoprenyl precursors, mevalonic acid, and geranylgeranyl pyrophosphate (GGpp) attenuated the statin-mediated downregulation of inflammatory markers. Prevention of protein isoprenylation by the GGpp transferase inhibitor (GGTI-286) or inhibition of Rho-family function with Clostridium difficile Toxin A blocked the inflammatory response similar to the effect of statin treatment. We argue that the statin-mediated decrease in AD risk arises from their pleiotropic actions, effecting a reduction in neuronal Abeta production and microglia-directed inflammation.
|Cyclin E overexpression obstructs infiltrative behavior in breast cancer: a novel role reflected in the growth pattern of medullary breast cancers. |
Berglund, P; Stighall, M; Jirström, K; Borgquist, S; Sjölander, A; Hedenfalk, I; Landberg, G
Cancer research 65 9727-34 2005
Cell cycle deregulation is a prerequisite in tumor development and overexpression of cyclin E, a major G1-S regulator, is often observed in breast cancer and is further linked to poor prognosis. By overexpressing cyclin E in a retinoblastoma-inactivated breast cancer cell line, we induced significant alterations in the expression of genes associated with proliferation and cell adhesion. Rearrangements of the actin cytoskeleton in addition to increased adhesive properties, decreased motility, and invasive potential in functional assays, indicated an overall abrogated mobility. Consistent in vivo findings were obtained upon investigation of 985 primary breast cancers, where cyclin E-high tumors predominantly (67%) displayed a low infiltrative, pushing growth pattern. Furthermore, medullary breast cancers, a subtype defined by its pushing, delimited growth, exhibited a remarkable frequency of cyclin E deregulation (87%) compared with other histologic subtypes (5-20%). Taken together, our results suggest the novel role of cyclin E in modeling infiltrative behavior. The consequences of cyclin E overexpression in breast cancer seems to be multiple, including effects on proliferation as well as growth patterns, a scenario that is indeed observed in the archetype of cyclin E-overexpressing medullary breast cancers.
|Mechanisms of statin-mediated inhibition of small G-protein function. |
Cordle, A; Koenigsknecht-Talboo, J; Wilkinson, B; Limpert, A; Landreth, G
The Journal of biological chemistry 280 34202-9 2005
3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been reported to reduce the risk of Alzheimer disease. We have shown previously that statins inhibit a beta-amyloid (Abeta)-mediated inflammatory response through mechanisms independent of cholesterol reduction. Specifically, statins exert anti-inflammatory actions through their ability to prevent the isoprenylation of members of the Rho family of small G-proteins, resulting in the functional inactivation of these G-proteins. We report that statin treatment of microglia results in perturbation of the cytoskeleton and morphological changes due to alteration in Rho family function. Statins also block Abeta-stimulated phagocytosis through inhibition of Rac action. Paradoxically, the statin-mediated inactivation of G-protein function was associated with increased GTP loading of Rac and RhoA, and this effect was observed in myeloid lineage cells and other cell types. Statin treatment disrupted the interaction of Rac with its negative regulator the Rho guanine nucleotide dissociation inhibitor (RhoGDI), an interaction that is dependent on protein isoprenylation. We propose that lack of negative regulation accounts for the increased GTP loading. Isoprenylation of Rac is also required for efficient interaction with the plasma membrane, and we report that statin treatment dramatically reduces the capacity of Rac to interact with membranes. These results suggest a mechanism by which statins inhibit the actions of Rho GTPases and attenuate Abeta-stimulated inflammation.
|Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis. |
Oceguera-Yanez, F; Kimura, K; Yasuda, S; Higashida, C; Kitamura, T; Hiraoka, Y; Haraguchi, T; Narumiya, S
The Journal of cell biology 168 221-32 2005
Although Rho regulates cytokinesis, little was known about the functions in mitosis of Cdc42 and Rac. We recently suggested that Cdc42 works in metaphase by regulating bi-orient attachment of spindle microtubules to kinetochores. We now confirm the role of Cdc42 by RNA interference and identify the mechanisms for activation and down-regulation of Cdc42. Using a pull-down assay, we found that the level of GTP-Cdc42 elevates in metaphase, whereas the level of GTP-Rac does not change significantly in mitosis. Overexpression of dominant-negative mutants of Ect2 and MgcRacGAP, a Rho GTPase guanine nucleotide exchange factor and GTPase activating protein, respectively, or depletion of Ect2 by RNA interference suppresses this change of GTP-Cdc42 in mitosis. Depletion of Ect2 also impairs microtubule attachment to kinetochores and causes prometaphase delay and abnormal chromosomal segregation, as does depletion of Cdc42 or expression of the Ect2 and MgcRacGAP mutants. These results suggest that Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis.Full Text Article
|Galpha13 stimulates cell migration through cortactin-interacting protein Hax-1. |
Radhika, V; Onesime, D; Ha, JH; Dhanasekaran, N
The Journal of biological chemistry 279 49406-13 2004
Galpha13, the alpha-subunit of the heterotrimeric G protein G13, has been shown to stimulate cell migration in addition to inducing oncogenic transformation. Cta, a Drosophila ortholog of G13, has been shown to be critical for cell migration leading to the ventral furrow formation in Drosophila embryos. Loss of Galpha13 has been shown to disrupt cell migration associated with angiogenesis in developing mouse embryos. Whereas these observations point to the vital role of G13-orthologs in regulating cell migration, widely across the species barrier, the mechanism by which Galpha13 couples to cytoskeleton and cell migration is largely unknown. Here we show that Galpha13 physically interacts with Hax-1, a cytoskeleton-associated, cortactin-interacting intracellular protein, and this interaction is required for Galpha13-stimulated cell migration. Hax-1 interaction is specific to Galpha13, and this interaction is more pronounced with the mutationally or functionally activated form of Galpha13 as compared with the wild-type Galpha13. Expression of Hax-1 reduces the formation of actin stress fibers and focal adhesion complexes in Galpha13-expressing NIH3T3 cells. Coexpression of Hax-1 also attenuates Galpha(13)-stimulated activity of Rho while potentiating Galpha13-stimulated activity of Rac. The presence of a quadnary complex consisting of Galpha13, Hax-1, Rac, and cortactin indicates the role of Hax-1 in tethering Galpha13 to the cytoskeletal component(s) involved in cell movement. Whereas the expression of Hax-1 potentiates Galpha13-mediated cell movement, silencing of endogenous Hax-1 with Hax-1-specific small interfering RNAs drastically reduces Galpha13-mediated cell migration. These findings, along with the observation that Hax-1 is overexpressed in metastatic tumors and tumor cell lines, suggest a novel role for the association of oncogenic Galpha13 and Hax-1 in tumor metastasis.
|Evaluation of Rho family small G-protein activity induced by integrin ligation on human leukocytes. |
Angela Gismondi, Fabrizio Mainiero, Angela Santoni
Methods in molecular biology (Clifton, N.J.) 239 69-76 2004
|Proline-rich tyrosine kinase 2 and Rac activation by chemokine and integrin receptors controls NK cell transendothelial migration. |
Gismondi, Angela, et al.
J. Immunol., 170: 3065-73 (2003) 2003
Protein tyrosine kinase activation is an important requisite for leukocyte migration. Herein we demonstrate that NK cell binding to endothelium activates proline-rich tyrosine kinase 2 (Pyk-2) and the small GTP binding protein Rac that are coupled to integrin and chemokine receptors. Chemokine-mediated, but not integrin-mediated, Pyk-2 and Rac activation was sensitive to pretreatment of NK cells with pertussis toxin, a pharmacological inhibitor of G(i) protein-coupled receptors. Both Pyk-2 and Rac are functionally involved in chemokine-induced NK cell migration through endothelium or ICAM-1 or VCAM-1 adhesive proteins, as shown by the use of recombinant vaccinia viruses encoding dominant negative mutants of Pyk-2 and Rac. Moreover, we found that Pyk-2 is associated with the Rac guanine nucleotide exchange factor Vav, which undergoes tyrosine phosphorylation upon integrin triggering. Finally, we provide direct evidence for the involvement of Pyk-2 in the control of both chemokine- and integrin-mediated Rac activation. Collectively, our results indicate that Pyk-2 acts as a receptor-proximal link between integrin and chemokine receptor signaling, and the Pyk-2/Rac pathway plays a pivotal role in the control of NK cell transendothelial migration.
|Androgen-stimulated DNA synthesis and cytoskeletal changes in fibroblasts by a nontranscriptional receptor action |
Castoria, G., et al
J Cell Biol, 161:547-56 (2003) 2003
|Receptor activation regulates cortical, but not vesicular localization of NDP kinase |
Gallagher, B. C., et al
J Cell Sci, 116:3239-50 (2003) 2003
|Loss of phosphatidylinositol 3-phosphate binding by the C-terminal Tiam-1 pleckstrin homology domain prevents in vivo Rac1 activation without affecting membrane targeting. |
Baumeister, MA; Martinu, L; Rossman, KL; Sondek, J; Lemmon, MA; Chou, MM
The Journal of biological chemistry 278 11457-64 2003
Dbl family guanine nucleotide exchange factors (GEFs) for Rho family small GTPases invariably contain a pleckstrin homology (PH) domain that immediately follows their Dbl homology (DH) domain. Although the DH domain is responsible for GEF activity, the role of the PH domain is less clear. We previously reported that PH domains from several Dbl family members bind phosphoinositides with very low affinity (K(d) values in the 10 microM range). This suggests that, unlike several other PH domains, those from Dbl proteins will not function as independent membrane-targeting modules. To determine the functional relevance of low affinity phosphoinositide binding, we mutated the corresponding PH domain from Tiam-1 to abolish its weak, specific binding to phosphatidylinositol 3-phosphate. We first confirmed in vitro that phosphoinositide binding by the isolated DH/PH domain was impaired by the mutations but that intrinsic GEF activity was unaffected. We then introduced the PH domain mutations into full-length Tiam-1 and found that its ability to activate Rac1 or serum response factor in vivo was abolished. Immunofluorescence studies showed that membrane targeting of Tiam-1 was essentially unaffected by mutations in the C-terminal PH domain. Our studies therefore indicate that low affinity phosphatidylinositol 3-phosphate binding by the C-terminal PH domain may be critical for in vivo regulation and activity of Tiam-1 but that the PH domain exerts its regulatory effects without altering membrane targeting. We suggest instead that ligand binding to the PH domain induces conformational and/or orientational changes at the membrane surface that are required for maximum exchange activity of its adjacent DH domain.
|Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators |
Seshiah, P. N., et al
Circ Res, 91:406-13 (2002) 2002
|SH2-Bbeta is a Rac-binding protein that regulates cell motility. |
Diakonova, Maria, et al.
J. Biol. Chem., 277: 10669-77 (2002) 2002
The Src homology 2 (SH2) domain-containing protein SH2-Bbeta binds to and is a substrate of the growth hormone (GH) and cytokine receptor-associated tyrosine kinase JAK2. SH2-Bbeta also binds, via its SH2 domain, to multiple activated growth factor receptor tyrosine kinases. We have previously implicated SH2-Bbeta in GH and platelet-derived growth factor regulation of the actin cytoskeleton. We extend these findings by establishing a potentiating effect of SH2-Bbeta on GH-dependent cell motility and defining regions of SH2-Bbeta required for this potentiation. Time-lapse video microscopy, phagokinetic, and/or wounding assays demonstrate reduced movement of cells overexpressing SH2-Bbeta lacking an intact SH2 domain because of a point mutation or a C-terminal truncation. An N-terminal proline-rich domain (amino acids 85-106) of SH2-Bbeta is required for inhibition of cellular motility by SH2 domain-deficient mutants. Co-immunoprecipitation experiments indicate that Rac binds to this domain. GH is shown to activate endogenous Rac, and dominant negative mutants of SH2-Bbeta are shown to inhibit membrane ruffling induced by constitutively active Rac. These findings suggest that SH2-Bbeta is an adapter protein that facilitates actin rearrangement and cellular motility by recruiting Rac and potentially Rac-regulating, Rac effector, or other actin-regulating proteins to activated cytokine (e.g. GH) and growth factor receptors.
|VE-cadherin regulates endothelial actin activating Rac and increasing membrane association of Tiam. |
Lampugnani, Maria Grazia, et al.
Mol. Biol. Cell, 13: 1175-89 (2002) 2002
Previously published reports support the concept that, besides promoting homotypic intercellular adhesion, cadherins may transfer intracellular signals. However, the signaling pathways triggered by cadherin clustering and their biological significance are still poorly understood. We report herein that transfection of VE-cadherin (VEC) cDNA in VEC null endothelial cells induces actin rearrangement and increases the number of vinculin positive adhesion plaques. VEC expression augments the level of active Rac but decreases active Rho. Microinjection of a dominant negative Rac mutant altered stress fiber organization, whereas inhibition of Rho was ineffective. VEC expression increased protein and mRNA levels of the Rac-specific guanosine exchange factor Tiam-1 and induced its localization at intercellular junctions. In addition, in the presence of VEC, the amounts of Tiam, Rac, and the Rac effector PAK as well as the level of PAK phosphorylation were found increased in the membrane/cytoskeletal fraction. These observations are consistent with a role of VEC in localizing Rac and its signaling partners in the same membrane compartment, facilitating their reciprocal interaction. Through this mechanism VEC may influence the constitutive organization of the actin cytoskeleton.
|Vav regulates activation of Rac but not Cdc42 during FcgammaR-mediated phagocytosis. |
Patel, Jayesh C, et al.
Mol. Biol. Cell, 13: 1215-26 (2002) 2002
Phagocytosis is the process whereby cells direct the spatially localized, receptor-driven engulfment of particulate materials. It proceeds via remodeling of the actin cytoskeleton and shares many of the core cytoskeletal components involved in adhesion and migration. Small GTPases of the Rho family have been widely implicated in coordinating actin dynamics in response to extracellular signals and during diverse cellular processes, including phagocytosis, yet the mechanisms controlling their recruitment and activation are not known. We show herein that in response to ligation of Fc receptors for IgG (FcgammaR), the guanine nucleotide exchange factor Vav translocates to nascent phagosomes and catalyzes GTP loading on Rac, but not Cdc42. The Vav-induced Rac activation proceeds independently of Cdc42 function, suggesting distinct roles for each GTPase during engulfment. Moreover, inhibition of Vav exchange activity or of Cdc42 activity does not prevent Rac recruitment to sites of particle attachment. We conclude that Rac is recruited to Fcgamma membrane receptors in its inactive, GDP-bound state and that Vav regulates phagocytosis through subsequent catalysis of GDP/GTP exchange on Rac.
|Rac activation upon cell-cell contact formation is dependent on signaling from the epidermal growth factor receptor. |
Betson, Martha, et al.
J. Biol. Chem., 277: 36962-9 (2002) 2002
Cadherins are transmembrane receptors that mediate cell-cell adhesion. They play an essential role in embryonic development and maintenance of tissue architecture. The Rho family small GTPases regulate actin cytoskeletal dynamics in different cell types. The function of two family members, Rho and Rac, is required for the stability of cadherins at cell-cell contacts. Consistent with the published data we have found that Rac is activated upon induction of intercellular adhesion in epithelial cells. This activation is dependent on functional cadherins (Nakagawa, M., Fukata, M., Yamaga, M., Itoh, N., and Kaibuchi, K. (2001) J. Cell Sci. 114, 1829-1838; Noren, N. K., Niessen, C. M., Gumbiner, B. M., and Burridge, K. (2001) J. Biol. Chem. 276, 3305-3308). Here we show for the first time that clustering of cadherins using antibody-coated beads is sufficient to promote Rac activation. In the presence of Latrunculin B, Rac can be partially activated by antibody-clustered cadherins. These results suggest that actin polymerization is not required for initial Rac activation. Contrary to what has been described before, phosphatidylinositol 3-kinases are not involved in Rac activation following cell-cell adhesion in keratinocytes. Interestingly, inhibition of epidermal growth factor receptor signaling efficiently blocks the increased Rac-GTP levels observed after contact formation. We conclude that cadherin-dependent adhesion can activate Rac via epidermal growth factor receptor signaling.
|Characterization of p190RhoGEF, a RhoA-specific guanine nucleotide exchange factor that interacts with microtubules |
van Horck, F. P., et al
J Biol Chem, 276:4948-56 (2001) 2001
|Rac mediates cytoskeletal rearrangements and increased cell motility induced by urokinase-type plasminogen activator receptor binding to vitronectin. |
Kjøller, L and Hall, A
J. Cell Biol., 152: 1145-57 (2001) 2001
The urokinase-type plasminogen activator receptor (uPAR) is involved in the regulation of cell motility in a variety of cell types. We show here that expression of human uPAR in growing murine fibroblasts leads to a dramatic reorganization of the actin cytoskeleton. uPAR expression induces multiple rapidly advancing protrusions that resemble the leading edge of migrating cells. The cytoskeletal changes are independent of uPA and activation of the RGD-binding activity of integrins but require uPAR binding to vitronectin (VN). The actin reorganization is blocked by coexpression of dominant negative versions of either Rac (N17Rac) or p130Cas, but not by inhibitors of Cdc42 or Rho, and is accompanied by a Rac-dependent increase in cell motility. In addition, a fourfold increase in the level of activated Rac is induced by uPAR expression. We conclude that uPAR interacts with VN both to initiate a p130Cas/Rac-dependent signaling pathway leading to actin reorganization and increased cell motility and to act as an adhesion receptor required for these responses. This mechanism may play a role in uPAR-mediated regulation of cell motility at sites where VN and uPAR are co-expressed, such as malignant tumors.
|Haematopoietic cell-specific CDM family protein DOCK2 is essential for lymphocyte migration. |
Fukui, Y, et al.
Nature, 412: 826-31 (2001) 2001
Cell migration is a fundamental biological process involving membrane polarization and cytoskeletal dynamics, both of which are regulated by Rho family GTPases. Among these molecules, Rac is crucial for generating the actin-rich lamellipodial protrusion, a principal part of the driving force for movement. The CDM family proteins, Caenorhabditis elegans CED-5, human DOCK180 and Drosophila melanogaster Myoblast City (MBC), are implicated to mediate membrane extension by functioning upstream of Rac. Although genetic analysis has shown that CED-5 and Myoblast City are crucial for migration of particular types of cells, physiological relevance of the CDM family proteins in mammals remains unknown. Here we show that DOCK2, a haematopoietic cell-specific CDM family protein, is indispensable for lymphocyte chemotaxis. DOCK2-deficient mice (DOCK2-/-) exhibited migration defects of T and B lymphocytes, but not of monocytes, in response to chemokines, resulting in several abnormalities including T lymphocytopenia, atrophy of lymphoid follicles and loss of marginal-zone B cells. In DOCK2-/- lymphocytes, chemokine-induced Rac activation and actin polymerization were almost totally abolished. Thus, in lymphocyte migration DOCK2 functions as a central regulator that mediates cytoskeletal reorganization through Rac activation.
|Redox regulation of human Rac1 stability by the proteasome in human aortic endothelial cells. |
Kovacic, H N, et al.
J. Biol. Chem., 276: 45856-61 (2001) 2001
Rac1 has been shown to activate a NADPH oxidase complex producing superoxide anions in a variety of mammalian cell types. We evaluated the impact of Rac1-induced reactive oxygen species production on the turnover of Rac1 itself in human aortic endothelial cells. The concentration of a constitutively active mutant of Rac1 (Rac1(V12)) was increased by treatment of the cells with diphenylene iodinium (DPI), an inhibitor of the NADPH oxidase. Such an effect was not observed for the dominant negative form of Rac1 (Rac1(N17)). We showed a decrease in proteolytic degradation of Rac1(V12) in the presence of DPI, and showed that short term treatment with H(2)O(2) reverses the effect of DPI. We found that proteasome inhibitors (lactacystin and MG132) increased Rac1(V12) protein level. In support of this finding, we have identified in the primary sequence of Rac1 a potential destruction box domain, which is known to be a signal for protein degradation mediated by the ubiquitin/proteasome system. We show that Rac1(V12) is ubiquitinated before degradation. By contrast Rac1(N17) induces an accumulation of the ubiquitinated form of Rac1. These results suggest that Rac1 activation of NADPH oxidase is necessary for the proteolytic degradation of Rac1 itself.
|Vav1/Rac-dependent actin cytoskeleton reorganization is required for lipid raft clustering in T cells. |
Villalba, M, et al.
J. Cell Biol., 155: 331-8 (2001) 2001
Formation of the immunological synapse (IS) in T cells involves large scale molecular movements that are mediated, at least in part, by reorganization of the actin cytoskeleton. Various signaling proteins accumulate at the IS and are localized in specialized membrane microdomains, known as lipid rafts. We have shown previously that lipid rafts cluster and localize at the IS in antigen-stimulated T cells. Here, we provide evidence that lipid raft polarization to the IS depends on an intracellular pathway that involves Vav1, Rac, and actin cytoskeleton reorganization. Thus, lipid rafts did not translocate to the IS in Vav1-deficient (Vav1-/-) T cells upon antigen stimulation. Similarly, T cell receptor transgenic Jurkat T cells also failed to translocate lipid rafts to the IS when transfected with dominant negative Vav1 mutants. Raft polarization induced by membrane-bound cholera toxin cross-linking was also abolished in Jurkat T cells expressing dominant negative Vav1 or Rac mutants and in cells treated with inhibitors of actin polymerization. However, Vav overexpression that induced F-actin polymerization failed to induce lipid rafts clustering. Therefore, Vav is necessary, but not sufficient, to regulate lipid rafts clustering and polarization at the IS, suggesting that additional signals are required.
|Endogenous, hyperactive Rac3 controls proliferation of breast cancer cells by a p21-activated kinase-dependent pathway |
Mira, J. P., et al
Proc Natl Acad Sci U S A, 97:185-9 (2000) 2000
|Dual stimulation of Ras/mitogen-activated protein kinase and RhoA by cell adhesion to fibronectin supports growth factor-stimulated cell cycle progression |
Danen, E. H., et al
J Cell Biol, 151:1413-22 (2000) 2000
|The p35/Cdk5 kinase is a neuron-specific Rac effector that inhibits Pak1 activity. |
Nikolic, M, et al.
Nature, 395: 194-8 (1998) 1998
Cyclin-dependent kinase 5 (Cdk5) and its neuron-specific regulator p35 are essential for neuronal migration and for the laminar configuration of the cerebral cortex. In addition, p35/Cdk5 kinase concentrates at the leading edges of axonal growth cones and regulates neurite outgrowth in cortical neurons in culture. The Rho family of small GTPases is implicated in a range of cellular functions, including cell migration and neurite outgrowth. Here we show that the p35/Cdk5 kinase co-localizes with Rac in neuronal growth cones. Furthermore, p35 associates directly with Rac in a GTP-dependent manner. Another Rac effector, Pak1 kinase, is also present in the Rac-p35/Cdk5 complexes and co-localizes with p35/Cdk5 and Rac at neuronal peripheries. The active p35/Cdk5 kinase causes Pak1 hyperphosphorylation in a Rac-dependent manner, which results in down-regulation of Pak1 kinase activity. Because the Rho family of GTPases and the Pak kinases are implicated in actin polymerization, the modification of Pak1, imposed by the p35/Cdk5 kinase, is likely to have an impact on the dynamics of the reorganization of the actin cytoskeleton in neurons, thus promoting neuronal migration and neurite outgrowth.
|Rac1 mediates collapsin-1-induced growth cone collapse. |
Jin, Z and Strittmatter, S M
J. Neurosci., 17: 6256-63 (1997) 1997
Collapsin-1 or semaphorin III(D) inhibits axonal outgrowth by collapsing the lamellipodial and filopodial structures of the neuronal growth cones. Because growth cone collapse is associated with actin depolymerization, we considered whether small GTP-binding proteins of the rho subfamily might participate in collapsin-1 signal transduction. Recombinant rho, rac1, and cdc42 proteins were triturated into embryonic chick (DRG) neurons. Constitutively active rac1 increases the proportion of collapsed growth cones, and dominant negative rac1 inhibits collapsin-1-induced collapse of growth cones and collapsin-1 inhibition of neurite outgrowth. DRG neurons treated with dominant negative rac1 remain sensitive to myelin-induced growth cone collapse. Similar mutants of cdc42 do not alter growth cone structure, neurite elongation, or collapsin-1 sensitivity. Whereas the addition of activated rho has no effect, the inhibition of rho with Clostridium botulinum C3 transferase stimulates the outgrowth of DRG neurites. C3 transferase-treated growth cones exhibit little or no lamellipodial spreading and are minimally responsive to collapsin-1 and myelin. These data demonstrate a prominent role for rho and rac1 in modulating growth cone motility and indicate that rac1 may mediate collapsin-1 action.