Key Spec Table
|Species Reactivity||Key Applications||Host||Format||Antibody Type|
|Po, Ca, H, M, R, Rb, F, Fe||WB, ELISA, IP, ICC, IF, IHC||M||Purified||Monoclonal Antibody|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Maintain at 2-8ºC in undiluted aliquots for up to 6 months after date of receipt.|
|Material Size||200 μg|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 -|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - 2145925||2145925|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - 2388833||2388833|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - 2430317||2430317|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - 1990892||1990892|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - 2271144||2271144|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - 2322571||2322571|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - 2506322||2506322|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - JC1604282||JC1604282|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - JC1641540||JC1641540|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - JC1682928||JC1682928|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - LV1555185||LV1555185|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - LV1581114||LV1581114|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - NG1721601||NG1721601|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - NG1740950||NG1740950|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - NG1741566||NG1741566|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - NG1780785||NG1780785|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - NG1839548||NG1839548|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - NG1857556||NG1857556|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 - NG1869834||NG1869834|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 -2470405||2470405|
|Anti-Glyceraldehyde-3-Phosphate Dehydrogenase, clone 6C5 -2521487||2521487|
|MOUSE ANTI-GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE MONOCLONAL ANTIBODY -|
References | 72 Available | See All References
|Reference overview||Pub Med ID|
|Partial restoration of cardiac function with ΔPDZ nNOS in aged mdx model of Duchenne cardiomyopathy. |
Lai, Yi, et al.
Hum. Mol. Genet., (2014) 2014
Transgenic gene deletion/over-expression studies have established the cardioprotective role of neuronal nitric oxide synthase (nNOS). However, it remains unclear whether nNOS-mediated heart protection can be translated to gene therapy. In this study, we generated an adeno-associated virus (AAV) nNOS vector and tested its therapeutic efficacy in the aged mdx model of Duchenne cardiomyopathy. A PDZ domain-deleted nNOS gene (ΔPDZ nNOS) was packaged into tyrosine mutant AAV-9 and delivered to the heart of ∼14-month-old female mdx mice, a phenotypic model of Duchenne cardiomyopathy. Seven months later, we observed robust nNOS expression in the myocardium. Supra-physiological ΔPDZ nNOS expression significantly reduced myocardial fibrosis, inflammation and apoptosis. Importantly, electrocardiography and left ventricular hemodynamics were significantly improved in treated mice. Additional studies revealed increased phosphorylation of phospholamban and p70S6K. Collectively, we have demonstrated the therapeutic efficacy of the AAV ΔPDZ nNOS vector in a symptomatic Duchenne cardiomyopathy model. Our results suggest that the cardioprotective role of ΔPDZ nNOS is likely through reduced apoptosis, enhanced phospholamban phosphorylation and improved Akt/mTOR/p70S6K signaling. Our study has opened the door to treat Duchenne cardiomyopathy with ΔPDZ nNOS gene transfer.
|Effect of pirfenidone on proliferation, TGF-β-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts. |
Conte, Enrico, et al.
Eur J Pharm Sci, 58C: 13-19 (2014) 2014
Pirfenidone is an orally active small molecule that has been shown to inhibit the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis. Although pirfenidone exhibits well documented antifibrotic and antiinflammatory activities, in vitro and in vivo, its molecular targets and mechanisms of action have not been elucidated. In this study, we investigated the effects of pirfenidone on proliferation, TGF-β-induced differentiation and fibrogenic activity of primary human lung fibroblasts (HLFs). Pirfenidone reduced fibroblast proliferation and attenuated TGF-β-induced α-smooth muscle actin (SMA) and pro-collagen (Col)-I mRNA and protein levels. Importantly, pirfenidone inhibited TGF-β-induced phosphorylation of Smad3, p38, and Akt, key factors in the TGF-β pathway. Together, these results demonstrate that pirfenidone modulates HLF proliferation and TGF-β-mediated differentiation into myofibroblasts by attenuating key TGF-β-induced signaling pathways.
|Control of lung vascular permeability and endotoxin-induced pulmonary oedema by changes in extracellular matrix mechanics. |
Mammoto, Akiko, et al.
Nat Commun, 4: 1759 (2013) 2013
Increased vascular permeability contributes to many diseases, including acute respiratory distress syndrome, cancer and inflammation. Most past work on vascular barrier function has focused on soluble regulators, such as tumour-necrosis factor-α. Here we show that lung vascular permeability is controlled mechanically by changes in extracellular matrix structure. Our studies reveal that pulmonary vascular leakage can be increased by altering extracellular matrix compliance in vitro and by manipulating lysyl oxidase-mediated collagen crosslinking in vivo. Either decreasing or increasing extracellular matrix stiffness relative to normal levels disrupts junctional integrity and increases vascular leakage. Importantly, endotoxin-induced increases of vascular permeability are accompanied by concomitant increases in extracellular matrix rigidity and lysyl oxidase activity, which can be prevented by inhibiting lysyl oxidase activity. The identification of lysyl oxidase and the extracellular matrix as critical regulators of lung vascular leakage might lead to the development of new therapeutic approaches for the treatment of pulmonary oedema and other diseases caused by abnormal vascular permeability.
|Identification of hamster inducible nitric oxide synthase (iNOS) promoter sequences that influence basal and inducible iNOS expression. |
Omar A Saldarriaga,Bruno L Travi,Goutam Ghosh Choudhury,Peter C Melby
Journal of leukocyte biology 92 2012
IFN-γ/LPS-activated hamster (Mesocricetus auratus) macrophages express significantly less iNOS (NOS2) than activated mouse macrophages, which contributes to the hamster's susceptibility to intracellular pathogens. We determined a mechanism responsible for differences in iNOS promoter activity in hamsters and mice. The HtPP (1.2 kb) showed low basal and inducible promoter activity when compared with the mouse, and sequences within a 100-bp region (-233 to -133) of the mouse and hamster promoters influenced this activity. Moreover, within this 100 bp, we identified a smaller region (44 bp) in the mouse promoter, which recovered basal promoter activity when swapped into the hamster promoter. The mouse homolog (100-bp region) contained a cis-element for NF-IL-6 (-153/-142), which was absent in the hamster counterpart. EMSA and supershift assays revealed that the hamster sequence did not support the binding of NF-IL-6. Introduction of a functional NF-IL-6 binding sequence into the hamster promoter or its alteration in the mouse promoter revealed the critical importance of this transcription factor for full iNOS promoter activity. Furthermore, the binding of NF-IL-6 to the iNOS promoter (-153/-142) in vivo was increased in mouse cells but was reduced in hamster cells after IFN-γ/LPS stimulation. Differences in the activity of the iNOS promoters were evident in mouse and hamster cells, so they were not merely a result of species-specific differences in transcription factors. Thus, we have identified unique DNA sequences and a critical transcription factor, NF-IL-6, which contribute to the overall basal and inducible expression of hamster iNOS.
|Mouse Tissues that Undergo Neoplastic Progression after K-Ras Activation Are Distinguished by Nuclear Translocation of phospho-Erk1/2 and Robust Tumor Suppressor Responses. |
Neha Parikh,Ryan L Shuck,Thuy-Ai Nguyen,Alan Herron,Lawrence A Donehower
Molecular cancer research : MCR 10 2012
Mutation of K-Ras is a frequent oncogenic event in human cancers, particularly cancers of lungs, pancreas, and colon. It remains unclear why some tissues are more susceptible to Ras-induced transformation than others. Here, we globally activated a mutant oncogenic K-Ras allele (K-Ras(G12D)) in mice and examined the tissue-specific effects of this activation on cancer pathobiology, Ras signaling, tumor suppressor, DNA damage, and inflammatory responses. Within 5 to 6 weeks of oncogenic Ras activation, mice develop oral and gastric papillomas, lung adenomas, and hematopoietic hyperproliferation and turn moribund. The oral, gastric, and lung premalignant lesions display activated extracellular signal-regulated kinases (Erk)1/2 and NF-ÎºB signaling as well as activated tumor suppressor and DNA damage responses. Other organs such as pancreas, liver, and small intestine do not exhibit neoplastic progression within 6 weeks following K-Ras(G12D) activation and do not show a potent tumor suppressor response. Even though robust Erk1/2 signaling is activated in all the tissues examined, the pErk1/2 distribution remains largely cytoplasmic in K-Ras(G12D)-refractory tissues (pancreas, liver, and intestines) as opposed to a predominantly nuclear localization in K-Ras(G12D)-induced neoplasms of lung, oral, and gastric mucosa. The downstream targets of Ras signaling, pElk-1 and c-Myc, are elevated in K-Ras(G12D)-induced neoplastic lesions but not in K-Ras(G12D)-refractory tissues. We propose that oncogenic K-Ras-refractory tissues delay oncogenic progression by spatially limiting the efficacy of Ras/Raf/Erk1/2 signaling, whereas K-Ras-responsive tissues exhibit activated Ras/Raf/Erk1/2 signaling, rapidly form premalignant tumors, and activate potent antitumor responses that effectively prevent further malignant progression. Mol Cancer Res; 10(6); 845-55. Â©2012 AACR.
|Disruption of RAB40AL function leads to Martin--Probst syndrome, a rare X-linked multisystem neurodevelopmental human disorder. |
Jirair Krikor Bedoyan,Valerie M Schaibley,Weiping Peng,Yongsheng Bai,Kajari Mondal,Amol C Shetty,Mark Durham,Joseph A Micucci,Arti Dhiraaj,Jennifer M Skidmore,Julie B Kaplan,Cindy Skinner,Charles E Schwartz,Anthony Antonellis,Michael E Zwick,James D Cavalcoli,Jun Z Li,Donna M Martin
Journal of medical genetics 49 2012
Martin--Probst syndrome (MPS) is a rare X-linked disorder characterised by deafness, cognitive impairment, short stature and distinct craniofacial dysmorphisms, among other features. The authors sought to identify the causative mutation for MPS.
|Elevated cyclin g2 expression intersects with DNA damage checkpoint signaling and is required for a potent g2/m checkpoint arrest response to Doxorubicin. |
Maike Zimmermann,Aruni S Arachchige-Don,Michaela S Donaldson,Robert F Dallapiazza,Colleen E Cowan,Mary C Horne
The Journal of biological chemistry 287 2012
To maintain genomic integrity DNA damage response (DDR), signaling pathways have evolved that restrict cellular replication and allow time for DNA repair. CCNG2 encodes an unconventional cyclin homolog, cyclin G2 (CycG2), linked to growth inhibition. Its expression is repressed by mitogens but up-regulated during cell cycle arrest responses to anti-proliferative signals. Here we investigate the potential link between elevated CycG2 expression and DDR signaling pathways. Expanding our previous finding that CycG2 overexpression induces a p53-dependent G(1)/S phase cell cycle arrest in HCT116 cells, we now demonstrate that this arrest response also requires the DDR checkpoint protein kinase Chk2. In accord with this finding we establish that ectopic CycG2 expression increases phosphorylation of Chk2 on threonine 68. We show that DNA double strand break-inducing chemotherapeutics stimulate CycG2 expression and correlate its up-regulation with checkpoint-induced cell cycle arrest and phospho-modification of proteins in the ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) signaling pathways. Using pharmacological inhibitors and ATM-deficient cell lines, we delineate the DDR kinase pathway promoting CycG2 up-regulation in response to doxorubicin. Importantly, RNAi-mediated blunting of CycG2 attenuates doxorubicin-induced cell cycle checkpoint responses in multiple cell lines. Employing stable clones, we test the effect that CycG2 depletion has on DDR proteins and signals that enforce cell cycle checkpoint arrest. Our results suggest that CycG2 contributes to DNA damage-induced G(2)/M checkpoint by enforcing checkpoint inhibition of CycB1-Cdc2 complexes.
|Effects of HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on NEU/HER2 overexpressing mammary tumours in MMTV-NEU-NT mice monitored by Magnetic Resonance Spectroscopy. |
Loreta M Rodrigues,Yuen-Li Chung,Nada M S Al Saffar,Swee Y Sharp,Laura E Jackson,Udai Banerji,Marion Stubbs,Martin O Leach,John R Griffiths,Paul Workman
BMC research notes 5 2012
|Impairments to the GH-IGF-I Axis in hSOD1G93A Mice Give Insight into Possible Mechanisms of GH Dysregulation in Patients with Amyotrophic Lateral Sclerosis. |
F J Steyn,S T Ngo,J D Lee,J W Leong,A J Buckley,J D Veldhuis,P A McCombe,C Chen,M C Bellingham
Endocrinology 153 2012
GH deficiency has been found in subjects with amyotrophic lateral sclerosis (ALS). Disrupted endocrine function could contribute to the progressive muscle loss and hypermetabolism seen in ALS. It is not possible to study all the elements of the GH-IGF-I axis in ALS patients. Consequently, it remains unclear whether dysfunctional GH secretion contributes to disease pathogenesis and why GH and IGF-I directed treatment strategies are ineffective in human ALS. The hSOD1(G93A) transgenic mouse model is useful for the detailed investigation of the pathogenesis of ALS. We report that symptomatic male hSOD1(G93A) transgenic mice exhibit a deficiency in GH secretion similar to that seen in human ALS. Further characterization of the GH-IGF-I axis in hSOD1(G93A) mice reveals central and peripheral abnormalities that are not found in wild-type age-matched controls. Specifically, we observe aberrant endogenous pulsatile GH secretion, reduced pituitary GH content, and decreased circulating levels of IGF-I, indicating global GH deficiency in hSOD1(G93A) mice. Furthermore, a reduction in the expression of the IGF-I receptor α-subunit in skeletal muscle and lumbar spinal cords of hSOD1(G93A) mice suggests impaired IGF-I signaling within these tissues. This is the first account of disrupted GH secretion in a transgenic mouse model of ALS. These observations are essential for the development of effective GH and IGF-I targeted therapies in ALS.
|Impaired contractility and detrusor hypertrophy in cavin-1-deficient mice. |
Mardjaneh Sadegh Karbalaei,Catarina Rippe,Sebastian Albinsson,Mari Ekman,Alva Mansten,Bengt Uvelius,Karl Swärd,Karl Sw
European journal of pharmacology 689 2012
Caveolae are membrane invaginations present in a variety of cell types. Formation of caveolae depends on caveolins and on the more recently discovered family of proteins known as the cavins. Genetic ablation of caveolin-1 was previously shown to give rise to a number of urogenital alterations, but the effects of cavin-1 deletion on urogenital function remain unknown. Here we characterised detrusor contractility and structure in cavin-1-deficient mice. Electron microscopy demonstrated essentially complete lack of caveolae in the knock-out detrusor, and immunoblotting disclosed reduced levels of cavin-3 and of all caveolin proteins. Bladder weight was increased in male knock-out mice, and length-tension relationships demonstrated a reduction in depolarisation-induced contraction. Contractility in response to muscarinic receptor activation was similarly reduced. Despite these functional changes, micturition patterns were similar in conscious and freely moving animals and diuresis was unchanged. Our breeding additionally disclosed that the number of knock-out mice generated in heterozygous crosses was lower than expected, suggesting embryonic/perinatal lethality. In conclusion, this is the first study to show that cavin-1 is critical for detrusor caveolae and for the overall contractility and structure of the urinary bladder.
|Molecular Characteristics of Clostridium perfringens TpeL Toxin and Consequences of Mono-O-GlcNAcylation of Ras in Living Cells. |
Gregor Guttenberg,Sven Hornei,Thomas Jank,Carsten Schwan,Wei Lü,Oliver Einsle,Panagiotis Papatheodorou,Klaus Aktories
The Journal of biological chemistry 287 2012
TpeL is a member of the family of clostridial glucosylating toxins produced by Clostridium perfringens type A, B, and C strains. In contrast to other members of this toxin family, it lacks a C-terminal polypeptide repeat domain, which is suggested to be involved in target cell binding. It was shown that the glucosyltransferase domain of TpeL modifies Ras in vitro by mono-O-glucosylation or mono-O-GlcNAcylation (Nagahama, M., Ohkubo, A., Oda, M., Kobayashi, K., Amimoto, K., Miyamoto, K., and Sakurai, J. (2011) Infect. Immun. 79, 905-910). Here we show that TpeL preferably utilizes UDP-N-acetylglucosamine (UDP-GlcNAc) as a sugar donor. Change of alanine 383 of TpeL to isoleucine turns the sugar donor preference from UDP-GlcNAc to UDP-glucose. In contrast to previous studies, we show that Rac is a poor substrate in vitro and in vivo and requires 1-2 magnitudes higher toxin concentrations for modification by TpeL. The toxin is autoproteolytically processed in the presence of inositol hexakisphosphate (InsP(6)) by an intrinsic cysteine protease domain, located next to the glucosyltransferase domain. A C-terminally extended TpeL full-length variant (TpeL1-1779) induces apoptosis in HeLa cells (most likely by mono-O-GlcNAcylation of Ras), and inhibits Ras signaling including Ras-Raf interaction and ERK activation. In addition, TpeL blocks Ras signaling in rat pheochromocytoma PC12 cells. TpeL is a glucosylating toxin, which modifies Ras and induces apoptosis in target cells without having a typical C-terminal polypeptide repeat domain.
|Self-renewal and differentiation of reactive astrocyte-derived neural stem/progenitor cells isolated from the cortical peri-infarct area after stroke. |
Issei S Shimada,Matthew D Lecomte,Jerrica C Granger,Noah J Quinlan,Jeffrey L Spees
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 2012
In response to stroke, subpopulations of cortical reactive astrocytes proliferate and express proteins commonly associated with neural stem/progenitor cells such as glial fibrillary acidic protein (GFAP) and Nestin. To examine the stem cell-related properties of cortical reactive astrocytes after injury, we generated GFAP-CreER(TM);tdRFP mice to permanently label reactive astrocytes. We isolated cells from the cortical peri-infarct area 3 d after stroke, and cultured them in neural stem cell medium containing epidermal growth factor and basic fibroblast growth factor. We observed tdRFP-positive neural spheres in culture, suggestive of tdRFP-positive reactive astrocyte-derived neural stem/progenitor cells (Rad-NSCs). Cultured Rad-NSCs self-renewed and differentiated into neurons, astrocytes, and oligodendrocytes. Pharmacological inhibition and conditional knock-out mouse studies showed that Presenilin 1 and Notch 1 controlled neural sphere formation by Rad-NSCs after stroke. To examine the self-renewal and differentiation potential of Rad-NSCs in vivo, Rad-NSCs were transplanted into embryonic, neonatal, and adult mouse brains. Transplanted Rad-NSCs were observed to persist in the subventricular zone and secondary Rad-NSCs were isolated from the host brain 28 d after transplantation. In contrast with neurogenic postnatal day 4 NSCs and adult NSCs from the subventricular zone, transplanted Rad-NSCs differentiated into astrocytes and oligodendrocytes, but not neurons, demonstrating that Rad-NSCs had restricted differentiation in vivo. Our results indicate that Rad-NSCs are unlikely to be suitable for neuronal replacement in the absence of genetic or epigenetic modification.
|Cyclic mechanical strain maintains Nanog expression through PI3K/Akt signaling in mouse embryonic stem cells. |
Rie Horiuchi,Takayuki Akimoto,Zhang Hong,Takashi Ushida
Experimental cell research 318 2012
Mechanical strain has been reported to affect the proliferation/differentiation of many cell types; however, the effects of mechanotransduction on self-renewal as well as pluripotency of embryonic stem (ES) cells remains unknown. To investigate the effects of mechanical strain on mouse ES cell fate, we examined the expression of Nanog, which is an essential regulator of self-renewal and pluripotency as well as Nanog-associated intracellular signaling during uniaxial cyclic mechanical strain. The mouse ES cell line, CCE was plated onto elastic membranes, and we applied 10% strain at 0.17Hz. The expression of Nanog was reduced during ES cell differentiation in response to the withdrawal of leukemia inhibitory factor (LIF); however, two days of cyclic mechanical strain attenuated this reduction of Nanog expression. On the other hand, the cyclic mechanical strain promoted PI3K-Akt signaling, which is reported as an upstream of Nanog transcription. The cyclic mechanical strain-induced Akt phosphorylation was blunted by the PI3K inhibitor wortmannin. Furthermore, cytochalasin D, an inhibitor of actin polymerization, also inhibited the mechanical strain-induced increase in phospho-Akt. These findings imply that mechanical force plays a role in regulating Nanog expression in ES cells through the actin cytoskeleton-PI3K-Akt signaling.
|Gene Silencing of the Mitochondrial Adaptor p66Shc Suppresses Vascular Hyperglycemic Memory in Diabetes. |
Paneni, Francesco, et al.
Circulation research, (2012) 2012
Rationale:Hyperglycemic memory may explain why intensive glucose control has failed to improve cardiovascular outcomes in patients with diabetes. Indeed, hyperglycemia promotes vascular dysfunction even after glucose normalization. However, the molecular mechanisms of this phenomenon remain to be elucidated.Objective:The present study investigated the role of mitochondrial adaptor p66(Shc) in this setting.Methods and Results:In human aortic endothelial cells (HAECs) exposed to high glucose and aortas of diabetic mice, activation of p66(Shc) by protein kinase C βII (PKCβII) persisted after returning to normoglycemia. Persistent p66(Shc) upregulation and mitochondrial translocation were associated with continued reactive oxygen species (ROS) production, reduced nitric oxide bioavailability, and apoptosis. We show that (Shc) gene overexpression was epigenetically regulated by promoter CpG hypomethylation and general control nonderepressible 5-induced histone 3 acetylation. Furthermore, p66(Shc)-derived ROS production maintained PKCβII upregulation and PKCβII-dependent inhibitory phosphorylation of endothelial nitric oxide synthase at Thr-495, leading to a detrimental vicious cycle despite restoration of normoglycemia. Moreover, p66(Shc) activation accounted for the persistent elevation of the advanced glycated end product precursor methylglyoxal. In vitro and in vivo gene silencing of p66(Shc), performed at the time of glucose normalization, blunted ROS production, restored endothelium-dependent vasorelaxation, and attenuated apoptosis by limiting cytochrome c release, caspase 3 activity, and cleavage of poly (ADP-ribose) polymerase.Conclusions:p66(Shc) is the key effector driving vascular hyperglycemic memory in diabetes. Our study provides molecular insights for the progression of diabetic vascular complications despite glycemic control and may help to define novel therapeutic targets.
|Smad2 Decelerates Re-epithelialization during Gingival Wound Healing. |
K Tomikawa,T Yamamoto,N Shiomi,M Shimoe,S Hongo,K Yamashiro,T Yamaguchi,H Maeda,S Takashiba
Journal of dental research 91 2012
During periodontal regeneration, inhibition of gingival downgrowth is necessary to promote migration of mesenchymal cells into the defects. Transforming growth factor (TGF)-β is a pleiotropic cytokine that has numerous cell functions, including regulation of epithelial growth. Recent studies have shown that Smad2, a downstream transcription factor of TGF-β, plays crucial roles in wound healing in the epithelia. Therefore, we investigated the effects of Smad2 overexpression on re-epithelialization of gingival wounds. Transgenic mice overexpressing smad2 driven by the keratin 14 promoter (k14-smad2) were confirmed to have significant Smad2 phosphorylation in gingival basal epithelia. Punch wounds were made in the palatal gingiva, and wound healing was assessed histologically for 7 days. Re-epithelialization was significantly retarded on day 2, while collagen deposition was enhanced on day 7 in k14-smad2 compared with wild-type mice. Moreover, expression of keratin 16 (K16), an indicator of keratinocyte migration, was significantly inhibited in wound-edge keratinocytes in k14-smad2. The inhibition of K16 coincided with the induction of Smad2 in the corresponding epithelia, while BrdU incorporation was unaffected. These results indicated that Smad2 has inhibitory effects in regulating keratinocyte migration during gingival wound healing. TGF-β/Smad2 signaling mediating alteration of K16 expression must be tightly regulated during periodontal regeneration.
|Imaging immune and metabolic cells of visceral adipose tissues with multimodal nonlinear optical microscopy. |
Yasuyo Urasaki,Mary G Johlfs,Ronald R Fiscus,Thuc T Le
PloS one 7 2012
Visceral adipose tissue (VAT) inflammation is recognized as a mechanism by which obesity is associated with metabolic diseases. The communication between adipose tissue macrophages (ATMs) and adipocytes is important to understanding the interaction between immunity and energy metabolism and its roles in obesity-induced diseases. Yet visualizing adipocytes and macrophages in complex tissues is challenging to standard imaging methods. Here, we describe the use of a multimodal nonlinear optical (NLO) microscope to characterize the composition of VATs of lean and obese mice including adipocytes, macrophages, and collagen fibrils in a label-free manner. We show that lipid metabolism processes such as lipid droplet formation, lipid droplet microvesiculation, and free fatty acids trafficking can be dynamically monitored in macrophages and adipocytes. With its versatility, NLO microscopy should be a powerful imaging tool to complement molecular characterization of the immunity-metabolism interface.
|NKCC1 upregulation disrupts chloride homeostasis in the hypothalamus and increases neuronal activity-sympathetic drive in hypertension. |
Zeng-You Ye,De-Pei Li,Hee Sun Byun,Li Li,Hui-Lin Pan
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 2012
Hypertension is a major risk factor for coronary artery disease, stroke, and kidney failure. However, the etiology of hypertension in most patients is poorly understood. Increased sympathetic drive emanating from the hypothalamic paraventricular nucleus (PVN) plays a major role in the development of hypertension. Na(+)-K(+)-2Cl(-) cotransporter-1 (NKCC1) in the brain is critically involved in maintaining chloride homeostasis and in neuronal responses mediated by GABA(A) receptors. Here we present novel evidence that the GABA reversal potential (E(GABA)) of PVN presympathetic neurons undergoes a depolarizing shift that diminishes GABA inhibition in spontaneously hypertensive rats (SHRs). Inhibition of NKCC1, but not KCC2, normalizes E(GABA) and restores GABA inhibition of PVN neurons in SHRs. The mRNA and protein levels of NKCC1, but not KCC2, in the PVN are significantly increased in SHRs, and the NKCC1 proteins on the plasma membrane are highly glycosylated. Inhibiting NKCC1 N-glycosylation restores E(GABA) and GABAergic inhibition of PVN presympathetic neurons in SHRs. Furthermore, NKCC1 inhibition significantly reduces the sympathetic vasomotor tone and augments the sympathoinhibitory responses to GABA(A) receptor activation in the PVN in SHRs. These findings suggest that increased NKCC1 activity and glycosylation disrupt chloride homeostasis and impair synaptic inhibition in the PVN to augment the sympathetic drive in hypertension. This information greatly improves our understanding of the pathogenesis of hypertension and helps to design better treatment strategies for neurogenic hypertension.
|Behavioral phenotype and BDNF differences related to apoE isoforms and sex in young transgenic mice. |
Ingrid Reverte,Anders Bue Klein,Cecilia Ratner,José L Domingo,Maria Teresa Colomina
Experimental neurology 237 2012
Human apolipoprotein E (apoE) plays an important role in lipid transport and distribution, being involved in neurite growth and neuroprotection in the brain. In humans, the apoE4 isoform is a risk factor for developing Azheimer's disease (AD), while apoE2 seems to provide neuroprotection. However, very little information is available on apoE2 genotype. In the present study, we have characterized behavioral and learning phenotypes in young transgenic mice apoE2, apoE3 and apoE4 of both sexes. We have also determined the levels of brain-derived neurotrophic factor (BDNF) and its receptor TrkB in cortex and hippocampus of male and female mice carrying either genotype. Our results show a worse performance of apoE4 and apoE2 mice in the acquisition of a spatial task compared to apoE3 mice, and a worse retention in apoE2 mice compared to the other two genotypes. On the other hand, an increase in the exploration of an open-field, which is compatible with a hyperactive behavior, was found in apoE2 females, while a decreased activity was observed in apoE4 mice. Increased BDNF levels in the frontal cortex were observed in apoE2 mice compared to apoE3. These results underscore behavioral differences between apoE genotypes in young mice, as well as the existence of interactions between genotype and gender, providing new valuable information on the apoE2 genotype.
|Vezatin is essential for dendritic spine morphogenesis and functional synaptic maturation. |
Lydia Danglot,Thomas Freret,Nicolas Le Roux,Nicolas Narboux Nême,Andrea Burgo,Vincent Hyenne,Anne Roumier,Vincent Contremoulins,François Dauphin,Jean-Charles Bizot,Guilan Vodjdani,Patricia Gaspar,Michel Boulouard,Jean-Christophe Poncer,Thierry Galli,Marie-Christine Simmler,Nicolas Narboux N,Fran Dauphin
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 2012
Vezatin is an integral membrane protein associated with cell-cell adhesion complex and actin cytoskeleton. It is expressed in the developing and mature mammalian brain, but its neuronal function is unknown. Here, we show that Vezatin localizes in spines in mature mouse hippocampal neurons and codistributes with PSD95, a major scaffolding protein of the excitatory postsynaptic density. Forebrain-specific conditional ablation of Vezatin induced anxiety-like behavior and impaired cued fear-conditioning memory response. Vezatin knock-down in cultured hippocampal neurons and Vezatin conditional knock-out in mice led to a significantly increased proportion of stubby spines and a reduced proportion of mature dendritic spines. PSD95 remained tethered to presynaptic terminals in Vezatin-deficient hippocampal neurons, suggesting that the reduced expression of Vezatin does not compromise the maintenance of synaptic connections. Accordingly, neither the amplitude nor the frequency of miniature EPSCs was affected in Vezatin-deficient hippocampal neurons. However, the AMPA/NMDA ratio of evoked EPSCs was reduced, suggesting impaired functional maturation of excitatory synapses. These results suggest a role of Vezatin in dendritic spine morphogenesis and functional synaptic maturation.
|The G60S Cx43 mutant enhances keratinocyte proliferation and differentiation. |
Jared M Churko,John J Kelly,Andrew Macdonald,Jack Lee,Jacinda Sampson,Donglin Bai,Dale W Laird
Experimental dermatology 21 2012
Transient knock-down of the gap junction protein Cx43 by antisense and siRNA, or gap junction block with mimetic peptides, have been shown to enhance epidermal wound healing. However, patients with oculodentodigital dysplasia (ODDD) express mutant Cx43 that leads to a chronic reduction in gap junctional intercellular communication. To determine whether mutant Cx43 in keratinocytes would impact upon the wound healing process, we localized Cx43 in human and mouse skin tissue expressing mutant Cx43 and assessed the ability of primary keratinocytes derived from a mouse model of ODDD to proliferate, migrate and differentiate. In the epidermis from an ODDD patient and in the epidermis of mice expressing the G60S mutant or in keratinocytes obtained from mutant mice, Cx43 was frequently found within intracellular compartments and rarely localized to punctate sites of cell-cell apposition. Primary keratinocytes derived from G60S mutant mice proliferated faster but migrated similarly to keratinocytes derived from wild-type control mice. Keratinocytes derived from mutant mice expressed abundant Cx43 and higher levels of involucrin and loricrin under low calcium conditions. However, after calcium-induced differentiation, similar levels of Cx43, involucrin and loricrin were observed. Thus, we conclude that during wound healing, mutant Cx43 may enhance keratinocyte proliferation and promote early differentiation of keratinocytes.
|Pax6 is a key component of regulated glucagon secretion. |
Yvan Gosmain,Claire Cheyssac,Mounia Heddad Masson,Audrey Guérardel,Caroline Poisson,Jacques Philippe
Endocrinology 153 2012
The Pax6 transcription factor is crucial for pancreatic α-cells. Indeed, Pax6-deficient mouse models are characterized by markedly altered α-cell differentiation. Our objective was to investigate the role of Pax6 in glucagon secretion process. We used a Pax6-deficient model in rat primary enriched-α cells with specific small interfering RNA leading to a 70% knockdown of Pax6 expression. We first showed that Pax6 knockdown decreases glucagon biosynthesis as well as glucagon release. Through physiological assays, we demonstrated that the decrease of Pax6 affects specifically acute glucagon secretion in primary α-cell in response to glucose, palmitate, and glucose-dependent insulinotropic peptide (GIP) but not the response to arginine and epinephrine. We identified in Pax6 knockdown model that genes involved in glucagon secretion such as the glucokinase (GCK), G protein-coupled receptor (GPR40), and GIP receptor (GIPR) as well as the corresponding proteins were significantly decreased whereas the insulin receptor (IR) Kir6.2/Sur1, and glucose transporter 1 genes were not affected. We demonstrated that Pax6 directly binds and activates specific elements on the promoter region of the GPR40, GCK, and GIPR genes. Finally, through site-directed mutagenesis experiments, we showed that disruption of Pax6 binding on the GCK, GPR40, and GIPR gene promoters led to specific decreases of their activities in the αTC1.9 glucagon-producing cell line. Hence our results indicate that Pax6 acts on the regulation of glucagon secretion at least through the transcriptional control of GCK, GPR40, and GIPR. We propose that Pax6 is not only critical for glucagon biosynthesis but also for glucagon secretion particularly in response to nutrients.
|Mice lacking urea transporter UT-B display depression-like behavior. |
Xin Li,Jianhua Ran,Hong Zhou,Tianluo Lei,Li Zhou,Jingyan Han,Baoxue Yang
Journal of molecular neuroscience : MN 46 2012
Urea transporter B is one of urea transporters that selectively transport urea driven by urea gradient across membrane and expressed abundantly in brain. To determine the physiological role of UT-B in brain, UT-B localization, urea concentration, tissue morphology of brain, and behavioral phenotypes were studied in UT-B heterozygous mice via UT-B null mice. UT-B mRNA was expressed in olfactory bulb, cortex, caudate nucleus, hippocampus and hypothalamus of UT-B heterozygous mice. UT-B null mice exhibited depression-like behavior, with urea accumulation, nitric oxide reduction, and selective neuronal nitric oxide synthase level increase in hippocampus. After acute urea loading, the urea level increased, NO production decreased in hippocampus from both types of mice. Moreover, urea level was higher, and NO concentration was lower consistently in UT-B null hippocampus than that in heterozygous hippocampus. In vitro, 25 mM urea inhibited NO production too. Furthermore, UT-B knockout induced a long-lasting notable decrease in regional cerebral blood flow and altered morphology, such as loss of neurons in CA3 region, swelling, and membranous myelin-like structure formation within myelinated and unmyelinated fibers in hippocampus. These results suggest that urea accumulation in the hippocampus induced by UT-B deletion can cause depression-like behavior, which possibly attribute to disturbance in NOS/NO system.
|Tetraploid cells from cytokinesis failure induce aneuploidy and spontaneous transformation of mouse ovarian surface epithelial cells. |
Lei Lv,Tianwei Zhang,Qiyi Yi,Yun Huang,Zheng Wang,Heli Hou,Huan Zhang,Wei Zhang,Qiaomei Hao,Zongyou Guo,Howard J Cooke,Qinghua Shi
Cell cycle (Georgetown, Tex.) 11 2012
Most ovarian cancers originate from the ovarian surface epithelium and are characterized by aneuploid karyotypes. Aneuploidy, a consequence of chromosome instability, is an early event during the development of ovarian cancers. However, how aneuploid cells are evolved from normal diploid cells in ovarian cancers remains unknown. In the present study, cytogenetic analyses of a mouse syngeneic ovarian cancer model revealed that diploid mouse ovarian surface epithelial cells (MOSECs) experienced an intermediate tetraploid cell stage, before evolving to aneuploid (mainly near-tetraploid) cells. Using long-term live-cell imaging followed by fluorescence in situ hybridization (FISH), we demonstrated that tetraploid cells originally arose from cytokinesis failure of bipolar mitosis in diploid cells, and gave rise to aneuploid cells through chromosome mis-segregation during both bipolar and multipolar mitoses. Injection of the late passage aneuploid MOSECs resulted in tumor formation in C57BL/6 mice. Therefore, we reveal a pathway for the evolution of diploid to aneuploid MOSECs and elucidate a mechanism for the development of near-tetraploid ovarian cancer cells.
|ASC-dependent RIP2 Kinase Regulates Reduced PGE2 Production in Chronic Periodontitis. |
D J Taxman,Y Lei,S Zhang,E Holley-Guthrie,S Offenbacher,J P-Y Ting
Journal of dental research 91 2012
Levels of prostaglandin E(2) (PGE(2)) and its processing enzyme, prostaglandin-endoperoxide-synthase-2/ cyclooxygenase-2 (PTGS2/COX-2), are elevated in actively progressing periodontal lesions, but suppressed in chronic disease. COX-2 expression is regulated through inflammatory signaling that converges on the mitogen-activated protein kinase (MAPK) pathway. Emerging evidence suggests a role for the inflammatory adaptor protein, ASC/Pycard, in MAPK activation. We postulated that ASC may represent a mediator of the MAPK-mediated regulatory network of PGE(2) production. Using RNAi-mediated gene slicing, we demonstrated that ASC regulates COX-2 expression and PGE(2) production in THP1 monocytic cells following infection with Porphyromonas gingivalis (Pg). Production of PGE(2) did not require the inflammasome adaptor function of ASC, but was dependent on MAPK activation. Furthermore, the MAP kinase kinase kinase CARD domain-containing protein RIPK2 was induced by Pg in an ASC-dependent manner. Reduced ASC and RIPK2 levels were revealed by orthogonal comparison of the expression of the RIPK family in ASC-deficient THP1 cells with that in chronic periodontitis patients. We show that pharmacological inhibition of RIPK2 represses PGE(2) secretion, and RNAi-mediated silencing of RIPK2 leads to diminished MAPK activation and PGE(2) secretion. These findings identify a novel ASC-RIPK2 axis in the generation of PGE(2) that is repressed in patients diagnosed with chronic adult periodontitis.
|HIV-1 infection and first line ART induced differential responses in mitochondria from blood lymphocytes and monocytes: the ANRS EP45 "Aging" study. |
Perrin, Sophie, et al.
PLoS ONE, 7: e41129 (2012) 2012
The ANRS EP45 "Aging" study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The data reported focus on mitochondria, organelles known to be involved in cell senescence.
|MicroRNA-133a regulates DNA methylation in diabetic cardiomyocytes. |
Vishalakshi Chavali,Suresh C Tyagi,Paras K Mishra
Biochemical and biophysical research communications 425 2012
We tested the hypothesis that miR-133a regulates DNA methylation by inhibiting Dnmt-1 (maintenance) and Dnmt-3a and -3b (de novo) methyl transferases in diabetic hearts by using Ins2(+/-) Akita (diabetic) and C57BL/6J (WT), mice and HL1 cardiomyocytes. The specific role of miR-133a in DNA methylation in diabetes was assessed by two treatment groups (1) scrambled, miR-133a mimic, anti-miR-133a, and (2) 5mM glucose (CT), 25 mM glucose (HG) and HG+miR-133a mimic. The levels of miR-133a, Dnmt-1, -3a and -3b were measured by multiplex RT-PCR, qPCR and Western blotting. The results revealed that miR-133a is inhibited but Dnmt-1 and -3b are induced in Akita suggesting that attenuation of miR-133a induces both maintenance (Dnmt-1) - and de novo - methylation (Dnmt-3b) in diabetes. The up regulation of Dnmt-3a in Akita hearts elicits intricate and antagonizing interaction between Dnmt-3a and -3b. In cardiomyocytes, over expression of miR-133a inhibits but silencing of miR-133a induces Dnmt-1, -3a and -3b elucidating the involvement of miR-133a in regulation of DNA methylation. The HG treatment up regulates only Dnmt-1 and not Dnmt-3a and -3b suggesting that acute hyperglycemia triggers only maintenance methylation. The over expression of miR-133a mitigates glucose mediated induction of Dnmt-1 illustrating the role of miR-133a in regulation of DNA methylation in diabetes.
|Porphyromonas gingivalis Mediates Inflammasome Repression in Polymicrobial Cultures through a Novel Mechanism Involving Reduced Endocytosis. |
Debra J Taxman,Karen V Swanson,Peter M Broglie,Haitao Wen,Elizabeth Holley-Guthrie,Max Tze-Han Huang,Justin B Callaway,Tim K Eitas,Joseph A Duncan,Jenny P Y Ting
The Journal of biological chemistry 287 2012
The interleukin (IL)-1β-processing inflammasome has recently been identified as a target for pathogenic evasion of the inflammatory response by a number of bacteria and viruses. We postulated that the periodontal pathogen, Porphyromonas gingivalis may suppress the inflammasome as a mechanism for its low immunogenicity and pathogenic synergy with other, more highly immunogenic periodontal bacteria. Our results show that P. gingivalis lacks signaling capability for the activation of the inflammasome in mouse macrophages. Furthermore, P. gingivalis can suppress inflammasome activation by another periodontal bacterium, Fusobacterium nucleatum. This repression affects IL-1β processing, as well as other inflammasome-mediated processes, including IL-18 processing and cell death, in both human and mouse macrophages. F. nucleatum activates IL-1β processing through the Nlrp3 inflammasome; however, P. gingivalis repression is not mediated through reduced levels of inflammasome components. P. gingivalis can repress Nlrp3 inflammasome activation by Escherichia coli, and by danger-associated molecular patterns and pattern-associated molecular patterns that mediate activation through endocytosis. However, P. gingivalis does not suppress Nlrp3 inflammasome activation by ATP or nigericin. This suggests that P. gingivalis may preferentially suppress endocytic pathways toward inflammasome activation. To directly test whether P. gingivalis infection affects endocytosis, we assessed the uptake of fluorescent particles in the presence or absence of P. gingivalis. Our results show that P. gingivalis limits both the number of cells taking up beads and the number of beads taken up for bead-positive cells. These results provide a novel mechanism of pathogen-mediated inflammasome inhibition through the suppression of endocytosis.
|Mouse model of muscleblind-like 1 overexpression: skeletal muscle effects and therapeutic promise. |
Christopher M Chamberlain,Laura P W Ranum
Human molecular genetics 21 2012
Myotonic dystrophy (DM) is a multisystemic disease caused by CTG or CCTG expansion mutations. There is strong evidence that DM1 CUG and DM2 CCUG expansion transcripts sequester muscleblind-like (MBNL) proteins and that loss of MBNL function causes alternative splicing abnormalities that contribute to disease. Because MBNL1 loss is thought to play an important role in disease and localized AAV delivery of MBNL1 partially rescues skeletal muscle pathology in DM mice, there is strong interest in MBNL1 overexpression as a therapeutic strategy. We developed the first transgenic MBNL1 overexpression mouse model (MBNL1-OE) to test the safety and efficacy of multisystemic MBNL1 overexpression. First, we demonstrate that MBNL1 overexpression is generally well-tolerated in skeletal muscle. Second, we show the surprising result that premature shifts in alternative splicing of MBNL1-regulated genes in multiple organ systems are compatible with life and do not cause embryonic lethality. Third, we show for the first time that early and long-term MBNL1 overexpression prevents CUG-induced myotonia, myopathy and alternative splicing abnormalities in DM1 mice. In summary, MBNL1 overexpression may be a valuable strategy for treating the skeletal muscle features of DM.
|Regulation of Nuclear Factor κB (NF-κB) Transcriptional Activity via p65 Acetylation by the Chaperonin Containing TCP1 (CCT). |
Nadja Pejanovic,Karin Hochrainer,Tao Liu,Birgit L Aerne,Miguel P Soares,Josef Anrather
PloS one 7 2012
The NF-κB family member p65 is central to inflammation and immunity. The purpose of this study was to identify and characterize evolutionary conserved genes modulating p65 transcriptional activity. Using an RNAi screening approach, we identified chaperonin containing TCP1 subunit η (CCTη) as a regulator of Drosophila NF-κB proteins, Dorsal and Dorsal-related immunity factor (Dif). CCTη was also found to regulate NF-κB-driven transcription in mammalian cells, acting in a promoter-specific context, downstream of IκB kinase (IKK). CCTη knockdown repressed IκBα and CXCL2/MIP2 transcription during the early phase of NF-κB activation while impairing the termination of CCL5/RANTES and CXCL10/IP10 transcription. The latter effect was associated with increased DNA binding and reduced p65 acetylation, presumably by altering the activity of histone acetyltransferase CREB-binding protein (CBP). We identified p65 lysines (K) 122 and 123 as target residues mediating the CCTη-driven termination of NF-κB-dependent transcription. We propose that CCTη regulates NF-κB activity in a manner that resolves inflammation.
|Humanized c-Myc Mouse. |
Frank M Lehmann,Samantha Feicht,Florian Helm,Anna Maurberger,Camilla Ladinig,Ursula Zimber-Strobl,Ralf Kühn,Josef Mautner,Armin Gerbitz,Georg W Bornkamm
PloS one 7 2012
A given tumor is usually dependent on the oncogene that is activated in the respective tumor entity. This phenomenon called oncogene addiction provides the rationale for attempts to target oncogene products in a therapeutic manner, be it by small molecules, by small interfering RNAs (siRNA) or by antigen-specific T cells. As the proto-oncogene product is required also for the function of normal cells, this raises the question whether there is a therapeutic window between the adverse effects of specific inhibitors or T cells to normal tissue that may limit their application, and their beneficial tumor-specific therapeutic action. To address this crucial question, suitable mouse strains need to be developed, that enable expression of the human proto-oncogene not only in tumor but also in normal cells. The aim of this work is to provide such a mouse strain for the human proto-oncogene product c-MYC.
|Classical Macrophage Activation Up-Regulates Several Matrix Metalloproteinases through Mitogen Activated Protein Kinases and Nuclear Factor-κB. |
Wei-Chun Huang,Graciela B Sala-Newby,Angela Susana,Jason L Johnson,Andrew C Newby
PloS one 7 2012
Remodelling of the extracellular matrix (ECM) and cell surface by matrix metalloproteinases (MMPs) is an important function of monocytes and macrophages. Recent work has emphasised the diverse roles of classically and alternatively activated macrophages but the consequent regulation of MMPs and their inhibitors has not been studied comprehensively. Classical activation of macrophages derived in vitro from un-fractionated CD16(+/-) or negatively-selected CD16(-) macrophages up-regulated MMP-1, -3, -7, -10, -12, -14 and -25 and decreased TIMP-3 steady-state mRNA levels. Bacterial lipopolysaccharide, IL-1 and TNFα were more effective than interferonγ except for the effects on MMP-25, and TIMP-3. By contrast, alternative activation decreased MMP-2, -8 and -19 but increased MMP -11, -12, -25 and TIMP-3 steady-state mRNA levels. Up-regulation of MMPs during classical activation depended on mitogen activated protein kinases, phosphoinositide-3-kinase and inhibitor of κB kinase-2. Effects of interferonγ depended on janus kinase-2. Where investigated, similar effects were seen on protein concentrations and collagenase activity. Moreover, activity of MMP-1 and -10 co-localised with markers of classical activation in human atherosclerotic plaques in vivo. In conclusion, classical macrophage activation selectively up-regulates several MMPs in vitro and in vivo and down-regulates TIMP-3, whereas alternative activation up-regulates a distinct group of MMPs and TIMP-3. The signalling pathways defined here suggest targets for selective modulation of MMP activity.
|Novel Insights into the Molecular Mechanism of Action of DNA Hypomethylating Agents: Role of Protein Kinase C δ in Decitabine-Induced Degradation of DNA Methyltransferase 1. |
Jharna Datta,Kalpana Ghoshal,Tasneem Motiwala,Samson T Jacob
Genes & cancer 3 2012
We have previously demonstrated proteasomal degradation of DNMT1 in mammalian cells following treatment with several DNA hypomethylating agents. Here, we demonstrate dose-dependent degradation of Dnmt1 in mouse embryonic stem (ES) cells expressing catalytic site mutant (cys-ser), confirming that the covalent bond formation between Dnmt1 and decitabine-incorporated DNA is not essential for this process. DNMT1o, the oocyte-specific isoform that lacks the N-terminal 118-amino acid domain, did not undergo decitabine-mediated degradation, which further proves the requirement of multiple domains including nuclear localization signal, KEN box, and BAH domains for this process. Analysis of glycerol density gradient fractions of micrococcal nuclease-digested nuclei showed that both nucleosomal and nucleoplasmic DNMT1 are degraded upon decitabine treatment. Among different inhibitors tested, the inhibitors of the proteasomal pathway and several protein kinases impeded decitabine-induced DNMT1 degradation. The maximal effect caused by inhibiting protein kinase C (PKC) persuaded us to investigate further its role in decitabine-mediated DNMT1 degradation. Blockage of the degradation process after treatment with rottlerin, an inhibitor of PKCδ, or after siRNA-mediated depletion of PKCδ, indicated that this protein kinase is involved in decitabine-mediated depletion of DNMT1. PKCδ interacted with and phosphorylated DNMT1 in vitro. Moreover, rottlerin inhibited both basal and decitabine-induced phosphorylation of DNMT1. These studies provide substantial evidence that decitabine-induced degradation of the maintenance methyltransferase DNMT1 does not require covalent bond formation with the substrate and also elucidate its underlying molecular mechanism.
|Age-related decrease in cerebrovascular-derived neuroprotective proteins: Effect of acetaminophen. |
Debjani Tripathy,Alma Sanchez,Xiangling Yin,Joseph Martinez,Paula Grammas
Microvascular research 84 2012
As the population ages, the need for effective methods to maintain brain function in older adults is increasingly pressing. Vascular disease and neurodegenerative disorders commonly co-occur in older persons. Cerebrovascular products contribute to the neuronal milieu and have important consequences for neuronal viability. In this regard vascular derived neuroprotective proteins, Such as vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), and pituitary adenylate cyclase activating peptide (PACAP) are important for maintaining neuronal viability, especially in the face of injury and disease. The objective of this study is to measure and compare levels of VEGF, PEDF and PACAP released from isolated brain microvessels of Fischer 344 rats at 6, 12, 18, and 24months of age. Addition of acetaminophen to isolated brain microvessels is employed to determine whether this drug affects vascular expression of these neuroprotective proteins. Experiments on cultured brain endothelial cells are performed to explore the mechanisms/mediators that regulate the effect of acetaminophen on endothelial cells. The data indicate cerebrovascular expression of VEGF, PEDF and PACAP significantly decreases with age. The age-associated decrease in VEGF and PEDF is ameliorated by addition of acetaminophen to isolated brain microvessels. Also, release of VEGF, PEDF, and PACAP from cultured brain endothelial cells decreases with exposure to the oxidant stressor menadione. Acetaminophen treatment upregulates VEGF, PEDF and PACAP in brain endothelial cells exposed to oxidative stress. The effect of acetaminophen on cultured endothelial cells is in part inhibited by the selective thrombin inhibitor hirudin. The results of this study suggest that acetaminophen may be a useful agent for preserving cerebrovascular function. If a low dose of acetaminophen can counteract the decrease in vascular-derived neurotrophic factors evoked by age and oxidative stress, this drug might be useful for improving brain function in the elderly.
|Gene network and pathway analysis of mice with conditional ablation of dicer in post-mitotic neurons. |
Véronique Dorval,Pascal Y Smith,Charlotte Delay,Ezequiel Calvo,Emmanuel Planel,Nadège Zommer,Luc Buée,Sébastien S Hébert,V Dorval,Nad Zommer,Luc Bu,S H
PloS one 7 2012
The small non-protein-coding microRNAs (miRNAs) have emerged as critical regulators of neuronal differentiation, identity and survival. To date, however, little is known about the genes and molecular networks regulated by neuronal miRNAs in vivo, particularly in the adult mammalian brain.
|Inhibition of damage-regulated autophagy modulator-1 (DRAM-1) impairs neutrophil differentiation of NB4 APL cells. |
Magali Humbert,Chantal Mueller,Martin F Fey,Mario P Tschan
Leukemia research 36 2012
The damage-regulator autophagy modulator 1 (DRAM-1) is a lysosomal protein that positively regulates autophagy in a p53-dependent manner. We aimed at analyzing the role of DRAM-1 in granulocytic differentiation of APL cells. We observed a significant increase of DRAM-1 expression during all-trans retinoic acid (ATRA)-induced neutrophil differentiation of NB4 APL cells but not in ATRA-resistant NB4-R2 cells. Next, knocking down DRAM-1 in NB4 APL cells was sufficient to impair neutrophil differentiation. Given that DRAM-1 is a transcriptional target of p53, we tested if DRAM-1 is regulated by the p53 relative p73. Indeed, inhibiting p73 prevented neutrophil differentiation and DRAM-1 induction of NB4 cells. In conclusion, we show for the first time that p73-regulated DRAM-1 is functionally involved in neutrophil differentiation of APL cells.
|Regulation of the tumor suppressor PTEN by SUMO. |
J Gonz,M Campagna,A Ortega-Molina,L Marcos-Villar,C F de la Cruz-Herrera,D Gonz,P Gallego,F Lopitz-Otsoa,M Esteban,M S Rodr,M Serrano,C Rivas,J González-Santamaría,D González,M S Rodríguez
Cell death & disease 3 2012
The crucial function of the PTEN tumor suppressor in multiple cellular processes suggests that its activity must be tightly controlled. Both, membrane association and a variety of post-translational modifications, such as acetylation, phosphorylation, and mono- and polyubiquitination, have been reported to regulate PTEN activity. Here, we demonstrated that PTEN is also post-translationally modified by the small ubiquitin-like proteins, small ubiquitin-related modifier 1 (SUMO1) and SUMO2. We identified lysine residue 266 and the major monoubiquitination site 289, both located within the C2 domain required for PTEN membrane association, as SUMO acceptors in PTEN. We demonstrated the existence of a crosstalk between PTEN SUMOylation and ubiquitination, with PTEN-SUMO1 showing a reduced capacity to form covalent interactions with monoubiquitin and accumulation of PTEN-SUMO2 conjugates after inhibition of the proteasome. Moreover, we found that virus infection induces PTEN SUMOylation and favors PTEN localization at the cell membrane. Finally, we demonstrated that SUMOylation contributes to the control of virus infection by PTEN.
|TGF-β-SMAD3 signaling mediates hepatic bile acid and phospholipid metabolism following lithocholic acid-induced liver injury. |
Tsutomu Matsubara,Naoki Tanaka,Misako Sato,Dong Wook Kang,Kristopher W Krausz,Kathleen C Flanders,Kazuo Ikeda,Hans Luecke,Lalage M Wakefield,Frank J Gonzalez
Journal of lipid research 53 2012
Transforming growth factor-β (TGFβ) is activated as a result of liver injury, such as cholestasis. However, its influence on endogenous metabolism is not known. This study demonstrated that TGFβ regulates hepatic phospholipid and bile acid homeostasis through MAD homolog 3 (SMAD3) activation as revealed by lithocholic acid-induced experimental intrahepatic cholestasis. Lithocholic acid (LCA) induced expression of TGFB1 and the receptors TGFBR1 and TGFBR2 in the liver. In addition, immunohistochemistry revealed higher TGFβ expression around the portal vein after LCA exposure and diminished SMAD3 phosphorylation in hepatocytes from Smad3-null mice. Serum metabolomics indicated increased bile acids and decreased lysophosphatidylcholine (LPC) after LCA exposure. Interestingly, in Smad3-null mice, the metabolic alteration was attenuated. LCA-induced lysophosphatidylcholine acyltransferase 4 (LPCAT4) and organic solute transporter β (OSTβ) expression were markedly decreased in Smad3-null mice, whereas TGFβ induced LPCAT4 and OSTβ expression in primary mouse hepatocytes. In addition, introduction of SMAD3 enhanced the TGFβ-induced LPCAT4 and OSTβ expression in the human hepatocellular carcinoma cell line HepG2. In conclusion, considering that Smad3-null mice showed attenuated serum ALP activity, a diagnostic indicator of cholangiocyte injury, these results strongly support the view that TGFβ-SMAD3 signaling mediates an alteration in phospholipid and bile acid metabolism following hepatic inflammation with the biliary injury.
|Activity-dependent local translation of matrix metalloproteinase-9. |
Magdalena Dziembowska,Jacek Milek,Aleksandra Janusz,Emilia Rejmak,Ewelina Romanowska,Tomasz Gorkiewicz,Adrian Tiron,Clive R Bramham,Leszek Kaczmarek
The Journal of neuroscience : the official journal of the Society for Neuroscience 32 2012
Local, synaptic synthesis of new proteins in response to neuronal stimulation plays a key role in the regulation of synaptic morphogenesis. Recent studies indicate that matrix metalloproteinase-9 (MMP-9), an endopeptidase that regulates the pericellular environment through cleavage of its protein components, plays a critical role in regulation of spine morphology and synaptic plasticity. Here, we sought to determine whether MMP-9 mRNA is transported to dendrites for local translation and protein release. First, dendritic transport of MMP-9 mRNA was seen in primary hippocampal neuronal cultures treated with glutamate and in dentate gyrus granule cells in adult anesthetized rats after induction of long-term potentiation. Second, rapid, activity-dependent polyadenylation of MMP-9 mRNA; association of the mRNA with actively translating polysomes; and de novo MMP-9 protein synthesis were obtained in synaptoneurosomes isolated from rat hippocampus. Third, glutamate stimulation of cultured hippocampal neurons evoked a rapid (in minutes) increase in MMP-9 activity, as measured by cleavage of its native substrate, β-dystroglycan. This activity was reduced by the polyadenylation inhibitor, thus linking MMP-9 translation with protein function. In aggregate, our findings show that MMP-9 mRNA is transported to dendrites and locally translated and that the protein is released in an activity-dependent manner. Acting in concert with other dendritically synthesized proteins, locally secreted MMP-9 may contribute to the structural and functional plasticity of the activated synapses.
|Effects of an Early Experience of Reward through Maternal Contact or its Denial on Laterality of Protein Expression in the Developing Rat Hippocampus. |
Androniki Raftogianni,Antonios Stamatakis,Angeliki Papadopoulou,Konstantinos Vougas,Athanasios K Anagnostopoulos,Fotini Stylianopoulou,George Th Tsangaris
PloS one 7 2012
Laterality is a basic characteristic of the brain which is detectable early in life. Although early experiences affect laterality of the mature brain, there are no reports on their immediate neurochemical effects during neonatal life, which could provide evidence as to the mechanisms leading to the lateralized brain. In order to address this issue, we determined the differential protein expression profile of the left and right hippocampus of 13-day-old rat control (CTR) pups, as well as following exposure to an early experience involving either receipt (RER) or denial (DER) of the expected reward of maternal contact. Proteomic analysis was performed by 2-dimensional polyacrylamide gel electrophoresis (PAGE) followed by mass spectroscopy. The majority of proteins found to be differentially expressed either between the three experimental groups (DER, RER, CTR) or between the left and right hemisphere were cytoskeletal (34%), enzymes of energy metabolism (32%), and heat shock proteins (17%). In all three groups more proteins were up-regulated in the left compared to the right hippocampus. Tubulins were found to be most often up-regulated, always in the left hippocampus. The differential expression of β-tubulin, β-actin, dihydropyrimidinase like protein 1, glial fibrillary acidic protein (GFAP) and Heat Shock protein 70 revealed by the proteomic analysis was in general confirmed by Western blots. Exposure to the early experience affected brain asymmetry: In the RER pups the ratio of proteins up-regulated in the left hippocampus to those in the right was 1.8, while the respective ratio was 3.6 in the CTR and 3.4 in the DER. Our results could contribute to the elucidation of the cellular mechanisms mediating the effects of early experiences on the vulnerability for psychopathology, since proteins shown in our study to be differentially expressed (e.g. tubulins, dihydropyrimidinase like proteins, 14-3-3 protein, GFAP, ATP synthase, α-internexin) have also been identified in proteomic analyses of post-mortem brains from psychiatric patients.
|HIV protease inhibitors do not cause the accumulation of prelamin A in PBMCs from patients receiving first line therapy: the ANRS EP45 "aging" study. |
Perrin, Sophie, et al.
PLoS ONE, 7: e53035 (2012) 2012
The ANRS EP45 "Aging" study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The present report focuses on lamin A processing, a pathway known to be altered in systemic genetic progeroid syndromes.
|ARNO regulates VEGF-dependent tissue responses by stabilizing endothelial VEGFR-2 surface expression. |
Hanna K Mannell,Joachim Pircher,Daniel I Chaudhry,Stefan K C Alig,Elisabeth G Koch,Ramona Mettler,Ulrich Pohl,Florian Krötz
Cardiovascular research 93 2012
The vascular endothelial growth factor (VEGF) stimulates angiogenesis by induction of vessel permeability, proliferation, and migration of endothelial cells, an important process in ischaemic diseases. ADP-ribosylation factor (ARF) nucleotide-binding site opener (ARNO) (cytohesin-2) is a guanine exchange factor important for cellular signalling through ARF GTPases. However, a role for ARNO in VEGF-dependent endothelial processes has so far not been documented. Therefore, we investigated whether ARNO has a role in VEGF-dependent activation of endothelial cells and thus vessel permeability.
|Functional and molecular evidence of myelin- and neuroprotection by thyroid hormone administration in experimental allergic encephalomyelitis. |
M L Dell'Acqua,L Lorenzini,G D'Intino,S Sivilia,P Pasqualetti,V Panetta,M Paradisi,M M Filippi,C Baiguera,M Pizzi,L Giardino,P M Rossini,L Calzà
Neuropathology and applied neurobiology 38 2012
Recent data in mouse and rat demyelination models indicate that administration of thyroid hormone (TH) has a positive effect on the demyelination/remyelination balance. As axonal pathology has been recognized as an early neuropathological event in multiple sclerosis, and remyelination is considered a pre-eminent neuroprotective strategy, in this study we investigated whether TH administration improves nerve impulse propagation and protects axons.
|Osteogenic differentiation of stem cells alters vitamin d receptor expression. |
Rene Olivares-Navarrete,Ken Sutha,Sharon L Hyzy,Daphne L Hutton,Zvi Schwartz,Todd McDevitt,Barbara D Boyan
Stem cells and development 21 2012
Pluripotent and multipotent stem cells adopt an osteoblastic phenotype when cultured in environments that enhance their osteogenic potential. Embryonic stem cells differentiated as embryoid bodies (EBs) in osteogenic medium containing β-glycerophosphate exhibit increased expression of bone markers, indicating that cells are osteoblastic. Interestingly, 1α,25-dihydroxyvitaminD3 (1,25D) enhances the osteogenic phenotype not just in EBs but also in multipotent adult mesenchymal stem cells (MSCs). 1,25D acts on osteoblasts via classical vitamin D receptors (VDR) and via a membrane 1,25D-binding protein [protein disulfide isomerase family A, member 3 (PDIA3)], which activates protein kinase C -signaling. The aims of this study were to determine whether these receptors are regulated during osteogenic differentiation of stem cells and if stem cells and differentiated progeny are responsive to 1,25D. mRNA and protein levels for VDR, PDIA3, and osteoblast-associated proteins were measured in undifferentiated cells and in cells treated with osteogenic medium. Mouse EBs expressed both VDR and PDIA3, but VDR increased as cells underwent osteogenic differentiation. Human MSCs expressed Pdia3 at constant levels throughout differentiation, but VDR increased in cells treated with osteogenic medium. These results suggest that both 1,25D signaling mechanisms are important, with PDIA3 playing a greater role during early events and VDR playing a greater role in later stages of differentiation. Understanding these coordinated events provide a powerful tool to control pluripotent and multipotent stem cell differentiation through induction medium.
|p53, a novel regulator of lipid metabolism pathways. |
Ido Goldstein,Osnat Ezra,Noa Rivlin,Alina Molchadsky,Shalom Madar,Naomi Goldfinger,Varda Rotter
Journal of hepatology 56 2012
In this study we aimed at characterizing the regulation of hepatic metabolic pathways by the p53 transcription factor.
|An aberrant cerebellar development in mice lacking matrix metalloproteinase-3. |
Inge Van Hove,Mieke Verslegers,Tom Buyens,Nathalie Delorme,Kim Lemmens,Stijn Stroobants,Ilse Gantois,Rudi D'Hooge,Lieve Moons
Molecular neurobiology 45 2012
Cell-cell and cell-matrix interactions are necessary for neuronal patterning and brain wiring during development. Matrix metalloproteinases (MMPs) are proteolytic enzymes capable of remodelling the pericellular environment and regulating signaling pathways through cleavage of a large degradome. MMPs have been suggested to affect cerebellar development, but the specific role of different MMPs in cerebellar morphogenesis remains unclear. Here, we report a role for MMP-3 in the histogenesis of the mouse cerebellar cortex. MMP-3 expression peaks during the second week of postnatal cerebellar development and is most prominently observed in Purkinje cells (PCs). In MMP-3 deficient (MMP-3(-/-)) mice, a protracted granule cell (GC) tangential migration and a delayed GC radial migration results in a thicker and persistent external granular layer, a retarded arrival of GCs in the inner granular layer, and a delayed GABAergic interneuron migration. Importantly, these neuronal migration anomalies, as well as the consequent disturbed synaptogenesis on PCs, seem to be caused by an abnormal PC dendritogenesis, which results in reduced PC dendritic trees in the adult cerebellum. Of note, these developmental and adult cerebellar defects might contribute to the aberrant motor phenotype observed in MMP-3(-/-) mice and suggest an involvement of MMP-3 in mouse cerebellar development.
|BRCA2 protein deficiency exaggerates doxorubicin-induced cardiomyocyte apoptosis and cardiac failure. |
Krishna K Singh,Praphulla C Shukla,Adrian Quan,Jean-François Desjardins,Fina Lovren,Yi Pan,Vinay Garg,Sumandeep Gosal,Ankit Garg,Paul E Szmitko,Michael D Schneider,Thomas G Parker,William L Stanford,Howard Leong-Poi,Hwee Teoh,Mohammed Al-Omran,Subodh Verma
The Journal of biological chemistry 287 2012
The tumor suppressor breast cancer susceptibility gene 2 (BRCA2) plays an important role in the repair of DNA damage, and loss of BRCA2 predisposes carriers to breast and ovarian cancers. Doxorubicin (DOX) remains the cornerstone of chemotherapy in such individuals. However, it is often associated with cardiac failure, which once manifests carries a poor prognosis. Because BRCA2 regulates genome-wide stability and facilitates DNA damage repair, we hypothesized that loss of BRCA2 may increase susceptibility to DOX-induced cardiac failure. To this aim, we generated cardiomyocyte-specific BRCA2 knock-out (CM-BRCA2(-/-)) mice using the Cre-loxP technology and evaluated their basal and post-DOX treatment phenotypes. Although CM-BRCA2(-/-) mice exhibited no basal cardiac phenotype, DOX treatment resulted in markedly greater cardiac dysfunction and mortality in CM-BRCA2(-/-) mice compared with control mice. Apoptosis in left ventricular (LV) sections from CM-BRCA2(-/-) mice compared with that in corresponding sections from wild-type (WT) littermate controls was also significantly enhanced after DOX treatment. Microscopic examination of LV sections from DOX-treated CM-BRCA2(-/-) mice revealed a greater number of DNA double-stranded breaks and the absence of RAD51 focus formation, an essential marker of double-stranded break repair. The levels of p53 and the p53-related proapoptotic proteins p53-up-regulated modulator of apoptosis (PUMA) and Bax were significantly increased in samples from CM-BRCA2(-/-) mice. This corresponded with increased Bax to Bcl-2 ratios and elevated cytochrome c release in the LV sections of DOX-treated CM-BRCA2(-/-) mice. Taken together, these data suggest a critical and previously unrecognized role of BRCA2 as a gatekeeper of DOX-induced cardiomyocyte apoptosis and susceptibility to overt cardiac failure. Pharmacogenomic studies evaluating cardiac function in BRCA2 mutation carriers treated with doxorubicin are encouraged.
|Targeting human epidermal growth factor receptor 2 by a cell-penetrating peptide-affibody bioconjugate. |
Srinath Govindarajan,Jeyarajan Sivakumar,Prathyusha Garimidi,Nandini Rangaraj,Jerald M Kumar,Nalam M Rao,Vijaya Gopal
Biomaterials 33 2012
Cell-penetrating peptide (CPP)-based delivery systems represent a strategy that facilitates DNA import efficiently and non-specifically into cells. To introduce specificity, we devised an approach that combines a cell-penetrating peptide, TAT-Mu (TM) and a targeting ligand, an HER2 antibody mimetic-affibody (AF), designated as TMAF to deliver nucleic acids into the cells. In this study, we synthesized TMAF protein and its truncated versions, i.e. MAF and AF, by expressing the corresponding plasmids in Escherichia coli BL21(DE3)pLysS cells. Purified TMAF binds DNA efficiently and protects plasmid DNA from DNaseI action. Transfection of HER2+ breast cancer cell lines MDA-MB-453, SK-OV-3, SK-BR-3 and an ovarian cancer cell line with plasmid DNA pCMV?-gal, resulted in enhanced ?-galactosidase activity when compared to control MDA-MB-231 cells. Maximal activity observed in MDA-MB-453 cells at DNA:TMAF:Protamine sulphate (PS) corresponding to 1:8:2 charge ratios. Further the observed gene transfection was resistant to serum, sensitive to the presence of free AF and non-toxic. Variants of TMAF although non-toxic, were far less efficient indicating the effective role of the TAT and Mu domains. The observed DNA uptake and reporter gene activity mediated by TMAFin vitro could be linked with the cell-surface density of tyrosine kinase receptor HER2 (ErbB2) levels estimated by Western blot. Further, we confirmed the efficacy of DNA transfer by TMAF protein in xenograft mouse models using MDA-MB-453 cells. Expression of ?-galactosidase as the reporter gene, upon intratumoral injection of DNA, in complex with TMAF, lends credence to specific DNA import and distribution within the tumor tissue that was attributed to high HER2 receptor overexpression in MDA-MB-453 cells. Through delivery of anti-TF hshRNA: TMAF: PS complex, we demonstrate specific knockdown of tissue factor (TF) in MDA-MB-453 cells in vitro. Most importantly, in a xenograft mouse model, we observe significant (P<0.05) and specific reduction of tumor volume when anti-TF hshRNA: TMAF: PS complex was injected intratumorally. Collectively our data indicate that AF-based chimeric peptides with nucleic acid binding properties may provide an effective tumor specific strategy to deliver therapeutic nucleic acids.
|Role of renal DJ-1 in the pathogenesis of hypertension associated with increased reactive oxygen species production. |
Santiago Cuevas,Yanrong Zhang,Yu Yang,Crisanto Escano,Laureano Asico,John E Jones,Ines Armando,Pedro A Jose
Hypertension 59 2012
The D(2) dopamine receptor (D(2)R) is important in the pathogenesis of essential hypertension. We have already reported that systemic deletion of the D(2)R gene in mice results in reactive oxygen species (ROS)-dependent hypertension, suggesting that the D(2)R has antioxidant effects. However, the mechanism of this effect is unknown. DJ-1 is a protein that has antioxidant properties. D(2)R and DJ-1 are expressed in the mouse kidney and colocalize and coimunoprecipitate in mouse renal proximal tubule cells. We hypothesized that D(2)Rs regulate renal ROS production in the kidney through regulation of DJ-1 expression or function. Heterozygous D(2)(+/-) mice have increased blood pressure, urinary 8-isoprostanes, and renal Nox 4 expression, but decreased renal DJ-1 expression. Silencing D(2)R expression in mouse renal proximal tubule cells increases ROS production and decreases the expression of DJ-1. Conversely, treatment of these cells with a D(2)R agonist increases DJ-1 expression and decreases Nox 4 expression and NADPH oxidase activity, effects that are partially blocked by a D(2)R antagonist. Silencing DJ-1 expression in mouse renal proximal tubule cells increases ROS production and Nox 4 expression. Selective renal DJ-1 silencing by the subcapsular infusion of DJ-1 siRNA in mice increases blood pressure, renal Nox4 expression, and NADPH oxidase activity. These results suggest that the inhibitory effects of D(2)R on renal ROS production are at least, in part, mediated by a positive regulation of DJ-1 expression/function and that DJ-1 may have a role in the prevention of hypertension associated with increased ROS production.
|Carbonic anhydrase XIV in the normal and hypertrophic myocardium. |
Lorena A Vargas,Bernardo V Alvarez
Journal of molecular and cellular cardiology 52 2012
Two AE3 transcripts, full-length (AE3fl) and cardiac (AE3c) are expressed in the heart. AE3 catalyzes electroneutral Cl(-)/HCO(3)(-) exchange across cardiomyocyte sarcolemma. AE proteins associate with carbonic anhydrases (CA), including CAII and CAIV, forming a HCO(3)(-) transport metabolon (BTM), increasing HCO(3)(-) fluxes and regulating cardiomyocytes pH. CAXIV, which is also expressed in the heart's sarcolemma, is a transmembrane enzyme with an extracellular catalytic domain. Herein, AE3/CAXIV physical association was examined by coimmunoprecipitation using rodent heart lysates. CAXIV immunoprecipitated with anti-AE3 antibody and both AE3fl and AE3c were reciprocally immunoprecipitated using anti-CAXIV antibody, indicating AE3fl-AE3c/CAXIV interaction in the myocardium. Coimmunoprecipitation experiments on heart lysates from a mouse with targeted disruption of the ae3 gene, failed to pull down AE3 with the CAXIV antibody. Confocal images demonstrated colocalization of CAXIV and AE3 in mouse ventricular myocytes. Functional association of AE3fl and CAXIV was examined in isolated hypertrophic rat cardiomyocytes, using fluorescence measurements of BCECF to monitor cytosolic pH. Hypertrophic cardiomyocytes of spontaneously hypertensive rats (SHR) presented elevated myocardial AE-mediated Cl(-)/HCO(3)(-) exchange activity (J(HCO3-) mM.min(-1)) compared to normal (Wistar) rats (7.5±1.3, n=4 versus 2.9±0.1, n=6, respectively). AE3fl, AE3c, CAII, CAIV, and CAIX protein expressions were similar in SHR and Wistar rat hearts. However, immunoblots revealed a twofold increase of CAXIV protein expression in the SHR myocardium compared to normal hearts (n=11). Furthermore, the CA-inhibitor, benzolamide, neutralized the stimulatory effect of extracellular CA on AE3 transport activity (3.7±1.5, n=3), normalizing AE3-dependent HCO(3)(-) fluxes in SHR. CAXIV/AE3 interaction constitutes an extracellular component of a BTM which potentiates AE3-mediated HCO(3)(-) transport in the heart. Increased CAXIV expression and consequent AE3/CAXIV complex formation would render AE3 hyperactive in the SHR heart.
|The role of notch 1 activation in cardiosphere derived cell differentiation. |
Lijuan Chen,Muhammad Ashraf,Yingjie Wang,Mi Zhou,John Zhang,Gangjian Qin,Jack Rubinstein,Neal L Weintraub,Yaoliang Tang
Stem cells and development 21 2012
Cardiosphere derived cells (CDC) are present in the human heart and include heterogeneous cell populations of cardiac progenitor cells, multipotent progenitors that play critical roles in the physiological and pathological turnover of heart tissue. Little is known about the molecular pathways that control the differentiation of CDC. In this study, we examined the role of Notch 1/J kappa-recombining binding protein (RBPJ) signaling, a critical cell-fate decision pathway, in CDC differentiation. We isolated CDC from mouse cardiospheres and analyzed the differentiation of transduced cells expressing the Notch1 intracellular domain (N1-ICD), the active form of Notch1, using a terminal differentiation marker polymerase chain reaction (PCR) array. We found that Notch1 primarily supported the differentiation of CDC into smooth muscle cells (SMC), as demonstrated by the upreguation of key SMC proteins, including smooth muscle myosin heavy chain (Myh11) and SM22α (Tagln), in N1-ICD expressing CDC. Conversely, genetic ablation of RBPJ in CDC diminished the expression of SMC differentiation markers, confirming that SMC differentiation CDC is dependent on RBPJ. Finally, in vivo experiments demonstrate enhanced numbers of smooth muscle actin-expressing implanted cells after an injection of N1-ICD-expressing CDC into ischemic myocardium (44±8/high power field (hpf) vs. 11±4/high power field (hpf), n=7 sections, P<0.05). Taken together, these results provide strong evidence that Notch1 promotes SMC differentiation of CDC through an RBPJ-dependent signaling pathway in vitro, which may have important implications for progenitor cell-mediated angiogenesis.
|Effect of acute administration of Pistacia lentiscus L. essential oil on rat cerebral cortex following transient bilateral common carotid artery occlusion. |
Quartu, Marina, et al.
Lipids Health Dis, 11: 8 (2012) 2012
Ischemia/reperfusion leads to inflammation and oxidative stress which damages membrane highly polyunsaturated fatty acids (HPUFAs) and eventually induces neuronal death. This study evaluates the effect of the administration of Pistacia lentiscus L. essential oil (E.O.), a mixture of terpenes and sesquiterpenes, on modifications of fatty acid profile and endocannabinoid (eCB) congener concentrations induced by transient bilateral common carotid artery occlusion (BCCAO) in the rat frontal cortex and plasma.
|Stability of reference proteins in human placenta: general protein stains are the benchmark. |
D Lanoix,J St-Pierre,A-A Lacasse,M Viau,J Lafond,C Vaillancourt,A A Lacasse
Placenta 33 2012
The stability of reference proteins in semi-quantitative Western blot experiments in normal and diseased placenta has never been studied. This study aims to determine the stability of five reference proteins and two general protein stains in placentas from preeclampsia, gestational diabetes mellitus and matched control pregnancies. The stability of the reference proteins was analysed using indicators of inter-group (P value) and intra-group (coefficient of variation) stability. The effect of different normalization strategies was determined by normalizing serotonin transporter (SERT) expression against the different reference protein markers. Results show significant expression variability of β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1), peptidylprolyl isomerase A (PPIA) and α-tubulin, and that amido black staining is the most stable reference protein marker. Furthermore, results show that SERT expression significantly differs according to the reference protein markers used for its normalization. The present study demonstrated the importance of using stable reference protein markers and normalization strategy in order to get correct results in semi-quantitative Western blot experiments in placental tissues.
|Consumption of oxygen: a mitochondrial-generated progression signal of advanced cancer. |
C C Cook,A Kim,S Terao,A Gotoh,M Higuchi
Cell death & disease 3 2012
Changes in mitochondrial genome such as mutation, deletion and depletion are common in cancer and can determine advanced phenotype of cancer; however, detailed mechanisms have not been elucidated. We observed that loss of mitochondrial genome reversibly induced overexpression and activation of proto-oncogenic Ras, especially K-Ras 4A, responsible for the activation of AKT and ERK leading to advanced phenotype of prostate and breast cancer. Ras activation was induced by the overexpression of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR), the rate-limiting enzyme of the mevalonate pathway. Hypoxia is known to induce proteasomal degradation of HMGR. Well differentiated prostate and breast cancer cells with high mitochondrial DNA content consumed a large amount of oxygen and induced hypoxia. Loss of mitochondrial genome reduced oxygen consumption and increased in oxygen concentration in the cells. The hypoxic-to-normoxic shift led to the overexpression of HMGR through inhibiting proteasomal degradation. Therefore, reduction of mitochondrial genome content induced overexpression of HMGR through hypoxic to normoxic shift and subsequently the endogenous induction of the mevalonate pathway activated Ras that mediates advanced phenotype. Reduction of mitochondrial genome content was associated with the aggressive phenotype of prostate cancer in vitro cell line model and tissue specimens in vivo. Our results elucidate a coherent mechanism that directly links the mitochondrial genome with the advanced progression of the disease.
|Human immunodeficiency virus type 1 envelope proteins traffic toward virion assembly sites via a TBC1D20/Rab1-regulated pathway. |
Dikla Nachmias,Ella H Sklan,Marcelo Ehrlich,Eran Bacharach
Retrovirology 9 2012
The cellular activity of many factors and pathways is required to execute the complex replication cycle of the human immunodeficiency virus type 1 (HIV-1). To reveal these cellular components, several extensive RNAi screens have been performed, listing numerous 'HIV-dependency factors'. However, only a small overlap between these lists exists, calling for further evaluation of the relevance of specific factors to HIV-1 replication and for the identification of additional cellular candidates. TBC1D20, the GTPase-activating protein (GAP) of Rab1, regulates endoplasmic reticulum (ER) to Golgi trafficking, was not identified in any of these screens, and its involvement in HIV-1 replication cycle is tested here.
|The carboxyl-terminal region of erythroid-specific 5-aminolevulinate synthase acts as an intrinsic modifier for its catalytic activity and protein stability. |
Senkottuvelan Kadirvel,Kazumichi Furuyama,Hideo Harigae,Kiriko Kaneko,Yoshiko Tamai,Yoji Ishida,Shigeki Shibahara
Experimental hematology 40 2012
Erythroid-specific 5-aminolevulinate synthase (ALAS2) is essential for hemoglobin production, and a loss-of-function mutation of ALAS2 gene causes X-linked sideroblastic anemia. Human ALAS2 protein consists of 587 amino acids and its carboxyl(C)-terminal region of 33 amino acids is conserved in higher eukaryotes, but is not present in prokaryotic ALAS. We explored the role of this C-terminal region in the pathogenesis of X-linked sideroblastic anemia. In vitro enzymatic activity was measured using bacterially expressed recombinant proteins. In vivo catalytic activity was evaluated by comparing the accumulation of porphyrins in eukaryotic cells stably expressing each mutant ALAS2 tagged with FLAG, and the half-life of each FLAG-tagged ALAS2 protein was determined by Western blot analysis. Two novel mutations (Val562Ala and Met567Ile) were identified in patients with X-linked sideroblastic anemia. Val562Ala showed the higher catalytic activity in vitro, but a shorter half-life in vivo compared to those of wild-type ALAS2 (WT). In contrast, the in vitro activity of Met567Ile mutant was about 25% of WT, while its half-life was longer than that of WT. However, in vivo catalytic activity of each mutant was lower than that of WT. In addition, the deletion of 33 amino acids at C-terminal end resulted in higher catalytic activity both in vitro and in vivo with the longer half-life compared to WT. In conclusion, the C-terminal region of ALAS2 protein may function as an intrinsic modifier that suppresses catalytic activity and increases the degradation of its protein, each function of which is enhanced by the Met567Ile mutation and the Val562Ala mutation, respectively.
|Tumor suppressive microRNA-1285 regulates novel molecular targets: aberrant expression and functional significance in renal cell carcinoma. |
Hiedo Hidaka,Naohiko Seki,Hirofumi Yoshino,Takeshi Yamasaki,Yasutoshi Yamada,Nijiro Nohata,Miki Fuse,Masayuki Nakagawa,Hideki Enokida
Oncotarget 3 2012
MicroRNAs (miRNA) are non-coding RNAs, approximately 22 nucleotides in length, which function as post-transcriptional regulators. A large body of evidence indicates that miRNAs regulate the expression of cancer-related genes involved in proliferation, migration, invasion, and metastasis. The aim of this study was to identify novel cancer networks in renal cell carcinoma (RCC) based on miRNA expression signatures obtained from RCC clinical specimens. Expression signatures revealed that 103 miRNAs were significantly downregulated (more than 0.5-fold change) in RCC specimens. Functional screening (cell proliferation assays) was performed to identify tumor suppressive activities of 20 downregulated miRNAs. Restoration of mature miRNAs in cancer cells showed that 14 miRNAs (miR-1285, miR-206, miR-1, miR-135a, miR-429, miR-200c, miR-1291, miR-133b, miR-508-3p, miR-360-3p, miR-509-5p, miR-218, miR-335, miR-1255b and miR-1285) markedly inhibited cancer cell proliferation, suggesting that these miRNAs were candidate tumor suppressive miRNAs in RCC. We focused on miR-1285 because it significantly inhibited cancer cell proliferation, invasion, and migration following its transfection. We addressed miR-1285-regulated cancer networks by using genome-wide gene expression analysis and bioinformatics. The data showed that transglutaminase 2 (TGM2) was directly regulated by miR-1285. Silencing of the target gene demonstrated significant inhibition of cell proliferation and invasion in the RCC cells. Furthermore, immunohistochemistry showed that TGM2 expression levels in RCC specimens were significantly higher than those in normal renal tissues. Downregulation of tumor suppressive miR-1285, which targets oncogenic genes including TGM2, might contribute to RCC development. Thus, miR-1285 modulates a novel molecular target and provides new insights into potential mechanisms of RCC oncogenesis.
|Glomerular MYH9 expression is reduced by HIV-1. |
Thomas Hays,Vivette D D'Agati,Jonathan A Garellek,Tjani Warren,Marc E Trubin,Deborah P Hyink,John Cijiang He,Paul E Klotman
AIDS (London, England) 26 2012
The continuing disease burden of HIV-associated nephropathy (HIVAN) warrants better elucidation of its pathogenic mechanisms. Given that loss of MYH9 function causes a Mendelian renal disease, we hypothesized that renal expression of MYH9 is down-regulated by HIV-1 in HIVAN pathogenesis.
|The transcription factor Erg controls endothelial cell quiescence by repressing activity of nuclear factor (NF)-κB p65. |
Nicola H Dryden,Andrea Sperone,Silvia Martin-Almedina,Rebecca L Hannah,Graeme M Birdsey,Samia Taufiq Khan,Janice A Layhadi,Justin C Mason,Dorian O Haskard,Berthold Göttgens,Anna M Randi
The Journal of biological chemistry 287 2012
The interaction of transcription factors with specific DNA sequences is critical for activation of gene expression programs. In endothelial cells (EC), the transcription factor NF-κB is important in the switch from quiescence to activation, and is tightly controlled to avoid excessive inflammation and organ damage. Here we describe a novel mechanism that controls the activation of NF-κB in EC. The transcription factor Erg, the most highly expressed ETS member in resting EC, controls quiescence by repressing proinflammatory gene expression. Focusing on intercellular adhesion molecule 1(ICAM)-1 as a model, we identify two ETS binding sites (EBS -118 and -181) within the ICAM-1 promoter required for Erg-mediated repression. We show that Erg binds to both EBS -118 and EBS -181, the latter located within the NF-κB binding site. Interestingly, inhibition of Erg expression in quiescent EC results in increased NF-κB-dependent ICAM-1 expression, indicating that Erg represses basal NF-κB activity. Erg prevents NF-κB p65 from binding to the ICAM-1 promoter, suggesting a direct mechanism of interference. Gene set enrichment analysis of transcriptome profiles of Erg and NF-κB-dependent genes, together with chromatin immunoprecipitation (ChIP) studies, reveals that this mechanism is common to other proinflammatory genes, including cIAP-2 and IL-8. These results identify a role for Erg as a gatekeeper controlling vascular inflammation, thus providing an important barrier to protect against inappropriate endothelial activation.
|RNA editing of the Q/R site of GluA2 in different cultured cell lines that constitutively express different levels of RNA editing enzyme ADAR2. |
Takenari Yamashita,Chieko Tadami,Yoshinori Nishimoto,Takuto Hideyama,Daisuke Kimura,Takeshi Suzuki,Shin Kwak
Neuroscience research 73 2012
Adenosine deaminase acting on RNA 2 (ADAR2) catalyzes RNA editing at the glutamine/arginine (Q/R) site of GluA2, and an ADAR2 deficiency may play a role in the death of motor neurons in ALS patients. The expression level of ADAR2 mRNA is a determinant of the editing activity at the GluA2 Q/R site in human brain but not in cultured cells. Therefore, we investigated the extent of Q/R site-editing in the GluA2 mRNA and pre-mRNA as well as the ADAR2 mRNA and GluA2 mRNA and pre-mRNA levels in various cultured cell lines. The extent of the GluA2 mRNA editing was 100% except in SH-SY5Y cells, which have a much lower level of ADAR2 than the other cell lines examined. The ADAR2 activity at the GluA2 pre-mRNA Q/R site correlated with the ADAR2 mRNA level relative to the GluA2 pre-mRNA. SH-SY5Y cells expressed higher level of the GluA2 mRNA in the cytoplasm compared with other cell lines. These results suggest that the ADAR2 expression level reflects editing activity at the GluA2 Q/R site and that although the edited GluA2 pre-mRNA is readily spliced, the unedited GluA2 pre-mRNA is also spliced and transported to the cytoplasm when ADAR2 expression is low.
|Differential expression of molecular markers of synaptic plasticity in the hippocampus, prefrontal cortex, and amygdala in response to spatial learning, predator exposure, and stress-induced amnesia. |
Phillip R Zoladz,Collin R Park,Joshua D Halonen,Samina Salim,Karem H Alzoubi,Marisa Srivareerat,Monika Fleshner,Karim A Alkadhi,David M Diamond
Hippocampus 22 2012
We have studied the effects of spatial learning and predator stress-induced amnesia on the expression of calcium/calmodulin-dependent protein kinase II (CaMKII), brain-derived neurotrophic factor (BDNF) and calcineurin in the hippocampus, basolateral amygdala (BLA), and medial prefrontal cortex (mPFC). Adult male rats were given a single training session in the radial-arm water maze (RAWM) composed of 12 trials followed by a 30-min delay period, during which rats were either returned to their home cages or given inescapable exposure to a cat. Immediately following the 30-min delay period, the rats were given a single test trial in the RAWM to assess their memory for the hidden platform location. Under control (no stress) conditions, rats exhibited intact spatial memory and an increase in phosphorylated CaMKII (p-CaMKII), total CaMKII, and BDNF in dorsal CA1. Under stress conditions, rats exhibited impaired spatial memory and a suppression of all measured markers of molecular plasticity in dorsal CA1. The molecular profiles observed in the BLA, mPFC, and ventral CA1 were markedly different from those observed in dorsal CA1. Stress exposure increased p-CaMKII in the BLA, decreased p-CaMKII in the mPFC, and had no effect on any of the markers of molecular plasticity in ventral CA1. These findings provide novel observations regarding rapidly induced changes in the expression of molecular plasticity in response to spatial learning, predator exposure, and stress-induced amnesia in brainregions involved in different aspects of memory processing.
|Immunohistochemical detection of glypican-5 in paraffin-embedded material: an optimized method for a novel research antibody. |
Khin Thway,Joanna Selfe,Janet Shipley
Applied immunohistochemistry & molecular morphology : AIMM / official publication of the Society for Applied Immunohistochemistry 20 2012
Glypican-5 (GPC5) is a cell surface heparan sulfate proteoglycan and 1 of 6 closely related members of the glypican family in mammals. Glypicans are predominantly expressed during development in cell-specific and tissue-specific contexts, and the expression of some is linked to developmental disorders and several visceral malignancies. We have previously shown that the region of amplification at 13q31.3 in a subset of rhabdomyosarcomas contains the GPC5 locus, and by copy number and gene expression analyses, that GPC5 is consistently expressed and upregulated in amplified tumors. As the immunohistochemical profile of GPC5 is untested, our aim was to optimize a commercially available anti-human GPC5 antibody for immunohistochemical use in formalin-fixed and paraffin-embedded (FFPE) tissue. Quantitative real-time polymerase chain reaction analyses of normal tissue samples indicated that the brain and testis highly expressed GPC5. High protein expression in these tissues and a cell line constructed to overexpress GPC5 were demonstrated by Western blotting. These normal tissues and the isogenic cell line were FFPE, and immunohistochemical expression of GPC5 was assessed using different methods of antigen retrieval, detection, and primary antibody concentration. The optimum conditions for detection were by heat-induced antigen retrieval, in sodium citrate buffer at pH 6. Enzyme-mediated retrieval did not produce effective detection, producing weaker, less well-localized GPC5 expression. We demonstrate that anti-human GPC5 antibody is amenable to use in FFPE tissue and with the optimized protocol we describe shows specific cellular localization and good staining intensity with minimal background staining.
|Proteomic and protein interaction network analysis of human T lymphocytes during cell-cycle entry. |
Stephen J Orr,Daniel R Boutz,Rong Wang,Constantinos Chronis,Nicholas C Lea,Thivyan Thayaparan,Emma Hamilton,Hanna Milewicz,Eric Blanc,Ghulam J Mufti,Edward M Marcotte,N Shaun B Thomas
Molecular systems biology 8 2012
Regulating the transition of cells such as T lymphocytes from quiescence (G(0)) into an activated, proliferating state involves initiation of cellular programs resulting in entry into the cell cycle (proliferation), the growth cycle (blastogenesis, cell size) and effector (functional) activation. We show the first proteomic analysis of protein interaction networks activated during entry into the first cell cycle from G(0). We also provide proof of principle that blastogenesis and proliferation programs are separable in primary human T cells. We employed a proteomic profiling method to identify large-scale changes in chromatin/nuclear matrix-bound and unbound proteins in human T lymphocytes during the transition from G(0) into the first cell cycle and mapped them to form functionally annotated, dynamic protein interaction networks. Inhibiting the induction of two proteins involved in two of the most significantly upregulated cellular processes, ribosome biogenesis (eIF6) and hnRNA splicing (SF3B2/SF3B4), showed, respectively, that human T cells can enter the cell cycle without growing in size, or increase in size without entering the cell cycle.
|Interactions between inflammatory signals and the progesterone receptor in regulating gene expression in pregnant human uterine myocytes. |
Yun Lee,Suren R Sooranna,Vasso Terzidou,Mark Christian,Jan Brosens,Kaisa Huhtinen,Matti Poutanen,Geraint Barton,Mark R Johnson,Phillip R Bennett
Journal of cellular and molecular medicine 16 2012
The absence of a fall in circulating progesterone levels has led to the concept that human labour is associated with 'functional progesterone withdrawal' caused through changes in the expression or function of progesterone receptor (PR). At the time of labour, the human uterus is heavily infiltrated with inflammatory cells, which release cytokines to create a 'myometrial inflammation' via NF-κB activation. The negative interaction between NF-κB and PR, may represent a mechanism to account for 'functional progesterone withdrawal' at term. Conversely, PR may act to inhibit NF-κB function and so play a role in inhibition of myometrial inflammation during pregnancy. To model this inter-relationship, we have used small interfering (si) RNA-mediated knock-down of PR in human pregnant myocytes and whole genome microarray analysis to identify genes regulated through PR. We then activated myometrial inflammation using IL-1β stimulation to determine the role of PR in myometrial inflammation regulation. Through PR-knock-down, we found that PR regulates gene networks involved in myometrial quiescence and extracellular matrix integrity. Activation of myometrial inflammation was found to antagonize PR-induced gene expression, of genes normally upregulated via PR. We found that PR does not play a role in repression of pro-inflammatory gene networks induced by IL-1β and that only MMP10 was significantly regulated in opposite directions by IL-1β and PR. We conclude that progesterone acting through PR does not generally inhibit myometrial inflammation. Activation of myometrial inflammation does cause 'functional progesterone withdrawal' but only in the context of genes normally upregulated via PR.
|PU.1 is linking the glycolytic enzyme HK3 in neutrophil differentiation and survival of APL cells. |
Elena A Federzoni,Peter J M Valk,Bruce E Torbett,Torsten Haferlach,Bob Löwenberg,Martin F Fey,Mario P Tschan
Blood 119 2012
The transcription factor PU.1 is a master regulator of myeloid differentiation and function. On the other hand, only scarce information is available on PU.1-regulated genes involved in cell survival. We now identified the glycolytic enzyme hexokinase 3 (HK3), a gene with cytoprotective functions, as transcriptional target of PU.1. Interestingly, HK3 expression is highly associated with the myeloid lineage and was significantly decreased in acute myeloid leukemia patients compared with normal granulocytes. Moreover, HK3 expression was significantly lower in acute promyelocytic leukemia (APL) compared with non-APL patient samples. In line with the observations in primary APL patient samples, we observed significantly higher HK3 expression during neutrophil differentiation of APL cell lines. Moreover, knocking down PU.1 impaired HK3 induction during neutrophil differentiation. In vivo binding of PU.1 and PML-RARA to the HK3 promoter was found, and PML-RARA attenuated PU.1 activation of the HK3 promoter. Next, inhibiting HK3 in APL cell lines resulted in significantly reduced neutrophil differentiation and viability compared with control cells. Our findings strongly suggest that HK3 is: (1) directly activated by PU.1, (2) repressed by PML-RARA, and (3) functionally involved in neutrophil differentiation and cell viability of APL cells.
|Release of the glucose-regulated protein 94 by baby hamster kidney cells. |
Yulia Evdokimovskaya,Yuri Skarga,Veronika Vrublevskaya,Oleg Morenkov
Cell biochemistry and function 30 2012
Glucose-regulated protein 94 (grp94) is a major component of the endoplasmic reticulum (ER) lumen of eukaryotic cells. We showed that grp94 is released from baby hamster kidney (BHK-21) cells into a serum-free medium. The exit of grp94 into the medium was not related to the protein discharge due to cell death and was independent of de novo protein synthesis. The treatment of cells with brefeldin A and monensin, the inhibitors of the classical pathway of protein secretion, did not decrease the extracellular level of grp94, indicating that the discharge of grp94 from cells does not occur through the ER/Golgi-dependent pathway. Exosomes, membrane vesicles secreted by several cell types, were not involved in the release of grp94 from cells. Methyl-β-cyclodextrin, a substance that disrupts the lipid raft organization, considerably reduced the extracellular level of grp94, indicating that lipid rafts are involved in the liberation of grp94 from BHK-21 cells. The results suggest that BHK-21 cells release grp94 into the serum-free medium via the nonclassical secretory pathway in which lipid rafts play an important role. Copyright
|Exons 5-15 of kazrin are dispensable for murine epidermal morphogenesis and homeostasis. |
Mariya K Chhatriwala,Sara Cipolat,Lisa M Sevilla,Rachida Nachat,Fiona M Watt
The Journal of investigative dermatology 132 2012
Kazrin binds to periplakin and ARVCF catenin, and regulates adhesion and differentiation of cultured human keratinocytes. To explore kazrin function in vivo, we generated a kazrin gene-trap mouse in which only exons 1-4 were expressed, fused to β-galactosidase. On transient transfection, the protein encoded by exons 1-4 did not enter the nucleus, but did cause keratinocyte shape changes. The mice had no obvious defects in skin development or homeostasis, and periplakin and desmoplakin localization was normal. Expression of the kazrin-β-galactosidase fusion protein faithfully reported endogenous kazrin expression. Kazrin was not expressed in embryonic epidermis and was first detected at postnatal day 1. In adult mice, epidermal kazrin expression was less widespread than in humans and Xenopus, being confined to the bulb of anagen hair follicles, the infundibulum, and parakeratotic tail epidermis. In anagen bulbs, kazrin was expressed by a band of cells with elongated morphology and low desmoplakin levels, suggesting a role in morphogenetic cell movements. We conclude that exons 5-15 of kazrin, encoding the nuclear localization signal and C-terminal domain, are not required for epidermal development and function. The previously reported role of kazrin in regulating cell shape appears to reside within the N-terminal coiled-coil domain encoded by exons 1-4.
|Rap1 can bypass the FAK-Src-Paxillin cascade to induce cell spreading and focal adhesion formation. |
Ross, Sarah H, et al.
PLoS ONE, 7: e50072 (2012) 2012
We developed new image analysis tools to analyse quantitatively the extracellular-matrix-dependent cell spreading process imaged by live-cell epifluorescence microscopy. Using these tools, we investigated cell spreading induced by activation of the small GTPase, Rap1. After replating and initial adhesion, unstimulated cells exhibited extensive protrusion and retraction as their spread area increased, and displayed an angular shape that was remodelled over time. In contrast, activation of endogenous Rap1, via 007-mediated stimulation of Epac1, induced protrusion along the entire cell periphery, resulting in a rounder spread surface, an accelerated spreading rate and an increased spread area compared to control cells. Whereas basal, anisotropic, spreading was completely dependent on Src activity, Rap1-induced spreading was refractory to Src inhibition. Under Src inhibited conditions, the characteristic Src-induced tyrosine phosphorylations of FAK and paxillin did not occur, but Rap1 could induce the formation of actomyosin-connected adhesions, which contained vinculin at levels comparable to that found in unperturbed focal adhesions. From these results, we conclude that Rap1 can induce cell adhesion and stimulate an accelerated rate of cell spreading through mechanisms that bypass the canonical FAK-Src-Paxillin signalling cascade.
|Neurodegeneration in an Abeta-induced model of Alzheimer's disease: the role of Cdk5. |
Lopes, Joao P, et al.
Aging Cell, 9: 64-77 (2010) 2010
Cdk5 dysregulation is a major event in the neurodegenerative process of Alzheimer's disease (AD). In vitro studies using differentiated neurons exposed to Abeta exhibit Cdk5-mediated tau hyperphosphorylation, cell cycle re-entry and neuronal loss. In this study we aimed to determine the role of Cdk5 in neuronal injury occurring in an AD mouse model obtained through the intracerebroventricular (icv) injection of the Abeta(1-40) synthetic peptide. In mice icv-injected with Abeta, Cdk5 activator p35 is cleaved by calpains, leading to p25 formation and Cdk5 overactivation. Subsequently, there was an increase in tau hyperphosphorylation, as well as decreased levels of synaptic markers. Cell cycle reactivation and a significant neuronal loss were also observed. These neurotoxic events in Abeta-injected mice were prevented by blocking calpain activation with MDL28170, which was administered intraperitoneally (ip). As MDL prevents p35 cleavage and subsequent Cdk5 overactivation, it is likely that this kinase is involved in tau hyperphosphorylation, cell cycle re-entry, synaptic loss and neuronal death triggered by Abeta. Altogether, these data demonstrate that Cdk5 plays a pivotal role in tau phosphorylation, cell cycle induction, synaptotoxicity, and apoptotic death in postmitotic neurons exposed to Abeta peptides in vivo, acting as a link between diverse neurotoxic pathways of AD.
|Blockade of IL-15 activity inhibits microglial activation through the NFkappaB, p38, and ERK1/2 pathways, reducing cytokine and chemokine release. |
Gomez-Nicola, Diego, et al.
Glia, 58: 264-76 (2010) 2010
Reactive glia formation is one of the hallmarks of damage to the CNS, but little information exists on the signals that direct its activation. Microglial cells are the main regulators of both innate and adaptative immune responses in the CNS. The proinflammatory cytokine IL-15 is involved in regulating the response of T and B cells, playing a key role in regulating nervous system inflammatory events. We have used a microglial culture model of inflammation induced by LPS and IFNgamma to evaluate the role of IL-15 in the proinflammatory response. Our results indicate that IL-15 is necessary for the reactive response, its deficiency (IL-15-/-) leading to the development of a defective proinflammatory response. Blockade of IL-15, both with blocking antibodies or with the ganglioside Neurostatin, inhibited the activation of the NFkappaB pathway, decreasing iNOS expression and NO production. Inhibiting IL-15 signaling also blocked the activation of the mitogen-activated protein kinase (MAPK) pathways ERK1/2 and p38. The major consequence of these inhibitory effects, analyzed using cytokine antibody arrays, was a severe decrease in the production of chemokines, cytokines and growth factors, like CCL17, CCL19, IL-12, or TIMP-1, that are essential for the development of the phenotypic changes of glial activation. In conclusion, activation of the IL-15 system seems a necessary step for the development of glial reactivity and the regulation of the physiology of glial cells. Modulating IL-15 activity opens the possibility of developing new strategies to control gliotic events upon inflammatory stimulation.
|Proteomic and histochemical analysis of proteins involved in the dying-back-type of axonal degeneration in the gracile axonal dystrophy (gad) mouse. |
Akiko Goto, Yu-Lai Wang, Tomohiro Kabuta, Rieko Setsuie, Hitoshi Osaka, Akira Sawa, Shoichi Ishiura, Keiji Wada, Akiko Goto, Yu-Lai Wang, Tomohiro Kabuta, Rieko Setsuie, Hitoshi Osaka, Akira Sawa, Shoichi Ishiura, Keiji Wada, Akiko Goto, Yu-Lai Wang, Tomohiro Kabuta, Rieko Setsuie, Hitoshi Osaka, Akira Sawa, Shoichi Ishiura, Keiji Wada
Neurochemistry international 54 330-8 2009
Local axonal degeneration is a common pathological feature of peripheral neuropathies and neurodegenerative disorders of the central nervous system, including Alzheimer's disease, Parkinson's disease, and stroke; however, the underlying molecular mechanism is not known. Here, we analyzed the gracile axonal dystrophy (gad) mouse, which displays the dying-back-type of axonal degeneration in sensory neurons, to find the molecules involved in the mechanism of axonal degeneration. The gad mouse is analogous to a null mutant of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1). UCH-L1 is a deubiquitinating enzyme expressed at high levels in neurons, as well as testis and ovary. In addition, we recently discovered a new function of UCH-L1-namely to bind to and stabilize mono-ubiquitin in neurons, and found that the level of mono-ubiquitin was decreased in neurons, especially in axons of the sciatic nerve, in gad mice. The low level of ubiquitin suggests that the target proteins of the ubiquitin proteasome system are not sufficiently ubiquitinated and thus degraded in the gad mouse; therefore, these proteins may be the key molecules involved in axonal degeneration. To identify molecules involved in axonal degeneration in gad mice, we compared protein expression in sciatic nerves between gad and wild-type mice at 2 and 12 weeks old, using two-dimensional difference gel electrophoresis. As a result, we found age-dependent accumulation of several proteins, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 14-3-3, in gad mice compared with wild-type mice. Histochemical analyses demonstrated that GAPDH and 14-3-3 were localized throughout axons in both gad and wild-type mice, but GAPDH accumulated in the axons of gad mice. Recently, it has been suggested that a wide range of neurodegenerative diseases are characterized by the accumulation of intracellular and extracellular protein aggregates, and it has been reported that oxidative stress causes the aggregation of GAPDH. Furthermore, histochemical analysis demonstrated that sulfonated GAPDH, a sensor of oxidative stress that elicits cellular dysfunction, was expressed in the axons of gad mice, and 4-hydroxy-2-nonenal, a major marker of oxidative stress, was also only detected in gad mice. Our findings suggest that GAPDH may participate in a process of the dying-back-type of axonal degeneration in gad mice and may provide valuable insight into the mechanisms of axonal degeneration.
|Role of Host Cell Polarity and Leading Edge Properties in Pseudomonas Type III Secretion. |
Bridge, Dacie R, et al.
Microbiology (Reading, Engl.), (2009) 2009
Type III secretion (T3S) functions in establishing infections in a large number of Gram-negative bacteria, yet little is known about how host cell properties might function in this process. We used the opportunistic pathogen, Pseudomonas aeruginosa, and the ability to alter host cell sensitivity to Pseudomonas T3S to explore this problem. HT-29 epithelial cells were used to study cellular changes associated with loss of T3S sensitivity, which could be induced by treatment with methyl-beta-cyclodextrin or perfringolysin O. HL-60 promyelocytic cells are innately resistant to Pseudomonas T3S and were used to study cellular changes occurring in response to induction of T3S sensitivity, which occurred following treatment with phorbol esters. Using both cell models, a positive correlation was observed between eukaryotic cell adherence to tissue culture wells and T3S sensitivity. In examining the type of adhesion process linked to T3S sensitivity in HT-29 cells, a hierarchal order of protein involvement was identified that paralleled the architecture of leading edge focal complexes. Conversely, in HL-60 cells induction of T3S sensitivity coincided with the onset of leading edge properties and the development of actin-rich projections associated with polarized cell migration. When leading edge architecture was examined by immunofluorescent staining for actin, Rac1, IQGAP1 and PI3 kinase, intact leading edge structure was found to closely correlate with host cell sensitivity to Pa-T3S. Our model for host cell involvement in Pseudomonas T3S proposes that cortical actin polymerization at the leading edge alters membrane properties to favor T3S translocon function and the establishment of infections, which is consistent with Pseudomonas infections targeting wounded epithelial barriers undergoing cell migration.
|Structure-activity-dependent regulation of cell communication by perfluorinated fatty acids using in vivo and in vitro model systems. |
Upham, Brad L, et al.
Environ. Health Perspect., 117: 545-51 (2009) 2009
BACKGROUND: Perfluoroalkanoates, [e.g., perfluorooctanoate (PFOA)], are known peroxisome proliferators that induce hepatomegaly and hepatocarcinogenesis in rodents, and are classic non-genotoxic carcinogens that inhibit in vitro gap-junctional intercellular communication (GJIC). This inhibition of GJIC is known to be a function of perfluorinated carbon lengths ranging from 7 to 10. OBJECTIVES: The aim of this study was to determine if the inhibition of GJIC by PFOA but not perfluoropentanoate (PFPeA) observed in F344 rat liver cells in vitro also occurs in F344 rats in vivo and to determine mechanisms of PFOA dysregulation of GJIC using in vitro assay systems. METHODS: We used an incision load/dye transfer technique to assess GJIC in livers of rats exposed to PFOA and PFPeA. We used in vitro assays with inhibitors of cell signaling enzymes and antioxidants known to regulate GJIC to identify which enzymes regulated PFOA-induced inhibition of GJIC. RESULTS: PFOA inhibited GJIC and induced hepatomegaly in rat livers, whereas PFPeA had no effect on either end point. Serum biochemistry of liver enzymes indicated no cytotoxic response to these compounds. In vitro analysis of mitogen-activated protein kinase (MAPK) indicated that PFOA, but not PFPeA, can activate the extracellular receptor kinase (ERK). Inhibition of GJIC, in vitro, by PFOA depended on the activation of both ERK and phosphatidylcholine-specific phospholipase C (PC-PLC) in the dysregulation of GJIC in an oxidative-dependent mechanism. CONCLUSIONS: The in vitro analysis of GJIC, an epigenetic marker of tumor promoters, can also predict the in vivo activity of PFOA, which dysregulated GJIC via ERK and PC-PLC.
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