|The ADAM15 ectodomain is shed from secretory exosomes. |
Lee, HD; Kim, YH; Koo, BH; Kim, DS
We demonstrated previously that a disintegrin and metalloproteinase 15 (ADAM15) is released into the extracellular space as an exosomal component, and that ADAM15-rich exosomes have tumor suppressive functions. However, the suppressive mechanism of ADAM15-rich exosomes remains unclear. In this study, we show that the ADAM15 ectodomain is cleaved from released exosomes. This shedding process of the ADAM15 ectodomain was dramatically enhanced in conditioned ovarian cancer cell medium. Proteolytic cleavage was completely blocked by phenylmethylsulfonyl fluoride, indicating that a serine protease is responsible for exosomal ADAM15 shedding. Experimental evidence indicates that the ADAM15 ectodomain itself has comparable functions with those of ADAM15-rich exosomes, which effectively inhibit vitronectininduced cancer cell migration and activation of the MEK/extracellular regulated kinase signaling pathway. We present a tumor suppressive mechanism for ADAM15 exosomes and provide insight into the functional significance of exosomes that generate tumor-inhibitory factors.
|miR-1247 functions by targeting cartilage transcription factor SOX9. |
Martinez-Sanchez, A; Murphy, CL
The Journal of biological chemistry
microRNAs are a large and essential class of gene regulators that play key roles in development, homeostasis, and disease. They are necessary for normal skeletal development, and their expression is altered in arthritis. However, the specific role of individual microRNAs is only beginning to be unraveled. Using microRNA expression profiling in healthy human articular cartilage cells (chondrocytes), we identified miR-1247 expression as highly correlated with that of the differentiated cell phenotype. Transcribed from the DLK1-DIO3 locus, the function of miR-1247 is completely unknown. In mice its expression level was relatively high in cartilage tissue, and correlated with cartilage-associated microRNA miR-675 across a range of 15 different mouse tissues. To further probe miR-1247 function, overexpression and inhibition studies were performed in isolated human chondrocytes. Modulation of miR-1247 was found to exert profound phenotypic effects altering expression levels of cartilage master regulator transcription factor SOX9. SOX9 is essential for cartilage development and subsequent function throughout life, and mutations in this gene result in severe dwarfism. Putative miR-1247 binding sites were further investigated using luciferase reporter assays, which indicated binding of miR-1247 to a highly conserved region in the coding sequence of SOX9 but not in its 3'-UTR. Interestingly, depletion of SOX9 in human chondrocytes resulted in increased levels of the mature, processed microRNA, suggesting a negative feedback loop between miR-1247 and its target SOX9.
|Exosome release of ADAM15 and the functional implications of human macrophage-derived ADAM15 exosomes. |
Lee, HD; Koo, BH; Kim, YH; Jeon, OH; Kim, DS
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
A disintegrin and metalloproteinase 15 (ADAM15), the only ADAM protein containing an Arg-Gly-Asp (RGD) motif in its disintegrin-like domain, is a widely expressed membrane protein that is involved in tumor progression and suppression. However, the underlying mechanism of ADAM15-mediated tumor suppression is not clearly understood. This study demonstrates that ADAM15 is released as an exosomal component, and ADAM15 exosomes exert tumor suppressive activities. We found that exosomal ADAM15 release is stimulated by phorbol 12-myristate 13-acetate, a typical protein kinase C activator, in various tumor cell types, and this results in a corresponding decrease in plasma membrane-associated ADAM15. Exosomes rich in ADAM15 display enhanced binding affinity for integrin αvβ3 in an RGD-dependent manner and suppress vitronectin- and fibronectin-induced cell adhesion, growth, and migration, as well as in vivo tumor growth. Exosomal ADAM15 is released from human macrophages, and macrophage-derived ADAM15 exosomes have tumor inhibitory effects. This work suggests a primary role of ADAM15 for exosome-mediated tumor suppression, as well as functional significance of exosomal ADAM protein in antitumor immunity.
|EGFR and ADAMs cooperate to regulate shedding and endocytic trafficking of the desmosomal cadherin desmoglein 2. |
Klessner, JL; Desai, BV; Amargo, EV; Getsios, S; Green, KJ
Molecular biology of the cell
Regulation of classic cadherins plays a critical role in tissue remodeling during development and cancer; however, less attention has been paid to the importance of desmosomal cadherins. We previously showed that EGFR inhibition results in accumulation of the desmosomal cadherin, desmoglein 2 (Dsg2), at cell-cell interfaces accompanied by inhibition of matrix metalloprotease (MMP)-dependent shedding of the Dsg2 ectodomain and tyrosine phosphorylation of its cytoplasmic domain. Here, we show that EGFR inhibition stabilizes Dsg2 at intercellular junctions by interfering with its accumulation in an internalized cytoplasmic pool. Furthermore, MMP inhibition and ADAM17 RNAi, blocked shedding and depleted internalized Dsg2, but less so E-cadherin, in highly invasive SCC68 cells. ADAM9 and 15 silencing also impaired Dsg2 processing, supporting the idea that this desmosomal cadherin can be regulated by multiple ADAM family members. In contrast, ADAM10 siRNA enhanced accumulation of a 100-kDa Dsg2 cleavage product and internalized pool of Dsg2. Although both MMP and EGFR inhibition increased intercellular adhesive strength in control cells, the response to MMP-inhibition was Dsg2-dependent. These data support a role for endocytic trafficking in regulating desmosomal cadherin turnover and function and raise the possibility that internalization and regulation of desmosomal and classic cadherin function can be uncoupled mechanistically.Full Text Article
|ADAM15 is an adherens junction molecule whose surface expression can be driven by VE-cadherin. |
Ham, Claire, et al.
Exp. Cell Res., 279: 239-47 (2002)
ADAM15 belongs to the family of proteins containing disintegrin and metalloprotease domains (ADAM) that have been implicated in cell adhesion via integrin binding and shedding of cell surface molecules. Here we provide the first report on the localization of an ADAM in adherens junctions. We show that ADAM15 colocalizes with a cell adhesion molecule, vascular endothelial (VE)-cadherin, which mediates endothelial cell adherens junction formation. In contrast, the distribution of ADAM15 correlates poorly with the localization in cell contacts of one of its proposed ligands, the beta1-integrin. Furthermore, ADAM15 accumulation in cell-cell contacts is preceded by VE-cadherin-mediated adherens junction formation. To investigate the dependence of ADAM15 surface expression on adherens junction formation, we coexpressed VE-cadherin with ADAM15 and an ADAM15 green fluorescence protein (GFP) fusion protein in Chinese hamster ovary cells. VE-cadherin coexpression results in the translocation of ADAM15-GFP to the cell periphery. Analysis of cell surface levels of ADAM15 and ADAM15-GFP, with or without VE-cadherin coexpression, clearly demonstrates that VE-cadherin can drive surface expression of ADAM15. Our data suggest that ADAM15 may be a novel component of adherens junctions and thus could play a role in endothelial functions that are mediated by these cell contacts.