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Objective: To investigate mechanisms underlying the capability of ADAM15 to transform FasL-mediated death-inducing signals into prosurvival activation of Src and focal adhesion kinase (FAK) in rheumatoid arthritis synovial fibroblasts (RASFs)., Methods: Caspase 3/7 activity and apoptosis rate were determined in RASFs and ADAM15-transfected T/C28a4 cells upon Fas/CD95 triggering using enzyme assays and annexin V staining. Phosphorylated Src and FAK were analyzed by immunoblotting. Interactions of ADAM15 and CD95 with calmodulin (CaM), Src, or FAK were analyzed by pull-downs using CaM-Sepharose and coimmunoprecipitations with specific antibodies. Protein binding assays were performed using recombinant CaM and ADAM15. Immunofluorescence was performed to investigate subcellular colocalization of ADAM15, Fas/CD95, and CaM., Results: The antiapoptotic effect of ADAM15 in FasL-stimulated cells was demonstrated either by increased apoptosis of cells transfected with an ADAM15 construct lacking the cytoplasmic domain compared to cells transfected with full-length ADAM15 or by reduced apoptosis resistance of RASFs upon RNA interference silencing of ADAM15. Fas ligation triggered a Ca 2+  release-activated Ca 2+ /calcium release-activated calcium channel protein 1 (CRAC/Orai1) channel-dependent CaM recruitment to Fas/CD95 and ADAM15 in the cell membrane. Simultaneously, Src associated with CaM was shown to become engaged in the ADAM15 complex also containing cytoplasmic-bound FAK. Accordingly, Fas ligation in RASFs led to ADAM15-dependent phosphorylation of Src and FAK, which was associated with increased survival. Pharmacologic interference with either the CaM inhibitor trifluoperazine or the CRAC/Orai inhibitor BTP-2 simultaneously applied with FasL synergistically enhanced Fas-mediated apoptosis in RASFs., Conclusion: ADAM15 provides a scaffold for formation of CaM-dependent prosurvival signaling complexes upon CRAC/Orai coactivation by FasL-induced death signals and a potential therapeutic target to break apoptosis resistance in RASFs., (© 2018, American College of Rheumatology.)

The regulation of temporo-spatial compartmentalization of protein synthesis is of crucial importance for a variety of physiologic cellular functions. Here, we demonstrate that the cell membrane-anchored disintegrin metalloproteinase ADAM15, upregulated in a variety of aggressively growing tumor cells, in the hyperproliferative synovial membrane of inflamed joints as well as in osteoarthritic chondrocytes, transiently binds to poly(A) binding protein 1 (PABP) in cells undergoing adhesion. The cytoplasmic domain of ADAM15 was shown to selectively interact with the proline-rich linker of PABP. Immunostainings of adhesion-triggered cells demonstrate an ADAM15-dependent recruitment of PABP to cell membrane foci coinciding with ongoing mRNA translation as visualized by the detection of puromycin-terminated polypeptides. Moreover, the increase in cell membrane-associated neosynthesis of puromycylated proteins upon induction of cell adhesion was proven linked to ADAM15 expression in HeLa and ADAM15-transfected chondrocytic cells. Thus, down regulation of ADAM15 by siRNA and/or the use of a cell line transfected with a mutant ADAM15-construct lacking the cytoplasmic tail resulted in a considerable reduction in the amount of cell membrane-associated puromycylated proteins formed during induced cell adhesion. These results provide first direct evidence for a regulatory role of ADAM15 on mRNA translation at the cell membrane that transiently emerges in response to triggering cell adhesion and might have potential implications under pathologic conditions of matrix remodeling associated with ADAM15 upregulation., Competing Interests: The authors have declared that no competing interests exist.

Objective: To study the contribution of ADAM15, a disintegrin metalloproteinase that is up-regulated in the rheumatoid arthritis (RA) synovial membrane, to the characteristic resistance of RA synovial fibroblasts (RASFs) to apoptosis induction by genotoxic stress or stimulation with proapoptotic FasL, which is present at high concentrations in RA synovial fluid., Methods: Caspase 3/7 activity and the total apoptosis rate in RASFs upon exposure to the DNA-damaging agent camptothecin or FasL were determined using enzyme assays and annexin V staining. Phosphorylated signaling proteins were analyzed by immunoblotting. RNA interference was used to silence ADAM15 expression. NF-κB activity was determined by enzyme-linked immunosorbent assay., Results: RASFs displayed significantly higher caspase 3/7 activity upon camptothecin and FasL exposure when ADAM15 had been down-regulated by specific small interfering RNAs. Upon FasL stimulation, RASFs phosphorylated focal adhesion kinase (FAK) and c-Src (Src), and activated phosphatidylinositol 3-kinase as well as the transcription factor NF-κB. This ADAM15-dependent, FasL-induced activation of antiapoptotic kinases and NF-κB was demonstrated by a marked reduction of apoptosis upon knockdown of ADAM15 protein expression. Inhibitors specifically interfering with FAK and Src signaling, such as FAK inhibitor 14 and dasatinib, potently induce apoptosis in RASFs, with significant enhancement by the silencing of ADAM15., Conclusion: ADAM15 contributes to apoptosis resistance in RASFs by activating the Src/FAK pathway upon FasL exposure, rendering the FAK/Src signaling pathway an interesting target for potential therapeutic intervention in RA., (Copyright © 2013 by the American College of Rheumatology.)

ADAM15, a disintegrin and metalloproteinase, is capable of counteracting genotoxic stress-induced apoptosis by the suppression of caspase-3 activation. A cell line expressing the membrane-bound ADAM15 without its cytoplasmic tail, however, lost this anti-apoptotic property, suggesting a crucial role of the intracellular domain as a scaffold for recruitment of survival signal-transducing kinases. Accordingly, an enhanced phosphorylation of FAK at Tyr-397, Tyr-576, and Tyr-861 was detected upon genotoxic stress by camptothecin in ADAM15-transfected T/C28a4 cells, but not in transfectants expressing an ADAM15 mutant without the cytoplasmic tail. Accordingly, a specific binding of the cytoplasmic ADAM15 domain to the C terminus of FAK could be shown by mammalian two-hybrid, pulldown, and far Western studies. In cells expressing full-length ADAM15, a concomitant activation of Src at Tyr-416 was detected upon camptothecin exposure. Cells transfected with a chimeric construct consisting of the extracellular IL-2 receptor α-chain and the cytoplasmic ADAM15 domain were IL-2-stimulated to prove that the ADAM15 tail can transduce a percepted extracellular signal to enhance FAK and Src phosphorylation. Our studies further demonstrate Src binding to FAK but not a direct Src interaction with ADAM15, suggesting FAK as a critical intracellular adaptor for ADAM15-dependent enhancement of FAK/Src activation. Moreover, the apoptosis induction elicited by specific inhibitors (PP2, FAK 14 inhibitor) of FAK/Src signaling was significantly reduced by ADAM15 expression. The newly uncovered counter-regulatory response to genotoxic stress in a chondrocytic survival pathway is potentially also relevant to apoptosis resistance in neoplastic growth.

ADAM15 belongs to a family of transmembrane multi-domain proteins implicated in proteolysis, cell-cell and cell-matrix interactions in various disease conditions. In osteoarthritis (OA), ADAM15 is up-regulated in the chondrocytes already at early stages of cartilage degeneration where it seems to exert homeostatic effects likely associated with its ability to enhance integrin-mediated chondrocyte adhesion to the surrounding collagen matrix. The aim of our present study was, therefore, to characterize functional domains of ADAM15 involved in collagen II (CII) interaction and to analyse associated outside-in signalling events. Accordingly, ADAM15 and respective deletion mutants were stably transfected into the chondrocyte cell line T/C28a4. Transfected cells were adhered to CII and phosphoproteins analysed by Western blotting. Co-immunoprecipitation served to identify protein binding to ADAM15. Our results elucidate the prodomain as critical for the capacity of ADAM15 to enhance CII adhesion, thereby identifying for the first time a cell-adhesive role of a metalloproteinase prodomain. Moreover, the cytoplasmic tail of ADAM15 confers a modulatory effect on the autophosphorylation site Y397 of the focal adhesion kinase (FAK) during chondrocyte-collagen interaction. In conclusion, the newly uncovered impact of ADAM15 on signalling events that arise from chondrocyte interactions with its collagen matrix might contribute to the elucidation of the mechanism underlying its proposed chondroprotective role in degenerative cartilage disease.

Objective: The membrane-anchored metalloproteinase disintegrin ADAM15 is up-regulated in osteoarthritis and has been implicated in proteolysis and cell-matrix interactions. To address its role in cartilage metabolism, we performed an analysis of joint morphology in aging mice with a targeted inactivation of the ADAM15 gene (ADAM15(-/-)). In addition, a human chondrocyte cell line overexpressing ADAM15 was used to investigate the role of ADAM15 in an in vitro model of chondrocyte-matrix interactions., Methods: Knee joint sections from 3-, 6-, and 12-14-month-old ADAM15(-/-) and wild-type (WT) 129/SvJ mice were examined for synovial hyperplasia, cartilage degradation, and osteophyte formation. Stable transfection of the human T/C28a4 chondrocyte cell line with full-length human ADAM15 complementary DNA led to the establishment of ADAM15-overexpressing chondrocytes that were further analyzed for their capability to adhere to and to survive on cartilage matrix molecules (fibronectin and types II and VI collagen) under conditions of serum starvation. ADAM15 expression was investigated by reverse transcription-polymerase chain reaction and Western blotting., Results: Aging ADAM15(-/-) mice exhibited accelerated development of osteoarthritic lesions compared with WT mice, and the difference was statistically significant at age 12 months. The osteoarthritic changes preferentially affected male ADAM15(-/-) mice. ADAM15 overexpression in T/C28a4 cells led to the specific reinforcement of chondrocyte adhesion to cartilage types II and VI collagen, and this was associated with enhanced cell viability under conditions of serum starvation., Conclusion: The accelerated development of murine osteoarthritis in ADAM15 deficiency as well as the proadhesive and cell survival-promoting in vitro effect of ADAM15 overexpression suggest a homeostatic rather than a destructive role of ADAM15 in cartilage remodeling.

Objective: The aim of the study was to analyze the expression of the disintegrin metalloproteinase MDC15 (metargidin, or ADAM15) at the messenger RNA (mRNA) and protein levels in synovial tissue from osteoarthritis (OA) and rheumatoid arthritis (RA) patients compared with normal specimens., Methods: Conventional immunohistochemistry and confocal laser scanning microscopy of immunofluorescently stained sections, as well as in situ hybridization experiments and reverse transcription-polymerase chain reaction were performed for analyses of MDC15 expression on normal, OA, and RA synovial tissue specimens., Results: In normal synovium, MDC15 expression was detectable at a very low level. MDC15 expression was considerably increased in OA-derived tissue samples, whereas a maximum of signal intensity for MDC15 mRNA and protein was seen in the RA lining layer. The CD68+ macrophage-like synoviocytes (type A) and the CD68- fibroblast-like synoviocytes (type B) were positive for MDC15. Moreover, a very strong expression of MDC15 was also found in CD138+ plasma cells in all RA tissues as well as in OA specimens that contained areas of mononuclear cell infiltrates. CD20+ B cells and CD4+ and CD8+ T cells, however, did not exhibit expression of MDC15, either in the synovial tissue in situ or in preparations of circulating lymphocytes made from the peripheral blood of RA patients or healthy controls., Conclusion: Our results demonstrate high levels of MDC15 expression in macrophage-like and fibroblast-like synoviocytes as well as in plasma cells as a histologic feature most prominent in RA synovial tissue compared with normal or OA synovial tissue. This suggests a potential role of MDC15 in the pathogenesis of cartilage destruction in inflammatory joint disease.

Objective: The aim of the study was to investigate the messenger RNA (mRNA) expression of the disintegrin metalloproteinase MDC15 (metargidin, or ADAM-15) in normal and osteoarthritic (OA) articular cartilage., Methods: In situ hybridization experiments and reverse transcription-polymerase chain reaction (RT-PCR) were performed on tissue samples of adult normal and OA articular cartilage., Results: MDC15 mRNA could be detected in normal articular cartilage by RT-PCR using tissue-extracted total RNA as a template. However, the mRNA level remained below the sensitivity of in situ hybridization. In contrast, in situ hybridizations of OA cartilage revealed an intense staining with the MDC15-specific riboprobes. The extension of the analysis to chondrosarcomas showed a strong up-regulation of MDC15 mRNA in these malignant transformed cells., Conclusion: Our results demonstrate a markedly strong up-regulation of MDC15 in adult OA and neoplastic cartilage compared with adult normal articular cartilage, indicating a potential role of the disintegrin metalloproteinase in cartilage remodeling.

Annexin V has been isolated from chicken muscle and cartilage either by EDTA extraction or by plasma membrane purification and solubilization with detergent to obtain the hydrophilic and hydrophobic variants. The hydrophobic variant of the cartilage annexin V associated with phosphatidylserine-containing liposomes in a Ca(2+)-independent manner, whereas the EDTA-extracted molecule required Ca2+ for association with the liposomes. The collagen-binding assay used is based on the principle of a cell attachment assay using mildly pepsinized collagen type II or intact collagen type I as the solid-phase substrate. Soluble intact collagen type I or II was added as competitive inhibitor. The lipophilic and the EDTA-extracted anchorins CII from cartilage were inhibited to the same extent by collagen type II on pepsinized collagen type II as the solid-phase substrate. The EDTA-extracted muscle annexin V exhibited a fivefold lower affinity to collagen type II than its counterpart from cartilage. Peptide mapping studies and amino acid sequencing of selected peptides from the hydrophobic cartilage annexin V and the hydrophilic cartilage and muscle annexin V revealed 100% identity to the established chicken annexin V protein sequence in the corresponding amino acids 7-29 and 118-126. These results indicate that annexin V may occur in multiple pools within one cell type and/or tissue and that its biological function may depend on the subcellular distribution as well as the microenvironment in the tissue.