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ADAM15 in Apoptosis Resistance of Synovial Fibroblasts

Converting Fas/CD95 Death Signals Into the Activation of Prosurvival Pathways by Calmodulin Recruitment
: Janczi, T.; Böhm, B.B.; Fehrl, Y.; DeGiacomo, P.; Kinne, R.W.; Burkhardt, H.


Arthritis & rheumatology 71 (2019), Nr.1, S.63-72
ISSN: 2326-5191
ISSN: 2326-5205
Deutsche Forschungsgemeinschaft DFG
BU 584-5/1
Fraunhofer IME ()

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 Ca2+ release‐activated Ca2+/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.