ADAM15 in Apoptosis Resistance of Synovial Fibroblasts: Converting Fas/CD95 Death Signals Into the Activation of Prosurvival Pathways by Calmodulin Recruitment

Arthritis Rheumatol. 2019 Jan;71(1):63-72. doi: 10.1002/art.40667. Epub 2018 Nov 20.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADAM Proteins / genetics*
  • ADAM Proteins / metabolism
  • Anilides / pharmacology
  • Apoptosis / drug effects
  • Apoptosis / genetics*
  • Arthritis, Rheumatoid / metabolism*
  • Calmodulin / antagonists & inhibitors
  • Calmodulin / metabolism*
  • Cell Line
  • Chondrocytes / metabolism*
  • Fas Ligand Protein / metabolism*
  • Fibroblasts / metabolism*
  • Focal Adhesion Kinase 1 / metabolism
  • Humans
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism
  • ORAI1 Protein / antagonists & inhibitors
  • ORAI1 Protein / metabolism
  • Phosphorylation
  • RNA Interference
  • Synovial Membrane / cytology
  • Thiadiazoles / pharmacology
  • Trifluoperazine / pharmacology
  • fas Receptor / metabolism
  • src-Family Kinases / metabolism


  • 4-methyl-4'-(3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl)-1,2,3-thiadiazole-5-carboxanilide
  • Anilides
  • Calmodulin
  • FAS protein, human
  • FASLG protein, human
  • Fas Ligand Protein
  • Membrane Proteins
  • ORAI1 Protein
  • ORAI1 protein, human
  • Thiadiazoles
  • fas Receptor
  • Trifluoperazine
  • Focal Adhesion Kinase 1
  • PTK2 protein, human
  • src-Family Kinases
  • ADAM Proteins
  • ADAM15 protein, human