P037 Multiple roles of phospholipase C-ETA2, as a novel C2 domain containing protein, in the pathogenesis of rheumatoid arthritis

Introduction The C2 domain is a Ca 2+ -dependent membrane-targeting motif found in many cellular proteins involved in signal transduction or inflammation pathway. However, the effects of C2 domain-containing proteins in the fibroblast-like synoviocyte (FLS) of rheumatoid arthritis (RA) have not yet been elucidated. Objectives The aim of this study was to screen novel C2 domain-containing proteins related to aggressiveness of FLS, and confirm the precise roles of target protein in RA. Methods We transduced RA-FLS with a recombinant adenovirus expressing a C2 domain library. To confirm the effect of phospholipase C-eta2 (PLCH2), as a candidate C2 domain-containing target protein, we established stable MH7A cell lines overexpressing GFP-tagged PLCH2. We also conducted in vitro and in vivo experiments using a recombinant adenovirus expressing the full length form of PLCH2 or C2 domain of PLCH2 (C2-PLCH2). Results Several C2 domain-containing proteins were identified whose over-expression resulted in reduced proliferation and NF-kB activity of RA-FLS. Among those, we focused on PLCH2 in this study. PLCH2 levels were significantly decreased in RA-FLS and synovium than OA. PLCH2 and C2-PLCH2 suppressed cell invasion and migration, cytokines production, and matrix metalloproteinase secretion in RA-FLS and stable MH7A cell lines compared to control. PLCH2 and C2-PLCH2 sensitised RA-FLS to apoptosis in vitro and in vivo model of matrigel implants engrafted into immunodeficient mice. PLCH2 and C2-PLCH2 further decreased arthritis severity in collagen-induced arthritis (CIA) mouse model. Conclusions Our results showed that invasive characteristics of RA-FLS and inflammatory arthritis of CIA are reduced by PLCH2 and C2 domain of PLCH2. Therefore C2 domain of PLCH2 may have therapeutic potential for RA. References . Farah CA, Sossin WS. The role of C2 domains in PKC signalling. Adv Exp Med Biol2012;740:663–83. . Yanez M, Gil-Longo J, Campos-Toimil M. Calcium binding proteins. Adv Exp Med Biol2012;740:461–82. . Friedrich R, Yeheskel A, Ashery U. DOC2B, C2 domains, and calcium: A tale of intricate interactions. Mol Neurobiol2010February;41(1):42–51. . Zhou Y, Sondek J, Harden TK. Activation of human phospholipase C-eta2 by Gbetagamma. Biochemistry2008April 15;47(15):4410–7. . Popovics P, Beswick W, Guild SB, Cramb G, Morgan K, Millar RP, Stewart AJ. Phospholipase C-η2 is activated by elevated intracellular Ca(2+) levels. Cell Signal2011November;23(11):1777–84. Acknowledgements This work was supported by grants (NRF-2014R1A2A1A11051360 and NRF-2015R1A5A2008833) from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIP). Disclosure of interest None declared