Fibroblastic Synoviocytes Secrete Plasma Proteins Via 2-Macroglobulins Serving as Intracellular and Extracellular Chaperones

Changes in plasma protein levels in synovial fluid (SF) have been implicated in osteoarthritis and rheumatoid arthritis. It was previously thought that the presence of plasma proteins in SF reflected ultrafiltration or extravasation from the vasculature, possibly due to retraction of inflamed endothelial cells. Recent proteomic analyses have confirmed the abundant presence of plasma proteins in SF from control and arthritic patients. Systematic depletion of high-abundance plasma proteins from SF and conditioned media from synoviocytes cultured in serum, and protein analysis under denaturing/reducing conditions have limited our understanding of sources and the native structures of plasma protein complexes in SF. Using Western blotting, qPCR, and mass spectrometry, we found that Hig-82 lapine fibroblastic synovicytes cultured under serum-free conditions expressed and secreted plasma proteins, including the cytokine-binding protein secreted phosphoprotein 24kDa (Spp24) and many of the proteases and protease inhibitors found in SF. Treating synoviocytes with TGF-1 or BMP-2 for 24h upregulated the expression of plasma proteins, including Spp24, (2)-HS-glycoprotein, (1)-antitrypsin, IGF-1, and C-reactive protein. Furthermore, many of the plasma proteins of mass <151kDa were secreted as disulfide-bound complexes with members of the (2)-macroglobulin (A2M) family, which serve as intracellular and extracellular chaperones, not protease inhibitors. Using brefeldin A to block vesicular traffic and protease inhibitors to inhibit endogenous activation of naive A2M, we demonstrated that the complexes were formed in the endoplasmic reticulum lumen and that Ca(2+)cysteine protease-dependent processes are involved. J. Cell. Biochem. 116: 2563-2576, 2015. Published 2015. This article is a U.S. Government work and is in the public domain in the USA