title: Fibroblastic synoviocytes secrete plasma proteins via α2 -macroglobulins serving as intracellular and extracellular chaperones.
abstract: 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 24 kDa (Spp24) and many of the proteases and protease inhibitors found in SF. Treating synoviocytes with TGF-β1 or BMP-2 for 24 h upregulated the expression of plasma proteins, including Spp24, α2 -HS-glycoprotein, α1 -antitrypsin, IGF-1, and C-reactive protein. Furthermore, many of the plasma proteins of mass <151 kDa were secreted as disulfide-bound complexes with members of the α2 -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 naïve A2M, we demonstrated that the complexes were formed in the endoplasmic reticulum lumen and that Ca(2+) cysteine protease-dependent processes are involved.
authors.affiliation: Research Service (151), Veterans Affairs Greater Los Angeles Healthcare System, Sepulveda, California, 91343.
authors.forename: Ke-Wei
authors.initials: KW
authors.lastname: Zhao
authors.affiliation: Geriatric Research, Education and Clinical Center (11E), Veterans Affairs Greater Los Angeles Healthcare System, Sepulveda, California, 91343.|Department of Medicine, University of California, Los Angeles, California, 90095.
authors.forename: Elsa J Brochmann
authors.initials: EJ
authors.lastname: Murray
authors.affiliation: Geriatric Research, Education and Clinical Center (11E), Veterans Affairs Greater Los Angeles Healthcare System, Sepulveda, California, 91343.|Department of Medicine, University of California, Los Angeles, California, 90095.|Interdepartmental Program in Biomedical Engineering, University of California, Los Angeles, California, 90095.
authors.forename: Samuel S
authors.initials: SS
authors.lastname: Murray
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000378:metabolism*; D055396:Bone Morphogenetic Protein 2 / Q000494:pharmacology; D018929:Cell Culture Techniques; D002460:Cell Line; D016895:Culture Media, Serum-Free / Q000737:chemistry; D057056:Cysteine Proteases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism*; D051381:Rats; D013582:Synovial Fluid / Q000166:cytology / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology; D000511:alpha-Macroglobulins / Q000378:metabolism*
chemical_list: D001798:Blood Proteins; D055396:Bone Morphogenetic Protein 2; D016895:Culture Media, Serum-Free; D018832:Molecular Chaperones; D053773:Transforming Growth Factor beta1; D000511:alpha-Macroglobulins; D057056:Cysteine Proteases
keywords: BLOOD: SYNOVIUM BARRIER; BONE MORPHOGENETIC PROTEIN-2; C-REACTIVE PROTEIN; CHAPERONE; HIG-82 CELLS; INSULIN-LIKE GROWTH FACTOR-1; INTER-α-TRYPSIN INHIBITOR HEAVY CHAIN H4; MATRIX METALLOPROTEINASE; Spp24; TRANSFORMING GROWTH FACTOR-β1; α1-ANTITRYPSIN (A1AT); α2-MACROGLOBULIN