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.

Alpha-2 macroglobulin in Alzheimer’s disease: a marker of neuronal injury through the RCAN1 pathway

“…Although A2M is a well-established extracellular chaperone capable of clearing misfolded, aggregated proteins including Aβ, recent evidence suggests that it may also function as an intracellular chaperone. 70 This finding, in combination with our results suggesting that A2M may respond to tau phsphorylation states, opens up a novel line of investigation to further study A2M-tau interactions in vivo . Our results additionally suggest the importance of better understanding mechanisms underlying the sex-specific A2M response in preclinical AD.…”

Low Mass Blood Peptides Discriminative of Inflammatory Bowel Disease (IBD) Severity: A Quantitative Proteomic Perspective

“…The identified differential peptides cement the role of neuroendocrine and enteric nervous systems as measured via hormones, peptides, protein fragments, naturally occurring within the circulating plasma. The nature and context of these peptides in the enriched low mass fraction of plasma may be related to the associated increase in vascular permeability (35,36), as well as the activated binding and packaging of peptides into complexes in the endoplasmic reticulum (ER) tagged for secretion (37). At the interface between the epithelium and lymphatic/circulatory system, the altered secretion of lyzozyme, defensins, IgA and mucins as well as the catabolism and breakdown of (intracellular) proteins become sequestered into the circulatory system and can be indicators of disease, phenotype and severity.…”

“…Our data has shown increased levels of kininogen in IBD patients, along with other acute phase proteins such as fibrinogen, ␤-2-microglobulin, ␣-2-macroglobulin, haptoglobin and the apolipoproteins. In particular ␣-2-macroglobulin has previously been implicated as an intra and extracellular chaperone of vesicular traffic in the ER (37,38). All proteins and small peptides have been measured initially from the enriched low mass component of plasma as well as in unfractionated plasma and serum using MRM.…”

HK2 Proximal Tubule Epithelial Cells Synthesize and Secrete Plasma Proteins Predominantly Through the Apical Surface

“…The ultimate validation of the gene expression findings was to prove that translation, biosynthesis and secretion of individual plasma proteins take place in HK2 cells. Newly synthesized secretory proteins in serum‐free cultured HK2 medium were directly evaluated by our sensitive non‐reducing Western blotting method [Zhao et al, ]. As PETC are polar cells similar to synoviocytes, any secretory protein synthesized will exit either from the apical side (lumen facing medium) or from the basolateral side (interstitial facing plastic side or two cells interacting each other) [Stoops and Caplan, ].…”

“…We have recently demonstrated that plasma proteins contained in the joint (synovial) fluid are not leaked from the circulation, but rather, are synthesized and secreted by the fibroblastic synoviocytes, polarized cells lining inside the synovium [Zhao et al, ]. This suggests that synoviocytes behave as hepatocytes in the production and secretion of plasma proteins into the synovial fluids.…”