Analyses of differential proteome of human synovial fibroblasts obtained from arthritis

A total of 49 spots were significantly overexpressed in either RA or OA in comparison to healthy control and were subjected to MALDI-TOF-MS. Among the spots, 25 proteins were successfully identified. The function of these proteins was involved in cell organization, energy, protein fate, cell stress, and metabolism, as described in Kumar’s work [6]. While, some unreported proteins were found in our study, i.e., S100A4, S100A10, proteasome activator PA28 beta chain, Ribosomal protein P0, Ribonucleoprotein La, and GDP dissociation inhibitors. Most importantly, our data proved that these total 25 proteins were significantly and quantitatively higher in SFs from RA and OA than that from healthy individuals. The most abundant differential proteins in SFs from RA and OA were cathepsin D, α-enolase, MnSOD, PRX2, Anx-1, and S100 calcium-binding protein A4 and A10.

Over the last 25 years, many autoantibody activities in RA have been described. Some of the RA-specific antibodies might be very helpful in the early diagnosis and prognosis of the disease [9]. The protein α-Enolase, a highly conserved multifunctional protein, is known to be upregulated by hypoxia and by proinflammatory stimuli, both of which are features of the synovial membrane microenvironment in RA. Antibodies against α-enolase have been found in the sera of RA, and citrullinated α-enolase has been described as a candidate autoantigen in RA [10]. Recently, triosephosphate isomerase (TPI) was demonstrated to be a pathogenic autoantigen in OA [11], and the anti-TPI autoantibodies were detected in a subset of patients with RA [12]. Our proteomics study show that α-enolase and TPI were upregulated in synovial fibroblasts from RA and OA, which is consistent with the results obtained from other routine methods. How an antibody against ubiquitous cytoplasmic enzyme to provoke joint-specific autoimmune disease should be further explained. But, our results indicated that α-enolase and TPI likely have potential diagnostic and prognostic value for RA.

Glucocorticoids (GCs) have been used as anti-inflammatory drugs for more than half a century. Anx-1, 37-kd member of the annexin superfamily of calcium and phospholipid binding proteins, has been suggested to function as a cellular mediator of the anti-inflammatory actions of GCs [13]. Moreover, recent evidences show that Anx-1 exerts endogenous anti-inflammatory effects on adjuvant-induced arthritis via the regulation of cytokine gene expression in the synovium. Endogenous Anx-1 is able to downregulate proinflammatory cytokines, such as IL-1β, TNFα, IL-6, and MIF expression [14]. Thus, it hints that Anx-1 could be one of the promising new agents for treatments specifically tailored to RA, and administration of Anx-1 analogs, or non-GC manipulation of endogenous Anx-1 expression, may be a valuable strategy against arthritis and other inflammatory diseases.

Increasing evidence demonstrated an important role of oxidative stress in the development of the chronic and destructive inflammation, such as RA or OA [15]. The overproduction of reactive oxygen species (ROS) molecules may contribute to the degradation of cartilage matrix [16]. To prevent toxicity by ROS, mammalian cells possess a well-coordinated antioxidant enzyme system. Examples of antioxidant enzymes are the superoxide dismutases (SOD), glutathione peroxidase, catalase, and a novel family of peroxidases, the peroxiredoxins (PRX). Increased SOD activity and increased expression of PRX5 has been reported in OA [17]. The present study show that MnSOD and PRX2 are more abundant in SFs of RA or OA, which suggests that they may play a protective role against oxidative stress in inflammatory arthritis. Furthermore, antioxidants and oxidative enzymes have been shown to ameliorate arthritis in animal models [18]. MnSOD that is upregulated to a higher level in OA than that in RA implied that inadequate control of ROS had been involved in the pathophysiology of RA. An understanding of the complex interactions involved in these pathways may allow the development of novel therapeutic strategies for RA.

It is now well understood that SFs contribute significantly to matrix degradation in RA through the expression of matrix degrading enzymes such as matrix metalloproteinases and cathepsins [19]. Cathepsins can be divided into three subgroups according to their active-site amino acid (i.e., cysteine B, C, H, F, K, L O, S, V, and W), aspartic (D and E), or serine (G) cathepsins. The cysteine proteases cathepsin B and cathepsin L, particularly, have been shown to be activated by proto-oncogenes and upregulated in RA synovium at sites of cartilage invasion [20]. The aspartate protease cathepsin D has not been studied in much detail with respect to arthritis in contrast to cathepsins B and L. However, cathepsin D has been purified from RA synovium and has been demonstrated in RA and OA synovial fluid [21]. Cathepsin D has also been found to be overexpressed in OA’s SFs as compared with that in RA’s SFs in our experiment. Thus, it is reasonable to think that cathepsin D plays an important role in the pathological alteration of joints. The correlation between cathepsin D expression and disease severity and the contribution of cathepsin D to fibroblast-mediated collagen degradation remains to be fully elucidated.

The question of whether the massive synovial hyperplasia characteristic for RA is mainly due to the proliferation of SFs or to defective apoptosis of SFs is still a matter for discussion. Over recent years, evidence has been accumulated for both hypotheses [22]. On the basis of the well-known phenomenon that SFs can grow very fast over multiple passages in vitro, cell proliferation has been considered the major mechanism for the hyperplastic growth of the RA synovium. S100A4 and S100A10 are members of the S100 calcium-binding protein family. To date, it has been demonstrated in many studies that S100A4 is involved in cancer growth and metastasis [23]. An increased expression of S100A4 mRNA has been confirmed in the lining and sublining layer of RA synovium [24]. Since several phenomena are similar between RA and malignant tumors [25], these data implied that S100A4 might be contribute to the aggressive, invasive, and tumor-like behavior of RA synovium. The mechanisms that associate S100A4 with synovitis are not well understood. The dysregulation of the transcription factors such as activator protein (AP-1), NF-κB, and the tumor suppressor gene, p53, have been implicated to play the role in the aggressive proliferative and invasive functions of SFs. The accumulation of p53 has been observed in SFs from rheumatoid patients, and an important role of p53 in regulation of cellular proliferation in rheumatoid synovium was supported by some experiments [26, 27]. This implicated that S100A4 might trigger a cascade of events through interaction with the intracellular target proteins such as tumor suppressor p53 to participate in the major mechanism for the hyperplastic growth of SFs.

In conclusion, the overexpressed proteins we found in SFs from RA and OA were included in enzymatic and structural proteins, signal transduction proteins, calcium binding protein, etc. With literature searches, our results indicated that (1) S100A4 might interact with the intracellular target proteins, such as tumor suppressor p53, to participate in the hyperplastic growth of SFs, (2) cathepsin D might play an important role in the pathological alteration of joints, (3) α-Enolase and TPI likely have potential diagnostic and prognostic value for RA, and (4) antioxidants and oxidative enzymes, as well as enhancing endogenous Anx-1 expression, might be the valuable strategies against arthritis. This proteomics analysis may significantly promote our understanding of the molecular biology of arthritis and will facilitate further studies on searching for markers of diagnosis and new therapeutic agents.