Objective: To clarify the pathogenesis of rheumatoid arthritis (RA) by comparing protein expression in fibroblast‐like synoviocytes (FLS) from patients with RA with that in FLS from normal subjects, using proteomics analysis.Methods: Proteins extracted from primary cultures of FLS obtained from 50 patients with RA and 10 normal subjects were analyzed by automated 2‐dimensional nano–electrospray ionization liquid chromatography tandem mass spectometry. Differentially expressed proteins were screened by 2‐samplet‐test (P< 0.05) and fold change (>1.5), based on the bioinformatics analysis. The expression of vasculature development–related proteins (Thy‐1, connective tissue growth factor [CTGF], and thrombospondin 1 [TSP‐1]) and redox‐related proteins (superoxide dismutase 2 [SOD2]) in synovial tissue was confirmed by real‐time polymerase chain reaction and Western blotting. The effect of Thy‐1 and CTGF knockdown on Thy‐1, CTGF, TSP‐1, and vascular endothelial growth factor (VEGF) was analyzed by RNA interference experiments.Results: According to the criteria of having >1 unique peptide per protein present and a false discovery rate of ≤5%, 1,060 proteins were identified from patients with RA, and 1,292 proteins were identified from normal subjects, from which 100 differentially expressed proteins were screened out from the RA proteins. Of these, 46 proteins were up‐regulated, and the remaining 54 proteins were down‐regulated. Gene ontology and pathway analyses showed that 6 vasculature development–related proteins were up‐regulated, including Thy‐1, CTGF, and TSP‐1, while 11 redox‐related proteins were down‐regulated, including SOD2. The results were consistent with those obtained using mass spectrometry. Thy‐1, VEGF, CTGF, and TSP‐1 were down‐regulated after Thy‐1 knockdown, and VEGF and CTGF were down‐regulated after CTGF knockdown. Recombinant human CTGF could enhance proliferation and Transwell migration of human umbilical vein endothelial cells.Conclusion: Up‐regulation of vasculature development–related proteins and down‐regulation of redox‐related proteins in FLS are predominant factors that may contribute to the pathogenesis of RA.

The effects of inflammation may not always benefit the individual. Its amplifying nature represents a highly regulated biological program, and the inflammatory microenvironment is its essential component. Growing evidence suggests that the ECM (extracellular matrix) is important for the early steps of inflammation. Versican, a ubiquitous component of the ECM, contributes to the formation of the inflammatory response and is highly regulated by cytokines. Certain cytokines exert their initial effects on versican to alter the homeostasis of the inflammatory milieu, and inappropriate production of versican may promote the next inflammatory response. Therefore, versican could be the first step in the amplification of the inflammatory response, and ongoing research of this molecule may help to explain the pathogenesis of inflammation.

Analyses of supernatants from apoptotic cells have helped in the identification of many signals that modulate the states of cell activation and differentiation. However, the current knowledge about the soluble factors that are released during apoptosis is rather limited. Previous studies have shown that S5a and angiocidin (both encoded by PSMD4) induce human acute monocytic leukemia cells (THP-1 cells) to differentiate into macrophages, but the cell-surface receptor of S5a has not been identified. In this study, we show that apoptotic THP-1 cells release endogenous S5a that binds to death receptor-6 (DR6, also known as TNFRSF1), which was identified as an orphan receptor, to induce THP-1 cells to differentiate. Furthermore, we found that the NF-κB pathway is activated, and that the transcription factors WT1 (Wilms9 tumor 1) and c-myb mediate S5a-induced THP-1 differentiation. We also show that differentiation is blocked by anti-DR6 antibody, DR6 siRNA, DR6-Fc, NF-κB inhibitor or WT1 siRNA treatment. Our findings indicate that the interaction between cells can determine their differentiation, and we provide evidence for a functional interaction between S5a and DR6, which provides a novel potential mechanism to induce the differentiation of cancer cells, especially during biotherapy for leukemia.

Angiopoietin-related protein 1 (ARP1) is one of the antiangiogenic factors and plays an important role in endothelial cell proliferation, migration, and blood vessel network formation. Here a rapid method to prepare ARP1 polyclonal antibody in 1 month was developed. The gene of fibrinogen homology domain (FD) for ARP1 was cloned and the protein was expressed in a soluble form of MBP-FD fused protein. The MBP-FD protein was purified using amylose affinity chromatography of maltose-binding protein. Polyclonal antibodies against MBP-FD were obtained through immunization in BALB/c mice. The titer was determined by indirect enzyme-linked immunosorbent assay (ELISA), and the antibody specificity was assessed by Western blot. The full-length ARP1 protein in stable form expressed in transfected human large lung cancer cell lines NCI-H460 was detected by immunocytochemistry (ICC) analysis using ARP1 polyclonal antibodies. The result shows that the antibody possesses good specificity and sensitivity. This work provides a substantial base for the further studies of ARP1 function and associated mechanisms. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.