Change in cellular localization of a rheumatoid arthritis-related antigen (RA-A47) with downregulation upon stimulation by inflammatory cytokines in chondrocytes

Abstract

We previously isolated a rheumatoid arthritis-related antigen (RA-A47) protein that had reactivity with RA sera from a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8. Sequencing analysis of ra-a47 cDNA revealed RA-A47 as a product of the colligin-2 gene, which is also known as the human heat shock protein (HSP) 47 gene. Expression of hsp47 has been shown to be cooperatively altered with that of collagen genes upon stimulation. In this study, it was confirmed that the mRNA expression of ra-a47 and COL2A1, a type II collagen gene, was upregulated on stimulation with transforming growth factor (TGF) beta in chondrocytes. However, in contrast, inflammatory cytokines such as tumor necrosis factor (TNF) alpha, interferon (IFN) beta, and interleukin (IL)-6 downregulated the expression of ra-a47 mRNA, whereas the expression of COL2A1 mRNA was not repressed, or even upregulated, in HCS-2/8 cells. Of note, inducible NO synthase (iNOS) and matrix metalloproteinase (MMP)-9 mRNAs were strongly stimulated by TNFalpha. We also found that cell-surface type II collagen disappeared upon such a stimulation, suggesting that decrement of RA-A47 may inhibit the secretion of type II collagen and lead to its accumulation inside the cells. RA-A47 was detected in the cultured medium of TNFalpha-treated HCS-2/8 cells and of IL-1-treated rabbit chondrocytes by Western blot analysis. Under the same conditions, RA-A47 was detected on the cell surface by immunofluorescence staining. These findings demonstrate that the RA-A47 chaperone protein is specifically downregulated, causing the intracellular accumulation of unsecretable type II collagen, while the extracellular matrix (ECM) is degraded by MMPs and iNOS through the stimulation of chondrocytes by TNFalpha. The altered localization of RA-A47 to the surface or outside of cells may represent the mechanism for the recognition of RA-A47 as an autoantigen during rheumatoid arthritis.

Interferon stimulates interleukin 1 receptor antagonist production in human articular chondrocytes and synovial fibroblasts

“…21 A recent report described the biological effects of IFNb in a human chondrosarcoma derived chondrocytic cell line, suggesting that chondrocytes are responsive to IFNb. 15 We report that human chondrocytes indeed express mRNA encoding type I interferon receptors and that IFNb modulates IL1b induced IL1Ra production in these cells. Although IFNb displayed no stimulatory effect on its own, it markedly enhanced IL1Ra secretion in response to IL1b.…”

Novel Transcription Factor-Like Function of Human Matrix Metalloproteinase 3 Regulating the CTGF/CCN2 Gene

“…The HCS-2/8 cell line was established from a chondrosarcoma, but the expression of CCN2 in cartilaginous tumors is negatively related with the level of their malignancy (45), indicating that HCS-2/8 cells can be a model of a normal phenotype of chondrocytes. This cell line also has been used for arthritis studies, because HCS-2/8 produces MMP3 and matrix degradation-related molecules in response to tumor necrosis factor alpha (16,17,41). In addition, MMP3 immunopositive cells were specifically expressed in normal articular cartilage of a 14-year-old human, while MMP9, MMP13, and ADAMTS4 were not detected (H. I. Roach, personal communication).…”

“…After being cultured for a further 24 h, cells were lysed in 200 l of 1ϫ passive lysis buffer (Promega, Madison, WI) with gentle rocking for 20 min and were collected. Luciferase assays were carried out by using a Dual Glo luciferase assay system (Promega), as described previously, on a smaller scale (17). Relative luciferase activities were calculated as the ratios of firefly luciferase activity to Renilla luciferase activity.…”

ER Stress Proteins in Autoimmune and Inflammatory Diseases

“…Its gene disruption in mice causes significant reductions in mature collagens in connective tissues, resulting in embryonic lethality (Nagai et al, 2000; Marutani et al, 2004; Matsuoka et al, 2004). Several studies showed that the levels of anti-hsp47 autoantibody are specifically increased in RA patients (Hattori et al, 1998, 2000, 2001, 2003, 2005). However, little is known about how hsp47 and its autoantibody correlate with RA pathogenesis.…”