Downregulation of a rheumatoid arthritis‐related antigen (RA‐A47) by ra‐a47 antisense oligonucleotides induces inflammatory factors in chondrocytes

Abstract

Previously we have shown that the expression of RA-A47 (rheumatoid arthritis-related antigen) which is identical to HSP47, a collagen-binding chaperon, is downregulated in chondrocytes by tumor necrosis factor alpha (TNFalpha). RA-A47 was also found on the surface of chondrocytes where it is recognized as an antigen in the serum of rheumatoid arthritis (RA) patients. Its translocation to the cell surface from endoplasmic reticulum membrane where it is normally located was also enhanced by TNFalpha. To understand the significance of RA-A47 downregulation in chondrocytes independent from other effects of TNFalpha, we used an antisense oligonucleotide approach and investigated the effect of this treatment on the expression of molecules related to matrix degradation and production of growth factors for chondrocytic, endothelial, and synovial cells. Here we show that treatment of rabbit chondrocyes and human chondrosarcoma cells HCS-2/8 by ra-a47 antisense S-oligonucleotides significantly reduced the expression of ra-a47 both at mRNA and protein level. Interestingly, this TNFalpha-independent RA-A47 downregulation was associated with a strong induction of matrix metalloproteinase (MMP)-9 mRNA and inducible NO synthase (iNOS) mRNA. The induction of active-type MMP-9 was further detected by gelatin zymography. Under the same conditions, the release of basic fibroblast growth factor (bFGF) and connective tissue growth factor (CTGF) from HCS-2/8 cells into the conditioned medium (CM) was strongly enhanced. These effects were not a result of TNFalpha upregulation, since the ra-a47 antisense oligonucleotide treatment did not enhance TNFalpha synthesis. These observations indicate that downregulation of RA-A47 induces TNFalpha-independent cartilage-degrading pathways involving iNOS and MMP-9. Furthermore, the stimulation of bFGF and CTGF release from chondrocytes may stimulate the proliferation of adjacent endothelial and/or synovial cells.

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).…”

Identification of a peripheral blood transcriptional biomarker panel associated with operational renal allograft tolerance

“…Interestingly, however, we found that TGF-␤ regulates the function of 27% of the peripheral blood genes that differentiate tolerance from CR. These TGF-␤-regulated genes include latent TGF-␤ binding protein 4 (LTBP4) (2.6-fold), which functions to convert latent TGF-␤ protein into the active form; N-cadherin (CDH2) (5-fold), which is known to enhance the ability of TGF-␤ to induce cell-cycle arrest in the G 1 phase (22, 23); and CD9, a surface antigen initiating the TGF-␤ signaling pathway (24) that was expressed at Ϸ40% higher levels in tolerance. Additional TGF-␤-regulated genes include ␣-fetoprotein, natural killer cell group 7 (NKG7) (25), connective tissue growth factor (CTGF), and fibronectin (FN1) (26), which are gene markers for apoptosis, immune suppression, growth arrest, and the stress-response, respectively, and are also involved in early T and NK cell activation (27).…”

Quantitative Serum Proteomics of Tryptophan Nutrition Using Bi-directional Heavy Oxygen Labeling with a RuBisCO Internal Standard

“…A flow rate of L/min was used for both SCX and RP columns. A salt gradient was applied in steps of 0, 10,15,20,25,30,35,40,45,50,57,64,90 and 700 mM ammonium acetate in 5% ACN, 0.1% formic acid and the resultant peptides loaded directly into the sample loop of a 0.18 x 100 mm BioBasic C18 RP LC column of a ProteomeX workstation (Thermo Electron). The RP gradient used 0.1% formic acid in ACN and increased the ACN concentration in a linear gradient from 5% to 30% in 30 min and then 30% to 65% in 9 min followed by 95% for 5 min and 5% for 15 min.…”

“…TGFB1 is involved in inflammation and numerous autoimmune disorders as well as tumorigenesis and metastasis of many types of cancer [23,24]. The link between inflammation and cancer is well known [25] and so this is perhaps not surprising. This first network pathway model suggests tryptophan deficiency is involved in autoimmune, dermatological, connective tissue disorders and cancer.…”