| The hyper-proliferation and invasiveness of synovial tissue is the main reason for the destruction of joint cartilage in rheumatoid arthritis (RA) patients. In order to know the mechanism of pathological process in RA joints, fibroblast-like synovial cells (synovial cells) were isolated from RA patients and cultured in vitro to look for proliferation- and (br) invasiveness-related molecules in synovial cells. Since most of protein tyrosine kinases (PTKs) were involved in the proliferation or apoptosis of normal and tumor cells, PTKs in synovial cells were chosen to be our research subject. Degenerate primers were designed according to some known PTKs and the 3?part of mRNA of PTKs were amplified by RT-PCR. The PCR products were then cloned directly into T vectors. Sequence analysis showed that at least six kinds of PTKs were expressed in synovial cells, including platelet-derived growth factor receptor A (PDGFRA), insulin-like growth factor I receptor (IGF-IR), Janus kinase 1 (JAKI), TYK2, discoidin domain receptor 2 (DDR2), and Lyn. RNA dot hybridization showed that the expression level of PDGFRA, Department of Biochenuistrj and Molecular Biology -7- k~m眎~ IGF-1R, and DDR2 were higher than that in osteoarthritis (OA, as control) synovial cells, whereas the expression level of TYK2, iAKI, and Lyn were not different between the two kinds of cells. DDR2 was a kind of wide-distributed receptor tyrosine kinase, and was thought to involve in the migration of some tumors. What is more interesting is that the ligand of DDR2 is fibrilic collagen (I, II, or Ill), and the activation of DDR2 by collagen could upregulate the expression of MMP-1 in many kinds of cells. As collagen II is always thought to be a critical element in the pathogenesis of RA, and MMP-l is a direct factor in the destruction of cartilage in RA patients, DDR2 might act as an important mediator or a bridge to link these two important factors. In order to know whether this process exists and whether DDR2 is a bridge in the process, we focused our study on the function of DDR2 in the over-expression of MMP-l in RA synovial cells. Without enough OA synovial cells, we chose NIH 3T3 cells as control in some experiments. The distribution of DDR2 in synovial tissue was studied by in situ hybridization and immunohistochemistry. The stain signal told us that DDR2 was expressed not only in synovial cell line on the surface of synovial tissue, but also in many other cells under the synovial line. Regretfully, the type of the positive cells could not be identified. DDR2 could also be detected in cultured RA synovial cells, and the positive signal mainly existed in cytoplasm and cell membrane. Western blotting showed that DDR2 were expressed both in RA synovial cells and NIH 3T3 cells, but only the DDR2 in RA synovial cells were activated. Gel Collagenase Analysis told that the activity of MMP-l could be detected out directly in culture supernatant of RA synovial cells but not in OA synovial cells and NIH 3T3 cells. After stimulation of collagen II, NIH 3T3 cells became able to express and secrete low level of MMP-l. At the meantime, DDR2 in NIH 3T3 cells became activated. All these results indicated that the over-expression of MMP-l in RA synovial cells and the expression of MMP-l in NIH 3T3 cells after collagen li-stimulation were related to the activation of DDR2; the induction of MMP-l by col...
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