| ObjectiveTo explore the effect of transforming growth factor - β ( TGF - β) on me-sangial cell proliferation and extracellular matrix (ECM) accumulation. To study whether extracellular signal - regulated kinase ( ERK) participate in TGF - β-induced ECM overproduction in mesangial cells.IntroductionThe incidence rate of diabetic mellitus is gradually increasing. Diabetic ne-phropathy ( DN) is one of the most serious microvascular complications. The major pathologic feather of DN are glomerular basal membrane thicken and excess ECM deposition. It is very important to investigate the pathogenesis of DN and find a new way for therapy.TGF - β1is a multifunctional cytokine. The critical role of TGF - β1 has been well recognized in several renal diseases, including diabetic nephropathy. Glomerular mesangial matrix expansion can be reversed by treatment with monoclonal antitransforming growth factor - beta antibody; in addition this process does not dependent on the glucose level in plasma. But TGF - β1 also regulates multiple cellular functions including inhibition and stimulation of cell growth, apoptosis and immune inhibition, we must find a specific way to block the bad effect only.ERK is a subtype of mitogen - activated protein kinase ( MAPK). Emergingevidence indicate that ERK participate in TGF - β1 - induced ECM overproduction in hepatic stellate cells, chondrogenic cells and in lung fibroblasts cells. PD98059 is a specific inhibitor of MAPK/ERK kinase. We use PD98059 to study the role of ERK in TGF - β1 - induced ECM overproduction.Materials and methodsMesangial cells were obtained from a Sprague — Dawley rat' s cell lines. All experiments were performed using cells between the subculturing 3th and 8th passages. Using trypan blue staining assay to detect the cytotoxic effect of various concentrations of TGF - β1 ( 0. 05 - 4.0ng/ml) and PD98059 (10 - 50umol/l). We added different concentrations of TGF - β1 with or without MEK1 inhibitor PD98059 to cell culture medium. Cell proliferation were assessed by MTT color-imetric assay. Fibronectin (FN) protein concentrations in culture supernatant were tested by Enzyme -linked immunoadsordent assay(ELISA) method.Result1, There are no obvious cytotoxic effect when TGF -pi(0.05-4.0ng/ml) and PD98059( 10 -50umol/l) act on mesangial cells for 48 hours.2,Compared with the 0.2% newborn bovine serum,5% newborn bovine serum can significant progress mesangial cell proliferation. TGF - β1 can inhibit the serum - induced cell proliferation effect( P <0.01) .3,PD98059 also can inhibit the serum - induced cell proliferation effect, the inhibitor effect increase according to the concentration ( P < 0. 01). When add TGF - β1 0. 25ng/ml and PD98059 together, they have double inhibitor effect(P<0.01).4,TGF -β1 can stimulate mesangial cells secrete more FN protein, it shows a dose - dependent and time - dependent way( P <0.01).5,PD98059 can inhibit the TGF - β1 stimulated FN protein secretion in mesangial cells, the inhibitor effect increase according to the concentration(P < 0.01).DiscussionTGF - (3 is multifunctional cytokines, including TGF - β1, TGF -β2 and TGF - β3. Normal kidney shows similar, weak expression for all three TGF - (3 isoforms. In contrast diabetic nephropathy showed significantly increased expression of all the three TGF - β isoforms in glomeruli and the tubulointerstitium, especially TGF - β1. One of the main pathologic finding in diabetic nephropathy is mesangial matrix area expansion, which caused by overproduction, and less degradation of matrix protein. Mesangial cells are the major cell to synthesis matrix protein, it regulated by cytokines and growth factors. In the present study, we demonstrated TGF - βi can significantly stimulate mesangial cells secrete FN protein in vitro and this effect shows a dose - dependent and time - dependent way.TGF - β1 can inhibit mesangial cells proliferation. It has been reported high glucose can inhibit mesangial cells proliferation and induce overexpression of TGF - β1. Our study shows the inhibitor effect on mesangial cells proliferation of high glucose at least partly through upregulates TGF - β1. Recently, several reports considered matrix expansion in diabetic nephropathy is mainly because of matrix protein deposition instead of cell proliferation, our results support this view.TGF - β1 exerts its multiple biologic actions by variable signaling pathway network. Mitogen -activated protein kinase(MAPK) is an important TGF - β1 signaling pathway which can translocate TGF - β1 signaling from the cell membrane to the nucleus. It has four subfamilies. ERK is the most classic one. When use the PD98059, a inhibitor of ERK,the TGF - β1 - induced inhibitor effect of mesangial cells proliferation can be enhanced. It suggests ERK pathway may associate with cell proliferation. Terada et al reported in pig proximal tubular epidermal cell lines Cyclin D1 and A promoter activity and cell cycle progression is positively regulated by the ERK pathway and can be inhibited by PD98059. In mesangial cells whether ERK involved in above process need more research.
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