TGF-Beta Auto-Induction and Connective Tissue Growth Factor Expression in Human Renal Tubule Epithelial Cells Requires N-Ras

Abstract

Background/Aims: Transforming growth factor (TGF) β is strongly implicated in the progression of renal fibrosis. TGFβ1 is reported to cause epithelial-mesenchymal transition, inhibition of epithelial cell proliferation, increased apoptosis, auto-induction of TGFβ production and induction of secondary mediators of tissue fibrosis such as connective tissue growth factor (CTGF, CCN2). The aims of this study were to investigate the role of the Ras/MAP kinase pathway in TGFβ1 inhibition of proliferation, TGFβ auto-induction and TGFβ1-induced CTGF expression in HKC human renal tubule epithelial cells. Methods and Results: TGFβ1 (0–25 ng/ml) inhibited proliferation of HKC cells and at 25 ng/ml also induced apoptosis. After 5–10 min of incubation, TGFβ1 increased cellular levels of phospho-ERK1/2 and phospho-AKT with a bell-shaped dose-response curve with a maximally effective concentration of 2.5 ng/ml. TGFβ3 caused an increase in extracellular TGFβ1, which was significantly reduced in the presence of PD 98059. TGFβ1 increased cellular and secreted CTGF protein in HKC cells in a MEK-dependent manner. To identify the Ras isoform involved, specific antisense oligonucleotides targeted to Ha-Ras, Ki-Ras and N-Ras were employed. Only inhibition of N-Ras resulted in a significant reduction of auto-induced TGFβ1 secretion and TGFβ1-induced cellular and secreted CTGF. Conclusion: These results establish that the Ras/MAP kinase pathway, specifically through N-Ras, mediates TGFβ1 auto-induction and TGFβ1-induced CTGF expression in human renal tubule epithelial cells.