Connective tissue growth factor regulates the key events in tubular epithelial to myofibroblast transition in vitro

Connective tissue growth factor (CTGF) has been reported to play an important role in mediating the profibrotic effects of transforming growth factor-beta (TGF-beta) in various renal diseases. To elucidate the role of CTGF in renal tubular epithelialmyofibroblast transdifferentiation, we examined the expression of alpha-smooth muscle actin (alpha-SMA), vimentin, tenascin-C, and collagen IV expression upon the stimulation of CTGF in cultured human proximal tubular epithelia] cell line (HKC, and further investigated the effects of endogenous CTGF blockade on the transdifferentiation process induced by TGF-beta. It is revealed that upon the stimulation of recombinant human CTGF (rhCTGF, 2.5 or 5.0 mug/L), the expression of alpha-SMA and tenascin-C mRNA increased significantly (p < 0.01), while collagen IV gene expression decreased significantly (p < 0.01), all in a dose-dependent manner. The percentage of alpha-SMA-positive cells was significantly larger in the rhCTGF-stimulated groups than that in negative control (38.9%, 65.5% vs. 2.4%, respectively, p < 0.01) as confirmed by flow cytometry. Both cytoplasmic and secretory tenascin-C expression was upregulated by the stimulation of rhCTGF (p < 0.01). Under this condition, collagen IV secreted into the culture media was lowered markedly (p < 0.01). On RT-PCR analysis, TGF-β1 upregulated CTGF gene expression, preceding that of α-SMA. The a-SMA mRNA expression induced by TGF-β1 was significantly inhibited by CTGF antisense oligodeoxynucleotide (ODN) transfection (p < 0.01). With prolonged incubation time, CTGF antisense ODN also inhibited intracellular alpha-SMA protein synthesis, as demonstrated by indirect immuno-fluorescence. So it is concluded that CTGF could promote the transdifferentiation of human renal tubular epithelial cells towards myofibroblasts in vitro, both directly and as a downstream mediator of TGF-beta, and CTGF blockade would be a possible therapeutic target against tubulointerstitial fibrosis. (C) 2004 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.