TGF-beta 1 induces proliferation in human renal fibroblasts via induction of basic fibroblast growth factor (FGF-2)

Background. The prognosis of primary renal disease is often dependent on th e degree of tubulointerstitial scarring. Scarring is caused by proliferatio n and excessive matrix production of renal fibroblasts and possibly other c ellular elements. Transforming growth factor-beta (TGF-beta) is the most im portant cytokine for the induction of matrix synthesis in the kidney. How e ver, its effects on renal fibroblast proliferation have not been determined . We have recently demonstrated that the expression of basic fibroblast gro wth factor (FGF-2) is robustly upregulated in human kidneys with tubulointe rstitial fibrosis and that FGF-2 is a potent inducer of fibroblast prolifer ation. The present study examined the interaction between TGF-beta1 and FGF -2 in human renal fibroblasts. Methods. Experiments were performed on a transformed medullary fibroblast l ine and on primary cortical kidney and skin fibroblasts isolated from human biopsies. mRNA levels of FGF-2 and TGF-beta1 were analyzed by Northern blo t analyses. Changes in protein expression were examined by immunoblots and enzyme-linked immunosorbent assay (ELISA). Bromodeoxyuridine incorporation assays and cell counts were used to analyze cell proliferation. The express ion of cell cycle-regulatory proteins cyclin-dependent kinase (cdk) 2 and t he cdk inhibitor p27(kipl) Were determined by immunoblots. Results. Stimulation of renal fibroblasts with FGF-2 resulted in no change of TGF-beta1 mRNA expression, whereas incubation of the cells with TGF-beta 1 induced FGF-2 mRNA up to 3.51 +/- 0.21-fold after six hours. This increas e could be blocked almost completely by the addition of cyclohexamide, indi cating that the process is in large part dependent on protein synthesis. Th e up-regulation in FGF-2 mRNA expression was paralleled by de novo detectio n of FGF-2 protein in the supernatant. peaking after 12 to 24 hours, as det ermined by Western blot and ELISA, whereas cellular protein was only increa sed up to 2.1-fold. Interestingly, both methods detected re lease of FGF-2 protein to the supernatant already at three hours, indicating a role for TG F-beta1 in directly releasing preformed FGF-2. Since TGF-beta1 induced FGF- 2, which results in fibroblast proliferation, we hypothesized that TGF-beta 1 may cause fibroblast proliferation mediated by FGF-2. This hypothesis was verified by cell proliferation assays demonstrating that stimulation of re nal fibroblasts with TGF-beta1 resulted in an up to 3.21 +/- 0.28-fold incr ease in bromodeoxyuridine incorporation and a 1.95 +/- 0.16-fold increase i n cell number after 72 hours. This mitogenic effect of TGF-beta1 could he b locked completely by the addition of a neutralizing antibody to FGF-2 or th e tyrosine kinase inhibitor tyrphostin AG1296, which blocks FGF receptor (F GFR) tyrosine kinase activity. Conversely, a neutralizing antibody to epide rmal growth factor (EGF) or the tyrphostin B42. which inhibits EGF receptor signal transduction, had no effect. interestingly, a neutralizing antibody to PDGF had only minor effects in primary kidney fibroblasts but reduced T GF-beta1-induced proliferation considerably in primary skin fibroblasts. Fi nally. TGF-beta1-induced proliferation in kidney fibroblasts was paralleled by a robust increase in cdk 2 protein expression up to 72 hours, whereas p 27(kipl), whose activity is maintained by TGF-beta in epithelial cells. was down-regulated up To 48 hours. Conclusions. Our studies demonstrate. to our knowledge for the first time. that TGF-beta1 induces proliferation in human renal fibroblasts and that th is process is mediated largely by FGF-2. The induction of proliferation by TGF-beta1 via induction of FGF-2 may play an important role in the autonomy of renal fibroblast growth and thus in the pathogenesis of human fibrogene sis.