TGF-ßs stimulate cell proliferation via an autocrine production of FGF-2 in corneal stromal fibroblasts

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Abstract
PURPOSE. Although transforming growth factor-ßs (TGF-ßs) inhibit epithelial cell proliferation, these same substances stimulate cell proliferation of fibroblasts. In order to elucidate the mechanism of stimulatory activity of TGF-ß on fibroblast, the present study was performed to determine whether TGF-ß might be an indirect mitogen acting through induction of an endogenous growth factor(s) that then acts as the direct mitogen in an autocrine manner in corneal stromal fibroblasts (CSFs). METHODS. Cell proliferation was determined either by counting cell numbers or by analyzing the incorporation of [ 3 H]-thymidine into DNA. The synthesis of TGF-ß, TGF-ß receptors, FGF-2 and p27 was analyzed by immunoprecipitation and immunoblotting. RESULTS. TGF-ß1, TGF-ß2, and TGF-ß3 significantly stimulated cell proliferation of CSFs in a dose-dependent manner. The medium conditioned by CSFs and subsequently activated by acid-inhibited cell proliferation of corneal endothelial cells by 40%. When the acid-activated media conditioned by CSFs were immunoprecipitated with either combined anti-TGF-ß1 and TGF-ß2 antibodies or anti-TGF-ß3 antibody, all three TGF-ßs, with an apparent molecular size of 25 kDa, were detected, whereas CSFs produced an 80-kDa latent form of TGF-ß1. These cells also express TGF-ß type II receptor and betaglycan. Interestingly, CSFs produced and secreted 18-kDa FGF-2, the synthesis of which is further stimulated by either TGF-ß1 or TGF-ß3, while both the neutralizing antibody to FGF-2 and the FGF-2 specific antisense oligonucleotide primers significantly inhibited the stimulatory activities of TGF-ß1 in CSFs. The expression of p27, a negative regulator in cell cycle, was not altered by TGF-ß. CONCLUSIONS. These findings indicate that CSFs produce both TGF-ßs and FGF-2 and that FGF-2 appears to be a direct stimulator for TGF-ß-mediated cell proliferation in CSFs.