Basic fibroblast growth factor stimulates the release of preformed transforming growth factor beta 1 from human proximal tubular cells in the absence of de novo gene transcription or mRNA translation

Abstract

Interstitial fibrosis is significantly correlated with the progression of renal impairment for most causes of renal insufficiency. Transforming growth factor beta 1 (TGF-beta 1) and basic fibroblast growth factor (bFGF) are two of a group of profibrotic cytokines that have been associated with the development of renal interstitial fibrosis. We have previously demonstrated that alterations in D-glucose concentrations modulate the synthesis of TGF-beta 1 by human renal proximal tubular cells (HPTC) in vitro. The aim of the present study was to examine the influence of bFGF on TGF-beta 1 synthesis by HPTC in culture and to examine any modulation of this response by changes in ambient glucose concentration. Incubation of growth-arrested HPTC (72 hours in serum-free medium) with bFGF resulted in a dose-dependent increase in latent TGF-beta 1 secretion. Maximal release of TGF-beta 1 was seen at a bFGF dose of 50 ng/ml in cells incubated in 5 mM D-glucose (7.48 +/- 2.5 ng/ml, mean +/- SEM; n = 3; p = 0.04). This release of TGF-beta 1 in response to bFGF was unaffected by increasing the concentration of glucose in the culture media to 25 mM (7.76 +/- 1.3, mean +/- SEM; n = 3; p < 0.02). It was also unaffected by pretreatment of cells with either actinomycin-D or cycloheximide. TGF-beta 1 secretion was, however, inhibited in a dose-dependent manner by the exposure of cells to the microtubule-disrupting agent vinblastine, indicating that the generation of TGF-beta 1 was dependent on the secretion of preformed, stored TGF-beta 1. In a separate series of experiments, exposure of HPTC to TGF-beta 1 (10 ng/ml) led to the induction of bFGF mRNA, which was first apparent at 12 hours and reached maximal levels 24 hours after stimulation (normalized bFGF/alpha-actin mRNA ratio was 1.5 times that of the control). This increase in bFGF mRNA was accompanied by a time-dependent increase in bFGF protein production, which was maximal after 24 hours (19.83 +/- 12.7 pg/ml versus 2.49 +/- 0.34 pg/ml, mean +/- SEM, stimulated versus control; n = 3; p = 0.03). These findings demonstrate that bFGF stimulates the secretion of preformed, latent TGF-beta 1 by HPTC but does not induce de novo TGF-beta 1 gene transcription or TGF-beta 1 protein synthesis. We have also demonstrated a positive-feedback loop involving TGF-beta 1 and bFGF and postulate that this may be involved in the progressive nature of renal fibrosis in vivo.