Translational regulation of renal proximal tubular epithelial cell transforming growth factor-beta 1 generation by insulin

We have previously demonstrated that the proximal tubular cell may contribu te to the pathogenesis of renal interstitial fibrosis in diabetes. Transfor ming growth factor (TGF)-beta1 is one of a group of pro-fibrotic cytokines and growth factors, which have been associated with the development of inte rstitial fibrosis. The aim of the current study was to examine the effect o f insulin on the generation of TGF-beta1. by proximal tubular cells. HK-2 c ells were grown to confluence in the absence of insulin, and serum deprived for 48 hours before all experimental manipulations. Addition of insulin (5 mug/ml) to the culture medium led to a time-dependent increase in TGF-beta 1 concentration in the cell culture supernatant, and increased incorporatio n of radiolabeled amino acids into TGF-beta1 suggestive of de novo TGF-beta 1 protein synthesis. Addition of insulin did not alter TGF-beta1 mRNA expre ssion as assessed by reverse transcriptase-polymerase chain reaction or Nor thern analysis. insulin-induced increase in TGF-beta1 concentration was not abrogated by actinomycin D, however, stimulation by insulin, in the presen ce of cycloheximide led to a dose-dependent decrease in TGF-beta1 productio n. Addition of insulin had no effect on TGF-beta1 mRNA stability as assesse d by actinomycin D chase, but led to increased binding of a cytoplasmic pro tein to a putative stem loop structure in the 5'-UTR of TGF-beta1 mRNA, pre viously implicated in the posttranscriptional control of TGF-beta1 synthesi s. To address the functional significance of insulin-induced alteration in TGF-beta1 synthesis, we examined its effect on matrix turnover. Insulin sti mulated type IV collagen gene expression and an increase in the concentrati ons of the type IV collagen laid down in the extracellular matrix. This inc rease in type IV collagen was abrogated when cells were stimulated by insul in in the presence of an anti-TGF-beta1-blocking antibody. in conclusion th e data demonstrate that insulin may directly alter the production of TGF-be ta1 by renal proximal tubular cells by a posttranscriptional mechanism, and that this may have implications for the increase in extracellular matrix t hat accompanies diabetic nephropathy.