Estradiol reverses TGF-beta1-stimulated type IV collagen gene transcription in murine mesangial cells.

We have previously shown that estradiol suppresses types I and IV collagen synthesis by mesangial cells grown in the presence of serum. In the present study, we examined the interaction between estradiol and transforming growth factor-beta (TGF-beta) on collagen IV synthesis. In a luciferase reporter gene construct containing the type IV collagen promoter and 1-chain regulatory sequences, we found that TGF-beta1 (2 ng/ml) stimulated alpha1-collagen IV gene transcription in serum-free media (140.5 +/- 6.2 relative luciferase units, expressed as a percent of control untreated cells, P < 0.001). Estradiol reversed the stimulatory effects of TGF-beta1 on reporter gene transcription in a dose-dependent manner [for 2.5 x 10(-9) M, 114.2 +/- 0.2, P < 0.002 vs. TGF-beta1; for 10(-7) M, 89.5 +/- 4.0, P < 0.001 vs. TGF-beta1 and P = not significant (NS) vs. control]. Using immunoprecipitation techniques, we found that estradiol (10(-7) M) reversed TGF-beta1-stimulated type IV collagen synthesis (175.3 +/- 14.7 vs. 111.6 +/- 7.1, expressed as a percent of control untreated cells, P < 0.001) but did not affect TGF-beta1-stimulated type I collagen synthesis (166.9 +/- 18.8 vs. 162.2 +/- 16.2, P = NS). These results were confirmed with Western blotting. Nuclear extracts from mesangial cells treated with TGF-beta1 showed increased binding to a Sp1 consensus binding sequence oligonucleotide and to an Sp1 binding site in the collagen IV promoter. Estradiol reversed this enhanced binding. These data suggest that estradiol antagonizes TGF-beta1-stimulated type IV collagen synthesis at a transcriptional level and that this effect may be mediated by interactions with the transcription factor Sp1.