ESTRADIOL REVERSES TGF-BETA-1-STIMULATED TYPE-IV COLLAGEN GENE-TRANSCRIPTION IN MURINE MESANGIAL CELLS

We have previously shown that estradiol suppresses types I and IV coll agen synthesis by mesangial cells grown in the presence of serum. In t he present study, we examined the interaction between estradiol and tr ansforming growth factor-beta (TGF-beta) on collagen IV synthesis. In a luciferase reporter gene construct containing the type IV collagen p romoter and alpha(1)-chain regulatory sequences, we found that TGF-bet a 1 (2 ng/ml) stimulated alpha(1)-collagen IV gene transcription in se rum-free media (140.5 +/- 6.2 relative luciferase units, expressed as a percent of control untreated cells, P < 0.001). Estradiol reversed t he stimulatory effects of TGF-beta 1 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-beta 1; for 10(-7) M, 89.5 +/- 4.0, P < 0.001 vs. TGF-beta 1 and P = not significant (NS) vs, control]. Using immunoprecipitation t echniques, we found that estradiol (10(-7) M) reversed TGF-beta 1-stim ulated type IV collagen synthesis (175.3 +/- 14.7 vs. 111.6 +/- 7.1, e xpressed as a percent of control untreated cells, P < 0.001) but did n ot affect TGF-pl-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-beta 1 showed increased binding to a Sp1 consensus binding sequence oligon ucleotide and to an Sp1 binding site in the collagen IV promoter. Estr adiol reversed this enhanced binding. These data suggest that estradio l antagonizes TGF-beta 1-stimulated type IV collagen synthesis at a tr anscriptional level and that this effect may be mediated by interactio ns with the transcription factor Sp1.