Transforming growth factor beta-1 enhances Smad transcriptional activity through activation of p8 gene expression

We report that exposure of mouse embryonic fibroblasts to transforming grow th factor beta -1 (TGF beta -1) (5 ng/ml) results in a strong activation of p8 mRNA expression that precedes the induction of cell growth. Involvement of the p8 promoter in the regulation was demonstrated by using a p8-chlora mphenicol acetyltransferase construct. We therefore speculated that p8 migh t be a mediator of TGF beta -1 in these cells. The incorporation of [H-3]th ymidine on treatment with TGF beta -1 was indeed significantly higher in p8 (+/+) fibroblasts than in p8(-/-) fibroblasts, Smad transcriptional activit y was used as marker of the TGF beta -1 signalling pathway, to probe the lo wer p8(-/-) response to TGF beta -1. Two Smad-binding elements (SBEs)-lucif erase constructs were transfected into p8(-/-) and p8(+/+) embryonic fibrob lasts before treatment with TGF beta -1. A lower level of Smad transactivat ion was observed in p8(-/-) embryonic fibroblasts, under basal conditions a nd after stimulation with TGF beta -1. To test whether Smad underexpression in p8(-/-) cells was actually due to p8 depletion, p8(-/-) embryonic fibro blasts were transfected with a human p8 expression plasmid together with an SBE-luciferase construct. The expression of p8 restored Smad transactivati on in unstimulated and TGF beta -1-treated cells to the level found in p8(- /+) cells. We concluded that TGF beta -1 activates p8 expression. which in turn enhances the Smad-transactivating function responsible for TGF beta -1 activity.