Adenovirus-mediated gene transfer of dominant-negative Smad4 blocks TGF-beta signaling in pancreatic acinar cells

Transforming growth factor-beta (TGF-beta) is a potent inhibitor of pancrea tic acinar cell growth. Smad4 is a central mediator in the TGF-beta signali ng pathway. To study the effect of Smad4 on pancreatic growth, cell cycle p rotein expression, and the expression of a TGF-beta -responsive promoter in vitro, we constructed an adenovirus containing dominant-negative COOH term inal truncated Smad4 (AddnSmad4) downstream of the rat elastase promoter. A cinar cells expressed dominant-negative Smad4 within 8 h after infection, a nd expression persisted for 72 h. Mouse pancreatic acini were infected with either AddnSmad4 or control adenovirus expressing green fluorescent protei n, and TGF-beta was added 8 h after infection. Acinar cells were then incub ated for 1, 2, or 3 days, and [H-3] thymidine incorporation was determined. AddnSmad4 significantly reduced TGF-beta inhibition of [H-3]thymidine inco rporation, with maximal effects on day 3. AddnSmad4 also completely blocked TGF-beta -mediated growth inhibition in the presence of basic fibroblast g rowth factor. We next examined the effects of AddnSmad4 on TGF-beta -induce d expression of the cell cycle regulatory proteins p21(Cip1) and p27(Kip1). TGF-beta induced upregulation of p21(Cip1), which was completely blocked b y AddnSmad4. AddnSmad4 also inhibited TGF-beta -induced expression of the T GF-beta -responsive luciferase reporter 3TP-Lux. These results show that Sm ad4 is essential in TGF-beta -mediated signaling in pancreatic acinar cells .