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 pancreatic acinar cell growth. Smad4 is a central mediator in the TGF-beta signaling pathway. To study the effect of Smad4 on pancreatic growth, cell cycle protein expression, and the expression of a TGF-beta-responsive promoter in vitro, we constructed an adenovirus containing dominant-negative COOH terminal truncated Smad4 (AddnSmad4) downstream of the rat elastase promoter. Acinar cells expressed dominant-negative Smad4 within 8 h after infection, and expression persisted for 72 h. Mouse pancreatic acini were infected with either AddnSmad4 or control adenovirus expressing green fluorescent protein, and TGF-beta was added 8 h after infection. Acinar cells were then incubated for 1, 2, or 3 days, and [(3)H]thymidine incorporation was determined. AddnSmad4 significantly reduced TGF-beta inhibition of [(3)H]thymidine incorporation, with maximal effects on day 3. AddnSmad4 also completely blocked TGF-beta-mediated growth inhibition in the presence of basic fibroblast growth factor. We next examined the effects of AddnSmad4 on TGF-beta-induced expression of the cell cycle regulatory proteins p21(Cip1) and p27(Kip1). TGF-beta induced upregulation of p21(Cip1), which was completely blocked by AddnSmad4. AddnSmad4 also inhibited TGF-beta-induced expression of the TGF-beta-responsive luciferase reporter 3TP-Lux. These results show that Smad4 is essential in TGF-beta-mediated signaling in pancreatic acinar cells.
Zhang
L
L
Department of Surgery, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
Graziano
K
K
Pham
T
T
Logsdon
C D
CD
Simeone
D M
DM
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000037:antagonists & inhibitors / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D018014:Gene Transfer Techniques; D005799:Genes, Dominant; D017930:Genes, Reporter / Q000502:physiology; D008156:Luciferases / Q000235:genetics; D008297:Male; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D010179:Pancreas / Q000166:cytology / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects*; D051901:Smad4 Protein; D013936:Thymidine / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000494:pharmacology*; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000494:pharmacology / Q000502:physiology*; D025521:Tumor Suppressor Proteins; D015854:Up-Regulation / Q000187:drug effects
C493957:Cdkn1a protein, mouse; C493961:Cdkn1b protein, mouse; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D008869:Microtubule-Associated Proteins; D051901:Smad4 Protein; C490810:Smad4 protein, mouse; C490811:Smad4 protein, rat; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D008156:Luciferases; D013936:Thymidine