Adenovirus-mediated gene transfer of dominant-negative Smad4 blocks TGF-beta signaling in pancreatic acinar cells.
Adenovirus-mediated gene transfer of dominant-negative Smad4 blocks TGF-beta signaling in pancreatic acinar cells.
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腺病毒介导的显性失活 Smad4 基因转移可阻断胰腺腺泡细胞中的 TGF-β 信号传导。
DOI：
10.1152/ajpgi.2001.280.6.g1247
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Simeone,DM
中科院分区：
文献类型：
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作者：
Zhang,L;Graziano,K;Pham,T;Logsdon,CD;Simeone,DM
转化生长因子-β(转化生长因子-β)是一种有效的胰腺腺泡细胞生长抑制因子。Smad4是转化生长因子-β信号通路中的中心调节因子。为了研究Smad4在体外对胰腺生长、细胞周期蛋白表达和转化生长因子-β反应启动子表达的影响，我们构建了在大鼠弹性蛋白酶启动子下游含有显性负端截短Smad4的腺病毒。腺泡细胞在感染后8h内表达显性阴性Smad4，并持续表达72h。小鼠胰腺腺泡细胞感染表达绿色荧光蛋白的腺病毒AddnSmad4或对照组，感染后8h加入转化生长因子β。然后将腺泡细胞孵育1、2或3天，测定[3 H]胸腺嘧啶核苷掺入。AddnSmad4可显著降低转化生长因子β对[3 H]胸腺嘧啶核苷掺入的抑制作用，以第3天作用最强。AddnSmad4还可完全阻断碱性成纤维细胞生长因子对转化生长因子β介导的生长抑制作用。接下来，我们研究了AddnSmad4对转化生长因子-β诱导的细胞周期调控蛋白p21Cip1和p27Kip1表达的影响。转化生长因子-β诱导的p21Cip1表达上调可被AddnSmad4完全阻断。AddnSmad4还抑制转化生长因子-β诱导的转化生长因子-β反应荧光素酶报告基因3TP-Lux的表达。这些结果表明，Smad4在转化生长因子-β介导的胰腺腺泡细胞信号转导中是必不可少的。
Transforming growth factor-β (TGF-β) is a potent inhibitor of pancreatic acinar cell growth. Smad4 is a central mediator in the TGF-β signaling pathway. To study the effect of Smad4 on pancreatic growth, cell cycle protein expression, and the expression of a TGF-β-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-β was added 8 h after infection. Acinar cells were then incubated for 1, 2, or 3 days, and [3H]thymidine incorporation was determined. AddnSmad4 significantly reduced TGF-β inhibition of [3H]thymidine incorporation, with maximal effects onday 3. AddnSmad4 also completely blocked TGF-β-mediated growth inhibition in the presence of basic fibroblast growth factor. We next examined the effects of AddnSmad4 on TGF-β-induced expression of the cell cycle regulatory proteins p21Cip1and p27Kip1. TGF-β induced upregulation of p21Cip1, which was completely blocked by AddnSmad4. AddnSmad4 also inhibited TGF-β-induced expression of the TGF-β-responsive luciferase reporter 3TP-Lux. These results show that Smad4 is essential in TGF-β-mediated signaling in pancreatic acinar cells.