Involvement of Smads in TGFbeta1-induced furin (fur) transcription.
Involvement of Smads in TGFbeta1-induced furin (fur) transcription.
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DOI：
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发表时间：
2001
影响因子：
5.6
通讯作者：
F. Blanchette;P. Rudd;F. Grondin;L. Attisano;C. Dubois
中科院分区：
文献类型：
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作者：
F. Blanchette;P. Rudd;F. Grondin;L. Attisano;C. Dubois
Furin被认为是细胞组成分泌途径的主要转化酶之一，但调控其表达的机制尚不清楚。我们之前已经证明TGFbeta1上调其自身的转换酶furin，在各种生理和病理生理条件下创造了一个潜在重要的新激活/调节循环。fur （fes上游区域）基因通过三个可选启动子调控；P1 P1A和P1B。为了深入了解这种上调的分子机制，我们对P1、P1A和P1B启动子荧光素酶进行了短暂的细胞转染。转染HepG2细胞的实验表明，P1启动子对TGFbeta1 （5 ng/ml）的刺激最强，最敏感（3.2倍，P1A 2.4倍，P1B 2.1倍）。Smad2显性阴性突变体[Smad2(3SA)]或已知的Smad抑制剂[Smad7]共转染可抑制组成型和tgfbeta1诱导的荧光素酶活性，表明内源性Smad的参与。tgfbeta1通过过表达Smad2和/或Smad4诱导P1启动子转录激活的水平升高，在Smad2（3SA）存在时大大降低，并被Smad7完全抑制，提示内源性Smad2/Smad4复合物参与。此外，已知与Smad2/Smad4复合物相互作用的叉头激活素信号换能器（FAST-1）是tgfbeta1诱导的fur P1启动子转激活的有效刺激物。该启动子的5′缺失分析确定了近端区域（-8734和-7925之间）是携带Smad2对P1启动子的大部分转录激活的核苷酸延伸。总的来说，目前的数据表明Smad2和Smad4可能与FAST-1或其他DNA结合伙伴复合物参与了P1启动子的组成性和可诱导的转激活。这是对皮毛基因转录调控的首次详细研究。
Furin is recognized as being one of the main convertases of the cellular constitutive secretion pathway but the mechanisms regulating its expression are still unknown. We have previously demonstrated that TGFbeta1 up-regulates its own converting enzyme, furin, creating a novel activation/regulation cycle of potential importance in a variety of physiological and pathophysiological conditions. The fur (fes upstream region) gene is regulated via three alternative promoters; P1, P1A, and P1B. To gain insight into the molecular mechanism(s) underlying this up-regulation, we performed transient cell transfections with P1, P1A, and P1B promoter luciferase constructs. Transfection experiments in HepG2 cells revealed that fur P1 promoter is the strongest and the most sensitive to TGFbeta1 stimulation (5 ng/ml) (3.2-fold vs. 2.4-fold for P1A and 2.1-fold for P1B). Cotransfection with either a dominant negative mutant form of Smad2 [Smad2(3SA)] or a known Smad inhibitor [Smad7] inhibit constitutive and TGFbeta1-induced luciferase activity indicating the participation of endogenous Smads. Increased levels of TGFbeta1-induced transcriptional activation of the P1 promoter by overexpression of Smad2 and/or Smad4 is greatly reduced in the presence of Smad2(3SA) and completely inhibited by Smad7, suggesting the participation of endogenous Smad2/Smad4 complexes. Furthermore, the fork-head activin signal transducer (FAST-1), known to interact with Smad2/Smad4 complexes, is a potent stimulator of TGFbeta1-induced transactivation of the fur P1 promoter. Five prime-deletion analysis of this promoter identified the proximal region (between positions -8734 and -7925), as the nucleotide stretch that carries most of the transcriptional activation of fur P1 promoter by Smad2. Overall, the present data demonstrate that Smad2 and Smad4 possibly in complex with FAST-1 or other DNA binding partners participate in the constitutive and inducible transactivation of the fur P1 promoter. This represents the first detailed study of the transcriptional regulation of the fur gene.