Requirement of Smad3 and CREB-1 in mediating transforming growth factor-β (TGFβ) induction of TGFβ3 secretion
Requirement of Smad3 and CREB-1 in mediating transforming growth factor-β (TGFβ) induction of TGFβ3 secretion
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DOI：
10.1074/jbc.m600579200
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发表时间：
2006-10-06
影响因子：
4.8
通讯作者：
Mulder, Kathleen M.
中科院分区：
文献类型：
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作者：
Liu, Guangming;Ding, Wei;Mulder, Kathleen M.
由于肿瘤细胞转化生长因子-β（TGF-β）产生的增加通过旁分泌机制促进癌症进展，因此识别可以靶向阻断TGF-β产生的关键点非常重要。先前的研究已经鉴定了TGF β诱导上皮细胞中TGF β 1表达所需的精确信号传导组分和启动子元件（Yue，J.，Mulder，K. M.（2000）J.Biol.Chem.275，30765-30773）。为了确定TGF β 3表达的调节与TGF β 1的调节有何不同，我们确定了TGF β 3基因表达所需的精确信号通路和转录因子结合位点。通过电泳迁移率变动分析（EMSA）中的突变分析，我们证明了TGF β 3启动子中的cAMP反应元件（CRE）位点是TGF β诱导型TGF β 3表达所必需的。电泳迁移率超移分析表明，CRE结合蛋白1（CREB 1）和Smad 3的主要成分存在于这个TGF β诱导复合物。此外，通过使用染色质免疫沉淀分析，我们证明CREB-1、ATF-2和c-Jun组成性结合在TGF β 3启动子（-100至+1），而Smad 3仅在TGF β刺激后结合在该位点。此外，抑制JNK和p38抑制TGF β 3反式激活的TGF β诱导，而抑制ERK和蛋白激酶A没有影响。小干扰RNA-CREB 1和小干扰RNA-Smad 3显著抑制TGF β 3启动子报告基因活性和TGF β 3产生的TGF β刺激。我们的研究结果表明，TGF β 3启动子CRE位点的TGF β激活，导致TGF β 3的产生，是TGF β RII，JNK，p38和Smad 3所必需的，但不依赖于蛋白激酶A，ERK和Smad 4。
Because increased transforming growth factor-beta (TGF beta) production by tumor cells contributes to cancer progression through paracrine mechanisms, identification of critical points that can be targeted to block TGF beta production is important. Previous studies have identified the precise signaling components and promoter elements required for TGF beta induction of TGF beta 1 expression in epithelial cells (Yue, J., and Mulder, K. M. (2000) J. Biol. Chem. 275, 30765-30773). To determine how regulation of TGF beta 3 expression differs from that of TGF beta 1, we identified the precise signaling pathways and transcription factor-binding sites that are required for TGF beta 3 gene expression. By using mutational analysis in electrophoresis mobility shift assays (EMSAs), we demonstrated that the c-AMP-responsive element (CRE) site in the TGF beta 3 promoter was required for TGF beta-inducible TGF beta 3 expression. Electrophoresis mobility supershift assays indicated that CRE-binding protein 1 (CREB1) and Smad3 were the major components present in this TGF beta-inducible complex. Furthermore, by using chromatin immunoprecipitation assays, we demonstrated that CREB-1, ATF-2, and c-Jun bound constitutively at the TGF beta 3 promoter (-100 to +1), whereas Smad3 bound at this site only after TGF beta stimulation. In addition, inhibition of JNK and p38 suppressed TGF beta induction of TGF beta 3 transactivation, whereas inhibition of ERK and protein kinase A had no effect. Small interfering RNA-CREB1 and small interfering RNA-Smad3 significantly inhibited TGF beta stimulation of TGF beta 3 promoter reporter activity and TGF beta 3 production. Our results indicate that TGF beta activation of the TGF beta 3 promoter CRE site, which leads to TGF beta 3 production, is required for TGF beta RII, JNK, p38, and Smad3 but was independent of protein kinase A, ERK, and Smad4.