Transforming growth factor-β inhibits pulmonary surfactant protein B gene transcription through SMAD3 interactions with NKX2.1 and HNF-3 transcription factors
Transforming growth factor-β inhibits pulmonary surfactant protein B gene transcription through SMAD3 interactions with NKX2.1 and HNF-3 transcription factors
复制标题
DOI：
10.1074/jbc.m203188200
复制
发表时间：
2002-10-11
影响因子：
4.8
通讯作者：
Minoo, P
中科院分区：
文献类型：
--
作者：
Li, CG;Zhu, NL;Minoo, P
转化生长因子-β（TGF-β）通过一种尚不清楚的机制抑制表面活性蛋白B（Sp-B）基因转录。同源结构域和叉头转录因子NKX2.1和HNF-3分别是已知的Sp-B转录激活因子。由于SMAD是TGF-β诱导的基因激活的效应物，我们研究了通过SMAD与NKX2.1和HNF-3的相互作用也可能发生TGF-β的基因抑制的可能性。我们发现肺上皮癌H441细胞含有SMAD 2/3和SMAD-4，其定位于细胞核以响应TGF-β处理。TGF-β以剂量依赖性方式降低转染的Sp-B启动子/报告基因构建体的活性。共转染突变体，组成型激活形式的TGF-β I型受体阻遏SP-B启动子活性的TGF-β配体的情况下。Smads的显性负突变体阻断了TGF-β的阻遏活性。SMAD 3而非SMAD 2介导TGF-β对Sp-B启动子的阻遏活性。包括NKX2.1、肝细胞核因子3（HNF-3）或cAMP反应元件结合蛋白（CREB）结合位点的70个碱基对结构域内的突变消除了SMAD 3依赖的Sp-B转录抑制。电泳迁移率变动分析表明，没有证据表明SMAD 3的Sp-B启动子的直接结合，和SMAD 3的DNA结合突变体也阻遏Sp-B，这表明SMAD 3的直接DNA结合可能是不需要的。使用哺乳动物双杂交测定，我们发现SMAD 3与NKX2.1和HNF-3之间的物理和功能相互作用。此外，谷胱甘肽S-转移酶融合SMAD 3直接结合到体外合成的NKX2.1或HNF-3，证明SMAD 3和两种转录因子之间的蛋白质-蛋白质相互作用。TGF-β处理后，NKX2.1与Sp-B启动子的DNA结合减少，但Nkx2.1的表达不受影响。我们的结论是SMAD 3与正调控因子NKX2.1和HNF-3的相互作用是TGF-β诱导的Sp-B基因转录抑制的分子基础。
Transforming growth factor-beta (TGF-beta) represses surfactant protein B (Sp-B) gene transcription through a mechanism that remains unknown. A homeodomain and a forkhead transcription factor, NKX2.1 and HNF-3, respectively, are known activators of Sp-B transcription. Because SMADs are the effectors of TGF-beta-induced gene activation, we examined the possibility that gene repression by TGF-beta may also occur through interactions of SMADs with NKX2.1 and HNF-3. We found that lung epithelial carcinoma H441 cells contain SMAD2/3 and -4, which localize to the nucleus in response to TGF-beta treatment. The activity of a transfected Sp-B promoter/reporter construct was reduced in a dose-dependent manner by TGF-beta. Cotransfection with a mutant, constitutively activated form of the Tgf-beta type I receptor repressed Sp-B promoter activity in the absence of TGF-beta ligand. Dominant negative mutants of Smads blocked the repressor activity of TGF-beta. SMAD3, but not SMAD2, mediated the repressor activity of TGF-beta on the Sp-B promoter. Mutations within a 70-base pair domain that includes binding sites for NKX2.1, hepatocyte nuclear factor 3 (HNF-3), or cAMP response element-binding protein (CREB) eliminated SMAD3-dependent repression of Sp-B transcription. Electrophoretic mobility shift analysis showed no evidence for direct binding of SMAD3 to the Sp-B promoter, and a DNA binding mutant of SMAD3 also repressed Sp-B, suggesting that direct DNA binding of SMAD3 may not be required. Using a mammalian two hybrid assay, we found physical and functional interactions between SMAD3 and both NKX2.1 and HNF-3. Also, a glutathione S-transferase-fused SMAD3 directly binds to in vitro synthesized NKX2.1 or HNF-3, demonstrating protein-protein interactions between SMAD3 and the two transcriptional factors. The DNA binding of NKX2.1 to Sp-B promoter was reduced in response to TGF-beta treatment, although expression of Nkx2.1 was not affected. We conclude that SMAD3 interactions with the positive regulators NKX2.1 and HNF-3 underlie the molecular basis for TGF-beta-induced repression of Sp-B gene transcription.