Smad3 interacts with JunB and Cbfa1/Runx2 for transforming growth factor-β-stimulated collagenase-3 expression in human breast cancer cells
Smad3 interacts with JunB and Cbfa1/Runx2 for transforming growth factor-β-stimulated collagenase-3 expression in human breast cancer cells
复制标题
DOI：
10.1074/jbc.m312870200
复制
发表时间：
2004-06-25
影响因子：
4.8
通讯作者：
Partridge, NC
中科院分区：
文献类型：
--
作者：
Selvamurugan, N;Kwok, S;Partridge, NC
我们之前已经证明，转化生长因子 (TGF)-β1（转移性骨癌的关键分子）可刺激人乳腺癌细胞系 MDA-MB231 中胶原酶 3 的表达。为了了解 TGF-β1 对胶原酶 3 启动子活性作出反应的分子机制，对胶原酶 3 基因的启动子区域进行了功能分析，我们鉴定了远端 runt 结构域 (RD) 和近端 RD/激活蛋白 1 (AP-1) 位点，这是完全 TGF-β1 刺激的胶原酶 3 启动子活性所必需的。凝胶位移、实时逆转录酶 PCR 和蛋白质印迹分析显示，MDA-MB231 细胞中经 TGF-β1 处理后，c-Jun、JunB 和 Cbfa1/Runx2 水平增加。体外免疫共沉淀研究发现 JunB 和 Cbfa1/Runx2 之间没有物理相互作用，而 Smad3 与两者都有相互作用。染色质免疫沉淀实验证实了 Smad3 与 JunB 和 Cbfa1/Runx2 的相互作用。在基础条件下，Cbfa1/Runx2 与近端 RD/AP-1 和远端 RD 位点结合。响应 TGF-β1，Cbfa1/Runx2 仅出现在远端 RD 位点，而 JunB 占据近端 RD/AP-1 位点。在 MDA-MB231 细胞中，胶原酶 3 启动子远端 RD 位点出现 Smad3、JunB 和 Cbfa1/Runx2 的组合，以响应 TGF-β1。 Smad3、JunB 和 Cbfa1/Runx2 构建体与组成型活性 TGF-β I 型受体构建体的共转染确定了这些蛋白质的功能相互作用以及 TGF-β1 对胶原酶 3 基因的转录激活。综上所述，我们的结果表明，TGF-β1 刺激 JunB 和 Cbfa1/Runx2 结合到各自的 DNA 共有位点，并且 Smad3 可能稳定它们的相互作用，从而赋予 MDA-MB231 细胞中胶原酶 3 表达的功能性 TGF-β1 刺激。
We have previously shown that transforming growth factor (TGF)-beta1, a crucial molecule in metastatic bone cancer, stimulates collagenase-3 expression in the human breast cancer cell line, MDA-MB231. To understand the molecular mechanisms responsible for TGF-beta1 response on collagenase-3 promoter activity, a functional analysis of the promoter region of the collagenase-3 gene was carried out, and we identified the distal runt domain (RD) and proximal RD/activator protein-1 (AP-1) sites as necessary for full TGF-beta1-stimulated collagenase-3 promoter activity. Gel shift, real time reverse transcriptase-PCR, and Western blot analyses showed increased levels of c-Jun, JunB, and Cbfa1/Runx2 upon TGF-beta1 treatment in MDA-MB231 cells. Co-immunoprecipitation in vitro studies identified no physical interaction between JunB and Cbfa1/Runx2, whereas Smad3 interacted with both. Chromatin immunoprecipitation experiments confirmed interaction of Smad3 with JunB and Cbfa1/Runx2. Under basal conditions, Cbfa1/Runx2 bound to both the proximal RD/AP-1 and distal RD sites. In response to TGF-beta1, Cbfa1/Runx2 was seen only at the distal RD site, whereas JunB occupied the proximal RD/ AP-1 site. An assemblage of Smad3, JunB, and Cbfa1/ Runx2 at the distal RD site of the collagenase-3 promoter occurred in response to TGF-beta1 in MDA-MB231 cells. Co-transfection of Smad3, JunB, and Cbfa1/ Runx2 constructs along with a constitutively active TGF-beta type I receptor construct identified functional interaction of these proteins and transcriptional activation of the collagenase-3 gene by TGF-beta1. Taken together, our results suggest that TGF-beta1 stimulated JunB and Cbfa1/ Runx2 to bind to their respective DNA consensus sites and that Smad3 is likely to stabilize their interaction to confer functional TGF-beta1-stimulation of collagenase-3 expression in MDA-MB231 cells.