Selvamurugan Nagarajan, Kwok Sukyee, Partridge Nicola C
Department of Physiology and Biophysics, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
J Biol Chem. 2004 Jun 25;279(26):27764-73. doi: 10.1074/jbc.M312870200. Epub 2004 Apr 14.
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.
我们之前已经表明，转化生长因子（TGF）-β1是转移性骨癌中的关键分子，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启动子活性产生反应的分子机制，我们对胶原酶-3基因的启动子区域进行了功能分析，并且我们确定了远端 runt 结构域（RD）和近端RD/激活蛋白-1（AP-1）位点对于TGF-β1充分刺激胶原酶-3启动子活性是必需的。凝胶迁移、实时逆转录酶-PCR和蛋白质印迹分析表明，在MDA-MB231细胞中，TGF-β1处理后c-Jun、JunB和Cbfa1/Runx2的水平升高。体外共免疫沉淀研究未发现JunB与Cbfa1/Runx2之间存在物理相互作用，而Smad3与二者均有相互作用。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位点均结合。对TGF-β1产生反应时，仅在远端RD位点观察到Cbfa1/Runx2，而JunB占据近端RD/AP-1位点。在MDA-MB231细胞中，对TGF-β1产生反应时，Smad3、JunB和Cbfa1/Runx2在胶原酶-3启动子的远端RD位点组装。将Smad3、JunB和Cbfa1/Runx2构建体与组成型活性TGF-β I型受体构建体共转染，确定了这些蛋白质之间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与其各自的DNA共有位点结合，并且Smad3可能稳定它们的相互作用，从而赋予TGF-β1对MDA-MB231细胞中胶原酶-3表达的功能性刺激。
