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A transcriptional factor induced by HGF

HGF 诱导的转录因子

基本信息

批准号：
10470138
负责人：
TSUBOUCHI Hirohito
金额：
$ 7.68万
依托单位：
Miyazaki Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B).
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

Growth factors induce cell proliferation in hepatocytes by stimulating progression through the G1 phase of the cell cycle. Induction of cyclin D1 expression is a critical feature of the mitogenic action of growth factors. Recently, we have reported that hepatocyte growth factor (HGF) and epidermal growth factor (EGF) additively stimulate a Ras/MAPK pathway, resulting in additive enhancement of cyclin D1 expression in primary cultured rat hepatocytes. In the present study, we isolated the rat cyclin D1 gene 5'-flanking sequence and identified two transcriptional regulatory regions by transient transfection studies using dRLh84 rat hepatoma cells. One (CD1CRE) was mapped to a potential cAMP-responsive element (CRE) at position -41 bp, while another (CD1E0.7) was located 753 bp upstream of the initiation site. Electrophoretic gel mobility shift assays (EMSAs) indicated that a protein binding to the CD1CRE was a CREB.In contrast, competition EMSA showed that CD1E0.7 did not interact with hepatocyte nuclear factor (HNF)-3β, nuclear factor of activated T cells (NF-AT), or the Ets family, although CD1E0.7 revealed high homology with a binding site for these nuclear factors through a computer search. Moreover, the specific interaction of CD1E0.7 with a protein extracted from primary cultured rat hepatocytes treated with HGF or EGF was detected. These results indicate that CREB binds to CD1CRE and mediates the activation of the cyclin D1 promoter in rat hepatoma cells, and suggest that CD1E0.7 is possibly occupied by a transcription factor induced by growth factors in rat hepatocytes.

生长因子通过刺激细胞周期的G1期进展来诱导肝细胞中的细胞增殖。细胞周期蛋白D1表达的诱导是生长因子促有丝分裂作用的关键特征。最近，我们报道了肝细胞生长因子（HGF）和表皮生长因子（EGF）加性刺激Ras/MAPK通路，导致在原代培养的大鼠肝细胞中细胞周期蛋白D1的表达的加性增强。在本研究中，我们分离的大鼠细胞周期蛋白D1基因5 '侧翼序列，并确定了两个转录调控区的瞬时转染研究使用dRLh 84大鼠肝癌细胞。其中一个（CD 1CRE）定位于一个潜在的cAMP反应元件（CRE）的-41 bp处，而另一个（CD1E0.7）定位于起始位点上游753 bp处。电泳凝胶迁移率变动分析（EMSA）显示，CD 1CRE结合的蛋白是CREB。相反，竞争EMSA显示，CD1E0.7不与肝细胞核因子（HNF）-3β、活化T细胞核因子（NF-AT）或Ets家族相互作用，但通过计算机检索发现，CD1E0.7与这些核因子的结合位点高度同源。此外，检测到CD1E0.7与从用HGF或EGF处理的原代培养的大鼠肝细胞中提取的蛋白质的特异性相互作用。这些结果表明CREB与CD 1CRE结合并介导大鼠肝癌细胞中细胞周期蛋白D1启动子的激活，并表明CD 1E0.7可能被大鼠肝细胞中生长因子诱导的转录因子占据。