A transcriptional factor induced by HGF

生长因子通过刺激细胞周期的G1期进程来诱导肝细胞的增殖。诱导细胞周期蛋白D1的表达是生长因子促有丝分裂作用的重要特征。最近，我们报道了肝细胞生长因子(HGF)和表皮生长因子(EGF)相加地刺激RAS/MAPK通路，导致原代培养的大鼠肝细胞周期蛋白D1表达相加增强。在本研究中，我们分离了大鼠细胞周期蛋白D1基因5‘侧翼序列，并利用dRLh84大鼠肝癌细胞进行瞬时转染研究，鉴定了两个转录调控区。其中一个(CD1CRE)定位于潜在的cAMP反应元件(Cre)，位于起始点的-41bp处，另一个(CD1E0.7)位于起始点上游753bp处。凝胶迁移率改变分析(EMSA)结果显示CD1E0.7与肝细胞核因子-3β、活化T细胞核因子(NF-AT)或Ets家族无相互作用，但经计算机检索发现CD1E0.7与这些核因子的结合部位有很高的同源性。此外，还检测了CD1E0.7与经HGF或EGF处理的原代培养大鼠肝细胞中提取的蛋白的特异性相互作用。这些结果表明，CREB与CD1CRE结合并介导大鼠肝癌细胞周期蛋白D1启动子的激活，提示CD1E0.7可能被大鼠肝细胞生长因子诱导的转录因子占据。

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.