EGF SIGNALING PATHWAYS & GENE EXPRESSION IN THE GH4 CELL

EGF 信号通路

• 批准号: 6155582
• 负责人: CHERYL A PICKETT
• 金额: $ 3.27万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6155582
• 关键词: HeLa cells; SDS polyacrylamide gel electrophoresis; biological signal transduction; cell differentiation; enzyme activity; enzyme inhibitors; epidermal growth factor; gel mobility shift assay; gene expression; genetic mapping; genetic promoter element; genetic regulation; genetic transcription; growth factor receptors; isozymes; nucleic acid sequence; phospholipase C; phosphorylation; pituitary gland; polymerase chain reaction; prolactin; protein kinase C; site directed mutagenesis; tissue /cell culture; transcription factor; western blottings

Epidermal growth factor (EGF) and its receptor (EGF-R) have a fundamental role in controlling cell growth, differentiation and neoplasia. The response of cells to EGF clearly involves multiple messengers (or "signal transducers") to conduct the initial signal at the membrane to the nuclear transcription machinery, resulting in regulation of specific gene expression. Although several signal transduction messengers and pathways are known to be altered by EGF treatment of cells, the precise functional role of these putative transducers and the specific which must transpire in the process of EGF regulation of gene transcription remain unclear. We have established a transient co-transfection model system in highly differentiated cultured GH4 rat pituitary cells whereby an EGF responsive prolactin promoter-luciferase construct (prPRLluc) provides a functional assay for the effects of EGF on lactotroph specific gene expression. This system also provides a model for EGF induced differentiation, in contrast to the mitogenic effects of EGF on other cell lines. In previous studies, I have discovered that EGF-mediated regulation of the rPRL promoter is Ras-independent and that EGF and Ras employ mutually antagonistic signal transduction pathways to regulate the prolactin gene promoter. Neither Raf and MAP kinase, nor Ets-1/GHF1 are required for the EGF response. Furthermore, the EGF response maps to the FP II region of the rPRL promoter, a region known to bind a repressor (F2F) of rPRL gene expression. These findings are novel and are likely to be important to our understanding of the mechanisms by which a ubiquitous growth factor/growth factor receptor is able to produce specific effects on cells of different function. The overall goal of this grant proposal is to define the mediators and pathway whereby EGF regulates PRL gene transcription in the GH4 pituitary cell. To achieve these goals, I propose four specific aims. First, I plan to precisely map the EGF response element using site-specific mutations and heterologous promoters. I also plan to identify and characterize the trans-activating factor responsible for EGF activation of the rPRL promoter, and examine the relationship of this factor to the previously identified F2F utilizing gel-shift assays, UV-crosslinking, Southwestern analysis. In addition, the role of protein kinase C (PKC) in mediating the EGF effect will be extensively explored. This will require the use of specific PKC inhibitors, the identification of specific EGF-induced PKC isozyme translocation, and subsequently the overexpression of these specific isozymes in the GH4 cell. Finally, I plan to explore the role of PLCgamma-1 in the EGF response. This will involve examining the effect of expression of both PLCgamma-1 inhibitory and wild-type constructs on the EGF response. In addition, PCR amplified and cloned SH2/SH3 domains of PLCgamma-1 will be utilized as inhibitors of the PLCgamma-1 component of the EGF signaling pathway. I will also employ immunoprecipitation and Western blotting to document EGF-induced recruitment of PLCgamma-1 to the EGF-R, and phosphorylation of PLCgamma-1. These studies should provide important insights into the mechanism of EGF-dependent gene regulation.

表皮生长因子（EGF）及其受体（EGF-R）具有基本 在控制细胞生长，分化和肿瘤中的作用。这 细胞对EGF的响应显然涉及多个Messenger（或“信号） 传感器”）以在膜上传导初始信号向核 转录机制，导致特定基因的调节 表达。尽管几个信号转导的使者和途径 已知通过EGF的细胞处理改变了精确的功能 这些推定的传感器的作用以及必须渗透的特定 在EGF调节基因转录的过程中，尚不清楚。我们 已经在高度的 分化的培养的GH4大鼠垂体细胞，EGF响应 催乳素启动子 - 卢西酶构建体（PRPRLLUC）提供了功能 EGF对乳营养特异性基因表达的影响的测定。这 相比之下，系统还为EGF诱导的分化提供了模型 EGF对其他细胞系的有丝分裂作用。在先前的研究中， 我发现RPRL启动子的EGF介导的调节是 与RAS无关，EGF和RAS采用相互拮抗的信号 调节催乳素基因启动子的转导途径。两位皇家空军 EGF响应需要MAP激酶，NOTS-1/GHF1。 此外，EGF响应映射到RPRL的FP II区域 启动子，一个已知结合RPRL基因阻遏物（F2F）的区域 表达。这些发现是新颖的，可能对我们很重要 了解无处不在的生长因子/生长的机制 因子受体能够对不同的细胞产生特定的影响 功能。 该赠款提案的总体目标是定义调解员和 EGF在GH4垂体中调节PRL基因转录的途径 细胞。为了实现这些目标，我提出了四个具体目标。首先，我计划 精确地绘制使用位点特异性突变的EGF响应元件 和异源启动子。我还计划识别和表征 负责RPRL EGF激活的反式激活因子 启动子，并检查该因素与先前的关系 确定了使用凝胶转移测定，紫外线链接，西南的F2F 分析。另外，蛋白激酶C（PKC）在介导 EGF效应将被广泛探索。这将需要使用 特定的PKC抑制剂，鉴定特定EGF诱导的PKC 同工酶易位，随后的过表达 GH4细胞中的特定同工酶。最后，我计划探索 EGF响应中的PLCGAMMA-1。这将涉及检查 Plcgamma-1抑制性和野生型构建体的表达 EGF响应。另外，PCR放大和克隆的SH2/SH3域 PLCGAMMA-1将被用作Plcgamma-1成分的抑制剂 EGF信号通路。我还将采用免疫沉淀和 Western印迹记录了EGF引起的Plcgamma-1招募 EGF-R和Plcgamma-1的磷酸化。这些研究应提供 对EGF依赖性基因调节机制的重要见解。