FGF SIGNALING AND PITUITARY TUMORIGENESIS

FGF 信号传导和垂体肿瘤发生

• 批准号: 6177554
• 负责人: ANDREW P BRADFORD
• 金额: $ 18.75万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-14 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6177554
• 关键词: biological signal transduction; cell growth regulation; fibroblast growth factor; gel mobility shift assay; gene expression; genetic promoter element; growth factor receptors; mitogen activated protein kinase; molecular oncology; neoplasm /cancer genetics; neoplastic transformation; phospholipase C; phosphorylation; pituitary neoplasms; polymerase chain reaction; prolactin; protein kinase C; receptor expression; southern blotting; tissue /cell culture; transcription factor; transfection; western blottings

DESCRIPTION (Adapted from the applicant's abstract): In preliminary studies done during his fellowship, the Dr. Bradford demonstrated a role for FGF in regulation of the prolactin (PRL) promoter in GH4 pituitary cells. These preliminary studies indicate that FGF-2 and FFG-4 activate PRL promoter activity in a Ras and Raf independent manner, but involve MAP kinase and members of the Ets family of transcription factors. This regulation is specific and not observed for the growth hormone promoter in the same cells. Preliminary studies are also presented to suggest that this pathway is protein kinase C (PKC) dependent through the use of inhibitors and down-regulation of the enzyme. Based on these preliminary data, four specific aims are described: To map precisely the FGF response elements in the PRL promoter, the investigator will use a combination of conventional techniques. These will include mutagenesis with functional analysis of the PRL promoter using a luciferase assay, production of heterologous FRE promoter constructs which can be inserted upstream of a minimal viral promoter, insertion of PRL promoter element in the GH promoter which is normally not FGF responsive, and analysis of the nuclear factors binding to the FRE by both electrophoretic mobility shift assay and southwestern analysis. Analysis of FGF receptors and their responses in GH4 pituitary cells. FGF receptor expression will be determined by RTPCR. Receptor activation in tyrosine phosphorylation will be characterized, as will the mitogenic response to FGF 2 and FGF 4. The investigator will also determine whether there are changes in synthesis and secretion of prolactin in response to FGF treatment. Attempts will be made to identify and characterize the cytoplasmic components of the FGF signaling pathway. Here the investigator will focus on novel, non-Ras components which might account for the preliminary data. He will specifically investigate the possibility that B-Raf and Rap I are involve as well as phospholipase C and protein kinase C. Several other pathways will also be tested for their participation. Finally, the investigator will attempt to identify and characterize nuclear components of the FGF signal transduction pathway with a focus on the Ets transcription factors. These will be done through a series of transfections with dominant negative Ets and other potential interacting factors, through studies of the roles of GHF-1 and GHF-2 in the process and functional studies of the potential role of Jun kinase, nuclear MAP kinase and FGF induced phosphorylation of Ets factors.

描述（改编自申请人的摘要）：初步 布拉德福德博士在他的研究期间所做的研究证明了其作用 FGF 调节 GH4 垂体催乳素 (PRL) 启动子 细胞。这些初步研究表明 FGF-2 和 FFG-4 激活 PRL 启动子活性以 Ras 和 Raf 独立的方式进行，但涉及 MAP 激酶和转录因子 Ets 家族的成员。这 生长激素促进剂的调节是特异的并且没有观察到 相同的细胞。初步研究还表明 该途径依赖于蛋白激酶 C (PKC) 抑制剂和酶的下调。基于这些初步 根据数据，描述了四个具体目标： 精确绘制 FGF 反应 PRL 启动子中的元件，研究者将使用以下组合 常规技术。这些将包括具有功能性的诱变 使用荧光素酶测定法分析 PRL 启动子，产生 异源 FRE 启动子构建体可以插入到 最小病毒启动子，在 GH 中插入 PRL 启动子元件 通常对 FGF 不敏感的启动子，以及核分析 通过电泳迁移率变动测定与 FRE 结合的因子 和西南分析。 GH4 垂体细胞中 FGF 受体及其反应的分析。纤维生长因子 受体表达将通过RTPCR测定。受体激活 将表征酪氨酸磷酸化，以及促有丝分裂 对 FGF 2 和 FGF 4 的反应。研究者还将确定是否 催乳素的合成和分泌发生变化以响应 FGF治疗。 将尝试鉴定和表征细胞质 FGF 信号通路的组成部分。调查员将重点关注这里 关于可能解释初步数据的新颖的非 Ras 组件。 他会专门调查B-Raf和Rap I的可能性 还涉及磷脂酶 C 和蛋白激酶 C。其他几个 还将测试他们的参与途径。 最后，研究者将尝试识别并表征 FGF信号转导通路的核成分，重点是 Ets转录因子。这些将通过一系列的工作来完成 显性负 Et 和其他潜在相互作用的转染 通过研究GHF-1和GHF-2在此过程中的作用 Jun 激酶、核 MAP 的潜在作用和功能研究 激酶和 FGF 诱导 Ets 因子磷酸化。