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 布拉德福德博士在奖学金中进行的研究表现出了角色 用于FGF在GH4垂体中催乳素（PRL）启动子的调节中 细胞。这些初步研究表明FGF-2和FFG-4激活 PRL启动子活动以RAS和RAF独立方式，但涉及MAP 激酶和ETS转录因子家族的成员。这 调节是特定的，在生长激素启动子中未观察到 相同的细胞。还提出了初步研究，以表明 该途径是蛋白质激酶C（PKC）依赖于使用 酶的抑制剂和下调酶。基于这些初步 数据，描述了四个具体目的：精确映射FGF响应 PRL启动子中的元素，研究人员将使用 传统技术。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，生产 异源FRE启动子构建体，可以插入 最小病毒启动子，在GH中插入PRL启动子元件 通常不是FGF响应的启动子，核分析 两种电泳迁移率分析的因素与FRE结合 和西南分析。 FGF受体及其在GH4垂体细胞中的反应分析。 FGF 受体表达将由RTPCR确定。受体激活 酪氨酸磷酸化将被表征，有丝分裂也将 对FGF 2和FGF 4的响应。研究人员还将确定是否是否 催乳素的合成和分泌发生了变化 FGF治疗。 将尝试识别和表征细胞质 FGF信号通路的组件。在这里，调查员将集中精力 在新颖的非RAS组件上，可能解释了初步数据。 他将特别研究B-Raf和Rap I是 涉及以及磷脂酶C和蛋白激酶C.其他几个 途径还将进行参与测试。 最后，调查人员将尝试识别和表征 FGF信号转导途径的核成分，重点 ETS转录因子。这些将通过一系列 具有主要负ET和其他潜在相互作用的转染 通过研究GHF-1和GHF-2在此过程中的作用的因素 以及关于Jun激酶核图的潜在作用的功能研究 激酶和FGF诱导ETS因子的磷酸化。