Role of integrins and activin in granulosa cell growth

描述（由申请人提供）：在卵泡发育的早期阶段，调节颗粒细胞生长的因素是未知的。初步研究表明，细胞外基质（extracellular matrix， ECM）与激活素- a协同作用于早期卵泡的生长，并提示整合素-和激活素/TFG -信号通路之间存在相互作用。阐明早期卵泡颗粒细胞生长的分子机制可能有助于制定预防卵巢早衰、治疗年龄相关性不孕症、延缓更年期和预防癌症药物引起的性腺损伤的治疗策略。申请人在完成生殖内分泌学临床培训后，主要研究卵泡早期发育的调控机制。在英国与国际公认的生殖生物学家进行了一年的研究培训后，候选人开始与该项目的拟议导师合作研究卵巢卵泡生长中的整合素信号。申请人和导师所在的机构是一个在研究和顶级研究资源方面拥有全球认可的三机构联合企业。导师是美国国立卫生研究院资助的世界公认的整合素信号专家。申请人的短期目标是加强在细胞和分子生物学知识的基础，成为有效的技术研究细胞信号和产生初步数据。候选人的长期目标是成为一名独立的临床科学家，专注于将早期卵泡生长的基础研究转化为临床应用。为了实现这些目标，申请人提出了一个分阶段的发展计划，其中教学培训将在前三年完成，同时进行Specific Aim-1的实验。第四年和第五年将重点放在第二和第三个目标的实验上。本研究的具体目的是研究和描述自发永生化（SIGC）和原代颗粒细胞中ECM和激活素- a相互作用的机制，并确定阻断这种相互作用是否可以调节颗粒细胞的增殖。为了实现这些特定的目标，SIGC将在存在或不存在激活素a的情况下在聚赖氨酸、纤维连接蛋白、层粘连蛋白或胶原蛋白上培养。通过Western blotting、体内磷酸化、激酶测定和免疫荧光等方法，研究整合素和激活素受体的表达，以及关键整合素和TGFa/激活素信号分子的表达和磷酸化，来研究相互作用的机制。受整合素信号调控的转录因子如c-jun和c-fos的表达将通过Western blotting和启动子实验进行研究。为了明确ECM和激活素- a相互作用的机制，将利用激活素- a与ECM相互作用的信号蛋白的显性阴性突变体和信号分子的特异性抑制剂转染技术。检测转染显性阴性突变体前后细胞增殖和存活情况。

DESCRIPTION (provided by applicant): The factors that regulate granulosa cell growth during the early stages of follicular development are unknown. Preliminary work showed that extracellular matrix (ECM) plays a role in growth of early stage follicles in concert with activin-A, and suggested an interaction between integrin- and activin/TFG a-signaling pathways. The elucidation of the molecular mechanisms involved in the growth of granulosa cells of early stage follicles may result in the development of therapeutic strategies that prevent premature ovarian failure, treat age-related infertility, delay menopause and prevent gonadal damage due to cancer drugs. The applicant focused on the mechanisms behind regulation of early stages of follicle growth since the completion of the clinical training in Reproductive Endocrinology. After a year of research training in the UK with an internationally recognized reproductive biologist, the candidate began studying integrin signaling in ovarian follicle growth in collaboration with the proposed mentor of this project. The applicant's and the mentor's institution is a tri-institutional conglamerate with worldwide recognition in research and top research resources. The mentor is an NIH-funded and world recognized expert in integrin-signaling. The applicant's short-term goals are to strengthen the fund of knowledge in cell and molecular biology, become efficient in techniques to study cell signaling and to generate preliminary data. The candidates' long-term goal is to become an independent clinician-scientist with focus on translating basic research on early follicle growth to clinical applications. To achieve these goals, the applicant proposes a phased development plan where didactic training will be completed during the first three years along with performance of the experiments of Specific Aim-1. The fourth and fifth years will focus on the experiments of second and third aims. The specific aims of this proposal are to investigate and delineate the mechanisms of the interaction between the ECM and activin-A in spontaneously immortalized (SIGC) and primary granulosa cells in parallel, and to determine whether granulosa cell proliferation can be modulated by blockage of this interaction. In pursuit of these specific aims, SIGC will be cultured on polylysine, fibronectin, laminin or collagen in the presence or absence of activin-A. The mechanisms of interaction will be investigated by studying the expression of integrins and activin receptors as well as the expression and phosphorylation of the key integrin and TGFa/activin signaling molecules by Western blotting, in vivo phosphorylation, kinase assays, and immunofluorescence. Expression of transcription factors such as c-jun and c-fos, which are regulated by integrin signaling, will be studied by Western blotting and promoter assays. To pinpoint the mechanism of interaction between the ECM and activin-A, transfection techniques with dominant-negative mutants of the signaling proteins implicated in the interaction between activin-A and the ECM, and specific inhibitors of signaling molecules will be utilized. Cell proliferation and survival will be determined before and after transfection with the dominant-negative mutants.