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尚不清楚。初步工作表明，细胞外基质（ECM）与激活素A一起在早期卵泡的生长中起作用，并表明整合素和激活素/TFG α信号传导途径之间的相互作用。阐明早期卵泡颗粒细胞生长的分子机制可能会导致治疗策略的发展，以预防卵巢早衰，治疗年龄相关性不孕症，延迟绝经和预防由于癌症药物引起的性腺损伤。申请人专注于自完成生殖内分泌学临床培训以来卵泡生长早期阶段调控背后的机制。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，该候选人开始与该项目的拟议导师合作研究卵泡生长中的整合素信号传导。申请人和导师的机构是一个三机构的联合体，在研究和顶级研究资源方面具有世界公认的地位。 导师是NIH资助的世界公认的整合素信号传导专家。申请人的短期目标是加强细胞和分子生物学知识的基金，成为研究细胞信号传导和产生初步数据的有效技术。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沿着具体目标-1的实验。第四年和第五年将侧重于第二和第三个目标的实验。本提案的具体目的是研究和描述ECM和激活素A在自发永生化（SIGC）和初级颗粒细胞中平行相互作用的机制，并确定颗粒细胞增殖是否可以通过阻断这种相互作用来调节。为了实现这些特定目标，将在存在或不存在激活素A的情况下在聚赖氨酸、纤连蛋白、层粘连蛋白或胶原蛋白上培养SIGC。通过Western印迹、体内磷酸化、激酶测定和免疫荧光研究整合素和激活素受体的表达以及关键整合素和TGF α/激活素信号传导分子的表达和磷酸化，研究相互作用的机制。将通过Western印迹和启动子测定研究由整合素信号调节的转录因子如c-jun和c-fos的表达。为了查明ECM和激活素A之间相互作用的机制，将利用利用涉及激活素A和ECM之间相互作用的信号传导蛋白的显性负突变体和信号传导分子的特异性抑制剂的转染技术。在用显性阴性突变体转染之前和之后测定细胞增殖和存活。

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.