Mechanism of Action of Prohibitin in Ovarian Cell Function

抑制素在卵巢细胞功能中的作用机制

• 批准号: 7942740
• 负责人: WINSTON E THOMPSON
• 金额: $ 29.53万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7942740
• 关键词: Activins; Address; Affect; Alleles; Animal Model; Animals; Apoptosis; Apoptotic; Biochemical; Biological Assay; Cell Communication; Cell Cycle; Cell Cycle Progression; Cell Death; Cell Death Signaling Process; Cell Differentiation process; Cell Fractionation; Cell Proliferation; Cell physiology; Cells; Chemistry; Clinical; Contraceptive methods; Data; Development; Diagnostic; Differentiation Antigens; Differentiation and Growth; Down-Regulation; Embryo; Endocrine; Equilibrium; Event; Experimental Designs; Failure; Female; Fertility; Fractionation; Functional disorder; Future; Genetic; Goals; Gonadotropins; Growth and Development function; Hormonal; In Vitro; Infertility; Intervention; Knock-out; Knowledge; Laboratories; Lead; Light; MAP2K1 gene; Mediating; Mediator of activation protein; Menopause; Mitochondria; Molecular; Mus; Mutagenesis; Oocytes; Outcome; Ovarian; Ovary; Pathogenesis; Pathway interactions; Phenotype; Phosphorylation; Physiological; Pituitary Hormones; Play; Polycystic Ovary Syndrome; Process; Puberty; Publishing; Ras/Raf; Reporting; Reproduction; Reproductive Biology; Reproductive Health; Research; Role; Screening for Ovarian Cancer; Signal Transduction; Stimulus; Testing; Testosterone; Therapeutic; Time; Tyrosine; Woman; activin A; adenoviral-mediated; cell growth; design; folliculogenesis; granulosa cell; in vivo; loss of function; mutant; novel; novel diagnostics; population based; prohibitin; public health relevance; reproductive; research study; response; scaffold; tool

DESCRIPTION (provided by applicant): Follicular development and differentiation, key to female reproduction, are sequential events that are tightly regulated by balanced cell proliferation, survival and cell death. Previously we reported the prohibitins, PHB1 and REA, as important mediators of the effects of gonadotropins on granulosa cell (GC) differentiation and apoptosis. Consistent with our long term goal of understanding their functions in folliculogenesis, new preliminary data here underscores their major role in GC growth and differentiation. We show that there is a clear association between PHB1 expression and follicular development in vivo. Furthermore, PHB1 expression is differentially regulated in response to FSH plus activin (A) in proliferation (FSH+A??PHB1) vs. FSH plus testosterone (T) in differentiation (FSH+T??PHB1) in vitro. Forced expression of PHB1 impairs cell cycle progression and down regulation results in a proliferative phenotype in GCs. PHB1 is phosphorylated on Y249 in a MEK1-dependent fashion while PHB1 is required for ERK1/2 activation defining a new regulatory paradigm in response to FSH+T in primary GCs. These observations support our central hypothesis that prohibitins are critical intracellular mediators of FSH stimulation of GCs, affecting follicular development, and the derived prediction that FSH+T-dependent MEK1-mediated phosphorylation of PHB1 is essential for its function in GCs differentiation. In this proposal we will address these two essential premises using a systematic experimental design that combines gain- and loss-of-function, mutagenesis, genetic approaches, biochemical fractionation and immunostaining in primary GCs in vitro in response to FSH- dependent physiological synergistic stimuli with either activin or testosterone. A GC specific conditional knock- out PHB1 animal model will be used to circumvent the embryonic lethality identified for PHB1 and address the in vivo role of PHB1. Specifically, we propose: Aim 1: To test the hypothesis that PHB1 and REA levels determine the differential effects of FSH in proliferation and differentiation of GCs in vitro; Aim 2: To test the hypothesis that PHB1 is obligatory for folliculogenesis in vivo; and Aim 3: To test the hypothesis that PHB1 functions as a scaffold and substrate to coordinate FSH+T signaling in GCs differentiation. Unraveling the mechanisms underlying the growth and development of a competent follicle with the capacity t release a fertilizable oocyte is a major goal of basic reproductive biology research. PUBLIC HEALTH RELEVANCE: Understanding of the molecular mechanism of prohibitins in GCs growth and differentiation in the normal physiological development of the follicle as well as its role in the pathogenesis of ovarian dysfunction could have profound implications in the clinical setting in the field of fertility (contraception or infertility), and lead to development of novel molecular diagnostic and therapeutic tools (population based screenings, ovarian cancer).

描述（由申请人提供）：卵泡发育和分化，是女性繁殖的关键，是由平衡的细胞增殖，生存和细胞死亡严格调节的顺序事件。以前，我们报道了促性腺激素对颗粒细胞（GC）分化和细胞凋亡的影响的重要介体，这是禁止的PHB1和REA。与我们理解其在卵泡发生中的功能的长期目标一致，这里的新初步数据强调了它们在GC生长和分化中的主要作用。我们表明，体内PHB1表达与卵泡发育之间存在明显的关联。此外，在体外，PHB1表达响应于FSH加激活素（a）在增殖（FSH+A ?? PHB1）中的差异调节（a）。 PHB1的强迫表达会损害细胞周期的进程和下调导致GC中的增殖表型。 PHB1以MEK1依赖性方式在Y249上磷酸化，而ERK1/2激活需要PHB1来定义新的调节范式，以响应初级GC中的FSH+T。这些观察结果支持了我们的中心假设，即禁止素是FSH刺激GC的关键细胞内介体，影响卵泡发育，并且得出的预测是，FSH+T依赖性MEK1介导的PHB1的PHB1磷酸化对GC的功能至关重要。在该提案中，我们将使用系统的实验设计来解决这两个基本前提，该设计结合了功能和丧失功能，诱变，遗传方法，生化分级分级和在初级GC中的免疫染色，以响应FSH依赖的生理协同刺激与激活蛋白或睾丸激素。 GC特异性的条件敲除PHB1动物模型将用于规避针对PHB1鉴定的胚胎致死性，并解决PHB1的体内作用。具体而言，我们提出：目标1：测试PHB1和REA水平确定FSH在体外GC的增殖和分化中的差异作用的假设；目的2：检验PHB1在体内的卵泡发生的假设；目标3：检验PHB1充当支架和底物以协调GCS分化中的FSH+T信号的假设。阐明具有肥大的卵泡生长卵泡的生长和发展的机制是基本生殖生物学研究的主要目标。公共卫生相关性：对卵泡正常生理发展中禁止素的分子机制的理解，以及其在卵巢功能障碍的发病机理中的作用，在临床环境中在临床环境中具有深远的影响（避孕或不育），并导致新型分子诊断的工具（概述）。