Transcriptional control of FSH expression

描述（由申请人提供）：垂体通过产生和随后释放卵泡刺激素（FSH）刺激卵泡成熟周期。FSH在整个人类月经周期中以两个“峰值”分泌，并且这种释放模式由内分泌激素下丘脑释放肽和旁分泌生长因子控制，所述内分泌激素下丘脑释放肽和旁分泌生长因子聚集在垂体促性腺细胞上以刺激或抑制FSH产生。本提案中描述的项目涉及一类调节因子激活素的作用机制。促性腺激素内产生的激活素刺激包含FSH的两个蛋白质亚基之一的产生，FSH β亚基。激活素与细胞表面受体结合，激活SMAD家族中的细胞内信号传导蛋白。具体而言，激活素刺激SMAD 2和SMAD 3磷酸化和核转位。一旦进入细胞核，SMAD 3刺激FSH b亚基基因的转录。早期迹象表明SMAD 2不参与该过程;然而，很明显SMAD 3单独不能解释激活素对FSH β的刺激。在特定目标1中，SMAD 2依赖性信号转导将被拮抗，并检查对激活素刺激的FSH β转录的影响。SMAD 3结合FSH β启动子中的顺式作用调节元件。该位点对于激活素和SMAD 3刺激的基因表达是必需的，但不是充分的。在更远端的启动子中的另一个区域也是转录应答所必需的。该调控区的位置将被定位，并确定在那里起作用的转录因子。为了证明SMAD 3在FSH a表达中的体内作用，将在特异性目的2中产生条件性转基因小鼠模型。在这里，四环素诱导系统将用于表达显性负（dn-）形式的SMAD 3在成年小鼠的促性腺激素。将检查dn-SMAD 3对FSH合成和分泌以及发情周期和生育力的影响。最后，FSH β在促性腺激素细胞内表达，但不在身体的其他细胞类型中表达。控制这种细胞限制性基因表达的机制尚不清楚。SMAD 3与锌指转录因子加塔-2协同作用，刺激在其他情况下不表达该基因的细胞中的基础FSH β转录。在具体目标3中，将阐明控制加塔-2和SMAD 3的协同效应的机制，并检查加塔-2在基础FSHb表达中的必要性。总的来说，这些实验的结果将揭示控制基础和激活素刺激的FSH β表达的机制。考虑到FSH对女性生育能力的重要性，这些结果可能有助于设计新型避孕药，或者可能查明迄今为止我们未能发现的不孕症原因。

DESCRIPTION (provided by applicant): The pituitary gland stimulates cycles of ovarian follicle maturation through its production and subsequent release of follicle-stimulating hormone (FSH). FSH is secreted in two "peaks" across the human menstrual cycle and this pattern of release is governed by endocrine hormones hypothalamic releasing peptides and paracrine growth factors that converge on pituitary gonadotrope cells to stimulate or inhibit FSH production. The projects described in this proposal concern the mechanisms of action of one class of regulatory factors, the activins. Activins, produced within gonadotropes, stimulate the production of one of the two protein subunits that comprise FSH, the FSHbeta subunit. Activins bind to cell-surface receptors that activate intracellular signaling proteins in the SMAD family. Specifically, activins stimulate SMAD2 and SMAD3 phosphorylation and nuclear translocation. Once in the nucleus, SMAD3 stimulates transcription of the FSHbsubunit gene. Early indications were that SMAD2 was not involved in this process; however, it is clear that SMAD3 alone cannot account for activin's stimulation of FSHbeta. In Specific Aim 1, SMAD2-dependent signal transduction will be antagonized and the resulting effects on activin-stimulated FSHbeta transcription examined. SMAD3 binds a cis-acting regulatory element in the FSHbeta promoter. This site is necessary but not sufficient for activin and SMAD3-stimulated gene expression. Another region in the more distal promoter is also required for the transcriptional response. The position of this regulatory region will be mapped and the transcription factors acting there will be identified. To demonstrate an in vivo role for SMAD3 in FSHa expression, a conditional transgenic mouse model will be produced in Specific Aim 2. Here, the tetracycline-inducible system will be used to express a dominant negative (dn-) form of SMAD3 in gonadotropes of adult mice. The effects of dn-SMAD3 on FSH synthesis and secretion as well as estrous cyclicity and fertility will be examined. Finally, FSHbeta is expressed within gonadotropes, but not in other cell types of the body. The mechanisms controlling this cell-restricted gene expression are not understood. SMAD3, in concert with a zinc-finger transcription factor, GATA-2, stimulates basal FSHbeta transcription in cells that under other circumstances do not express this gene. In Specific Aim 3, the mechanisms controlling the synergistic effects of GATA-2 and SMAD3 will be elucidated and the necessity for GATA-2 in basal FSHb expression examined. Collectively, the results of these experiments will shed new light on the mechanisms controlling basal and activin-stimulated FSHbeta expression. Given the critical importance of FSH for fertility in women, these results may aid in the design of novel contraceptives or may pinpoint causes of infertility that have thus far eluded our detection.