Transcriptional control of FSH expression

描述（由申请人提供）：脑垂体通过产生和随后释放促卵泡激素（FSH）来刺激卵巢卵泡成熟周期。FSH在人类月经周期中有两个“高峰”分泌，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，这些激素聚集在垂体促性腺激素细胞上刺激或抑制FSH的产生。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。促性腺激素中产生的激活素，刺激构成FSH的两种蛋白质亚基之一——FSH β亚基的产生。激活素与细胞表面受体结合，激活SMAD家族中的细胞内信号蛋白。具体来说，激活素刺激SMAD2和SMAD3磷酸化和核易位。一旦进入细胞核，SMAD3刺激fshb亚基基因的转录。早期迹象表明SMAD2不参与这一过程；然而，很明显，SMAD3不能单独解释激活素对FSHbeta的刺激。在Specific Aim 1中，smad2依赖的信号转导将被拮抗，并由此对激活素刺激的fshβ转录产生影响。SMAD3结合FSHbeta启动子中的顺式调控元件。这个位点对于激活素和smad3刺激的基因表达是必要的，但不是充分的。转录反应也需要远端启动子中的另一个区域。这个调控区域的位置将被绘制出来，在那里起作用的转录因子将被识别出来。为了证明SMAD3在FSHa表达中的体内作用，将在Specific Aim 2中建立一个条件转基因小鼠模型。在这里，四环素诱导系统将用于表达SMAD3的显性阴性（dn-）形式的促性腺激素的成年小鼠。我们将研究dn-SMAD3对卵泡刺激素合成和分泌、发情周期和生育的影响。最后，fshβ在促性腺激素中表达，但在身体的其他细胞类型中不表达。控制这种细胞限制性基因表达的机制尚不清楚。SMAD3与锌指转录因子GATA-2协同作用，刺激在其他情况下不表达该基因的细胞中基础FSHbeta转录。在Specific Aim 3中，将阐明控制GATA-2和SMAD3协同作用的机制，并研究GATA-2在FSHb基础表达中的必要性。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FSHbeta表达的机制。鉴于卵泡刺激素对女性生育能力至关重要，这些结果可能有助于设计新型避孕药具，或可能查明迄今为止尚未发现的不孕原因。

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.