Transcriptional control of FSH expression

描述(申请人提供)：脑下垂体通过产生和随后释放卵泡刺激素(FSH)来刺激卵巢卵泡成熟周期。在人类月经周期中，FSH在两个“高峰”中分泌，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。激活素是在促性腺激素中产生的，它刺激组成FSH的两个蛋白质亚基之一的产生，即FSHβ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蛋白。具体地说，激活素刺激Smad2和Smad3的磷酸化和核转位。一旦进入细胞核，Smad3就会刺激FSHb亚单位基因的转录。早期的迹象表明，Smad2不参与这一过程；然而，很明显，Smad3本身不能解释激活素对FSHbeta的刺激。在特定的目标1中，Smad2依赖的信号转导将被拮抗，并检测其对激活素刺激的FSHbeta转录的影响。Smad3结合FSHbeta启动子中的顺式作用调控元件。这个位置是激活素和Smad3刺激的基因表达所必需的，但不是充分的。更远端启动子的另一个区域也是转录反应所必需的。这个调控区域的位置将被绘制出来，作用于那里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，针对特定的目的2建立了一个条件转基因小鼠模型。在这里，我们将使用四环素诱导系统在成年小鼠的促性腺激素中表达显性阴性(DN-)形式的Smad3。本研究将检测DN-Smad3对FSH的合成和分泌以及发情周期和受精率的影响。最后，FSHbeta在促性腺激素中表达，但不在身体的其他细胞类型中表达。控制这种受细胞限制的基因表达的机制尚不清楚。Smad3与锌指转录因子GATA-2结合，在其他情况下不表达该基因的细胞中刺激基础FSHbeta转录。在具体目标3中，将阐明GATA-2和Smad3协同作用的机制，并研究GATA-2在基础FSHB表达中的必要性。总而言之，这些实验的结果将为控制基础和激活素刺激的FSHbeta表达的机制提供新的线索。鉴于卵泡刺激素对女性生育力的关键重要性，这些结果可能有助于设计新的避孕药，或者可能精确定位到我们迄今未能发现的不孕原因。

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.