Activin signaling mechanisms in pituitary cells

描述（由申请人提供）：人类和遗传小鼠模型中自然发生的突变清楚地表明垂体促卵泡激素（FSH）在女性生殖周期和生育力中的必要性。FSH受体基因或FSH β亚基（FSH β）蛋白的破坏或缺失会在窦前阶段阻止卵泡成熟，从而阻止排卵。激活素和促性腺激素是垂体FSH的有效调节剂。激活素B亚型在垂体促性腺激素内产生，并以自分泌或旁分泌方式刺激FSH β亚基基因转录。激活素介导这种效应的机制尚不清楚。在其他细胞系统中，激活素结合由两种激活素II型受体之一和激活素IB型受体（ALK 4）组成的异聚受体复合物。在配体结合后，II型受体磷酸化ALK 4，ALK 4磷酸化细胞内信号传导分子SMAD 2和SMAD 3。小鼠促性腺细胞系LbetaT 2产生FSH β mRNA并分泌FSH以响应激活素。SMAD 3而非SMAD 2的过表达类似地刺激该细胞系中的FSH β基因表达。这些数据表明，激活素可能通过SMAD 3信号刺激FSH β基因。本提案中的第一个项目旨在通过阻断SMAD 3依赖性过程来验证这一假设。首先，SMAD 2或SMAD 3蛋白质的产生将使用RNA干扰（RNAi）在LbetaT 2细胞中被抑制。据预测，这些细胞中SMAD 3而非SMAD 2产生的减弱将阻断激活素刺激的FSH β表达。第二，细胞将用不能被ALK 4激活的SMAD 2或SMAD 3的显性阴性（dn）形式转染，从而破坏下游信号传导。预计dn-SMAD 3而不是dn-SMAD 2会破坏激活素刺激的FSH β基因表达。一旦进入细胞核，激活的SMAD通过与细胞特异性DNA结合伴侣相互作用影响基因转录。假设SMAD 3与促性腺激素限制性转录因子相互作用以刺激FSH β亚基基因表达。本提案的第二个项目旨在通过使用酵母双杂交筛选鉴定促性腺细胞中SMAD 3相互作用蛋白来验证这一假设。然后将研究候选蛋白的促性腺激素特异性表达及其在哺乳动物细胞中与SMAD 3相互作用的能力。拟议研究的结果将增加我们对激活素调节FSH机制的理解，可能会突出避孕设计的新目标，并可能为某些形式的不孕症的原因提供见解。

DESCRIPTION (provided by applicant): Naturally occurring mutations in humans as well as genetic mouse models clearly indicate the necessity of pituitary follicle-stimulating hormone (FSH) in female reproductive cyclicity and fertility. Disruption or deletion of the FSH receptor gene or the beta subunit of the FSH (FSH beta) protein arrest ovarian follicle maturation at the pre-antral stage, thereby preventing ovulation. The activins and inhibins are potent regulators of pituitary FSH. The activin B isoform is produced within pituitary gonadotropes and acts in an autocrine or paracrine manner to stimulate FSHbeta subunit gene transcription. The mechanisms through which the activins mediate this effect are not understood. In other cell systems, activins bind to a heteromeric receptor complex consisting of one of the two activin type II receptors and the activin type IB receptor (ALK4). Upon ligand binding, the type II receptor phosphorylates ALK4, which phosphorylates the intracellular signaling molecules, SMAD2 and SMAD3. The mouse gonadotrope cell line, LbetaT2, produces FSHbeta mRNA and secretes FSH in response to activins. Over-expression of SMAD3, but not SMAD2, similarly stimulates FSHbeta gene expression in this cell line. These data suggest that activins may signal through SMAD3 to stimulate the FSHbeta gene. The first project in this proposal is designed to test this hypothesis by blocking SMAD3-dependent processes. First, SMAD2 or SMAD3 protein production will be inhibited in LbetaT2 cells using RNA interference (RNAi). It is predicted that the attenuation of SMAD3, but not SMAD2, production in these cells will block activin-stimulated FSHbeta expression. Second, cells will be transfected with dominant-negative (dn) forms of SMAD2 or SMAD3 that cannot be activated by ALK4 and thereby disrupt downstream signaling, dn-SMAD3, but not dn-SMAD2, is predicted to disrupt activinstimulated FSHbeta gene expression. Once in the nucleus, activated SMADs affect gene transcription through interaction with cell-specific DNA binding partners. It is hypothesized that SMAD3 interacts with gonadotrope-restricted transcription factors to stimulate FSHbeta subunit gene expression. The second project in this proposal is designed to test this hypothesis by using a yeast two-hybrid screen to identify SMAD3-interacting proteins in gonadotrope cells. Candidate proteins will then be investigated for their gonadotrope specific expression and their ability to interact with SMAD3 in mammalian cells. The results of the proposed studies will increase our understanding of mechanisms of FSH regulation by activins, may highlight novel targets for contraceptive design, and may provide insights into causes of some forms of infertility.