TRANSCRIPTIONAL REGULATION OF LH BETA GENE EXPRESSION

垂体促性腺激素，促黄体生成素（LH）和促卵泡激素（FSH）是配子成熟和性腺甾体形成的关键调节剂。最近的证据表明，转录因子类固醇生成因子-1 （SF-1）和早期生长反应基因1 （Egr-1）调节lhβ亚基基因的表达。我们工作的长期目标是充分了解调节LHbeta基因表达的转录因子和顺式元件，这些信息将为了解正常和异常生殖功能提供信息。本提案的具体目的是：1)表征bicoid相关转录因子Ptx1在介导组织特异性LHbeta基因表达中的作用，2)确定LHbeta-Ptx1顺式元件在培养和体内初级促性腺激素中的生理重要性，以及3)确定介导cAMP/ pka诱导的LHbeta基因启动子活性刺激的分子机制。在Aim 1中，基于Drouin及其同事的一份报告，我们假设Ptx1单独作用并与SF-1协同作用以增加LHbeta启动子活性，这将通过电泳迁移量转移试验、瞬时转染永生化细胞系和产生Ptx1“敲低”细胞系进行测试。在Aim 2A中，将使用高效的重组腺病毒方法感染垂体原代细胞培养物，以确定具有促性腺激素特异性表达的5'侧翼区域。定义的促性腺激素特异性区域将在报告基因构建中使用，作为野生型序列或在Aim 1中鉴定的ptx1位点突变。我们预测，Ptx1位点突变的存在将显著减弱培养的垂体原代细胞（Aim 2B）和转基因小鼠系（Aim 2C）中报告基因的表达，从而验证Ptx1对lhβ基因表达的生理重要性。在Aim 3中，我们提出验证cAMP/ pka诱导的LHbeta启动子活性增加的假设是由于：A) pka调节的SF-1和/或Egr-1基因表达或翻译后修饰的变化，和/或B) LHbeta基因启动子序列中假定的AP-2顺式元件。通过进一步表征介导组织特异性和激素调节的LHbeta基因表达的因素，这些研究将有助于我们理解正常生殖生理，以及不孕症、多囊卵巢综合征、下丘脑性腺功能减退、青春期早熟和延迟等疾病的病理生理。

The pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), are critical modulators of gamete maturation and gonadal steroidogenesis. Recent evidence has demonstrated that the transcription factors, steroidogenic factor-1 (SF-1) and early growth response gene 1 (Egr-1), regulate expression of the LHbeta-subunit gene. The long-term objective of our work is to understand fully the transcription factors and cis-elements which modulate LHbeta gene expression, information which will provide insight into both normal and abnormal reproductive function. The Specific Aims of this proposal are: 1) to characterize the role of the bicoid-related transcription factor, Ptx1, in mediating tissue-specific LHbeta gene expression, 2) to define the physiologic importance of the LHbeta-Ptx1 cis-element(s) in primary gonadotropes in culture and in vivo, and 3) to identify the molecular mechanisms which mediate cAMP/PKA-induced stimulation of LHbeta gene promoter activity. In Aim 1, based on a report by Drouin and co-workers, we hypothesize that Ptx1 acts alone and in synergy with SF-1 to increase LHbeta promoter activity, as will be tested by electrophoretic mobility shift assay, transient transfection of immortalized cell lines, and generation of Ptx1 "knockdown" cell lines. In Aim 2A, primary pituitary cell cultures will be infected using a highly efficient, recombinant adenovirus approach in order to define the 5'flanking region which confers gonadotrope-specific expression. The defined gonadotrope-specific region will then be used in reporter constructs as either the wild-type sequence or with a mutation in the Ptx1-site(s) identified in Aim 1. We predict that the presence of a mutation in the Ptx1 site(s) will markedly blunt reporter expression in both cultured primary pituitary cells (Aim 2B) and transgenic mouse lines (Aim 2C), verifying the physiologic importance of Ptx1 to LHbeta gene expression. In Aim 3, we propose to test the hypothesis that cAMP/PKA-induced increases in LHbeta promoter activity are due to: A) PKA-regulated changes in SF-1 and/or Egr-1 gene expression or post-translational modifications, and/or B) putative AP-2 cis-element(s) in the LHbeta gene promoter sequence. By further characterizing factors which mediate tissue-specific and hormonally-regulated LHbeta gene expression, these studies will contribute to our understanding of normal reproductive physiology, as well as the pathophysiology of disorders including infertility, polycystic ovarian syndrome, hypothalamic hypogonadism, and premature and delayed puberty.