TRANSCRIPTIONAL REGULATION OF LH BETA GENE EXPRESSION

垂体促性腺激素黄体生成素(LH)和卵泡刺激素(FSH)是配子成熟和性腺类固醇生成的重要调节因子。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LHbeta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，这些信息将为深入了解正常和异常生殖功能提供信息。本研究的具体目的是：1)研究类双核转录因子Ptx1在组织特异性LHbeta基因表达中的作用；2)确定LHbeta-Ptx1顺式元件(S)在原代培养和体内促性腺激素细胞中的生理意义；3)确定cAMP/PKA刺激LHbeta基因启动子活性的分子机制。在目标1中，根据Drouin和他的同事的一份报告，我们假设Ptx1单独作用并与SF-1协同作用增加LHbeta启动子的活性，这将通过凝胶迁移率改变分析、永生化细胞系的瞬时转染和Ptx1“敲除”细胞系的产生来验证。在Aim 2a中，原代培养的垂体细胞将用高效的重组腺病毒方法感染，以确定提供促性腺激素特异性表达的5‘侧翼区。已定义的促性腺激素特异区将作为野生型序列或与目标1中确定的Ptx1位点突变(S)一起用于报告构建。我们预测，Ptx1位点突变(S)的存在将显著钝化培养的原代垂体细胞(Aim 2B)和转基因小鼠系(Aim 2C)中的报告表达，验证Ptx1对LHbeta基因表达的生理重要性。在目标3中，我们提出了一种假设，即cAMP/PKA诱导的LHbeta启动子活性的增加是由于：A)PKA调控的SF-1和/或Egr-1基因表达的变化或翻译后的修饰，和/或B)LHbeta基因启动子序列中可能的AP-2顺式元件(S)。通过进一步确定调节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的生殖生理学，以及不孕不育、多囊卵巢综合征、下丘脑性腺功能低下、早熟和青春期延迟等疾病的病理生理学。

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.