HORMONAL REGULATION OF GALANIN GENE EXPRESSION

甘丙肽是一种广泛分布的调节垂体的肽， 胰腺和胃的激素分泌。 在大鼠脑垂体内， 甘丙肽的产生仅限于乳营养细胞和促生长细胞 其中合成和分泌都受到循环雌激素的影响。 雌激素对垂体甘丙肽基因表达有显着影响。 在正常的发情周期中，稳定状态的mRNA水平变化30倍。 成年雌性的比率，并在怀孕期间增加10倍。 甘丙肽特异性mRNA占雌激素总mRNA的0.5% 在这些细胞中对甘丙肽基因表达进行转录控制。 这些观察结果表明甘丙肽基因提供了一个极好的 研究激素和细胞之间特定相互作用的模型， 特定的转录调节因子。 拟议的研究将调查大鼠 甘丙肽基因在垂体细胞中受到调节。 雌激素对 甘丙肽基因转录将被进一步表征。 的DNA 具有赋予雌激素反应性的序列将通过以下方法鉴定： 使用缺失突变体的瞬时表达测定。 我们将评估 这些序列结合并被雌激素激活的能力 受体蛋白 细胞谱系特异性表达的基础将通过以下方法进行检查： 甘丙肽生产细胞系和不生产细胞系的转染。 我们将 试图辨别甘丙肽基因内的DNA序列， 类型特异性 一旦确定，这些序列将用于分离 负责观察到的特异性的蛋白质，并鉴定 编码这种蛋白质的cDNA克隆。 将特别注意 Pit-1（GHF-1）在控制 甘丙肽表达。 Pit-1/GHF-1是一种转录激活因子， 似乎决定催乳素和生长的细胞类型特异性 激素基因在大鼠垂体中的表达。 Pit-1/GHF-1基因表达 仅限于垂体催乳素和生长激素， 受到雌激素的负调节 我们将确定这种蛋白质 调节甘丙肽表达及其活性如何受雌激素影响。 我们还将评估甘丙肽是否响应雌激素分泌 调节pit-1/GHF-1基因表达。 细胞特异性调节蛋白和细胞特异性调节蛋白之间的相互作用将被研究。 在瞬时表达试验中通过顺式- 元素 如果我们成功地鉴定出一种细胞特异性调节因子， 甘丙肽基因表达，我们将检查是否表达的 调节蛋白本身受雌激素调节， 影响甘丙肽基因的雌激素反应性 表情

Galanin is a widely-distributed peptide which modulates pituitary, pancreatic, and gastric hormone secretion. Within the rat pituitary, galanin production is limited to the lactotrophic and somatotrophic cells where both synthesis and secretion are influenced by circulating estrogens. Estrogens have a dramatic effect on pituitary galanin gene expression. Steady-state mRNA levels vary 30-fold during the normal estrous cycle of adult female rates, and increase another 10-fold during gestation. Galanin-specific mRNA accounts for up to 0.5% of total mRNA in estrogen exerts transcriptional control over galanin gene expression in these cells. These observations suggest that the galanin gene provides an excellent model for the study of the specific interaction of hormonal and cell- specific regulators of transcription. The proposed studies will investigate the mechanisms by which the rat galanin gene is regulated in pituitary cells. The effects of estrogen on galanin gene transcription will be further characterized. The DNA sequences with confer estrogen-responsiveness will be identified by transient expression assays using deletion mutants. We will then evaluate the ability of these sequences to bind and be activated by estrogen receptor protein. The basis for cell lineage specificity of expression will be examined by transfection of galanin-producing and on-producing cell lines. We will attempt to discern DNA sequences within the galanin gene which confer cell type specificity. Once identified, those sequences will be used to isolate the protein(s) responsible for the observed specificity and to identify cDNA clones which encode such protein(s). Particular attention will be given to the evaluation of the role of pit-1(GHF-1) in the control of galanin expression. Pit-1/GHF-1 is a transcriptional activator which appears to determine the cell-type specificity of prolactin and growth hormone gene expression in the rat pituitary. Pit-1/GHF-1 gene expression is limited to pituitary lactotrophs and somatotrophs and appears to be negatively regulated by estrogen. We will determine whether this protein regulates galanin expression and how its activity is affected by estrogen. We will also evaluate whether galanin secreted in response to estrogen regulates pit-1/GHF-1 gene expression. The interaction of cell- and hormone-specific regulatory proteins will be evaluated in transient expression assays by mutational analysis of cis- elements. If we are successful in identifying a cell-specific regulator of galanin gene expression, we will examine whether the expression of the regulatory protein is itself regulated by estrogen and whether production of the protein affects the estrogen-responsiveness of galanin gene expression.