TRANSCRIPTIONAL CONTROL OF GASTRIN BY GROWTH FACTORS

生长因子对胃泌素的转录控制

• 批准号: 2144951
• 负责人: JUANITA L. MERCHANT
• 金额: $ 13.07万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2144951
• 关键词: DNA binding protein; RNA splicing; bombesin; epidermal growth factor; gastrins; gel mobility shift assay; gene induction /repression; genetic regulatory element; genetic transcription; laboratory rabbit; molecular cloning; northern blottings; nuclear runoff assay; phorbols; phosphatase inhibitor; phosphorylation; posttranslational modifications; transcription factor; transfection; transforming growth factors

Gastrin is a potent regulator of normal gastrointestinal cell growth and acid secretion; therefore elucidation of mechanisms that control its synthesis may be applied to understanding such disease states as neoplastic transformation and peptic ulceration. Although gastrin release from enriched antral G cell populations is known to be regulated by cholinergic agonists and the neuropeptide bombesin, the direct cellular mechanisms controlling gastrin synthesis at the level of transcription are poorly understood. This proposal addresses the hypothesis that direct activation of the Epidermal Growth Factor (EGF) receptor on antral G cells stimulates gastrin gene expression. The specific aims focus on EGF as a physiologic regulator of gastrin gene expression and use this observation to dissect the nuclear events that occur in response to growth factor stimulation. Enriched primary G cells will be isolated from dog and stimulated with EGF, Transforming Growth Factor alpha (TGFalpha), bombesin, and phorbol esters prior to measuring changes in gastrin mRNA on Northern blots. Nuclear runoff assays and intron probe analysis will be used to determine if the increase in gastrin mRNA levels is the result of an increase in new transcript synthesis or mRNA stability. To demonstrate that the antral G cell is capable of responding directly to EGF, the EGF receptor and gastrin antibodies were co-localized to the G cell in dog antral tissue. Prior work in a pituitary cell line (GH4) using gastrin-reporter gene constructs identified a novel DNA element, -68GGGGCGGGGTGGGGGG-53, that is required for both EGF and phorbol ester regulation of gastrin gene expression. Two nuclear proteins bind specifically to this element: Sp1 and a previously uncharacterized transcription factor called gERP (gastrin EGF Responsive Protein). The cDNA for Sp1 has been previously cloned. Therefore, the gERP cDNA will be cloned by screening an expression lambdagt11 phage library (Southwester method). To determine whether Sp1 and gERP bind separately or cooperatively to confer the EGF response point mutations of gERE will be tested in gel shift and transient transfection assays. Transcriptional control by both EGF and phorbol ester-activated protein kinase C (PKC) converge upon the gastrin EGF Response Element (gERE); therefore, it is likely that changes in the phosphorylation state of these regulatory proteins are responsible for activation of the gastrin gene. To determine if changes in the phosphorylation state of Sp1 or gERP alter binding to gERE, specific phosphatase inhibitors (e.g. okadaic acid) will be added to the gel shift assays. If blocking phosphatase activity does indeed alter transcription factor binding in vitro, then the effect of these phosphatase inhibitors on transient transformants containing gastrin-reporter constructs will be examined. Therefore these studies hope to explore the molecular mechanisms by which the gastrin is regulated, in particular by growth factors.

胃蛋白是正常胃肠道细胞生长和 酸分泌；因此阐明了控制其机制的机制 合成可以应用于理解此类疾病状态 肿瘤转化和消化性溃疡。 虽然胃蛋白 已知从富集的肛门G细胞种群中释放受到调节 由胆碱能激动剂和神经肽bombesin，直接 控制胃蛋白合成的细胞机制 转录知之甚少。 该提议解决了 假设表皮生长因子（EGF）直接激活 肛门G细胞上的受体刺激胃蛋白基因表达。 这 具体目的专注于EGF作为胃蛋白基因的生理调节剂 表达并利用此观察结果来剖析核事件 响应生长因子刺激而发生。 富集原代G细胞 将从狗中隔离并用EGF刺激，从而改变生长 在测量之前 北部印迹上胃mRNA的变化。 核径流测定法和 内含子探针分析将用于确定是否增加 胃蛋白mRNA水平是新转录本增加的结果 合成或mRNA稳定性。 证明鼻g细胞是 能够直接响应EGF，EGF受体和胃蛋白 将抗体共定位于狗肛门组织中的G细胞。 事先的 使用胃蛋白 - 重生基因在垂体细胞系（GH4）中工作 构造确定了一种新颖的DNA元素，-68Gggggggggggggggggggggggggg -53， EGF和Phorbol酯的胃蛋白基因调节所必需 表达。 两个核蛋白与该元素特别结合：SP1 和先前未表征的转录因子称为GERP （胃泌素EGF反应蛋白）。 SP1的cDNA以前是 克隆。 因此，将通过筛选的GERP cDNA克隆 表达lambdagt11噬菌体库（西南法）。确定 SP1和GERP是单独或合作结合以授予EGF GERE的响应点突变将在凝胶转移和 瞬态转染测定法。 EGF和 佛波酯激活的蛋白激酶C（PKC）会收敛于胃蛋白 EGF响应元素（gere）;因此，可能会改变 这些调节蛋白的磷酸化状态负责 胃蛋白基因的激活。 确定是否改变 SP1或GERP的磷酸化状态改变了与Gere的结合， 磷酸酶抑制剂（例如冈田酸）将添加到凝胶移位中 测定。 如果阻断磷酸酶活性确实确实改变了转录 因子在体外结合，然后这些磷酸酶抑制剂的作用 关于瞬时转化体，含有胃蛋白 - 重复蛋白的构建体将 被检查。 因此，这些研究希望探索分子 胃蛋白受到调节的机制，特别是通过生长 因素。