POMC EXPRESSION AND PROCESSING IN PITUITARY

垂体中 POMC 的表达和加工

基本信息

批准号：
2673872
负责人：
DEAN MYERS
金额：
$ 11.02万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-08-01 至 1999-07-31
项目状态：
已结题

项目摘要

During the final weeks of gestation in sheep there is maturation of adrenocortical steroidogenesis resulting in the prepartum increase in plasma cortisol that induces parturition and organ maturation essential for neonatal survival. The fetal pituitary and hypothalamic paraventricular nucleus (PVN) are integral in initiating maturation of the adrenocortical stress response. Identifying the neurohumoral regulation of POMC gene expression and processing in the fetal pituitary is paramount in understanding neuroendocrine regulation the fetal HHAA. This proposal has three related goals: 1) to determine the role of corticotropin releasing hormone (CRH) and arginine vasopressin (AVP) in stimulating POMC gene expression in the ovine fetal anterior pituitary (AP); 2) identify the roles of AVP and CRH in regulating the biosynthetic processing of POMC to ACTH; 3) to identify the potential of the fetal NIL to produce ACTH during the final weeks of gestation in sheep. The following hypotheses address my goals: I: CRH is the primary hypothalamic neuropeptide stimulating POMC gene expression in the fetal AP at the critical stage of gestation proposed. I hypothesize that AVP, while acting as a major ACTH-secretagogue in the ovine fetus, does not enhance POMC gene expression, either alone or in synergy with CRH; II: CRH and AVP both stimulate the processing of POMC to ACTH by enhancing the ratio of expression of PC1 to PC2 necessary for ACTH production in the corticotrope. I hypothesize that CRH and AVP stimulate expression of PC1 and suppress expression of PC2 in the AP corticotrope. I hypothesize that CRH and AVP stimulate expression of CPE, an enzyme critical for the final processing of ACTH containing peptides to ACTH; III: ACTH is a major POMC derived peptide synthesized in the late gestation fetal NIL. I hypothesize that enhanced expression of PC1 in the late gestation sheep fetus NIL compared to the adult pattern of enzyme expression favors ACTH production in the fetal NIL. Specific Aims (SA) 1 and 2 address Hypothesis I: To quantify CRH and AVP stimulated POMC gene transcription, POMC RNA splicing (heteronuclear [hn] RNA processing), and steady-state levels of cytoplasmic POMC mRNA in fetal AP. SA 2 addresses Hypothesis II: To quantify CRH and AVP induced changes in levels of PC1, PC2 and CPE mRNA, protein and enzymatic activities in corticotropes of the fetal AP. SA 3 addresses both Hypothesis I and II: During the final 30 days of gestation, I will quantify changes in PC1, PC2, an CPE mRNA, protein expression and POMC processing. I will also quantify the ratio of POMC hnRNA to POMC mRNA in the fetal anterior pituitary corticotrope. SA 4 and 5 address Hypothesis III: During the final 30 dGA, I will quantify POMC gene expression as a function of the ratio and quantity of POMC hnRNA processing intermediates to cytoplasmic mRNA in the fetal NIL to determine the capacity of the NIL to synthesize POMC. SA 5: During the final 30 dGA, I will analyze POMC processing to determine if ACTH is a major product of the fetal NIL. I will concurrently determine levels of PC1, PC2 and CPE gene expression (mRNA and protein) and enzymatic activities in the fetal NIL. Studies proposed to achieve Specific Aim 4 and 5 will determine the molecular identity of ACTH immunoreactive forms and the ACTH potential of the fetal NIL during a critical stage of gestation in the ovine fetus. The proposed experiment will establish for the first time in sheep the roles of the two major PVN neuropeptides regulating the adrenocortical stress axis (CRH and AVP) in stimulating POMC gene expression and the expression and activities of genes necessary for the post-translational processing of POMC to ACTH in the fetal corticotrope.

在绵羊妊娠的最后几周，肾上腺皮质类固醇生成导致产前增加血浆皮质醇对诱导分娩和器官成熟至关重要为了新生儿的生存。胎儿垂体和下丘脑室旁核（PVN）对于启动成熟至关重要肾上腺皮质应激反应。识别神经体液胎儿垂体中 POMC 基因表达和加工的调控对于理解胎儿 HHAA 的神经内分泌调节至关重要。该提案具有三个相关目标：1）确定促肾上腺皮质激素释放激素 (CRH) 和精氨酸加压素 (AVP) 刺激绵羊胎儿垂体前叶的 POMC 基因表达（美联社）； 2) 确定AVP和CRH在调节生物合成中的作用将 POMC 加工成 ACTH； 3）识别胎儿NIL的潜力在绵羊妊娠的最后几周内产生 ACTH。这以下假设实现了我的目标： I：CRH 是主要的下丘脑神经肽刺激胎儿 AP 中的 POMC 基因表达建议妊娠关键阶段。我假设 AVP，同时作为绵羊胎儿中主要的 ACTH 分泌剂，不会增强 POMC基因表达，单独或与CRH协同表达；二：CRH 和 AVP 均通过提高比率来刺激 POMC 向 ACTH 的加工产生 ACTH 所必需的 PC1 至 PC2 的表达皮质激素。我假设 CRH 和 AVP 刺激 PC1 的表达并抑制 AP 促肾上腺皮质激素中 PC2 的表达。我假设 CRH 和 AVP 刺激 CPE 的表达，CPE 是一种对将含有ACTH的肽最终加工成ACTH； III：促肾上腺皮质激素是主要的 POMC 衍生肽在妊娠晚期胎儿 NIL 中合成。我推测妊娠晚期绵羊中 PC1 的表达增强与成人相比，胎儿 NIL 的酶表达模式有利于 ACTH 胎儿 NIL 中的产生。具体目标 (SA) 1 和 2 地址假设 I：为了量化 CRH 和 AVP 刺激的 POMC 基因转录， POMC RNA 剪接（异核 [hn] RNA 处理）和稳态胎儿 AP 中细胞质 POMC mRNA 的水平。 SA 2 地址假设 II：量化 CRH 和 AVP 引起的 PC1、PC2 和 CPE 水平的变化胎儿 AP 促肾上腺皮质激素中的 mRNA、蛋白质和酶活性。 SA 3 解决了假设 I 和 II：在最后 30 天内妊娠期间，我将量化 PC1、PC2、CPE mRNA、蛋白质的变化表达和 POMC 处理。我也会量化POMC的比例胎儿垂体前叶促肾上腺皮质激素中的 hnRNA 到 POMC mRNA。 SA 4 和 5 解决假设 III：在最后 30 dGA 期间，我将量化 POMC 基因表达作为 POMC 比例和数量的函数 hnRNA 将中间体加工成胎儿 NIL 中的细胞质 mRNA 确定 NIL 合成 POMC 的能力。 SA 5：期间最后 30 dGA，我将分析 POMC 处理以确定 ACTH 是否是主要产物为胎儿NIL。我将同时确定 PC1、PC2 和 CPE 基因表达（mRNA 和蛋白质）和酶促胎儿 NIL 中的活动。为实现具体目标而提出的研究 4和5将确定ACTH免疫反应性的分子身份胎儿 NIL 在关键阶段的形式和 ACTH 电位绵羊胎儿的妊娠。拟议的实验将建立首次在绵羊身上研究两种主要 PVN 神经肽的作用调节肾上腺皮质应激轴（CRH 和 AVP） POMC基因表达及必需基因的表达和活性用于胎儿中 POMC 转化为 ACTH 的翻译后加工皮质激素。