ACTIONS OF CALCIUM REGULATING HORMONES IN THE GUT

这项拟议的工作提出的总体假设是，钙- 调节肽，特别是甲状旁腺激素相关肽 甲状旁腺素受体(PTHrP)在肠道中起着重要的调节作用。世界上最大的推动力 拟议的项目是确定和阐明PTHrP 在肠道上皮起调节生长和分化的作用。一 具体目的是严格测试PTHrP是一种 自分泌/旁分泌调节剂在ITS部位附近发挥本地作用 产生，甚至可能是在产生多肽的细胞上。研究 在正常大鼠中，设计检查肠道中PTHrP的共定位 及其受体mRNAs和多肽。随着年龄的增长，怀孕， 哺乳，以及肠道细胞生长的其他生理状态 自然改变将被研究。PTHrP及其ITS联产 同一细胞系和同一细胞中的受体将通过In 原位杂交，分析基因在单细胞和 多肽免疫细胞化学。PTHrP的生长调节作用将是 在同时产生和反应的上皮肠道细胞系中进行检测 PTHrP。抗血清中和生物(cAMP刺激物) PTHrP的作用将被用来验证初步研究表明 抗血清对细胞生长有促进作用。甲状旁腺素受体拮抗剂的减慢作用 细胞周期或促进细胞程序性死亡(凋亡)是 概述。第二个具体目标将追求新颖的初步发现， PTHrP在肝脏中表达，具有调节肝脏生长的作用。 将研究PTHrP mRNA和多肽在肝再生中的作用 部分肝切除大鼠及妊娠、哺乳期和增龄期。一个 我们发现的高分化人肝细胞系(Hep G2) 为了生产大量的PTHrP，将进行研究。初步调查结果 PTHrP抗血清对细胞生长的促进作用 将继续研究中和PTHrP生物活性的方法 检查了机械装置。初步研究发现，这些细菌产生的甲状旁腺素 细胞可受氢化可的松、维生素D和其他物质的调节 将被研究并阐明所涉及的机制。Hep G2细胞将 瞬时转染含有不同种类的PTHrP/CAT构建体 PTHrP启动子上游的5‘-DNA长度 甲状旁腺素受体基因增强型顺式元件的特征 氢化可的松、维生素D、转化生长因子-β等的表达 监管因素。第三个目标是探索结肠癌 细胞系(LoVo)解释这一事实的细胞内机制 VIP和PTHrP都能刺激cAMP，鸟氨酸脱羧酶(ODC) MRNA和ODC活性，但它们对细胞生长的净影响是相反的- VIP是刺激性的，而PTHrP是抑制的。建议的研究应 确定PTHrP在肠上皮和肠上皮细胞中的新的生长调节作用 并阐明其作用机制及调节因素 肠道相关组织中甲状旁腺素相关蛋白的产生。总体而言，这项工作应该推动 我们对这种多肽在健康和健康中的重要作用的理解 疾病。

The overall hypothesis addressed by the proposed work is that Ca- regulatory peptides, particularly parathyroid hormone-related peptide (PTHrP) play important regulatory roles in the gut. A major thrust in the proposed project is to identify and elucidate mechanisms by which PTHrP functions in gut epithelium to regulate growth and differentiation. One specific aim is designed to test rigorously the idea that PTHrP is an autocrine/paracrine regulatory agent acting locally near the site of its production, perhaps even on the very cell producing the peptide. Studies in normal rats are designed to examine gut for co-localization of PTHrP and its receptor mRNAs and peptide. Changes with aging, pregnancy, lactation, and other physiologic states where gut cell growth is naturally altered will be studied. Co-production of PTHrP and its receptor in the same cell lines and the same cell will be studied by in situ hybridization, analysis of gene expression in a single cell and peptide immunocytochemistry. The growth regulatory role of PTHrP will be examined in epithelial gut cell lines that both produce and respond to PTHrP. Antisera shown to neutralize the biologic (cAMP-stimulatory) effect of PTHrP will be used to verify initial studies indicating enhanced growth of cells treated with antiserum. Effects of PTHrP to slow the cell cycle or to enhance programmed cell death (apoptosis) are outlined. A second specific aim will pursue novel initial findings that PTHrP is expressed in liver and can act to regulate growth of the liver. PTHrP mRNA and peptide will be studied in regenerating liver from partially hepatectomized rats and in gestation, lactation, and aging. A well differentiated human liver cell line (Hep G2), which we have found to produce prodigious amounts of PTHrP, will be studied. Initial findings showing enhanced cell growth in cells treated with antiserum to PTHrP which neutralizes PTHrP biologic activity will be pursued and the mechanisms examined. Preliminary findings that PTHrP production by these cells can be regulated by hydrocortisone, vitamin D, and other agents will be studied and the mechanisms involved elucidated. Hep G2 cells will be transiently transfected with PTHrP/CAT constructs containing varying lengths of 5 '-DNA upstream of PTHrP promoters to identify and characterize cis-elements which account for enhanced PTHrP gene expression in response to hydrocortisone, vitamin D, TGF-Beta, and other regulatory factors. A third aim is designed to explore, in a colon cancer cell line (LoVo) the intracellular mechanisms which account for the fact that VIP and PTHrP both stimulate cAMP, ornithine decarboxylase (ODC) mRNA, and ODC activity, but their net effect on cell growth is opposite - VIP being stimulatory and PTHrP inhibitory. The proposed studies should identify new growth regulatory roles for PTHrP in gut epithelium and in liver and clarify the mechanisms involved and the factors regulating PTHrP production in gut-related tissues. Overall the work should promote our understanding of the important roles this peptide plays in health and disease.