ACTIONS OF CALCIUM REGULATING HORMONES IN THE GUT

所提出的工作的总体假设是，钙- 调节肽，特别是甲状旁腺素相关肽 （PTHrP）在肠道中发挥重要的调节作用。一个主要的推力在 拟议的项目是确定和阐明PTHrP的机制， 在肠上皮中起调节生长和分化的作用。一 具体目标旨在严格测试PTHrP是一种 自分泌/旁分泌调节剂，其作用于局部靠近其 生产，甚至可能在产生肽的细胞上。研究 在正常大鼠中，设计用于检查肠道中PTHrP的共定位 及其受体mRNA和肽。随着年龄的增长，怀孕， 哺乳期和其他生理状态，其中肠细胞生长是 自然变化将被研究。PTHrP及其衍生物的联合生产 受体在相同的细胞系和相同的细胞将研究在 原位杂交、单细胞基因表达分析以及 肽免疫细胞化学PTHrP的生长调节作用将是 在肠上皮细胞系中检测， PTHrP。 抗血清显示可中和生物制剂（cAMP刺激） PTHrP的影响将用于验证初步研究表明， 用抗血清处理的细胞生长增强。PTHrP对减缓 细胞周期或增强程序性细胞死亡（凋亡）的方法是 概述。第二个具体目标是追求新的初步发现， PTHrP在肝脏中表达，可以调节肝脏的生长。 PTHrP mRNA和肽将在再生肝中进行研究。 部分肝切除大鼠和妊娠期、哺乳期和衰老期。一 高分化的人肝细胞系（Hep G2），我们发现， 产生大量PTHrP的方法。初步调查结果 显示在用PTHrP的抗血清处理的细胞中细胞生长增强 中和PTHrP生物活性的药物， 检查机制。初步研究结果表明，PTHrP生产这些 细胞可以通过氢化可的松、维生素D和其他药物来调节 将进行研究，并阐明所涉及的机制。Hep G2细胞 瞬时转染PTHrP/CAT构建体， PTHrP启动子上游的5 ′-DNA长度以鉴定和 表征解释增强的PTHrP基因的顺式元件 氢化可的松、维生素D、TGF-β和其他激素的表达 调节因素。第三个目标是探索，在结肠癌中， 细胞系（LoVo）的细胞内机制，占的事实， VIP和PTHrP均能刺激cAMP、ODC mRNA和ODC活性，但它们对细胞生长的净效应是相反的- VIP是刺激性的，PTHrP是抑制性的。拟议的研究应 鉴定PTHrP在肠上皮细胞和肠上皮细胞中新的生长调节作用， 阐明其作用机制和调控因素 肠道相关组织中PTHrP的产生。总的来说，这项工作应促进 我们对这种肽在健康中发挥的重要作用的理解， 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.