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 i s 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 i s 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.
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