The overall hypothesis addressed by the proposed work is that the Ca- regulatory/cell growth peptide, parathyroid hormone-related protein (PTHrP), plays important regulatory cells in the gut and liver. The major thrust is to identify and elucidate molecular mechanisms by which PTHrP functions in gut epithelium and hepatocytes to regulate cell growth and turnover.
Specific aim 1 will utilize GI and liver cells treated with exogenous PTHrP analogs or stably transfected with PTHrP cDNA in a sense or antisense or antisense orientation to study mechanisms and modes of action or PTHrP on cell proliferation and cell death (apoptosis). PTHrP cDNA bearing mutations of the peptide's nuclear targeting sequences, as well as neutralizing PTHrP antisera will be employed by discern whether the protein acts in an intracrine fashion by targeting directly to the nucleus or largely by autocrine/paracrine mechanisms following secretion. Mechanisms underlying changes in cell proliferation or cell death will be examined systematically using a variety of methods already in hand.
Specific aim 2 will extend the vitro findings in aim 1 to the intact animal by establishing transgenic mice that over- or under- express PTHrP in the gut and liver. The animals will receive a transgene construct containing the coding sequence for full-length PTHrP driven by a fatty acid binding protein promoter sequence that targets protein expression to the intestinal epithelium and liver. Studies of peptide expression using histological, immunoassay, and RNA detection methods will be coupled to studies assessing biological functioning of the gut and complex context of the whole organism.
Specific aim 3 is designed to express sufficient amounts of recombinant PTHrP to enable study of its 3D structure. An intein-mediated purification of expressed protein using an affinity chitin-binding column will be used to generate the mg quantities of protein required for 1D, 2D, and eventual 3D analysis. The overall goal is to understand the fundamental structure of the peptide and relate that to findings from eventual studies of its interaction with receptor proteins that mediate its effects or with enzymes that process it. Overall the work will provide insight into the fundamental actions of PTHrP in the gut and liver and should enhance our understanding of the important roles this protein plays in health and disease.
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