The acute regulation of POMC gene expression by the polypeptide hormone CRH in AtT20 cells.
Item
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Title
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The acute regulation of POMC gene expression by the polypeptide hormone CRH in AtT20 cells.
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Identifier
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AAI9130344
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identifier
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9130344
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Creator
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Lorang, Dominique.
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Contributor
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Adviser: James L. Roberts
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Date
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1991
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular | Biology, Neuroscience
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Abstract
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The goal of this thesis project is to elucidate the second messenger pathways mediating corticotropin releasing hormone (CRH)-regulated proopiomelanocortin (POMC) gene expression in the mouse AtT-20 D16/16 anterior pituitary cell line. In this study we demonstrate that the polypeptide hormone CRH and the synthetic glucocorticoid dexamethasone differentially regulate POMC gene transcription in AtT20 cells similar to the rat corticotrope in primary culture. In order to examine the role of second messengers in the regulation of POMC gene expression by CRH, we measured the levels of POMC heteronuclear RNA (hnRNA) in nuclear RNA samples by a solution hybridization/nuclease protection assay and the rate of POMC gene transcription by a nuclear transcription run-on assay after short-term treatment with various Ca{dollar}\sp{lcub}2+{rcub}{dollar}- and cAMP-elevating secretagogues in AtT20 cells. In all run-on assays, the rate of POMC gene transcription reflected changes observed in POMC hnRNA levels measured under identical treatment conditions. Acutely elevating intracellular Ca{dollar}\sp{lcub}2+{rcub}{dollar} with the ionophore ionomycin or the dihydropyridine agonist Bay K is sufficient for activating POMC gene transcription and elevating POMC hnRNA levels with a magnitude of induction comparable to that elicited by cAMP-elevating agents. Therefore, both Ca{dollar}\sp{lcub}2+{rcub}{dollar} and cAMP are important second messengers regulating POMC gene expression. Combined Bay K and CRH treatment resulted in a greater increase in POMC hnRNA levels over treatment with either agent alone suggesting an interaction between Ca{dollar}\sp{lcub}2+{rcub}{dollar}-and cAMP-related second messenger pathways. Activating the protein kinase C pathway with phorbol esters had no effect on endogenous POMC gene transcription or the expression of transiently transfected POMC-CAT fusion genes in AtT20 cells. In a series of POMC promoter/deletion experiments, we identified a Ca{dollar}\sp{lcub}2+{rcub}{dollar}/cAMP regulatory region of the POMC 5{dollar}\sp\prime{dollar} flanking sequence located between {dollar}-{dollar}236 and {dollar}-{dollar}133 bp relative to the transcription initiation start site. Cytosolic elevations of either cAMP or Ca{dollar}\sp{lcub}2+{rcub}{dollar} stimulate POMC gene transcription to a lesser extent than does the combination of cAMP and Ca{dollar}\sp{lcub}2+{rcub}{dollar} through this discrete POMC promoter region, indicating an interaction between these signaling pathways perhaps at the level of binding of trans-acting factors to DNA.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
