Title

Regulation of proopiomelanocortin in granulosa cells

Date of Award

1991

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Michael H. Melner, Committee Chair

Abstract

The regulation of the Pro-opiomelanocortin (POMC) gene promoter was examined by transfer of promoter-reporter constructs into primary cultures of rat granulosa cells. The POMC promoter was shown to be stimulated by both gonadotropins and a $\beta$-adrenergic agonist. A similar stimulation was seen with both cAMP analogues and phosphodiesterase inhibitors. Since gonadotropins and $\beta$-adrenergic agonists are known to raise intracellular cAMP levels in granulosa cells, these data suggested a mechanism of action for these agents involving cAMP-dependent protein kinase (PKA). In order to test this hypothesis more directly, an expression vector coding for a mutant PKA regulatory subunit was co-transfected with a POMC promoter-reporter construct. The mutant regulatory subunit, acting as a specific inhibitor of the PKA catalytic subunit, abolished gonadotropin and $\beta$-adrenergic stimulation. A control promoter, that of the SV40 virus early region, was not affected by either treatment with gonadotropin or co-transfection with the PKA regulatory mutant.Deletion mutagenesis of the promoter/reporter construct was performed to delineate DNA sequences important in basal and gonadotropin regulation of POMC expression. Deletion of regions from $-$704 to $-$44 had little effect on expression; however deletion from $-$44 to $-$37 significantly reduced both basal and cAMP-stimulated expression. Deletion of a large part of the 5$\sp\prime$ untranslated region, +3 to +63, also had this effect. These results suggest that two separate regions, $-$44 to $-$37 and +3 to +63. are necessary for both basal and gonadotropin-regulated expression.The ability of dihydrotestosterone (DHT), a non-aromatizable androgen, to stimulate the POMC promoter was also investigated. Preliminary evidence indicates that DHT synergizes with gonadotropin or $\beta$-adrenergic stimulation of both steroidogenesis and POMC promoter activity. However, no independent action of androgens was detected.Two novel methods of measuring the reporter gene, chloramphenicol acetyltransferase (CAT), were also developed. One of the methods depends on high performance liquid chromatographic separation and UV absorption measurement of non-radioactive chloramphenicol. The other method involved the design of a novel fluorescent substrate for CAT which is separated from its acetylated derivatives by thin-layer chromatography.

Keywords

Biology, Molecular; Biology, Cell; Health Sciences, Medicine and Surgery

Link to Full Text

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