Pituitary-Specific and Hormonally Regulated Gene Expression Directed by the Rat Proopiomelanocortin Promoter in Transgenic Mice

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Abstract
All aspects of POMC biosynthesis exhibit tissue-specific regulation. The single copy gene is highly expressed in anterior lobe (AL) corticotrophs and intermediate lobe (IL) melanotrophs of the pituitary gland and in the arcuate nucleus of the hypothalamus. POMC gene transcription in corticotrophs is induced by hypothalamic CRH and vasopressin and inhibited by adrenal glucocorticoids, while in melanotrophs it is predominantly regulated by .beta.-adrenergic neural input and dopamine. To identify the rat POMC (rPOMC) gene sequence necessary and sufficient to target expression and hormonal regulation in corticotrophs and melanotrophs, we generated 13 transgenic mice carrying rPOMC fusion genes. The genes consisted of 706 or 480 basepairs of rPOMC 5'' flanking sequences ligated to either the Escherichia coli LacZ gene encoding .beta.-galactosidase or the K1 mutant of the SV40 large T-antigen gene. Overall, half of the transgenic lines had reporter gene expression in their AL and IL in a pattern indistinguishable from ACTH immunohistochemistry. In three of these lines, .beta.-galactosidase or K-1 T-antigen was localized by double immunofluorescence exclusively to ACTH-positive corticotrophs and melanotrophs. Transcriptional regulation of the rPOMC-LacZ fusion gene in response to hormonal manipulation was quantified by a fluorescence assay for .beta.-galactosidase enzyme activity in pituitary extracts. There was a 15-fold increase in AL enzyme activity after adrenalectomy and a 3-fold increase in IL activity after haloperidol treatment. X-gal histochemistry of pituitaries from hormonally treated mice confirmed the cellular specificity of these effects. Expression of the transgenes was not detected in the arcuate nucleus of the hypothalamus, nucleus of the tractus solitarius, or the reproductive tract of any of the transgenic mice. These data suggest that 706 basepairs or less of rPOMC 5'' flanking sequence are sufficient for the following aspects of POMC gene regulation: corticotroph- and melanotroph-specific expression, glucocorticoid-mediated inhibition in the AL, and dopaminergic inhibition in the IL.

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