HORMONAL REGULATION OF GALANIN GENE EXPRESSION

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Galanin is a widely-distributed peptide which modulates pituitary, pancreatic, and gastric hormone secretion. Within the rat pituitary, galanin production is limited to the lactotrophic and somatotrophic cells where both synthesis and secretion are influenced by circulating estrogens. Estrogens have a dramatic effect on pituitary galanin gene expression. Steady-state mRNA levels vary 30-fold during the normal estrous cycle of adult female rates, and increase another 10-fold during gestation. Galanin-specific mRNA accounts for up to 0.5% of total mRNA in estrogen exerts transcriptional control over galanin gene expression in these cells. These observations suggest that the galanin gene provides an excellent model for the study of the specific interaction of hormonal and cell- specific regulators of transcription. The proposed studies will investigate the mechanisms by which the rat galanin gene is regulated in pituitary cells. The effects of estrogen on galanin gene transcription will be further characterized. The DNA sequences with confer estrogen-responsiveness will be identified by transient expression assays using deletion mutants. We will then evaluate the ability of these sequences to bind and be activated by estrogen receptor protein. The basis for cell lineage specificity of expression will be examined by transfection of galanin-producing and on-producing cell lines. We will attempt to discern DNA sequences within the galanin gene which confer cell type specificity. Once identified, those sequences will be used to isolate the protein(s) responsible for the observed specificity and to identify cDNA clones which encode such protein(s). Particular attention will be given to the evaluation of the role of pit-1(GHF-1) in the control of galanin expression. Pit-1/GHF-1 is a transcriptional activator which appears to determine the cell-type specificity of prolactin and growth hormone gene expression in the rat pituitary. Pit-1/GHF-1 gene expression is limited to pituitary lactotrophs and somatotrophs and appears to be negatively regulated by estrogen. We will determine whether this protein regulates galanin expression and how its activity is affected by estrogen. We will also evaluate whether galanin secreted in response to estrogen regulates pit-1/GHF-1 gene expression. The interaction of cell- and hormone-specific regulatory proteins will be evaluated in transient expression assays by mutational analysis of cis- elements. If we are successful in identifying a cell-specific regulator of galanin gene expression, we will examine whether the expression of the regulatory protein is itself regulated by estrogen and whether production of the protein affects the estrogen-responsiveness of galanin gene expression.