Differential regulation and action of estrogen receptors alpha and beta in GH3 cells.

The pituitary lactotroph, a well established target for estrogens, expresses estrogen receptor-alpha (ER alpha) and -beta (ER beta). A truncated isoform of ER alpha, named TERP, is expressed in the pituitary, but not in the uterus. In this study we used the somatolactotroph cell line, GH3 cells, to examine 1) the expression of ER alpha, TERP, or ER beta and their regulation by estradiol; 2) the presence of receptor proteins; and 3) the effects of overexpressing ER beta or TERP on estrogen induction of the PRL gene and activation of the estrogen response element (ERE). Incubation of GH3 cells with estradiol (0.1-10 nM) produced dose-dependent increases in messenger RNA levels of ER beta and TERP, but not ER alpha, as determined by quantitative RT-PCR. Cell incubation with 1 nM estradiol resulted in a time-dependent biphasic increase in TERP and a delayed rise in ER beta, suggesting activation by both direct and indirect mechanisms. A polyclonal ER beta antibody directed against an N-terminal synthetic peptide was generated. This antibody detected ER beta-positive cells in ovarian granulosa cells and in many cells throughout the pituitary; its specificity was demonstrated by preabsorption with the synthetic peptide. The antibody detected a 58- to 60-kDa protein by Western blotting of ovarian, pituitary, and GH3 cell extracts. Cotransfection of ER beta and reporter genes (PRL promoter/luciferase or ERE/luciferase) into GH3 cells resulted in a dose-dependent increase in estrogen-induced PRL gene expression, with a lesser activation of the ERE. A 20-kDa TERP protein was undetectable in untreated GH3 cells and was weakly induced by estradiol. Overexpression of TERP had no effect on estrogen induction of either PRL or ERE. We conclude that 1) both ER beta and TERP messenger RNAs in GH3 cells are increased by estradiol in a dose- and time-dependent manner, whereas ER alpha is not altered; 2) a 58-kDa ER beta protein is expressed in both the pituitary and GH3 cells; and 3) overexpression of ER beta increases estrogen-induced PRL gene expression.

Transcription of prolactin gene in milk secretory cells of the rat mammary gland.

In-situ hybridization and Northern blot hybridization were used to identify mRNA for pituitary prolactin in mammary tissue obtained from female rats 1 day before expected parturition, 1 day after parturition and on day 7 of lactation. Prolactin cDNA was labelled with 32P for Northern analysis and with digoxigenin for in-situ hybridization. Total and poly(A)+ RNA from pituitary, mammary and control (fat and kidney) tissues were analysed by agarose gel electrophoresis with transfer to nitrocellulose and hybridization to a cDNA for rat prolactin. Although present in much smaller amounts than the 1.0 kb transcript in pituitary RNA homogenates, mammary RNA homogenates from all three stages contained mRNA of approximately 1.0 kb which hybridized with the prolactin probe. Similar analyses of fat and kidney failed to reveal any hybridization at the 1.0 kb size. When tissue sections were hybridized to the cDNA probe, specific hybridization was observed in the milk secretory cells of the mammary alveoli and the lactotroph cells of the anterior pituitary, but not in liver cells or in RNAase-treated sections of mammary tissue. In summary, these results demonstrate that milk secretory cells of the rat mammary gland transcribe the gene for prolactin, and they raise the possibility that a primary target tissue for blood-borne prolactin may also synthesize prolactin.