Differential Regulation and Action of Estrogen Receptors α and β in GH3 Cells.

The pituitary lactotroph, a well established target for estrogens, expresses estrogen receptor-a (ERa) and -b (ERb). A truncated isoform of ERa, 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 ERa, TERP, or ERb and their regulation by estradiol; 2) the presence of receptor proteins; and 3) the effects of overexpressing ERb 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 dosedependent increases in messenger RNA levels of ERb and TERP, but not ERa, 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 ERb, suggesting activation by both direct and indirect mechanisms. A polyclonal ERb antibody directed against an N-terminal synthetic peptide was generated. This antibody detected ERb-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 58to 60-kDa protein by Western blotting of ovarian, pituitary, and GH3 cell extracts. Cotransfection of ERb 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 ERb and TERP messenger RNAs in GH3 cells are increased by estradiol in a doseand time-dependent manner, whereas ERa is not altered; 2) a 58-kDa ERb protein is expressed in both the pituitary and GH3 cells; and 3) overexpression of ERb increases estrogen-induced PRL gene expression. (Endocrinology 140: 2651–2658, 1999) T PITUITARY lactotroph is a well established target cell of estrogens. Estrogen increases PRL gene expression, synthesis, storage, and release as well as lactotroph proliferation (1–4). Other estrogen-responsive genes in the lactotroph include c-fos (5), galanin (6), and vascular endothelial growth factor (7), which are up-regulated by estrogen, and transforming growth factor-b, which is down-regulated (8). The actions of estrogens could be mediated by three estrogen receptors, ERa, its truncated isoform, and ERb. ERb, first identified in the rat prostate and ovary (9), shares several similarities with ERa. Both receptors have a high binding affinity for estrogen and activate the same estrogen response element (ERE) (10), but differ in their trans-activation domains, suggesting distinct roles in gene activation. At the messenger RNA (mRNA) level, ERb has been detected in many tissues, including rat (11, 12) and human (13), but not mouse (14), pituitaries. Using a combined immunocytochemistry/in situ hybridization approach, we detected ERb expression in most pituitary cell types, including 30% of the lactotrophs (12). However, ERb has not been well demonstrated at the protein level due to the limited availability of specific antibodies. In addition, the specific actions of this receptor within the pituitary gland have not been delineated. The truncated estrogen receptor product (TERP), discovered by Shupnik et al. (15, 16), lacks exons 1–4 of ERa but contains most of the hormone-binding domain and the second trans-activation domain. Given these structural properties, TERP may act either as a dominant negative regulator or as an enhancer of estrogenic action. TERP is expressed in the pituitary, but not in the uterus, and its mRNA levels increase in response to estrogen (12, 16). Pituitary TERP gene expression is altered during the estrous cycle, with the highest mRNA levels seen on the morning of proestrus (17). Although not all of the pituitary cell types that express TERP have been identified, cell separation experiments revealed that the lactotroph-enriched fraction had the highest amount