Differential Regulation and Action of Estrogen Receptors   and   in GH3 Cells

Abstract

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.

The role of IGF1 on the differentiation of prolactin secreting cells in the mouse anterior pituitary

“…EGF induces the expression of dopamine receptor and transdifferentiation into lactotroph-like cells in the GH3 cell line (Missale et al 1991) and PRL cell differentiation in the MtT/S cell line (Goda et al 1998). Ben-Jonathan et al (2009) show that the ability of EGF to stimulate Prl gene expression and release is mediated by Erk1/2-induced phosphorylation of Esr1 in GH3 cells, which express both Esr1 and Esr2 (Mitchner et al 1999). Thus, E 2 and EGF can share a common pathway in the control of Prl gene expression.…”

Gonadotrope oestrogen receptor-α and -β and progesterone receptor immunoreactivity after ovariectomy and exposure to oestradiol benzoate, tamoxifen or raloxifene in the rat: correlation with LH secretion

“…Thus, the disruption of ER gene has little effect on pituitary function in ER-knockout models (Couse & Korach 1999), and oestrogen treatment has little or no effect on ER in pituitary cells (Mitchner et al 1999). Much more is known about the ER subtype, which is the predominant isoform in most pituitary secretory cells (Scully et al 1999).…”

Estrogen Receptors α and β in Human Testis: Both Isoforms are Expressed

“…The specificity of these antibodies detecting ERa and ERb proteins has been previously described in tissues known to express these receptors [Kruijver et al 2003;Mitchner et al 1999]. As secondary antibody against ERa and ERb, a biotinylated goat anti-rabbit IgG antibody (Sigma-Aldrich) was used at a dilution of 1:20.…”