Ligand-Independent Activation of Pituitary ER: Dependence on PKA-Stimulated Pathways

Abstract

In pituitary and other target tissues, estrogen acts through ERs, which are ligand-activated nuclear transcription factors. ERs can also be activated by intracellular signaling pathways in a ligand-independent manner in some cells. Because the pituitary is the target of several cAMP-activating factors, we examined the ability of cAMP to activate ERs in the alphaT3 gonadotrope cell line. Forskolin, 8-bromo-cAMP, and pituitary adenylate cyclase-activating polypeptide all enhanced ER-dependent promoter activity, which was inhibited by antiestrogen or a pituitary-specific inhibitory ER variant. Activation was PKA dependent and was blocked by the PKA inhibitor H89 or cotransfection of the inhibitor PKI. Although cAMP activated MAPK in alphaT3 cells, inhibition of MAPK with the MEK inhibitor PD98059 did not prevent forskolin-induced ER activation. Similarly, epidermal growth factor did not stimulate ER activity, although it increased MAPK activation. Forskolin-induced activation of ER was enhanced by cotransfection of steroid receptor coactivator-1 and was inhibited by the repressor of ER action, suggesting that cAMP does not alter the normal interactions between ER and cofactors. In contrast to results with estrogen, cAMP treatment did not decrease ER protein levels. These results demonstrate that in the pituitary, cAMP activates ER in a ligand-independent manner exclusively through PKA.

Effects of FSH and 17β-estradiol on the transactivation of estrogen-regulated promoters and cell proliferation in L cells

“…As shown in Fig. 1a, non-radiolabelled E 2 , ICI182,780 and TAM efficiently competed with [ 3 H]17␤-estradiol for specific binding to ERs present in cytosol preparations from L-(hFSHR − ) cells (IC 50 16 ± 0.10, 21 ± 0.13 and 15 ± 0.09 nM, for E 2 , ICI182,780 and TAM, respectively), whereas increasing concentrations of the synthetic progestin norethisterone was unable to displace [ 3 H]17␤-estradiol from the receptor. The presence of both ER␣ and ER␤ in these cells was confirmed by immunocytochemical studies (Fig.…”

“…Estrogen receptor activation modulates transcription of specific genes by engaging a number of transcription factors (co-activators or co-repressors) in target cells, which in turn negatively or positively regulate transcriptional activity of ligand-activated receptors [7,8] In addition to estrogen-mediated activation, the transcriptional effects of ERs can be mediated through several mechanisms other than estradiol (E 2 )-ER complexes binding to DNA [9,10]. It is now accepted that ERs may be activated in the absence of E 2 by a variety of extracellular signals, including growth factors [11][12][13] and other cell surface membrane-targetted signals that trigger activation of a number of kinase-regulated signaling cascades including the cAMP/protein kinase A (PKA) pathway [12,[14][15][16][17], protein kinase C (PKC) [14,18,19], phosphatidylinositol 3 (PI3)-dependent kinase (PI3-K)/Akt [20,21], and mitogenactivated protein kinase (MAPK) pathways [11,[22][23][24]. Selective expression of receptors for these ligands, along with differences in ER isoform and coactivator/corepressor protein expression allow for target cell-and promoter-specific effects of activated ERs [6,8,[25][26][27].…”

Estrogen and progesterone receptors: from molecular structures to clinical targets

“…The involvement of PKA in ER phosphorylation remains controversial and cannot explain the enhancement of transcription by itself. While some researchers claim that a contribution could also be due to phosphorylation of SRC-1 by PKA on the amino acid residues T1179 and S1185 [126], others state that the co-activator SRC-1 cannot be phosphorylated directly by PKA [127]. Recent evidence suggests that phosphorylation of ER-S305, within the D-Domain of ERa, leads to an altered orientation between ER and its co-activator SRC-1.…”

Absence of ligand-independent transcriptional activation of the estrogen receptor via the estrogen response element in pituitary lactotrophs in primary culture

“…These findings indicate that growth factors exert their actions through the ER. Indeed, a number of studies using ERE-containing promoter-driven reporter genes have shown ligand-independent ERE-mediated transcriptional activation in a variety of cell lines in response to EGF [9][10][11][12], transforming growth factor (TGF)-␣ [9,12], insulin-like growth factor-1 (IGF-1) [9,[12][13][14], insulin [13][14][15], heregulin [16], cAMP [10,11,17], dopamine [10,18,19], and tetradecanoyl 12-phorbol 13-acetate [12]. The physiological and pathological significance of ligand-independent ER activation, however, remains to be clarified.…”

“…We developed an adenovirus-mediated, Cre/loxP-based, lactotrophspecific, ER transcriptional assay, which we named LASETA. This assay specifically targeted the lactotrophs within a primary culture of pituitary cells which also contains estrogen-responsive gonadotrophs suggested to exhibit ligand-independent ER activation in response to cAMP administration [17]. The Cre/loxP system was used to restrict the luciferase reporter gene expression to lactotrophs by regulating the expression of Cre recombinase by PRL promoter activity.…”