Molecular mechanism of estrogen receptor (ER)alpha-specific, estradiol-dependent expression of the progesterone receptor (PR) B-isoform.

The physiological effects of progesterone are mediated by the progesterone receptor (PR) isoforms PRA and PRB, transcribed from a single gene, under control of two distinct promoters. Both the isoforms display different, promoter- and cell line-specific transactivation properties. Upregulation of both isoforms in response to estradiol stimulation has been described, although the two promoters contain no classical estrogen response element (ERE). Therefore, we decided to investigate the regulation of PRB-expression through distinct estrogen receptor (ER)-isoforms: ERalpha and ERbeta We demonstrate, that in HeLa cells treated with E2, PRB promoter activity was enhanced (five-fold) by ERalpha, but not by ERbeta. ERbeta was also unable to stimulate activity of the PRB promoter in BT20 and Ishikawa cells, where ERalpha induced reporter activity by two-fold. Deletion of the AF1-but not AF2 domain from ERalpha resulted in loss of the transactivation potential in all cell lines tested. Furthermore, in BT20 cells deletion of the AF2 domain of ERalpha resulted in stronger transcriptional activation than that mediated through wild-type ERalpha. In SK-BR-3 cells both ERs repressed PRB promoter activity and this repression was enhanced by co-transfection of SRC1. However, strong estrogen-dependent stimulation was observed after deletion of AF2. We conclude that PRB expression is stimulated by ERalpha but not ERbeta in an unique, AF1-dependent but AF2-independent mechanism.

Insights into the molecular biology of the estrogen receptor define novel therapeutic targets for breast cancer.

Evidence for a role of ovarian factors in the growth of metastatic breast cancer was first recognized over 100 years ago. Today, anti-estrogens are central to the treatment of breast cancer of all stages. We now understand that the action of estrogen is mediated by the estrogen receptors (ER) which are members of the nuclear receptor family of ligand-regulated transcription factors. In this article we review the molecular mechanisms through which ER activates transcription of target genes and through which available anti-estrogens mediate their therapeutic effects. We discuss possible mechanisms of failure of treatment with current anti-estrogens and how newer anti-estrogens under development attempt to address these problems. In addition an expanded view of the molecular mechanisms of estrogen action is leading to the development of novel selective ER modulators or SERMs.

Herpes gestationis may present itself as a paraneoplastic syndrome of choriocarcinoma-a case report.

BACKGROUND: Herpes gestationis (HG) is a rare, recurrent, pruritic, vesicobullous dermatosis occurring predominantly in pregnancy and seldom in early puerperium. Reports of the association of HG with choriocarcinoma are extremely rare and this case highlights such a possible link. CASE: This case focuses on the late postpartal manifestation of HG being associated with metastatic high-risk choriocarcinoma. Direct immunofluorescence was used to verify the diagnosis of HG. Symptomatic therapy with corticosteroids and antihistamines alleviated the pruritic symptoms associated with HG, but only the recurrent course of chemotherapy for the choriocarcinoma resulted in complete disappearance of all signs and symptoms of the HG. CONCLUSION: The time course of this case highlights once again the possible association of HG as a paraneoplastic syndrome of choriocarcinoma.

Hormones and hormone antagonists: mechanisms of action in carcinogenesis of endometrial and breast cancer.

Proliferation of breast and endometrial cells is under the control of ovarian steroid hormones (SHs) such as oestrogen and progesterone. They mediate diverse physiological functions via interaction with nuclear-localised steroid hormone receptors (HRs). The SH receptor complex modifies the expression of SH-regulated genes by binding to conserved binding sites in their promoter region or through cross-talk with other transcription factors. In non-malignant tissues, HRs are in balance with other factors regulating proliferation, differentiation and apoptosis. While dysfunction of the regulatory mechanisms is a part of malignant transformation, functional SH receptors can promote growth of SH-responsive tumours. Therefore, anti-hormones that block the interaction of steroid hormones with the SH receptor are useful tools for the treatment of SH-responsive carcinomas. However, a portion of ER-positive breast cancers and most endometrial cancers do not respond to anti-oestrogens and continued treatment results in hormone resistance, mostly without loss of the ER. This review focuses on the mechanisms of action of hormones and anti-hormones in breast and endometrial carcinomas.

Gene amplification and expression of the steroid receptor coactivator SRC3 (AIB1) in sporadic breast and endometrial carcinomas.

Steroid receptor coactivator 3 (SRC3) functions as a coactivator for nuclear receptor mediated transcriptional activation. It binds to nuclear receptors in a ligand-dependent fashion and recruits other factors such as CBP and p300 to the transactivation complex. Due to its function as activator of nuclear receptors, overexpression of SRC3 might enhance their effects. Gene amplification is a common mechanism that causes overexpression, already described for oncogenes like c-erbB2, c-myc and int2. In this study, SRC3 gene amplification and expression levels were analyzed in 127 sporadic breast carcinomas, 30 endometrial carcinomas and different cell lines (MCF7, HeLa, Ishikawa, T47D, BT-20, SK-BR-3, HEC-1a, RL 95-2, OVCAR3 and A-431). To determine gene amplification and mRNA expression levels, quantitative differential PCR and RT-PCR were performed in combination with fluorescent DNA technology. Gene amplification was not found in any of the breast and endometrial carcinomas, but was found in the carcinoma cell lines MCF7 (10-fold) and HeLa (3-fold). SRC3 overexpression was detected in 13% (3/23) of breast carcinomas and 17% (5/30) of endometrial carcinomas, as well as in MCF7 and HeLa cells. Thus, SRC3 overexpression found in breast and endometrial tumors is not caused by SRC3 gene amplification. A carcinogenic potential provided by SRC3 overexpression has to be elucidated in further studies.

Ribosomal subunit kinase-2 is required for growth factor-stimulated transcription of the c-Fos gene.

Ribosomal subunit kinases (Rsk) have been implicated in the regulation of transcription by phosphorylating and thereby activating numerous transcription factors, such as c-Fos, cAMP responsive element binding protein (CREB), and nuclear receptors. Here we describe the generation and characterization of immortalized embryonic fibroblast cell lines from mice in which the Rsk-2 gene was disrupted by homologous recombinant gene targeting. Rsk-2-deficient (knockout or KO) cell lines have no detectable Rsk-2 protein, whereas Rsk-1 expression is unaltered as compared with cell lines derived from wild-type control mice. KO cells exhibit a major reduction in platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF)-1-stimulated expression of the immediate-early gene c-Fos. This results primarily from a reduced transcriptional activation of the ternary complex factor Elk-1 and reduced activation of the serum response factor. The reduced Elk-1 activation in KO cells occurs despite normal activation of the mitogen-activated protein kinase pathway and normal PDGF- and IGF-1-stimulated Elk-1 phosphorylation. By contrast, PDGF- and IGF-1-stimulated phosphorylation and transcriptional activation of CREB is unaltered in KO cells. Thus Rsk-2 is required for growth factor-stimulated expression of c-Fos and transcriptional activation of Elk-1 and the serum response factor, but not for activation of CREB or the mitogen-activated protein kinase pathway in response to PDGF and IGF-1 stimulation.

Functional analysis of a novel estrogen receptor-beta isoform.

A new level of complexity has recently been added to estrogen signaling with the identification of a second estrogen receptor, ERbeta. By screening a rat prostate cDNA library, we detected ERbeta as well as a novel isoform that we termed ERbeta2. ERbeta2 contains an in-frame inserted exon of 54 nucleotides that results in the predicted insertion of 18 amino acids within the ERbeta hormone-binding domain. We also have evidence for the expression of both ERbeta1 and ERbeta2 in human cell lines. Competition ligand binding analysis of bacterially expressed fusion proteins revealed an 8-fold lower affinity of ERbeta2 for 17beta-estradiol (E2) [dissociation constant (Kd approximately 8 nM)] as compared with ERbeta1 (Kd approximately 1 nM). In vitro transcribed and translated ERbeta1 and ERbeta2 bind specifically to a consensus estrogen responsive element in a gel mobility shift assay. Furthermore, we show heterodimerization of ERbeta1 and ERbeta2 with each other as well as with ERalpha. In affinity interaction assays for proteins that associate specifically with the hormone-binding domain of these receptors, we demonstrate that the steroid receptor coactivator SRC-1 interacts in an estrogen-dependent manner with ERalpha and ERbeta1, but not with ERbeta2. In cotransfection experiments with expression plasmids for ERalpha, ERbeta1, and ERbeta2 and an estrogen-responsive element-containing luciferase reporter, the dose response of ERbeta1 to E2 was similar to that of ERalpha although the maximal stimulation was approximately 50%. In contrast, ERbeta2 required 100- to 1000-fold greater E2 concentrations for maximal activation. Thus, ERbeta2 adds yet another facet to the possible cellular responses to estrogen.

Inhibition of estrogen receptor action by the orphan receptor SHP (short heterodimer partner).

SHP (short heterodimer partner) is an unusual orphan receptor that lacks a conventional DNA-binding domain. Previous results have shown that it interacts with several other nuclear hormone receptors, including the retinoid and thyroid hormone receptors, and inhibits their ligand-dependent transcriptional activation. Here we show that SHP also interacts with estrogen receptors and inhibits their function. In mammalian and yeast two-hybrid systems as well as glutathione-S-transferase pull-down assays, SHP interacts specifically with estrogen receptor-alpha (ERalpha) in an agonist-dependent manner. The same assay systems using various deletion mutants of SHP map the interaction domain with ERalpha to the same SHP sequences required for interaction with the nonsteroid hormone receptors such as retinoid X receptor and thyroid hormone receptor. In transient cotransfection assays, SHP inhibits estradiol -dependent activation by ERalpha by about 5-fold. In contrast, SHP interacts with ERbeta independent of ligand and reduces its ability to activate transcription by only 50%. These data suggest that SHP functions to regulate estrogen signaling through a direct interaction with ERalpha.

p300 is a component of an estrogen receptor coactivator complex.

The estrogen receptor (ER) is a ligand-dependent transcription factor that regulates expression of target genes in response to estrogen in concert with other cellular signaling pathways. This suggests that the mechanism by which ER transmits an activating signal to the general transcription machinery may include factors that integrate these diverse signals. We have previously characterized the estrogen receptor-associated protein, ERAP160, as a factor that complexes with ER in an agonist-dependent manner. We have now found that the transcriptional coactivator p300 associates with agonist bound ER and augments ligand-dependent activation by ER. Our studies show that an ER coactivator complex involves a direct hormone-dependent interaction between ER and ERAP160, resulting in the recruitment of p300. In addition, antibodies directed against the cloned steroid receptor coactivator 1 (SRC1) recognize ERAP160. The known role of p300 in multiple signal transduction pathways, including those involving the second messenger cAMP, suggests p300 functions as a point of integration between ER and these other pathways.