Expression of estrogen receptors in human corpus cavernosum and male urethra.

Estrogen, largely produced in testis and adrenal gland, may play important roles in male reproduction. Most of the effects of estrogens are mediated by binding of estrogen to one or both of the two estrogen receptor (ER) subtypes alpha and beta. Recently, they have been described in testis, prostate, and efferent ducts, mostly in rodents. The goal of this study was to prove the evidence of ERs in human corpus cavernosum and male urethra, exploring the protein expression of these receptors by immunohistochemistry. Corpus cavernosum and corpus spongiosum smooth muscle was immunoreactive for the androgen receptor (AR), ER alpha, and strongly for ER beta. Endothelial cells were negative for AR, sporadically positive for ER alpha, and positive for ER beta. Urethral epithelium showed strong nuclear expression of AR, predominantly in the basal cell layer, and nuclear expression of ER alpha in the intermediate cells. ER beta was highly expressed in almost all urethral nuclei and, much more weakly, in cytoplasm. Progesterone receptor (PGR) was negative in all cases and all tissues. These results represent the first report that ER alpha and particularly ER beta are regularly expressed in human penile tissue.     

Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein-coupled receptor homolog, GPR30, and occurs via trans-activation of the epidermal growth factor receptor through release of HB-EGF

Estrogen rapidly activates the mitogen-activated protein kinases, Erk-1 and Erk-2, via an as yet unknown mechanism. Here, evidence is provided that estrogen-induced Erk-1/-2 activation occurs independently of known estrogen receptors, but requires the expression of the G protein-coupled receptor homolog, GPR30. We show that 17beta-estradiol activates Erk-1/-2 not only in MCF-7 cells, which express both estrogen receptor alpha (ER alpha) and ER beta, but also in SKBR3 breast cancer cells, which fail to express either receptor. Immunoblot analysis using GPR30 peptide antibodies showed that this estrogen response was associated with the presence of GPR30 protein in these cells. MDA-MB-231 breast cancer cells (ER alpha-, ER beta+) are GPR30 deficient and insensitive to Erk-1/-2 activation by 17beta-estradiol. Transfection of MDA-MB-231 cells with a GPR30 complementary DNA resulted in overexpression of GPR30 protein and conversion to an estrogen-responsive phenotype. In addition, GPR30-dependent Erk-1/-2 activation was triggered by ER antagonists, including ICI 182,780, yet not by 17alpha-estradiol or progesterone. Consistent with acting through a G protein-coupled receptor, estradiol signaling to Erk-1/-2 occurred via a Gbetagamma-dependent, pertussis toxin-sensitive pathway that required Src-related tyrosine kinase activity and tyrosine phosphorylation of tyrosine 317 of the Shc adapter protein. Reinforcing this idea, estradiol signaling to Erk-1/-2 was dependent upon trans-activation of the epidermal growth factor (EGF) receptor via release of heparan-bound EGF (HB-EGF). Estradiol signaling to Erk-1/-2 could be blocked by: 1) inhibiting EGF-receptor tyrosine kinase activity, 2) neutralizing HB-EGF with antibodies, or 3) down-modulating HB-EGF from the cell surface with the diphtheria toxin mutant, CRM-197. Our data imply that ER-negative breast tumors that continue to express GPR30 may use estrogen to drive growth factor-dependent cellular responses.     

Epitope-dependent localization of estrogen receptor-alpha,but not -beta,in en face arterial endothelium

Rapid, nongenomic effects of 17 beta-estradiol (E(2)) in endothelial cells are postulated to arise from membrane-associated estrogen receptors (ERs), which have not been visualized in vascular tissue. To identify membrane ERs, we used multiple site-directed ER alpha or ER beta antibodies to label en face rat cerebral and coronary arterial endothelia. Western blots revealed a novel 55-kDa ER alpha isoform. Three-dimensional images of cells labeled with these antibodies and markers for the nucleus and caveolin-1 were acquired with a wide-field microscope, deconvolved, and numerically analyzed. We found ER alpha in the nucleus and cell periphery, where one-third colocalized with caveolin-1. The receptor location was dependent on the epitope of the antibody. Human ovarian surface epithelium produced similar results; but in rat myometrium, the distribution was epitope independent and nuclear. ER beta distribution was predominantly intranuclear and epitope independent. A small amount of ER alpha colocalized with ER beta within the nucleus. The results were identical in both arterial preparations and insensitive to E(2). We postulate that the different ER alpha conformations at the membrane, in the nucleus, and between different cell types allow E(2) to trigger cell- and location-specific signaling cascades.     

Estrogen receptor beta in health and disease

Estrogens, acting through its two receptors, ESR1 (hereafter designated ER alpha) and ESR2 (hereafter designated ER beta), have diverse physiological effects in the reproductive system, bone, cardiovascular system, hematopoiesis, and central and peripheral nervous systems. Mice with inactivated ER alpha, ER beta, or both show a number of interesting phenotypes, including incompletely differentiated epithelium in tissues under steroidal control (prostate, ovary, mammary, and salivary glands) and defective ovulation reminiscent of polycystic ovarian syndrome in humans (in ER beta-/- mice), and obesity, insulin resistance, and complete infertility (both in male and female ER alpha-/- mice). Estrogen agonists and antagonists are frequently prescribed drugs with indications that include postmenopausal syndrome (agonists) and breast cancer (antagonists). Because the two estrogen receptors (ERs) have different physiological functions and have ligand binding pockets that differ enough to be selective in their ligand binding, opportunities now exist for development of novel ER subtype-specific selective-ER modulators.     

Nuclear location of hormone-free estrogen receptors by monoclonal antibodies could be a tissue-fixation dependent artifact

The 'two-step' model proposed by Jensen and his collaborators for explaining estrogen action conceptualized hormone-free estrogen receptors (ER) to be cytoplasmic, and hormone-filled, transformed ER to be nuclear. Applying monoclonal antibodies which recognized epitopes in ER and formaldehyde-fixed tissues, King et al demonstrated exclusively nuclear staining in target tissues utilizing immunoperoxidase technique. Recently these antibodies have become commercially available enabling other investigators to conduct studies. In this report, using these monoclonal antibodies we have demonstrated that a change in the concentration of formaldehyde alters the staining pattern yielding cytoplasmic instead of nuclear staining in calf uterus, MCF-7 cells, and ER(+) human breast cancer. In addition, neutralization of the antibody activity was not achieved with freshly prepared ER(+) cytosols. Formaldehyde-treated cytosols were essential. These results ought to caution investigators in determining in vivo location of antigens based on the staining pattern obtained in fixed tissues. Furthermore, this effect of formaldehyde on estrogen receptors may be applicable to other steroid hormone receptors.