Dennis B. Lubahn

Vascular estrogen receptors and endothelium-derived nitric oxide production in the mouse aorta. Gender difference and effect of estrogen receptor gene disruption.

The present study was designed to test the hypothesis that estrogen receptors (ER) in the blood vessel wall play a role in the modulation of the release of endothelium-derived nitric oxide (EDNO). Both basal and stimulated release of EDNO were determined in aortic rings isolated from female and male wild-type and male homozygous estrogen receptor knock-out (ERKO) mice. 125I-17beta-estradiol binding in aortic tissue showed significantly more high affinity cytosolic- nuclear-binding sites in male compared with female wildtype mice. Estrogen receptor transcripts were present in the aorta of male wild-type mice, but they were absent in male ERKO animals. Basal release of EDNO (determined by endothelium-dependent contraction caused by NG-nitro-arginine) was significantly higher in aorta of wild-type male mice compared with wild-type female mice, and significantly lower in the aorta of male ERKO compared with male wild-type mice. Acetylcholine-induced endothelium-dependent relaxation was similar in all groups studied. No difference was observed in the activity of calcium-dependent nitric oxide synthase in homogenates of lungs and brain taken from male wild-type and ERKO mice. These studies show a significant association between the number of estrogen receptors and basal release of EDNO in the aorta of mice, and suggest that decreased vascular estrogen receptor number may represent a novel risk factor for cardiovascular diseases.

Masculine Sexual Behavior Is Disrupted in Male and Female Mice Lacking a Functional Estrogen Receptor α Gene

Masculine sexual behavior is regulated by testosterone (T). However, T can be metabolized to form estrogens or other androgens, which then activate their own receptors. We used knockout mice lacking a functional estrogen receptor alpha (ER alpha) gene to test the hypothesis that, following aromatization, T acts via the ER alpha to activate normal masculine sexual behavior. After gonadectomy and T replacement, wild-type (WT) male and female mice displayed masculine behavior. However, given the same T treatment, little masculine behavior was displayed by mice of either sex that lack a normal copy of the ER alpha gene. In particular, the latency to display masculine sex behavior and the number of mount attempts per trial were significantly reduced in the ER alpha- mice compared to WT littermates (P < 0.05). In addition, we found that in both sexes, ER alpha- mice have a smaller cluster of androgen receptor immunoreactivity in the bed nucleus of the stria terminalis. Using adult ER alpha- mice we were unable to determine whether these genotypic differences are due to organizational or activational effects. However, it is clear that the ER alpha plays a key role in the expression of masculine sexual behavior and in the regulation of androgen receptors in a neuronal cell population involved in the display of motivated behaviors.

Estrogen Receptor Function as Revealed by Knockout Studies: Neuroendocrine and Behavioral Aspects☆

Estrogens are an important class of steroid hormones, involved in the development of brain, skeletal, and soft tissues. These hormones influence adult behaviors, endocrine state, and a host of other physiological functions. Given the recent cloning of a second estrogen receptor (ER) cDNA (the ER beta), work on alternate spliced forms of ER alpha, and the potential for membrane estrogen receptors, an animal with a null background for ER alpha function is invaluable for distinguishing biological responses of estrogens working via the ER alpha protein and those working via another ER protein. Data generated to date, and reviewed here, indicate that there are profound ramifications of the ER alpha disruption on behavior and neuroendocrine function. First, data on plasma levels of estradiol (E2), testosterone (T), and luteinizing hormone (LH) in wild-type (WT) versus ER alpha- mice confirm that ER alpha is essential in females for normal regulation of the hypothalamic-pituitary gonadal axis. Second, ovariectomized female ER alpha- mice do not display sexual receptivity when treated with a hormonal regime of estrogen and progesterone that induces receptivity in WT littermates. Finally, male sexual behaviors are disrupted in ER alpha- animals. Given decades of data on these topics our findings may seem self-evident. However, these data represent the most direct test currently possible of the specific role of the ER alpha protein on behavior and neuroendocrinology. The ER alpha- mouse can be used to ascertain the specific functions of ER alpha, to suggest functions for the other estrogen receptors, and to study indirect effects of ER alpha on behavior via actions on other receptors, neurotransmitters, and neuropeptides.

Paracrine Regulation of Epithelial Progesterone Receptor by Estradiol in the Mouse Female Reproductive Tract

Abstract Regulation of progesterone receptor (PR) by estradiol-17β (E2) in mouse uterine and vaginal epithelia was studied. In ovariectomized mice, PR expression was low in both vaginal stroma and epithelium, but high in uterine epithelium. E2 induced PR in vaginal epithelium and stroma, but down-regulated PR in uterine epithelium. Analysis of estrogen receptor α (ERα) knockout (ERKO) mice showed that ERα is essential for E2-induced PR expression in both vaginal epithelium and stroma, and for E2-induced down-regulation, but not constitutive expression of PR in uterine epithelium. Regulation of PR by E2 was studied in vaginal and uterine tissue recombinants made with epithelium and stroma from wild-type and ERKO mice. In the vaginal tissue recombinants, PR was induced by E2 only in wild-type epithelium and/or stroma. Hence, in vagina, E2 induces PR directly via ERα within the tissue. Conversely, E2 down-regulated epithelial PR only in uterine tissue recombinants constructed with wild-type stroma. Therefore, down-regulation of uterine epithelial PR by E2 requires stromal, but not epithelial, ERα. In vitro, isolated uterine epithelial cells retained a high PR level with or without E2, which is consistent with an indirect regulation of uterine epithelial PR in vivo. Thus, E2 down-regulates PR in uterine epithelium through paracrine mechanisms mediated by stromal ERα.

Regulation of Progesterone Receptors and Decidualization in Uterine Stroma of the Estrogen Receptor-α Knockout Mouse

Abstract Regulation of progesterone receptor (PR) in uterine stroma (endometrial stroma plus myometrium) by estrogen was investigated in estrogen receptor-α (ERα) knockout (αERKO) mice. 17β-Estradiol (E2) increased PR levels in uterine stroma of ovariectomized αERKO mice, and ICI 182 780 (ICI) inhibited this E2-induced PR expression. Estrogen receptor-β (ERβ) was detected in both uterine epithelium and stroma of wild-type and αERKO mice by immunohistochemistry. In organ cultures of αERKO uterus, both E2 and diethylstilbestrol induced stromal PR, and ICI inhibited this induction. These findings suggest that estrogen induces stromal PR via ERβ in αERKO uterus. However, this process is not mediated exclusively by ERβ, because in ERβ knockout mice, which express ERα, PR was up-regulated by E2 in uterine stroma. In both wild-type and αERKO mice, progesterone and mechanical traumatization were essential and sufficient to induce decidual cells, even though E2 and ERα were also required for increase in uterine weight. Progesterone receptor was strongly expressed in decidual cells in αERKO mice, and ICI did not inhibit decidualization or PR expression. This study suggests that up-regulation of PR in endometrial stroma is mediated through at least three mechanisms: 1) classical estrogen signaling through ERα, 2) estrogen signaling through ERβ, and 3) as a result of mechanical stimulation plus progesterone, which induces stromal cells to differentiate into decidual cells. Each of these pathways can function independently of the others.

Sex steroid receptor analysis in human melanoma.

Melanomas from 20 patients were evaluated for estrogen and progesterone receptors. No restriction as to patients age, sex, race, or menstrual status was made. Fifteen of the tumors were melanin producing. Of the 20 tumors examined, seven contained more than 3 fm/mg protein of specifically inhibitable estrogen binding. None of the tumors reported here demonstrated either a 4S or 8S binding protein as shown by sucrose density gradient analysis. All tumors in which estradiol binding was observed were melanin producing, whereas none of the amelanotic melanomas (five tumors) bound this steroid. Purified tyrosinase appeared to mimic the estrogen binding detected in melanoma cytosols, as demonstrated with the dextran‐coated charcoal technique. Although the binding of estradiol to tyrosinase was inhibited by DOPA, the binding of estradiol to estrogen receptor preparations was not. These studies represent an extension of previous studies of sex steroid binding in melanomas and suggest that gradient analysis and DOPA inhibition studies should be included in the evaluation of the estrogen binding phenomenon in human melanoma.

Disruption of estrogen receptor signaling enhances intestinal neoplasia in ApcMin/+ mice

Estrogen receptors (ERs) [ERalpha (Esr1) and ERbeta (Esr2)] are expressed in the human colon, but during the multistep process of colorectal carcinogenesis, expression of both ERalpha and ERbeta is lost, suggesting that loss of ER function might promote colorectal carcinogenesis. Through crosses between an ERalpha knockout and Apc(Min) mouse strains, we demonstrate that ERalpha deficiency is associated with a significant increase in intestinal tumor multiplicity, size and burden in Apc(Min/+) mice. Within the normal intestinal epithelium of Apc(Min/+) mice, ERalpha deficiency is associated with an accumulation of nuclear beta-catenin, an indicator of activation of the Wnt-beta-catenin-signaling pathway, which is known to play a critical role in intestinal cancers. Consistent with the hypothesis that ERalpha deficiency is associated with activation of Wnt-beta-catenin signaling, ERalpha deficiency in the intestinal epithelium of Apc(Min/+) mice also correlated with increased expression of Wnt-beta-catenin target genes. Through crosses between an ERbeta knockout and Apc(Min) mouse strains, we observed some evidence that ERbeta deficiency is associated with an increased incidence of colon tumors in Apc(Min/+) mice. This effect of ERbeta deficiency does not involve modulation of Wnt-beta-catenin signaling. Our studies suggest that ERalpha and ERbeta signaling modulate colorectal carcinogenesis, and ERalpha does so, at least in part, by regulating the activity of the Wnt-beta-catenin pathway.

Silver staining of histones in Triton-acid-urea gels

A reliable method for silver staining histones in Triton-acid-urea gels was developed. Optimum staining is achieved by treating the gels either with amido black or a colorless, water-soluble analog of amido black, 2,7-naphthalenedisulfonic acid, prior to staining with ammoniacal silver. Staining of purified calf thymus histones H2A, H2B, H3, and H4 by this method is 30 times more sensitive than staining with amido black alone, allowing the detection of each histone and its modified forms down to the nanogram level. The use of 2,7-naphthalenedisulfonic acid dramatically shortens the procedure permitting histone patterns to be visualized within 5 h.

Stromal-Epithelial Cell Communication in the Female Reproductive Tract

The female reproductive organs undergo well-characterized structural and functional changes during the estrous or menstrual cycles and the establishment and maintenance of pregnancy. These reproductive tract changes are primarily under the control of hormones such as estrogens, specifically estradiol 17-β (E2), and progesterone (P). Owing to the critical role of E2 and P in the normal functioning of the female reproductive tract, as well as the role of these hormones in the etiology and progression of various diseases of the reproductive organs, it is important to understand the cellular and molecular mechanisms by which these hormones act.

Enhanced Induction of Mucin-Depleted Foci in Estrogen Receptor β Knockout Mice

The role of the estrogen receptor β (ERβ) in the colon has received considerable interest, yet in vivo models are needed to better define its protective actions. In the present study, wild-type (WT), ERα, and ERβ knockout (αERKO and βERKO) mice were injected with azoxymethane, a colon chemical carcinogen. Fourteen weeks after azoxymethane exposure, the incidence of aberrant crypt foci (ACF) was assessed by methylene blue staining. βERKO mice showed significantly higher incidence (P < 0.001) of ACF (15.0 ± 2.5) compared with αERKO (3.4 ± 1.0) and WT (4.6 ± 1.0) mice. The colons in several βERKO mice had increased thickness and loss of normal morphology. It has been reported that ERβ plays a role in the maintenance of the colonic crypt architecture; this may explain the loss of crypt organization in the colonic epithelium of βERKO mice. The presence of mucin-depleted foci (MDF) has been shown, both in humans and in rodents, as an early event in colon cancer. Therefore, to surpass the limitations with ACF scoring, we performed Alcian blue-neutral red staining to assess the presence of MDF. This assay allowed the assessment of precancerous lesions on all the βERKO mice colons (38.3 ± 4.0; P < 0.001), comparing to WT and αERKO mice (6.6 ± 1.5 and 10.0 ± 1.9, respectively), and served to confirm the ACF results. Together, these data support the use of MDF staining as a biomarker for precancerous lesions and the protective role of ERβ in colon carcinogenesis. Cancer Prev Res; 3(9); 1198–204. ©2010 AACR.