Shigehira Saji

A role for estrogen receptor β in the regulation of growth of the ventral prostate

In normal rats and mice, immunostaining with specific antibodies revealed that nuclei of most prostatic epithelial cells harbor estrogen receptor β (ERβ). In rat ventral prostate, 530- and 549-aa isoforms of the receptor were identified. These sediment in the 4S region of low-salt sucrose gradients, indicating that prostatic ERβ does not contain the same protein chaperones that are associated with ERα. Estradiol (E2) binding and ERβ immunoreactivity coincide on the gradient, with no indication of ERα. In prostates from mice in which the ERβ gene has been inactivated (BERKO), androgen receptor (AR) levels are elevated, and the tissue contains multiple hyperplastic foci. Most epithelial cells express the proliferation antigen Ki-67. In contrast, prostatic epithelium from wild-type littermates is single layered with no hyperplasia, and very few cells express Ki-67. Rat ventral prostate contains an estrogenic component, which comigrates on HPLC with the testosterone metabolite 5α-androstane-3β,17β-diol (3βAdiol). This compound, which competes with E2 for binding to ERβ and elicits an estrogenic response in the aorta but not in the pituitary, decreases the AR content in prostates of wild-type mice but does not affect the elevated levels seen in ERβ knockout (BERKO) mice. Thus ERβ, probably as a complex with 3βAdiol, is involved in regulating the AR content of the rodent prostate and in restraining epithelial growth. These findings suggest that ligands specific for ERβ may be useful in the prevention and/or clinical management of prostatic hyperplasia and neoplasia.

Estrogen receptors and proliferation markers in primary and recurrent breast cancer

To elucidate the clinical importance of estrogen receptor (ER) β in breast cancer, 29 archival primary breast cancer specimens, six locally recurrent cancers, and five benign mammary tumors were examined histochemically for ERα, ERβ and the proliferation markers Ki67 and cyclin A. In benign tumors, most epithelial cells contained ERβ, but ERα was rare. In primary cancers, both ERα and ERβ occurred in epithelial cells, the presence of ERβ being associated with elevated expression of Ki67 and cyclin A, and ERα with decreased levels. Thus, the highest content of proliferation markers was seen in primary cancers that were ERα− ERβ+. Most Ki67-containing cells coexpressed ERβ, but few showed ERα. In locally recurring cancers, ERα, ERβ, and Ki67 were more highly expressed than in the corresponding primary tumors, and many cells containing ERβ, but few with ERα, expressed Ki67. Surprisingly, ERβ, but not ERα, was seen in the stromal cells of both primary and recurrent cancers. Because the response of breast cancers to tamoxifen therapy is correlated with the presence of ERα, cancer cells that lack ERα but contain ERβ and proliferation markers represent a novel population of apparently proliferating cells that probably are not targeted by the current antiestrogens. Thus, appropriate ERβ-specific ligands, perhaps in combination with tamoxifen, may be useful in improving the treatment of breast cancers.

A Role for the Androgen Receptor in Follicular Atresia of Estrogen Receptor Beta Knockout Mouse Ovary

Abstract Estrogen receptor beta (ERβ) is highly expressed, but ERα is not detectable in granulosa cells in the mouse ovary. In ERβ knockout (BERKO) mice, there is abnormal follicular development and very reduced fertility. At 3 wk of age, no significant morphologic differences were discernable between wild type (WT) and BERKO mouse ovaries, but by 5 mo of age, atretic follicles were abundant in BERKO mice and there were very few healthy late antral follicles or corpora lutea. At 2 yr of age, unlike the ovaries of their WT littermates, BERKO mouse ovaries were devoid of healthy follicles but had numerous large, foamy lipid-filled stromal cells. The late antral and atretic follicles in BERKO mice were characterized by a high level of expression of the androgen receptor (AR) and IGF-1 receptor. These proteins were abundantly expressed in granulosa cells of preantral and early antral follicles in both genotypes, but their expression was extinguished in late antral follicles of WT mice. Healthy late antral follicles and corpora lutea were restored in BERKO ovaries after 15 days of treatment of mice with the antiandrogen flutamide. The results suggest that in the absence of ERβ there was a loss of regulation of AR. Because androgens enhance recruitment of primordial follicles into the growth pool and cause atresia of late antral follicles, the inappropriately high level of AR probably is related to the follicular atresia and to the early exhaustion of follicles in BERKO mice.

Quantitative Analysis of Estrogen Receptor Proteins in Rat Mammary Gland

Estrogen receptor a and b proteins (ERa and ERb) at various stages of development of the rat mammary gland were quantified by Western blotting. ERa and ERb recombinant proteins were used as standards, and their molar concentrations were measured by ligand binding assays. In 3-week-old pregnant, lactating, and postlactating rats the ERa content ranged from 0.30 ‐1.55 fmol/mg total protein (mean values). The ERb content of the same samples ranged between 1.06 ‐7.50 fmol/mg total protein. At every developmental stage, the ERb content of the mammary gland was higher than that of ERa. When receptor levels were normalized against b-actin, it was evident that ER expression changed during development, with maximum expression of both receptors during the lactation period. With an antibody raised against the 18-amino acid insert of the ERb variant, originally called ERb2 but named ERbins in this paper, Western blots revealed that ERbins protein was up-regulated during the lactation period. RT-PCR showed that the levels of messenger RNA of ERbins paralleled those of the protein. Double immunohistochemical staining with anti-ERa and anti-ERbins antibodies revealed that ERbins protein colocalized with ERa in 70 ‐ 80% of the ERa-expressing epithelial cells during lactation and with 30% of these cells during pregnancy. These observations indicate that expression of ERbins is regulated not only quantitatively, but also with regard to its cellular distribution. As ERbins acts as the dominant repressor of ERa, we suggest that its coexpression with ERa quenches ERa function and may be one of the factors that contribute to the previously described insensitivity of the mammary gland to estrogens during lactation. (Endocrinology 142: 3177‐3186, 2001)

Involvement of Androgen Receptor in 17β-Estradiol-Induced Cell Proliferation in Rat Uterus

Abstract Although it is known that, in the uterus, estrogen receptor α (ERα) is involved in proliferation and progesterone receptor in differentiation, the role of the two other gonadal-hormone receptors expressed in the uterus, androgen receptor (AR) and estrogen receptor β (ERβ), remains undefined. In this study, the involvement of AR in 17β-estradiol (E2)-induced cellular proliferation in the immature rat uterus was investigated. AR levels were low in the untreated immature uterus, but 24 h after treatment of rats with E2, there was an increase in the levels of AR and of two androgen-regulated genes, IGF-I and Crisp (cysteine-rich secretory protein). As expected, E2 induced proliferation of luminal epithelial cells. These actions of E2 were all blocked by both the antiestrogen tamoxifen and the antiandrogen flutamide. The E2-induced AR was found by immunohistochemistry to be localized exclusively in the stroma, mainly in the myometrium, where it colocalized with ERα but not with ERβ. ERβ, detected with two different ERβ-specific antibodies, was expressed in both stromal and epithelial cells either alone or together with ERα. Treatment with E2 caused down-regulation of ERα and ERβ in the epithelium. The data suggest that, in E2-induced epithelial cell proliferation, ERα induces stromal AR and AR amplifies the ERα signal by induction of IGF-I. Because AR is never expressed in cells with ERβ, it is unlikely that ERβ signaling is involved in this pathway. These results indicate an important role for AR in proliferation of the uterus, where estrogen and androgen do not represent separate pathways but are sequential steps in one pathway.

The Normal and Malignant Mammary Gland: A Fresh Look with ERβ Onboard

Estrogens are important for the development and function of the normal mammary gland as well as for development of mammary cancer. The frontline therapy for treatment of estrogen receptor (ERα)4 positive breast cancer is antiestrogens. A second estrogen receptor (ERβ) is also expressed in the breast but it has not been measured because it is not detected by the immunoassays used to detect ERα. In many cell systems ERβ has actions which are opposite to those of ERα and this finding has raised questions about the role of ERβ in the development and treatment of breast cancer.