Association of increased estrogen receptor beta2 expression with parity-induced alterations in the rat mammary gland.

In this study, we investigated the cellular and molecular events involved in parity-related alterations in mammary gland (MG) proliferation and differentiation. Rat MGs were removed on day 9 of either first (nulliparous), second (primiparous) or third (multiparous) pregnancy. Expression of steroid hormone receptors along with cellular biomarkers of proliferation and differentiation were quantified in all MG tissue compartments by immunohistochemistry. Wnt-4 (a Wingless-like morphogenic gene involved in MG development), ERbeta and ERbeta2 mRNA were evaluated by RT-PCR analysis. Serum levels of mammotrophic hormones were measured. In comparison to nulliparous and primiparous rats, multiparous animals exhibited decreased luminal cell proliferation and PR levels, whereas alpha-lactalbumin, ERalpha, ERbeta and ERbeta2 expression were increased. In myoepithelial cells, while parity induced a decrease in proliferative activity, subsequent pregnancies and lactations lead to an increased state of differentiation. Our results showed that at least two periods of pregnancy and lactation were necessary to modify the studied parameters. The lower proliferative activity and higher differentiation state of the multiparous MG are associated with both a decreased PR expression and increased ERalpha and ERbeta expression. Since ERbeta and/or ERbeta2 isoform expression was related to parity history, results suggest that the decreased proliferative activity and PR expression observed in the MG of multiparous animals may be associated with overexpression of ERbeta and/or the ERbeta2 isoform, thereby antagonizing the proliferative effects associated with ERalpha.

Characterization of a plasma membrane-resident albumin-binding protein associated with the proliferation of estrogen-target, serum-sensitive cells.

Estrogens control the proliferation of their target cells through a receptor-mediated pathway. Recently presented evidence suggests that estradiol cancels the proliferative inhibition exerted by human albumin (HA) and recombinant human albumin (rHA) on estrogen-target serum-sensitive cells (indirect-negative hypothesis). We postulate that this mechanism requires the presence of a plasma membrane estrogen receptor (mER) and a plasma membrane albumin-binding protein (mABP). Direct evidence confirming the presence of mERalpha in MCF7 cells has recently been presented. Herein, we now show that Western blot analysis of purified T47D membrane proteins with the C542 ERalpha specific monoclonal antibody also revealed specific, multiple M(r) mERs (67, 110, and 130k M(r)). In addition, Western blot analysis with an ABP antiserum revealed a potential 60k M(r) ABP in both MCF7 and T47D plasma membrane extracts. No such evidence was observed in similar extracts from ER-negative, serum-insensitive MDA-MB231 cells. Ligand blot analysis of similar plasma membrane extracts with bovine serum albumin confirmed the presence of a 60k M(r) ABP in MCF7 and T47D cells; again, no such evidence was observed in comparable extracts from MDA-MB231 cells. Fluorescence and confocal microscopy of MCF7 cells fixed in 2.0% paraformaldehyde/0.1% glutaraldehyde identified specific membrane ABP antigenic sites by immunocytochemistry. Serum-insensitive MDA-MB231 cells fixed and labeled similarly did not exhibit this mABP. These results suggest that the proposed mABP is expressed only in serum-sensitive estrogen-target cells and is not expressed in cells insensitive to the proliferative inhibition of HA and rHA. Also, the present data suggest that the proposed mABP may be the recognition mechanism by which both HA and rHA inhibit MCF7 and T47D cell proliferation.

Mechanism of androgen action on cell proliferation: AS3 protein as a mediator of proliferative arrest in the rat prostate.

Androgens control the proliferation of their target cells first by increasing cell proliferation and later by inhibiting the proliferation of those same cells. Recently, we reported that the AS3 protein mediates the androgen-induced quiescence in androgen-target human cell lines. Our aims were to investigate the expression of the AS3 protein in the rat prostate in situ and in human cells in culture. Adult rats were separated into four groups (intact, castrated, castrated plus 3-d testosterone propionate replacement, and castrated plus 7-d testosterone propionate replacement). S9 cells expressing a tetracycline-regulated sense AS3 were also used. AS3 was expressed in the nuclei of over 90% of the epithelial cells and about 40% of the smooth muscle cells of the intact rat prostate. AS3 was not expressed in castrated rats or during the proliferative phase of androgen-induced regeneration. It was expressed in intact and castrated animals when the prostate has reached adult organ size. The AS3 protein was not expressed in cells that incorporate bromodeoxyuridine. These data suggest that AS3 is a mediator of the proliferative arrest in the normal rat prostate in situ and human prostate cell lines and that its expression is androgen-induced.