The role of estrogens in normal and abnormal development of the prostate gland

Abstract

Estrogens play a physiologic role during prostate development with regard to programming stromal cells and directing early morphogenic events. However, if estrogenic exposures are abnormally high during the critical developmental period, permanent alterations in prostate branching morphogenesis and cellular differentiation will result, a process referred to as neonatal imprinting or developmental estrogenization. These perturbations are associated with an increased incidence of prostatic lesions with aging, which include hyperplasia, inflammation, and dysplasia. To understand how early estrogenic exposures can permanently alter the prostate and predispose it to neoplasia, we examined the effects of estrogens on prostatic steroid receptors and key developmental genes. Transient and permanent alterations in prostatic AR, ERalpha, ERbeta, and RARs are observed. We propose that estrogen-induced alterations in these critical transcription factors play a fundamental role in initiating prostatic growth and differentiation defects by shifting the prostate from an androgen-dominated gland to one whose development is regulated by estrogens and retinoids. This in turn leads to specific disruptions in the expression patterns of key prostatic developmental genes that normally dictate morphogenesis and differentiation. Specifically, we find transient reductions in Nkx3.1 and permanent reductions in Hoxb-13, which lead to differentiation defects particularly within the ventral lobe. Prolonged developmental expression of Bmp-4 contributes to hypomorphic growth throughout the prostatic complex. Reduced expression of Fgf10 and Shh and their cognate receptors in the dorsolateral lobes leads to branching defects in those specific regions in response to neonatal estrogens. We hypothesize that these molecular changes initiated early in life predispose the prostate to the neoplastic state upon aging.

FIGURE 1

Schematic representation of steroid receptor expression in postnatal day 5–10 developing prostates from oil-treated control rats (A) and neonatally estrogenized rats (B). In the normal developing prostate (A), androgen receptor (AR) is the dominant steroid receptor in both epithelial and stromal cells. Under the influence of androgens, the stromal cells produce and secrete specific paracrine factors that dictate growth and differentiation of the gland. As epithelial cells differentiate, AR levels markedly increase and ERβ expression is induced. Other steroid receptors are expressed in a cell-specific manner and regulate cell-specific gene expression during critical developmental windows. Estrogen receptor α (ERα) is expressed at low levels in periductal stromal cells in the proximal region of the elongating ducts. RARβ is expressed in a subpopulation of basal epithelial cells, whereas RARα and γ are localized to periductal stromal cells along the ductal length. RXRα and RXRγ are expressed by basal cells, while RXRβ localizes to periductal stromal cells. Following a brief exposure to high levels of estrogens during the neonatal critical period, the prostatic steroid receptor profile is drastically altered (B). AR is absent in epithelial cells and is present at very low levels in stromal cells, thus dampening the androgen signaling pathway in the developing prostate. ERα is upregulated and expressed at high levels in periductal stromal cells along the length of the ducts, and progesterone receptor (PR) is induced in those same cells under the influence of estrogen. The number of cells expressing RARα and RARβ is markedly increased. Thus, estrogen exposure has switched the developing prostate from an androgen-dominated tissue to one that is regulated by estrogen, progesterone, and retinoids.

FIGURE 2

Real-time qRT-PCR of Nkx3.1expression in the developing rat prostate lobes. Samples were amplified in duplex using a SYBR-green assay and data were normalized to the ribosomal protein, RPL19 mRNA co-amplified for each tissue. A: Ventral lobe (VP) expression levels of days 1, 3, 6, 10, 15, 30, and 90 of life show a peak of expression at postnatal day 10 with low steady-state expression observed in adulthood. B: Postnatal days 6 and 10 expression levels in the VP, dorsal (DP), and lateral (LP) prostate lobes of rats exposed to oil (hatched bars) or 25 μg estradiol benzoate on days 1, 3, and 5 of life (solid bars). N = 4–10 assays/group. *P < 0.01 compared to estradiol-treated rats at same time point. (from Pu et al. Reprinted by permission.)

FIGURE 3

Expression and role of Bmp4 in the developing rat prostate. A: Whole-mount in situ hybridization of Bmp4 mRNA expression in a day 1 prostate complex shows broad mesenchymal Bmp4 expression in dorsal (DP) and lateral (LP) lobes at the early budding stage and signal condensation in periductal mesenchyme as ducts elongate and branch in the more advanced ventral (VP) prostate. B: VP Bmp4 mRNA levels over time as measured by real time qRT-PCR. Expression declined in control rats (hatched bar) as morphogenesis proceeds with significant loss by day 15 (*P < 0.05 vs. day 6 oil-treated tissue). Following neonatal estradiol exposure (solid bars), Bmp4 expression remained at high perinatal levels until after day 30 (+ P < 0.01 vs. day 6 estrogen-treated tissue). Bars represent mean ± SEM of 4–6 samples. C: VP organ culture for 8 days in the presence of 10⁻⁸ M testosterone (T), T + noggin (a Bmp4 anatgonist), T plus 20 μg estradiol (T + E) and T + E + noggin. Contralateral lobes were used for the T-only group and the T + E groups. A bar graph shows the area on day 8 normalized to day 0 (bars represent mean ± SEM for 6 experiments), while representative photos from each group at start of culture (day 0) and after 8 days are shown below. *P < 0.05 vs. T + E + noggin.

FIGURE 4

A schematic representation of the lobe-specific, estrogen-induced alterations in critical morphoregulatory genes in the rodent prostate. Brief neonatal exposure to high-dose estradiol results in alterations in expression, of key developmental genes in a lobe-specific manner that produces lobe-specific phenotypes. In the dorsoalateral lobes, estrogens suppress Shh signaling and Fgf 10 signaling, transiently reduce Nkx3.1 expression, and increase Bmp-4 levels. This results in phenotype X, which consists of branching deficiencies, hypomorphic growth, and mild epithelial differentiation defects. In the ventral lobe, estrogens permanently suppress Hoxb-13, transiently suppress Nkx3.1, and increase Bmp-4 expression postnatally. This leads to phenotype Y, which consists of severe differentiation defects and hypomorphic growth. In total, both common and unique phenotypes are proposed to result from differential regulation of key morphoregulatory genes by early estrogenic exposures.