Brief exposure of rats to high-dose estrogen during the neonatal period interrupts prostate development in a lobe-specific manner and predisposes the gland to dysplasia with aging, a phenomenon referred to as developmental estrogenization. Our previous studies have revealed that these effects are initiated through altered steroid receptor expression; however, the immediate downstream targets remain unclear. We have recently shown that developmental expression of Shh-ptc-gli is downregulated in the dorsolateral prostate following estrogenization, and this is responsible, in part, for branching deficits observed in that prostatic region specifically. In the present study, we examine the role of Fgf10 signaling during rat prostate development and as a mediator of the developmental estrogenized phenotype. Fgf10 and FgfR2iiib localize to the distal signaling center of elongating and branching ducts in separate prostate lobes where they regulate the expression of multiple morphoregulatory genes including Shh, ptc, Bmp7, Bmp4, Hoxb13, and Nkx3.1. Ventral and lateral lobe organ cultures and mesenchyme-free ductal cultures demonstrate a direct role for Fgf10/FgfR2iiib in ductal elongation, branching, epithelial proliferation, and differentiation. Based on these findings, a model is proposed depicting the localized expression and feedback loops between several morphoregulatory factors in the developing prostate that contribute to tightly regulated branching morphogenesis. Similar to Shh-ptc-gli, neonatal estrogen exposure downregulates Fgf10, FgfR2iiib, and Bmp7 expression in the dorsolateral prostate while ventral lobe expression of these genes is unaffected. Lateral prostate organ culture experiments demonstrate that growth and branching inhibition as well as Fgf10/FgfR2iiib suppression are mediated directly at the prostatic level. Furthermore, exogenous Fgf10 fully rescues the growth and branching deficits due to estrogen exposure. Together, these studies demonstrate that alterations in Fgf10 signaling are a proximate cause of Shh-ptc-gli and Bmp7 downregulation that together result in branching inhibition of the dorsolateral prostate following neonatal estrogen exposure.
While prostate gland development is dependent on androgens, other hormones including retinoids and estrogens can influence this process. Brief exposure to high-dose estrogen during the neonatal period in rats leads to permanent, lobe-specific aberrations in the prostate gland, a phenomenon referred to as developmental estrogenization. We have previously shown that this response is mediated through alterations in steroid receptor expression; however, further downstream mechanisms remain unclear. Herein, we examined Sonic hedgehog (Shh)-patched (ptc)-gli in the developing rat prostate gland, its role in branching morphogenesis, and the effects of neonatal estrogens on its expression and localization to determine whether a disturbance in this signaling pathway is involved in mediating the estrogenized phenotype. Shh was expressed in epithelial cells at the distal tips of elongating ducts in discreet, heterogeneous foci, while ptc and gli1-3 were expressed in the adjacent mesenchymal cells in the developing gland. The addition of Shh protein to cultured neonatal prostates reduced ductal growth and branching, decreased Fgf10 transcript, and increased Bmp4 expression in the adjacent mesenchyme. Shh-induced growth suppression was reversed by exogenous Fgf10, but not noggin, indicating that Fgf10 suppression is the proximate cause of the growth inhibition. A model is proposed to show how highly localized Shh expression along with regulation of downstream morphogens participates in dichotomous branching during prostate morphogenesis. Neonatal exposure to high-dose estradiol suppressed Shh, ptc, gli1, and gli3 expressions and concomitantly blocked ductal branching in the dorsal and lateral prostate lobes specifically. In contrast, ventral lobe branching and Shh-ptc-gli expression were minimally affected by estrogen exposure. Organ culture studies with lateral prostates confirmed that estradiol suppressed Shh-ptc-gli expression directly at the prostatic level. Taken together, the present findings indicate that lobe-specific decreases in Shh-ptc-gli expression are involved in mediating estradiol-induced suppression of dorsal and lateral lobe ductal growth and branching during prostate morphogenesis.
Axis positioning and tissue determination during development involve coordinated expression of Hox genes throughout the body. The most posterior Hox gene clusters are involved in prostate organogenesis. In the present study, we characterized and compared the expression profiles of posterior (5') Hox genes in the separate lobes of the adult rat prostate gland, the coagulating gland, seminal vesicles, and epididymis using quantitative real-time RT-PCR. These genes include Hoxa9-11, Hoxa13, Hoxd13, and Hoxb13. We identified a unique Hox code for each of these organs and propose that this contributes to the organ-specific and prostate lobe-specific identities in the adult rat. Using the ventral prostate (VP) as a model, we characterized the Hox genes expression patterns over time from birth through adulthood. Expression levels of the three Hox13 genes and Hoxa10 were significantly higher in the adult VP compared with the neonatal developing VP suggesting an important role during adult homeostasis. In contrast, Hoxa9 and Hoxa11 levels declined after morphogenesis suggesting a specific developmental role. Overall, the Hoxb13 gene exhibited the most striking temporal and organ-specific differences. Using in situ hybridization and immunohistochemistry, a distinct Hoxb13 anterior-to-posterior expression gradient was observed with the highest expression levels in the VP luminal epithelial cells, moderate levels in the lateral prostate, and low expression in the dorsal prostate. An expression gradient was also observed along the ductal length in all three prostate lobes with strongest expression at the distal tips and limited expression in the proximal ducts. After infection with a lentivirus expressing the Hoxb13 gene, NRP-152 cells cultured under nondifferentiating conditions exhibited robust cytokeratin 8 immunostain indicating that Hoxb13 expression drives luminal cell differentiation in the rat epithelium. Androgen regulation of prostatic Hox gene expression was examined during development in vitro and after castration in the adult rat. In the neonatal VP, all six Hox genes were significantly up-regulated by androgens, whereas none of the genes were affected by testosterone in the lateral prostate. In the adult rat, castration resulted in up-regulation of Hoxa9 and Hoxa13 in the VP and down-regulation of Hoxb13 in the dorsal prostate and lateral prostate. Taken together, we conclude that the prostatic Hox genes reach a destined expression level at specific developmental time points in the prostate gland and possess differential androgenic regulation in a temporal and lobe-specific manner. We suggest that this timely Hox code participates in determining lobe-specific prostatic identity and cellular differentiation.
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, ERα, ERβ, and RARs are observed. We propose that estrogeninduced 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 Fgf 10 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.
The Wnt genes encode a large family of secreted glycoproteins that play important roles in controlling tissue patterning, cell fate and proliferation during development. Currently, little is known regarding the role(s) of Wnt genes during prostate gland development. The present study examines the role of the noncanonical Wnt5a during prostate gland development in rat and murine models. In the rat prostate, Wnt5a mRNA is expressed by distal mesenchyme during the budding stage and localizes to periductal mesenchymal cells with an increasing proximal-to-distal gradient during branching morphogenesis. Wnt5a protein is secreted and localizes to periductal stroma, extracellular matrix and epithelial cells in the distal ducts. While Wnt5a expression is high during active morphogenesis in all prostate lobes, ventral prostate (VP) expression declines rapidly following morphogenesis while dorsal (DP) and lateral lobe (LP) expression remains high into adulthood. Steroids modulate prostatic Wnt5a expression during early development with testosterone suppressing Wnt5a and neonatal estrogen increasing expression. In vivo and ex vivo analysis of developing mouse and rat prostates were used to assess the functional roles of Wnt5a. Wnt5a−/− murine prostates rescued by organ culture exhibit disturbances in bud position and directed outgrowth leading to large bulbous sacs in place of elongating ducts. In contrast, epithelial cell proliferation, ductal elongation and branchpoint formation is suppressed in newborn rat prostates cultured with exogenous Wnt5a protein. While renal grafts of Wnt5a−/− murine prostates revealed that Wnt5a is not essential for cyto- and functional differentiation, a role in luminal cell polarity and lumenization of the ducts was indicated. Wnt5a suppresses prostatic Shh expression while Shh stimulates Wnt5a expression in a lobe-specific manner during early development indicating that Wnt5a participates in cross-talk with other members of the gene regulatory network that control prostate development. Although Wnt5a does not influence prostatic expression of other Wnt morphogens, it suppresses Wif-1 expression and can thus indirectly modulate Wnt signaling. In summary, the present finds demonstrate that Wnt5a is essential for normal prostate development where it regulates bud outgrowth, ductal elongation, branching, cell polarity and lumenization. These findings contribute to the growing body of knowledge on regulatory mechanisms involved in prostate gland development which are key to understanding abnormal growth processes associated with aging.
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