Paracrine regulation of apoptosis by steroid hormones in the male and female reproductive system

In males, androgens are essential in maintaining the integrity of the prostate. Androgen-ablation induces apoptosis of the prostatic epithelium. In females, ovariectomy induces apoptosis in uterine epithelium while progesterone inhibits this process. The objective of this study was to determine whether androgen and progesterone inhibit apoptosis, respectively, in mouse prostatic and uterine epithelia via steroid receptors in the epithelium or in the stroma. To address this question, prostatic tissue recombinants were prepared with rat urogenital sinus mesenchyme plus bladder epithelium from wild-type or testicular feminization mutant (Tfm) mice. Thus, prostatic tissue was generated having androgen receptor (AR) in both epithelium and stroma or in the stroma only. Castration of hosts induced apoptosis in the AR-negative Tfm prostatic epithelium with an epithelial apoptotic index virtually identical to prostatic tissue recombinants containing wild-type epithelium. Moreover, this castration-induced prostatic epithelial apoptosis was blocked by testosterone and dihydrotestosterone in both wild-type and Tfm prostatic tissue recombinants. Likewise, uterine tissue recombinants were prepared in which epithelium and/or stroma was devoid of progesterone receptor (PR) by using uterine epithelium and stroma of wild-type and PR knockout mice. Progesterone inhibited uterine epithelial apoptosis only in tissue recombinants prepared with PR-positive stroma. The PR status of the epithelium did not affect epithelial apoptotic index. Therefore, the apoptosis in prostatic and uterine epithelia is regulated by androgen and progesterone via stromal AR and PR, respectively. In both cases, epithelial AR or PR is not required for hormonal regulation of epithelial apoptosis in prostatic and uterine epithelium.     

Transforming growth factor beta 1 and androgen receptors in prostate neoplasia

OBJECTIVE: To investigate the interplay between transforming growth factor (TGF) beta 1, androgen receptors and stromal-epithelial interactions in benign prostatic hyperplasia (BPH), prostate intraepithelial neoplasia (PIN) and prostate carcinoma areas of prostate neoplasia. STUDY DESIGN: In this immunohistochemical study we investigated staining patterns and then determined the correlation between TGF-beta 1 expression and androgen receptor status in the epithelium and stroma of 60 paraffin-embedded tissues from radical prostatectomies. RESULTS: Staining patterns differed in the epithelium and stroma of tumor and peritumor prostatic tissue. TGF-beta 1 immunostaining (H-scores) in the epithelium and stroma increased significantly from BPH to PIN and from BPH to prostate carcinoma in the epithelium (P < .05), whereas androgen receptor (AR) immunoreactivity significantly (P < .05) increased from BPH to PIN to prostatic carcinoma in epithelium and stroma. TGF-beta 1 did not correlate with histologic grade of differentiation, whereas AR proteins were more strongly expressed in Gleason score 5 and 6 than score 7 tumors (P < .05). Nonlinear regression showed a significant correlation (P < .01) between TGF-beta 1 and AR expression only in the stromal compartment of PIN. CONCLUSION: These findings argue in favor of an interaction between TGF-beta 1 and AR in the early stages of prostate carcinogenesis and suggest that TGF-beta 1 plays a central role in stromal-epithelial interactions during the early stages of malignant transformation.     

Mesenchymal-epithelial interactions: past, present, and future

Abstract This review summarizes the history of research on mesenchymal–epithelial interactions in prostatic development from the first studies in 1970 to the present. From this study we have learned that prostatic development requires a reciprocal interaction between epithelium and mesenchyme in which urogenital sinus mesenchyme induces and patterns epithelial development and differentiation, while developing prostatic epithelium induces and patterns mesenchymal differentiation into smooth muscle and other resident cell types in the stroma. Prostatic development requires androgen action mediated by the androgen receptor (AR). Through analysis of tissue recombinants composed of wild-type and AR-null epithelium and mesenchyme, we have learned that many "androgenic effects" on prostatic epithelium do not require epithelial AR, but instead are elicited by the paracrine action of AR-positive mesenchyme. Present and future studies reviewed in this issue deal with the molecular mechanisms in this developmental communication between epithelium and mesenchyme.     

Hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development

This review on normal and neoplastic growth of the prostate emphasizes the importance of epithelial-mesenchymal/stromal interactions. Accordingly, during prostatic development urogenital sinus mesenchyme (a) specifies prostatic epithelial identity, (b) induces epithelial bud formation, (c) elicits prostatic bud growth and regulates ductal branching, (d) promotes differentiation of a secretory epithelium, and (e) specifies the types of secretory proteins expressed. In reciprocal fashion, prostatic epithelium induces smooth muscle differentiation in the mesenchyme. Epithelial-mesenchymal interactions during development continue postnatally into adulthood as stromal-epithelial interactions which play a homeostatic role and in so doing reciprocally maintain epithelial and stromal differentiation and growth-quiescence. Prostatic carcinogenesis involves perturbation of these reciprocal homeostatic cell-cell interactions. The central role of mesenchyme in prostatic epithelial development has been firmly established through analysis of tissue recombinants composed of androgen-receptor-positive wild-type mesenchyme and androgen-receptor-negative epithelium. These studies revealed that at the very least ductal morphogenesis, epithelial cytodifferentiation, epithelial apoptosis and epithelial proliferation are regulated by stromal and not epithelial androgen receptors. Likewise, progression from non-tumorigenesis to tumorigenesis elicited by testosterone plus estradiol proceeds via paracrine mechanisms. Thus, stromal-epithelial interactions play critical roles in the hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development.     

Stromal-epithelial interactions and heterogeneity of proliferative activity within the prostate

Growth and functional activity within the prostate gland is known to be regulated by androgens whose effects are thought to be mediated via androgen receptors. This concept has been derived in large part through analysis of whole organ homogenates, an approach which ignores potential heterogeneity of biological activity within the gland and the importance of cell-cell interactions. In this review recent findings are summarized which demonstrate that growth of the prostatic ductal network during prepubertal periods, as well as during prostatic regeneration in androgen-treated adult castrates, is nonuniform, with ductal growth being highest at the ductal tips and much lower in proximal ducts closer to the urethra. Androgen dependency for maintenance of ductal architecture following castration follows a similar pattern in that castration results in total destruction of distal ductal architecture, while proximal ducts are maintained albeit in an atrophic state. Thus, striking differences in biological properties are found in distal versus proximal prostatic ducts. Morphogenesis, growth, and secretory cytodifferentiation within the developing prostate is elicited by androgens which act via mesenchymal-epithelial interactions. Through analysis of chimeric prostates constructed with androgen-receptor-positive wild-type mesenchyme and androgen-receptor-negative Tfm (testicular feminization) bladder epithelium, it is now evident that androgenic effects can be elicited in androgen-receptor-deficient (androgen-insensitive) Tfm prostatic epithelium, provided that the connective tissue component of the chimeric prostate is wild type. This observation has been made for both the developing and adult prostate. From this data it is evident that certain androgenic effects (ductal morphogenesis, epithelial growth, and secretory cytodifferentiation) do not require the presence of intraepithelial androgen receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

前列腺癌雄激素受体和PI3K/Akt信号通路相互作用研究进展

前列腺癌的发生、进展依赖于雄激素,因此去势手术成为治疗晚期前列腺癌的标准疗法。但是去势后大多前列腺癌最终将转化为雄激素非依赖性前列腺癌,甚至进展为激素难治性前列腺癌,使得肿瘤的进展不受低水平雄激素的影响。即使如此,大多数激素非依赖性前列腺癌,依然阳性表达雄激素受体。因而雄激素受体在前列腺癌发生发展中起着重要作用。而PI3K/Akt信号通路能够通过维持细胞生存、抑制细胞凋亡、促进细胞代谢及血管生成等促进前列腺癌进展。本综述旨在总结前人研究,阐述雄激素受体和PI3K/Akt信号通路之间相互作用关系。研究表明Akt信号通路能够正性或者负性调控AR蛋白表达、蛋白的稳定性及其转录活性,从而维持细胞的生存、代谢。而AR即可以通过基因转录途径抑制Akt活化又能通过非转录基因途径激活Akt及其下游蛋白。因此,AR和Akt信号通路相互协同促进前列腺癌的发生及其向雄激素非依赖性前列腺癌进展。     

Conditional expression of the androgen receptor induces oncogenic transformation of the mouse prostate

The androgen signaling pathway, mediated through the androgen receptor (AR), is critical in prostate tumorigenesis. However, the precise role of AR in prostate cancer development and progression still remains largely unknown. Specifically, it is unclear whether overexpression of AR is sufficient to induce prostate tumor formation in vivo. Here, we inserted the human AR transgene with a LoxP-stop-loxP (LSL) cassette into the mouse ROSA26 locus, permitting "conditionally" activated AR transgene expression through Cre recombinase-mediated removal of the LSL cassette. By crossing this AR floxed strain with Osr1-Cre (odd skipped related) mice, in which the Osr1 promoter activates at embryonic day 11.5 in urogenital sinus epithelium, we generated a conditional transgenic line, R26hAR(loxP):Osr1-Cre+. Expression of transgenic AR was detected in both prostatic luminal and basal epithelial cells and is resistant to castration. Approximately one-half of the transgenic mice displayed mouse prostatic intraepithelial neoplasia (mPIN) lesions. Intriguingly, four mice (10%) developed prostatic adenocarcinomas, with two demonstrating invasive diseases. Positive immunostaining of transgenic AR protein was observed in the majority of atypical and tumor cells in the mPIN and prostatic adenocarcinomas, providing a link between transgenic AR expression and oncogenic transformation. An increase in Ki67-positive cells appeared in all mPIN and prostatic adenocarcinoma lesions of the mice. Thus, we demonstrated for the first time that conditional activation of transgenic AR expression by Osr1 promoter induces prostate tumor formation in mice. This new AR transgenic mouse line mimics the human disease and can be used for study of prostate tumorigenesis and drug development.     

Immunocytochemical Detection of the Androgen Receptor In Fine Needle Aspirates From Benign and Malignant Human Prostate

A monoclonal antibody to the androgen receptor was applied to fine needle aspirates from patients with benign and malignant prostatic disease. The series includes six patients with benign hyperplasia and 24 patients with prostatic carcinomas. The androgen receptor was detected in most nuclei of both benign and malignant epithelial cells. The intensity of immunostaining varied. No obvious relation was observed between the intensity of the staining in benign versus malignant cells. In addition no clear differences were found in the proportion of androgen receptor positive cells in benign aspirates as compared with aspirates from well differentiated or moderately well differentiated prostatic carcinomas. The relative number of androgen receptor positive cells was highest in smears from poorly differentiated prostatic carcinomas.     

Androgen Receptor Splice Variant AR3 Promotes Prostate Cancer via Modulating Expression of Autocrine/Paracrine Factors

Deregulation of androgen receptor (AR) splice variants has been implicated to play a role in prostate cancer development and progression. To understand their functions in prostate, we established a transgenic mouse model (AR3Tg) with targeted expression of the constitutively active and androgen-independent AR splice variant AR3 (a.k.a. AR-V7) in prostate epithelium. We found that overexpression of AR3 modulates expression of a number of tumor-promoting autocrine/paracrine growth factors (including Tgfβ2 and Igf1) and expands prostatic progenitor cell population, leading to development of prostatic intraepithelial neoplasia. In addition, we showed that some epithelial-mesenchymal transition-associated genes are up-regulated in AR3Tg prostates, suggesting that AR3 may antagonize AR activity and halt the differentiation process driven by AR and androgen. This notion is supported by our observations that the number of Ck5⁺/Ck8⁺ intermediate cells is increased in AR3Tg prostates after castration, and expression of AR3 transgene in these intermediate cells compromises prostate epithelium regeneration upon androgen replacement. Our results demonstrate that AR3 is a driver of prostate cancer, at least in part, through modulating multiple tumor-promoting autocrine/paracrine factors.     

Genistein represses the induction of prostatic buds by testosterone]

Genistein, a phytoestrogen and a kind of endocrine disrupters, inhibits tyrosine-specific protein kinase activity of the epidermal growth factor (EGF) receptor. It is also effective both in the suppression of the prostatic cell proliferation and the prostate carcinogenesis. We have recently demonstrated that several growth factors, like EGF, transforming growth factor-alpha (TGF-alpha), or keratinocyte growth factor (KGF), can induce prostatic bud formation in the absence of androgen. The present study was performed to investigate whether genistein can suppress testosterone-induced prostatic bud formation. Urogenital sinuses of 16.5-day male rat fetuses were cultured organotypically for 5 days in a serum-free medium containing 10 or 100 ng/ml genistein and 50 ng/ml testosterone. The number and total volume of prostatic buds were analyzed by laser scanning microscopy and computerized. We found that genistein inhibits significantly testosterone-induced prostatic bud formation. In the presence of genistein, cell proliferation of the sinus epithelium was suppressed and the number of prostatic buds and total volume of the buds were reduced as compared with those in the sinuses cultured with testosterone alone. Genistein did not appear to cause necrosis of the sinus. These results support our hypothesis that growth factors like EGF secreted from the sinus mesenchyme activated by testosterone are involved in the induction and stimulation of growth of the prostatic buds.     

The potential significance of aromatase in the etiology and treatment of prostatic disease

Prior to the present conference on aromatase, reports in the literature on prostatic aromatase have been scattered over time, few in number, and the results have been widely divergent. Moreover, several participants at this conference have reported unpublished data that failed to detect the existence of androgen aromatase in the prostate of man and other species. While papers and posters presented at this conference have added new information to this field, there would still appear to be no consensus as to the biological significance, if any, of the putative androgen aromatase system or the practical importance of inhibitors of prostatic and/or peripheral aromatase as a treatment modality for benign prostatic hyperplasia (BPH). Thus, it would be difficult to predict at this time the ultimate impact which current prostatic aromatase investigations will eventually have on our understanding and treatment of prostatic disease. To summarize the status of our current understanding of aromatase as it relates to prostatic function and disease, it would be safe to note that this field is virtually wide open for researchers to explore, both in terms of the future role that aromatase inhibitors may have in clinical investigations and in terms of the functional significance of aromatase, if any, in the normal prostate as well as in the pathogenesis of BPH and prostate cancer. Clearly, the widely divergent results currently available in the literature must reflect, in part, differences in methodology, anatomy, tissue types, the relative amounts of stroma and epithelium in specimens analyzed, the cellular and tissular (normal, BPH, and carcinomatous) heterogeneity encountered in clinical specimens, and the pharmacologic features of aromatase inhibitors tested.     

Stem/progenitor and intermediate cell types and the origin of human prostate cancer

Theories of cell lineage in human prostatic epithelium, based on protein expression, propose that basal and luminal cells: 1) are either independently capable of self-renewal or 2) arise from stem cells expressing a full spectrum of proteins (p63, cytokeratins CK5/14, CK8/18, and glutathione-S-transferase-pi [GST-pi]) similar to cells of the embryonic urogenital sinus (UGS). Such embryonic-like stem cells are thought to give rise to mature basal cells and secretory luminal cells. By single cell cloning of an immortalized, normal human prostate-derived, non-tumorigenic RWPE-1 cell line, we isolated and characterized two epithelial cell types, WPE-stem and WPE-int. WPE-stem cells show: i) strong, sixfold greater nuclear expression of p63; ii) nearly twofold greater expression of CK14; iii) threefold less CK18, and iv) low androgen receptor (AR) expression as compared with WPE-int cells. WPE-stem cells are androgen-independent for growth and survival. WPE-int cells express very low p63 and CK5/14, and high CK18. WPE-int cells are androgen-independent for growth and survival but are highly responsive as shown by androgen induction of AR and prostate specific antigen (PSA). Compared with WPE-int cells, WPE-stem cells are smaller and show more rapid growth. WPE-stem cells can grow in an anchorage-independent manner in agar with 4.5-fold greater cloning efficiency and as free floating "prostaspheres" in liquid medium; and express over 40-fold higher matrix metalloproteinase-2 activity. These results indicate that WPE-stem cells express several features characteristic of stem/progenitor cells present in the UGS and in adult prostatic epithelium. In contrast, WPE-int cells have an intermediate, committed phenotype on the pathway to luminal cell differentiation. We propose that in normal prostatic epithelium, cells exist at many stages in a continuum of differentiation progressing from stem cells to definitive basal and luminal cells. Establishment and characterization of clones of human prostatic epithelial cells provide novel models for determining cell lineages, the origin of prostate cancer, and for developing new strategies for tumor prevention and treatment.     

Androgen receptor localization in the human prostate: demonstration of heterogeneity using a new method of steroid receptor autoradiography

We have used a novel receptor labeling and autoradiographic technique to identify the cell types in human benign prostatic hyperplasia (BPH) that contain androgen receptors, and we have found that androgen receptor localization is heterogeneous. Prostatic androgen receptors were labeled by incubating slide-mounted frozen tissue sections (10 micron thickness) with [3H]R1881 in vitro. Tissue sections labeled in this way were subjected to concurrent biochemical and autoradiographic analysis. Some of the sections were wiped from the slides for scintillation counting to validate that the procedure indeed measures total cellular androgen receptors of appropriate high affinity and androgen steroid specificity. Replicate labeled slide-mounted tissue sections were dried rapidly, apposed to dry emulsion-coated coverslips, and exposed in the dark for autoradiography. Autoradiograms were developed, fixed, and stained; silver grains were counted over nuclei or cytoplasm of epithelium or stroma to evaluate specific androgen receptor location. Autoradiographic analysis of human glandular BPH demonstrated androgen receptor localization almost exclusively in the epithelial nuclei, with little or none in the stroma. We anticipate that data obtained using this new method of steroid receptor autoradiography may provide fresh insight into the mechanism of hormonal regulation of the prostate.     

A new exchange procedure for the quantitation of prostatic androgen receptor complexes formed in vivo

A procedure is described for the measurement of rat prostatic androgen receptor saturated in vivo with non-radioactive androgen. While NaSCN alone induces irreversible dissociation (denaturation) of androgen from the receptor, the combination of this chaotropic salt (0.15 M) with sucrose (15%) and sodium molybdate (10 mM) allows the exchange of R DHT with [3H]DHT at 0 degrees C with only minimal receptor denaturation. The validity of the present exchange assay is based on the following: a similar quantity of androgen receptor was detected when binding was measured directly after in vivo treatment with radioactive androgen or indirectly by [3H]DHT exchange after treatment with non-radioactive androgen. Steroid specificity, sedimentation analysis and equilibrium association constants indicated that this exchange assay labels the androgen receptor without interference from other prostatic steroid binding proteins. With this method it is now possible to quantitate not only prostatic androgen receptors bound to androgens in vitro but also hormone-receptor complexes formed in intact animals under the influence of endogenous androgen.     

Catalog of mRNA expression patterns for DNA methylating and demethylating genes in developing mouse lower urinary tract

The mouse prostate develops from a component of the lower urinary tract (LUT) known as the urogenital sinus (UGS). This process requires androgens and signaling between mesenchyme and epithelium. Little is known about DNA methylation during prostate development, including which factors are expressed, whether their expression changes over time, and if DNA methylation contributes to androgen signaling or influences signaling between mesenchyme and epithelium. We used in situ hybridization to evaluate the spatial and temporal expression pattern of mRNAs which encode proteins responsible for establishing, maintaining or remodeling DNA methylation. These include DNA methyltrasferases, DNA deaminases, DNA glycosylases, base excision repair and mismatch repair pathway members. The mRNA expression patterns were compared between male and female LUT prior to prostatic bud formation (14.5 days post coitus (dpc)), during prostatic bud formation (17.5 dpc) and during prostatic branching morphogenesis (postnatal day (P) 5). We found dramatic changes in the patterns of these mRNAs over the course of prostate development and identified examples of sexually dimorphic mRNA expression. Future investigation into how DNA methylation patterns are established, maintained and remodeled during the course of embryonic prostatic bud formation may provide insight into prostate morphogenesis and disease.     

Catalog of mRNA expression patterns for DNA methylating and demethylating genes in developing mouse lower urinary tract

The mouse prostate develops from a component of the lower urinary tract (LUT) known as the urogenital sinus (UGS). This process requires androgens and signaling between mesenchyme and epithelium. Little is known about DNA methylation during prostate development, including which factors are expressed, whether their expression changes over time, and if DNA methylation contributes to androgen signaling or influences signaling between mesenchyme and epithelium. We used in situ hybridization to evaluate the spatial and temporal expression pattern of mRNAs which encode proteins responsible for establishing, maintaining or remodeling DNA methylation. These include DNA methyltrasferases, DNA deaminases, DNA glycosylases, base excision repair and mismatch repair pathway members. The mRNA expression patterns were compared between male and female LUT prior to prostatic bud formation (14.5 days post coitus (dpc)), during prostatic bud formation (17.5 dpc) and during prostatic branching morphogenesis (postnatal day (P) 5). We found dramatic changes in the patterns of these mRNAs over the course of prostate development and identified examples of sexually dimorphic mRNA expression. Future investigation into how DNA methylation patterns are established, maintained and remodeled during the course of embryonic prostatic bud formation may provide insight into prostate morphogenesis and disease.     

Proprotein convertases modulate budding and branching morphogenesis of rat ventral prostate

The onset of prostate morphogenesis is involved in the interaction between mesenchyme and epithelium. Proprotein convertases (PCs) activate a variety of growth and differentiation factors including mesenchymal and epithelial factors, such as insulin-like growth factor (IGF) and transforming growth factor-beta (TGF-beta), which induce ductal budding and branching. In this study, we provide evidence that PCs play a critical role in prostatic budding from the urogenital sinus (UGS) and ductal branching morphogenesis of the neonatal rat ventral prostate. PCs were expressed only in the epithelial cells of neonatal rat prostate. PC activity in the ventral prostate was modulated by endogenous androgen. PC inhibition suppressed prostatic budding and branching. Taken together, our data indicates that androgen-induced PCs initiate the development of the prostate.     

Role of p63 and basal cells in the prostate

The prostate contains two major epithelial cell types - luminal and basal cells - both of which develop from urogenital sinus epithelium. The cell linage relationship between these two epithelial types is not clear. Here we demonstrate that luminal cells can develop independently of basal cells, but that basal cells are essential for maintaining ductal integrity and the proper differentiation of luminal cells. Urogenital sinus (UGS) isolated from p63(+/+) and p63(-/-) embryos developed into prostate when grafted into adult male nude mice. Prostatic tissue that developed in p63(-/-) UGS grafts contained neuroendocrine and luminal cells, but basal cells were absent. Therefore, p63 is essential for differentiation of basal cells, but p63 and thus basal cells are not required for differentiation of prostatic neuroendocrine and luminal epithelial cells. p63(-/-) prostatic grafts also contained atypical mucinous cells, which appeared to differentiate from luminal cells via activation of Src. In the response to castration, regression of p63(-/-) prostate was inordinately severe with almost complete loss of ducts, resulting in the formation of residual cystic structures devoid of epithelium. Therefore, basal cells play critical roles in maintaining ductal integrity and survival of luminal cells. However, regressed p63(-/-) prostate did regenerate in response to androgen administration, indicating that basal cells were not essential for prostatic regeneration.     

Epithelial-mesenchymal interactions in prostatic development. II. Biochemical observations of prostatic induction by urogenital sinus mesenchyme in epithelium of the adult rodent urinary bladder

Adult bladder epithelium (BLE) is induced to differentiate into glandular epithelium after association with urogenital sinus mesenchyme (UGM) and subsequent in vivo growth in syngeneic male hosts. Alteration of epithelial cytodifferentiation is associated with the expression of prostate-specific antigens, histochemical and steroid metabolic activities. These observations suggest that the inductive influence of the UGM has reprogrammed both the morphological and functional characteristics of the urothelium. In this report, differences regarding the mechanisms and effects of androgenic stimulation of prostate and bladder are exploited to determine the extent to which UGM plus BLE recombinants express a prostatelike, androgen-dependent phenotype. Results from cytosolic and autoradiographic binding studies suggest that androgen binding is induced in UGM plus BLE recombinants and that this activity is accounted for by the induced urothelial cells. In UGM plus BLE recombinants, androgen-induced [3H]thymidine or [35S]-methionine uptake analyzed by two-dimensional gel electrophoresis was qualitatively and quantitatively similar to that of prostate as opposed to bladder. These studies indicate that expression within BLE of prostatic phenotype is associated with a loss of urothelial characteristics and that androgen sensitivity is presumably a function of the inductive activities of the stroma.