Mouse Ventral Prostate Research Articles

Possible role of transient receptor potential melastatin 4 channels in adrenergic contractions in mouse prostate smooth muscles.

Transient receptor potential melastatin 4 (TRPM4) cation channels are expressed in prostate glands. However, the precise role of these channels in prostate contractility remains unclear. In this study, we examined whether TRPM4 channels were involved in adrenergic contractions in the mouse prostate gland. Adrenergic contractile responses elicited by noradrenaline or electrical field stimulation of the sympathetic nerve were isometrically recorded, and the effects of 9-phenanthrol, a specific TRPM4 channel inhibitor, on those contractile responses were investigated in mouse ventral prostate preparations. 9-phenanthrol (10 or 30 μM) inhibited noradrenaline- and sympathetic nerve-evoked contractions in a concentration-dependent manner. A similar inhibitory effect was observed with another TRPM4 channel inhibitor, 4-chloro-2-(2-(naphthalene-1-yloxy) acetamido) benzoic acid (NBA; 10 μM). Inhibition by 9-phenanthrol and NBA were much greater at lower noradrenaline concentrations and lower stimulus frequencies than those of higher concentrations or frequencies. However, 9-phenanthrol did not inhibit the noradrenaline-induced contractile response when the membrane potential was decreased to approximately 0 mV in the 140 mM K+ medium. Moreover, 9-phenanthrol does not affect noradrenaline-induced increases in spontaneous contractions of cardiac atrial preparation. This agent inhibited noradrenaline-induced contractions in the posterior aorta preparation. However, the inhibitory effect was significantly weaker than that observed in the prostate gland. These results suggest that TRPM4 channels are involved in adrenergic contractions in the mouse prostate gland, possibly through membrane depolarization by their opening; therefore, they might be potential candidates for treating benign prostatic hyperplasia.

Abstract 2228: Altering rictor with diosmetin reduces tumor progression in orthotopic prostate cancer model

Abstract Rapamycin insensitive companion of mTOR (Rictor) is an essential subunit of mTOR complex 2, maintains the integrity of the complex and functions as full activator of Akt. Rictor has been implicated to be involved in growth and progression of malignancies. Reports suggested that overexpression of Rictor in prostate cancer tissues, which may have potential role in prostate cancer progression. We demonstrated silencing Rictor in PC-3 cells decreased p- Akt (Ser-473) expression; conversely p-Akt (Thr-308) and p-PKCα (Ser-657) expression was increasing. Additionally to observe the molecular mechanism of Rictor regulation, we took HAP1 cells (derived from male chronic myelogenous leukemia (CML) cell line KBM-7) were edited and silenced by CRISPR/Cas9 with 2 and 10 base pair Rictor in a coding exon 5. HAP1 haploid human cell line contains a single copy of all chromosomes except for a heterozygous 30-megabase fragment of Chromosome 15; these cells are instrumental for genetic screening, as gene inactivation is greatly facilitated by the absence of a second gene copy. In these cells we observed p-Akt (Ser-473) expression was significantly less with more pronounced in 10 base pair than 2 base pair. Further to determine potential role of diosmetin (5, 7-Trihydroxy-4′-methoxyflavone; a natural flavonoid present in citrus plant, has anti-mutagenic properties) on Rictor silenced 2 base pair and 10 base pair HAP1 cells, we treated these cells in concentration response fashion, and observed decrease in p-Akt (Ser-473) expression than control cells. Next, we determine invivo potential of Rictor silencing, we used luciferase tag PC-3 cells silenced with Rictor gene orthotopically implanted in ventral prostate of mice. After 8 weeks of Rictor silenced cells implanted mice, we observed less luciferase activity in prostate and occasional metastasis than control luciferase PC-3 cells. Moreover, diosmetin fed mice in dose dependent fashion (20 and 50µg/animal/day) represented decrease in tumor luciferase activity with reduced tumor volume and no metastasis than control mice. We observed metastasis only in control and rapamycin (8 mg/kg body weight intraperitoneally every alternate day) treated mice in spine, lung and kidney. Taken together, our studies demonstrate that diosmetin fed animals resulted in growth inhibition and induction of apoptosis by altering Rictor signaling cascade. We are demonstrating that diosmetin modulates molecular targets viz., Rictor and Akt to alter cellular events and elicit anticancer effects in prostate cancer cells. Citation Format: Rebecca Pakradooni, Ahmad Khalifa, Riddhi Patel, Sanjeev Shukla. Altering rictor with diosmetin reduces tumor progression in orthotopic prostate cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2228. doi:10.1158/1538-7445.AM2017-2228

Estrogen receptor β, a regulator of androgen receptor signaling in the mouse ventral prostate.

As estrogen receptor β-/- (ERβ-/-) mice age, the ventral prostate (VP) develops increased numbers of hyperplastic, fibroplastic lesions and inflammatory cells. To identify genes involved in these changes, we used RNA sequencing and immunohistochemistry to compare gene expression profiles in the VP of young (2-mo-old) and aging (18-mo-old) ERβ-/- mice and their WT littermates. We also treated young and old WT mice with an ERβ-selective agonist and evaluated protein expression. The most significant findings were that ERβ down-regulates androgen receptor (AR) signaling and up-regulates the tumor suppressor phosphatase and tensin homolog (PTEN). ERβ agonist increased expression of the AR corepressor dachshund family (DACH1/2), T-cadherin, stromal caveolin-1, and nuclear PTEN and decreased expression of RAR-related orphan receptor c, Bcl2, inducible nitric oxide synthase, and IL-6. In the ERβ-/- mouse VP, RNA sequencing revealed that the following genes were up-regulated more than fivefold: Bcl2, clusterin, the cytokines CXCL16 and -17, and a marker of basal/intermediate cells (prostate stem cell antigen) and cytokeratins 4, 5, and 17. The most down-regulated genes were the following: the antioxidant gene glutathione peroxidase 3; protease inhibitors WAP four-disulfide core domain 3 (WFDC3); the tumor-suppressive genes T-cadherin and caveolin-1; the regulator of transforming growth factor β signaling SMAD7; and the PTEN ubiquitin ligase NEDD4. The role of ERβ in opposing AR signaling, proliferation, and inflammation suggests that ERβ-selective agonists may be used to prevent progression of prostate cancer, prevent fibrosis and development of benign prostatic hyperplasia, and treat prostatitis.

Abstract 2118: Differential efficiency in the deletion of estrogen receptor β DNA binding domain generates distinct phenotypes in the mouse ventral prostate and ovary

Abstract The anti-proliferative properties of estrogen receptor β (ERβ) and its ability to regulate growth and differentiation of the prostate epithelium make it interesting as a therapeutic target in prostate cancer. In 1998, the first ERβ knockout (ERβ-/-) mouse was created by inserting a neocassette into the ERβ gene to interrupt the gene at the DNA binding domain (DBD). In this original knockout, aging ERβ-/- mice develop prostate hyperplasia and upon estradiol treatment, they also develop prostatic intraepithelial neoplasia lesions, which are believed to be premalignant. These mice were also characterized by a reduction in the ability to ovulate and disruption of neuronal migration during fetal development. In 2008, another ERβ mutant mouse was generated by inserting LoxP sites around the third exon of the gene. Upon deletion of exon 3, the female mice were infertile, due to the inability of the female mice to ovulate but there were no other defects in either males or females. Thus it was concluded that, ERβ has very limited physiological function and the phenotype of the original ERβ-/- mouse deemed to be due to the presence of the neocassette which remained in the gene. In the present study, a new ERβ knockout mouse was generated by inserting Lox P sites around the third exon and this exon was deleted by crossing this mouse with either a CMV-Cre or a Rosa-Cre deleter mouse. The only abnormality found in the new mutant ERβ mouse was the inability of the female mice to ovulate. Sequencing of the ovary mRNA showed that the knockout strategy worked i.e., exon 3 was deleted and a shift in the reading frame was generated, resulting in the creation of two in frame stop codons. Nevertheless, in this new mutant mouse, there were no detectable defects in the prostate, the lungs or the brain. We demonstrated that although two stop codons should have prevented translation of the protein, ventral prostates, lungs and skeletal muscle of these mice produce a full length ERβ protein. This protein was detected in the nuclei of prostate epithelial cells by immunohistochemistry and western blot assays, using a set of four anti-ERβ antibodies directed against different epitopes. In contrast to the original knockout mouse, the expression of Ki67 and androgen receptor in the prostate epithelium was unchanged in the new ERβ-/- mouse. Moreover, DNA binding of prostate nuclear extracts from these mice to Estrogen Responsive Elements and AP-1 responsive elements was inhibited by ERβ-specific antibodies. We conclude that differential efficiency in the deletion of ERβ DBD generates divergent phenotypes in the mouse ventral prostate and ovary and this discrepancy may be mediated by tissue variations in the efficiency of the Cre recombinase system. Citation Format: Laure Maneix, Per Antonson, Sabrina S. Rochel-Maia, Hyun-Jin Kim, Margaret Warner, Jan-Ake Gustafsson. Differential efficiency in the deletion of estrogen receptor β DNA binding domain generates distinct phenotypes in the mouse ventral prostate and ovary. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2118. doi:10.1158/1538-7445.AM2014-2118

Gene Expression Profiling Reveals Regulation of ERK Phosphorylation by Androgen-Induced Tumor Suppressor U19/EAF2 in the Mouse Prostate

U19/EAF2 is regulated by androgens in the prostate and capable of regulating transcriptional elongation of RNA Pol II via interaction with the ELL family proteins. Inactivation of U19/EAF2 induces tumorigenesis in multiple organs; however the mechanism of U19/EAF2 tumor suppression remains unclear. To elucidate potential mechanisms of U19/EAF2 action, we performed cDNA microarray analysis and identified 164 mRNA transcripts regulated by U19/EAF2 in the mouse ventral prostate. Bioinformatics analysis indicated that U19/EAF2 knockout activates the RAS-BRAF-ERK signaling pathway, which is known to play important roles in carcinogenesis. qPCR verified increased expression of BRAF mRNA, and immunostaining and Western blot analysis demonstrated increased expression of p-ERK at the protein level suggested U19/EAF2 knockout activates this important pathway. These findings indicate that loss of EAF2 up-regulates transcription of RAS cascade genes including Grb2, PI3K, and BRAF, leading to elevated p-ERK levels, which may represent a major functional role of U19/EAF2 in the prostate. Furthermore, these observations suggest that U19/EAF2 is a key player in crosstalk between androgen receptor and the RAS-BRAF-ERK signaling pathway.

731 HIGH FAT DIET REDUCES THE EXPRESSION OF GLUTATHIONE PEROXIDASE 3 IN MOUSE PROSTATE

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011731 HIGH FAT DIET REDUCES THE EXPRESSION OF GLUTATHIONE PEROXIDASE 3 IN MOUSE PROSTATE Yoshitaka Sekine, Yosuke Furuya, Hidekazu Koike, Hiroshi Matsui, Kazuhiro Suzuki, and Alan Remaley Yoshitaka SekineYoshitaka Sekine Bethesda, MD More articles by this author , Yosuke FuruyaYosuke Furuya Maebashi, Japan More articles by this author , Hidekazu KoikeHidekazu Koike Maebashi, Japan More articles by this author , Hiroshi MatsuiHiroshi Matsui Maebashi, Japan More articles by this author , Kazuhiro SuzukiKazuhiro Suzuki Maebashi, Japan More articles by this author , and Alan RemaleyAlan Remaley Bethesda, MD More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1700AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES High fat diets are known to be a risk factor for prostate cancer. In this study, we investigated the effect of high fat diet on mouse prostate gene expression. METHODS C57BL/6J mice were fed either a control or high fat diet for 12 weeks. Total RNA was extracted from mouse ventral prostate (VP), dorsolateral prostate (DLP) and anteriol prostate (AP) separately. Microarray analyses were performed on VP and DLP, followed by canonical pathway analysis and regulatory network identification. For validation, quantitative RT-PCR was performed. H2O2 levels in culture medium were measured using Amplex Red Hydrogen Peroxide/Peroxidase Assay kit. To knockdown a candidate gene, cells were transfected with siRNA. Prostate biopsy samples from 64 patients were collected at Gunma University hospital between 2002–2007. RESULTS C57BL/6J mice on the high fat diet had nearly a 2-fold increase in total cholesterol, but decreased serum triglycerides. Approximately 2,125, and 1,194 genes responded significantly to the high fat diet in VP, DLP, respectively (p < 0.05, fold change > 1.2). Pathways and networks related to oxidative stress; glutathione metabolism, NRF-mediated oxidative stress response and NF-kappaB were all regulated by high fat diet. 4 genes (GPx3, Crabp1, Cyp2b10 and Yipf5) were either up regulated and down regulated more than 2-fold in VP by the high fat diet. Although in most cases they showed the same trends, none of these genes showed statistically significant changes when DLP was analyzed by microarray analysis. In the validation, GPx3 mRNA levels were decreased by approximately 2-fold by high fat diet in not only in VP, but also in DLP and AP. In human non-transformed prostate cells (PrSC, PrEC and BPH-1), cholesterol loading decreased GPx3 expression, and increased H2O2 levels of culture medium. When GPx3 expression was reduced by a siRNA transfection, H2O2 was found to be increased in cell culture media of PrSC and BPH-1 cells, consistent with the known role of GPx3 in lowering H2O2 levels. Troglitazone increased GPx3 expression in 3 normal prostate cells, and decreased H2O2 levels. In addition, troglitazone attenuated cholesterol-induced H2O2 increase. Tissue from prostate cancer biopsies had decreased GPx3 mRNA and was inversely related to the Gleason score. CONCLUSIONS High fat diet alters pathways related to many genes concerned with the increase of oxidative stress. GPx3, a gene identified by this analysis, was found to be down regulated by high fat diet and appears be decreased as well in human prostate cancers, suggesting that GPx3 may have a possible role in modulating carcinogenesis. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e293-e294 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yoshitaka Sekine Bethesda, MD More articles by this author Yosuke Furuya Maebashi, Japan More articles by this author Hidekazu Koike Maebashi, Japan More articles by this author Hiroshi Matsui Maebashi, Japan More articles by this author Kazuhiro Suzuki Maebashi, Japan More articles by this author Alan Remaley Bethesda, MD More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

High fat diet reduces the expression of glutathione peroxidase 3 in mouse prostate

High fat diets are known to be a risk factor for prostate cancer. In this study, we investigated the effect of high fat diet on mouse prostate gene expression. C57BL/6J mice were fed either a control or high fat diet for 12 weeks. Microarray analyses were performed on mouse ventral prostate (VP) and dorsolateral prostate (DLP), followed by canonical pathway analysis and regulatory network identification. mRNA changes were confirmed by real time PCR. Approximately 2,125, and 1,194 genes responded significantly to the high fat diet in VP, DLP, respectively. Pathways and networks related to oxidative stress, glutathione metabolism, NRF-mediated oxidative stress response and NF-kappaB were all differentially regulated by high fat diet. Glutathione peroxidase 3 (GPx3) mRNA levels were decreased by approximately twofold by high fat diet in all three prostate lobes. In human non-transformed prostate cells (PrSC, PrEC, and BPH-1), cholesterol loading decreased GPx3 expression, and increased H2 O2 levels of culture medium. Troglitazone increased GPx3 expression in three normal prostate cells, and decreased H2 O2 levels. In addition, troglitazone attenuated cholesterol-induced H2 O2 increase. Tissue from prostate cancer biopsies had decreased GPx3 mRNA and its level was inversely related to the Gleason score. High fat diet alters pathways related to many genes concerned with oxidative stress. GPx3, a gene identified by this analysis, was found to be down-regulated by high fat diet and appears be decreased in human prostate cancers, suggesting that GPx3 may have a possible role in modulating carcinogenesis. Prostate 71:1499-1509, 2011. © 2011 Wiley-Liss, Inc.

MMP‐2 contributes to the development of the mouse ventral prostate by impacting epithelial growth and morphogenesis

Epithelial growth, branching, and canalization are important morphogenetic events of the rodent ventral prostate (VP) that take place during the first postnatal week. In this study, we evaluated the effect of knocking out MMP-2 (MMP-2(-/-)), by examining developmental and structural aspects of the VP in MMP-2(-/-) mice. Neonate (day 6) MMP-2(-/-) mice showed fewer epithelial tips, a lower epithelial cell proliferation rate, and also reticulin fiber accumulation. The VP of adult MMP-2(-/-) mice showed lower relative weight, smaller epithelial and smooth-muscle cell volume, and a larger amount of thicker reticulin fibers. No differences in cell proliferation or apoptotic index were noted between adult MMP-2(-/-) and wild-type mice. MMP-9 was found in the adult MMP-2(-/-), but not in the wild-type. In conclusion, MMP-2 function is essential for the epithelial morphogenesis of the mouse VP, and expression of MMP-9 is not sufficient for acquisition of the normal adult histology.

Abstract 943: Obesity, P13k/Akt/mTOR signaling and prostate carcinogenesis in HiMyc mice

Abstract Obesity has increased over the last 30 years in the U.S. and is associated with increased mortality rates for various cancers, including prostate cancer. In contrast, calorie restriction (CR) has been shown to act as a potent inhibitor of tumorigenesis. While this association between dietary energy balance and cancer has been clearly established, the mechanisms underlying these effects are poorly understood. To study the role of obesity and dietary fat in prostate cancer, we have used the HiMyc transgenic mouse model, a well-characterized model for the study of prostate carcinogenesis. For the current studies, HiMyc mice at weaning were placed on three dietary regimens [30% CR, 10kcal% fat (AIN76A), 60 kcal% fat], therefore generating lean, overweight and obese phenotypes. Groups of mice were then sacrificed at 3 and 6 months of age and urogenital tracts were removed and embedded in paraffin for histological analyses. In separate groups of mice, ventral prostate (VP) was removed and used to generate protein lysates for Western blot analyses. Histopathologic evaluation revealed a variety of lesions, with the severity of the lesion correlating with caloric intake. All diet groups had approximately similar incidence of hyperplasia and low grade PIN at 3 and 6 months of age. However, the different energy diets primarily affected the progression of premalignant lesions to malignant lesions in the ventral prostate. At 6 months, diet induced obesity (DIO; 60 kcal% fat) significantly increased the incidence of adenocarcinoma with aggressive stromal invasion, as compared to the overweight control group (96% vs. 65% respectively, p=0.0221), while only in situ carcinomas but no invasive adenocarcinomas were observed in mice maintained on the CR diet. Immunohistochemistry was performed on additional sections to evaluate differences in activation or total levels of Akt, mTOR, P-S6 ribosomal, angiogenesis markers and cell cycle markers. DIO increased, while CR reduced activation of signaling through both Akt and mTOR. Similar effects were observed for levels of cyclin D1 and CD31 proteins. Western blot analyses performed on protein lysates from VP of mice maintained on the same dietary regimen for 6 months confirmed these differential effects of dietary energy balance on Akt and mTOR signaling. The observed changes in signaling appeared to be due, at least in part, to changes in signaling through the insulin-like growth factor 1 receptor (IGF-1R) as a result of altered levels of circulating IGF-1. Taken together, these findings suggest that pathways activated by obesity, such as the Akt and mTOR pathways, may be key targets for preventing and controlling obesity-related prostate cancer progression. Supported by grant CA 107588 and MD Anderson Prostate Spore grant CA 140388. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 943.

WNT5A selectively inhibits mouse ventral prostate development

The establishment of prostatic budding patterns occurs early in prostate development but mechanisms responsible for this event are poorly understood. We investigated the role of WNT5A in patterning prostatic buds as they emerge from the fetal mouse urogenital sinus (UGS). Wnt5a mRNA was expressed in UGS mesenchyme during budding and was focally up-regulated as buds emerged from the anterior, dorsolateral, and ventral UGS regions. We observed abnormal UGS morphology and prostatic bud patterns in Wnt5a null male fetuses, demonstrated that prostatic bud number was decreased by recombinant mouse WNT5A protein during wild type UGS morphogenesis in vitro, and showed that ventral prostate development was selectively impaired when these WNT5A-treated UGSs were grafted under under kidney capsules of immunodeficient mice and grown for 28 d. Moreover, a WNT5A inhibitory antibody, added to UGS organ culture media, rescued prostatic budding from inhibition by a ventral prostatic bud inhibitor, 2,3,8,7-tetrachlorodibenzo- p-dioxin, and restored ventral prostate morphogenesis when these tissues were grafted under immunodeficient mouse kidney capsules and grown for 28 d. These results suggest that WNT5A participates in prostatic bud patterning by restricting mouse ventral prostate development.

In vitro and in vivo modulation of testosterone mediated alterations in apoptosis related proteins by [6]‐gingerol

Ginger (Zingiber officinale, Zingiberaceae) has been widely used as a dietary spice, and as a traditional oriental medicine. The rhizome of ginger contains pungent vanillyl ketones, including [6]-gingerol and [6]-paradol, and have been credited with therapeutic and preventive health benefits, including anti-cancer activity. Prostate cancer is an attractive target for chemoprevention because of its ubiquity, treatment-related morbidity, long latency between premalignant lesions and clinically evident cancer, and defined molecular pathogenesis. Here we are reporting the modulatory effects of [6]-gingerol on testosterone-induced alterations on apoptosis related proteins in both in vitro, androgen sensitive LNCaP cells and in vivo, ventral prostate of Swiss albino mice. [6]-gingerol treatment resulted apoptosis in LNCaP cells, as indicated by depolarization of mitochondrial membrane potential, increase in sub G1 cell population by flow cytometry and the appearance of DNA laddering pattern in agarose gel electrophoresis. Results of western blot analysis showed that [6]-gingerol upregulated the testosterone depleted levels of p53 in mouse prostate and upregulated its downstream regulator Bax and further activated Caspase-9 and Caspase-3 in both LNCaP cells and in mouse prostate. We also found downregulation of testosterone induced antiapoptotic proteins, Bcl-2 and Survivin expression by [6]-gingerol in both LNCaP cells and in mouse ventral prostate. Thus, [6]-gingerol shows its protective effects in both in vivo and in vitro prostate cancer models by modulation of proteins involved in apoptosis pathway.

Castration rapidly decreases local insulin-like growth factor-1 levels and inhibits its effects in the ventral prostate in mice

The mechanisms by which castration induces prostate involution are largely unknown. Early responses to castration in mouse ventral prostate (VP) were explored by quantitative microscopy, cDNA array expression, quantitative RT-PCR, and Western blot analysis. As several changes occurred in the insulin-like growth factor (IGF) system this was studied in more detail. Laser micro-dissection was used to localize sites of IGF-1 and IGF-1 receptor (IGF-R1) production. IGF-1 protein levels and IGF-R1 mediated signaling via insulin regulated substrate 1 and 2 (IRS-1 and 2) were examined. IGF-1 was injected into the VP in intact, and castrated mice and effects studied 1 day later. IGF-1 and IGF binding protein 2 (IGFBP-2) mRNA were rapidly reduced whereas IGFBP-3 and IGF-R1 mRNA were increased after castration. IGF-1 was principally produced in the stromal compartment, while IGF-R1 was produced in both epithelial and stromal cells. IGF-1 and IRS-1 protein levels were decreased 1 and 3 days after castration, respectively, while IRS-2 was unchanged. Inactivating phosphorylation of IRS-1 at serine 307 was increased 1 day after castration, and activating phosphorylation at tyrosine 612 was decreased 2 days later. These changes were accompanied by decreased cell proliferation and increased cell death in the glandular and vascular compartment. Local injection of IGF-1 increased vascular density and epithelial cell proliferation in intact mice, but had no effect in castrated animals. Decreased IGF-1 levels and action may mediate some of the key features of castration-induced prostate involution.

Estrogen Receptor Alpha and Imprinting of the Neonatal Mouse Ventral Prostate by Estrogen

Estrogen Receptor Alpha and Imprinting of the Neonatal Mouse Ventral Prostate by Estrogen

Estrogen receptor alpha and imprinting of the neonatal mouse ventral prostate by estrogen.

Exposure to estrogen in the neonatal period affects prostatic growth and leads to an increased incidence of prostatic intraepithelial neoplasia in later life. The effects of neonatal estrogen are clearly dependent on estrogen receptor (ER) alpha because they do not occur in ERalpha-knockout mice. Because ERalpha is expressed in the stroma, but not in the epithelium, of the adult ventral prostate, the concept of indirect estrogen effects through stromal signaling has been proposed. Here, we show that during the first 4 weeks of life, there are profound and rapid changes in the ER profile in the mouse ventral prostate. ERalpha is abundant in the stroma during week 1, but by week 2 it is exclusively epithelial, and then by week 4, ERalpha is lost and ERbeta is dominant in the prostatic epithelium. The presence of ERalpha is associated with a high proliferation index, and ERbeta is associated with quiescence. Branching morphogenesis was altered in ERalpha-/-, but not in ERbeta-/-, mice. We conclude that imprinting and branching morphogenesis of the ventral prostate are mediated by estrogen acting directly on epithelial and stromal ERalpha during the first 2 weeks of life.

Characterization of a lymph node within the mouse prostate: Detailed analysis using whole mount histology.

Due to the prevalence of prostate disease, there is a growing interest in the immunobiology of the prostate and its contribution to such pathologies. Further study is needed to fully characterize immune responses within the prostate. Mouse ventral prostates were removed and analyzed using conventional immunohistochemistry, or by a novel whole mount technique, which allows the visualization of complete structures within the prostate. A lymphoid structure was detected within the base of the mouse ventral prostate. Whole mount and cryostat sections revealed an organized arrangement of lymphocytes, along with dendritic cells interspersed throughout the node. It possessed a rich network of lymph node specific high endothelial venules (HEVs) and adjoining lymphatics containing immune cells. Our results demonstrate the presence of a lymphoid structure within the base of the mouse ventral prostate possessing traits characteristic of an organized peripheral lymph node.

Estrogen receptor beta regulates epithelial cellular differentiation in the mouse ventral prostate.

We have previously reported epithelial cellular hyperplasia in ventral prostates (VP) of mice lacking estrogen receptor beta (ER beta). To investigate the causes of this phenomenon, we measured cellular proliferation and apoptosis in VP of ER beta(-/-) and WT mice. With BrdUrd labeling, the number of proliferating cells was 3.6-fold higher in ER beta(-/-) mice. There was also a decrease in apoptosis as measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay and an increase in expression of the anti-apoptotic bcl-2. The state of differentiation of the epithelial cells of the VP was studied by immunohistochemical staining, Western blotting, and fluorescence-activated cell sorting (FACS). In ER beta(-/-) mouse VP, the number of p63-positive cells (basal phenotype) was 2.6-fold higher, and expression level of cytokeratin (CK) 8, a luminal cell marker, was lower. FACS analysis with p63 showed that in WT mice the ratio of basal to intermediate/luminal cell populations expressing p63 was 1:2.5, whereas in ER beta(-/-) mice it was 1:9. The expression of basal/intermediate marker CK 19 in three FACS areas, g1, g2, and g3, gated according to cellular size and granularity, was 1:0.6:2 in WT and 1:4:6.7 in ER beta(-/-) mice, showing a shift of CK 19-positive cells toward a cell population of intermediate size and granularity. We conclude that, in ER beta(-/-) mouse VP, there is increased epithelial proliferation, decreased apoptosis, and accumulation of incompletely differentiated cells in an intermediate pool. The continued proliferation of intermediate cells leads to the prostatic epithelial hyperplasia observed in the absence of ER beta signaling.

HEPATOCYTE GROWTH FACTOR SUPPORTS ANDROGEN STIMULATION OF GROWTH OF MOUSE VENTRAL PROSTATE EPITHELIAL CELLS IN COLLAGEN GEL MATRIX CULTURE

To assess the role of hepatocyte growth factor (HGF) and androgen in growth of prostate epithelial cells, we isolated mouse ventral prostate epithelial cells and cultured them in a three-dimensional type I collagen gel matrix under serum-free conditions. Although the prostate epithelial cells tended to die in the insulin-supplemented basal medium, 5α-dihydrotestosterone (DHT) prevented the cell death, and HGF slightly stimulated the growth. By contrast, coexistence of DHT and HGF greatly augmented the growth and branching morphogenesis of the epithelial cells. Some of the outgrowths formed under these conditions showed enlarged structures resembling the prostate ducts or alveoli. Examination of the stromal cell-conditioned medium revealed that a growth-stimulating activity is present in the conditioned medium. A major portion of this activity was abolished by anti-HGF IgG. These observations suggest that HGF is produced by the stromal cells of the prostate gland and supports the androgen stimulation of growth of the epithelial cells.

Ultrastructural Study of the Specialized Structural Secretory Granules Observed in the Mouse Ventral Prostate

Ultrastructural Study of the Specialized Structural Secretory Granules Observed in the Mouse Ventral Prostate

Androgen-regulated expression of secretory protein synthesis in mouse ventral prostate

Two proteins of molecular weights 25 and 12 kDa (p25 and p12 respectively), whose expression is regulated by testosterone, were identified in mouse ventral prostate. An antiserum raised to mouse ventral prostate secretion was used to demonstrate that p25 corresponds to the major secretory glycoprotein in mouse prostatic fluid. This antiserum does not cross-react with the major secretory proteins of the rat ventral prostate. Western blot analysis of mouse ventral prostate proteins using the prostatic secretion antiserum demonstrates that p12 and p25 are detectable at 3 weeks of age, but the maximum level of both proteins is not attained until 5 weeks of age. In addition, synthesis of p25 was also observed in prostate tissue derived from differentiated embryonic urogenital sinus tissue growing as implants under the renal capsule of syngeneic male hosts.

Tissue interactions and prostatic growth: II. Morphological and biochemical characterization of adult mouse prostatic hyperplasia induced by fetal urogenital sinus implants.

Current hypotheses regarding the causes of human benign prostatic hyperplasia have implicated both steroid hormone imbalance and tissue interactions. To examine the role of the latter we have further investigated the phenomenon of urogenital sinus-induced hyperplasia of the adult mouse ventral prostate. Urogenital sinuses (UGS) or purified urogenital mesenchyme (UGM) from C3H mice were implanted into the ventral prostates or coagulating glands of 50- to 90-day-old BALB/c-nu/nu hosts. The animals were sacrificed at 15, 30, and 180 days postimplantation to establish time dependence. Wet weight and DNA content were used as measures of net growth. Glucose phosphate isomerase (GPI) isozyme analysis was used to determine the relative contributions of C3H and BALB/c cells to the enlarged chimeric ventral prostates. It was determined that the induced growth is time-dependent and that the UGS induces 2- to 4-fold more growth than UGM. GPI analysis shows that UGM-induced growth was composed primarily of host-derived cells whereas the UGS is composed nearly equally of host- and implant-derived cells. Histologic analysis reveals that the UGS implants induce marked epithelial proliferation. The proliferating glands occur in clusters, and the epithelium within the glands appears cribriform. Foci of postobstructive cystic atrophy are also found. Remnants of the implanted UGS are still present even at 180 days postimplantation. UGM-induced growth is of a more subtle nature and appears morphologically similar to the sham-operated controls. In view of the morphologic similarity with human disease, as well as the time and hormonal dependence of UGS-induced ventral prostatic hyperplasia, this model represents the basis for a unified hypothesis regarding the roles of tissue interaction and hormonal milieu in human benign prostatic hyperplasia.