Epithelial Androgen Receptor Research Articles

Abstract B04: Epithelial nuclear androgen receptor (AR) in radical prostatectomy samples as assessed by quantitative immunofluorescence is differentially expressed in African American versus Caucasian cases

Abstract Introduction: Numerous biomarkers are being evaluated as potential predictors of prostate cancer (PCa) recurrence after definitive surgical treatment with radical prostatectomy (RP). In particular, there is a significant literature on the importance of androgen receptor (AR) protein expression in predicting the aggressiveness of PCa as well as the probability of response to secondary therapeutic interventions (e.g. androgen deprivation therapy. The AR axis has been a primary target for researchers seeking treatment options for patients whose prostate cancer is not cured by surgery alone. By utilizing quantitative immunofluorescence (IF) multiplexed biomarkers assessd via spectral imaging, we evaluated whether AR expression is deferentially expressed by race. Methods: We performed multiplex immunoflorescence immunohistochemistry on tissue microarrays (TMA) made from representative cores of tumor tissue from formalin-fixed, paraffin-embedded prostatectomy samples from cases treated for PCa by RP at the Henry Ford Health System between 2001 and 2005. There were 400 stained cores sampled from 149 African American (AA) cases and 453 cores from 195 Caucasian American (CM) cases. The samples were stained by a multiplex IF assay which contained the nuclear marker DAPI, along with AR and CK18. The co-localization of DAPI, CK18 and AR permitted quantitative analysis of AR expression in only the epithelial nuclei, a known cellular compartment of prognostic expression. Differences in expression were evaluated using the Wilcoxon rank sum test. The association between race and AR expression level was adjusted for potential confounding covariates using non-parametric generalized additive models. Results: There was a statistically significant difference in the expression of AR signal in epithelial nuclei between Caucasian men and African American men, p-value = 0.03. The association between Race and AR remained statistically significant (p=0.02) even after adjusting for established clinical risk factors of PCa (age, grade, stage and PSA). It is also noteworthy that there were no statistically significant differences in age, grade, stage or PSA when comparing Caucasian and African American men in our dataset. Conclusions: AR levels in RP epithelial nuclei have previously been found to be a useful tool in guiding therapeutic response for men who have failed surgical therapy for their prostate cancer. However, our results suggest that even with other clinical risk factors being equal, such molecular metrics may be different across racial subgroups of patients. It is possible that expression of these molecular features may be affected by external exposures, or genetic factors which differ by race, but are not necessarily related to the underlying disease mechanisms we seek to measure. Additional research into such differences is necessary as the African American community would benefit from the development of molecular diagnostic assays specifically tailored to this population. Current practices in biomarker discovery rarely address the possibility of racial variation, thereby leading to a potentially inaccurate or incorrect assessment of disease severity and consequent therapy for African American PCa patients. Citation Format: Russell Bailey McBride, Faisal M. Khan, Benjamin A. Rybicki, Russell Cordon-Cardo. Epithelial nuclear androgen receptor (AR) in radical prostatectomy samples as assessed by quantitative immunofluorescence is differentially expressed in African American versus Caucasian cases. [abstract]. In: Proceedings of the Seventh AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 9-12, 2014; San Antonio, TX. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2015;24(10 Suppl):Abstract nr B04.

THE DUAL ROLE OF ANDROGEN RECEPTOR IN MESENCHYMAL CELLS

The androgen receptor (AR) mediates differentiation, proliferation and transformation of target tissues. These processes require a crosstalk between epithelial and stromal cells. Prostate cancer (PCa) represents a major cause of cancer-related mortality in men, and is often associated with deregulation of androgen/AR axis. Clinical and molecular findings have highlighted the role of epithelial AR in PCa progression. In contrast, the functions of AR in mesenchymal cells are still unclear. We previously reported that low androgen concentration (1 pM) triggers interaction of AR with the Src tyrosine kinase and PI3-K, thus driving cell cycle progression in fibroblasts. In contrast, stimulation of fibroblasts and fibrosarcoma cells with physiological (10 nM) androgen concentration leads to interaction of AR with full-length filamin A (FLNa) and does not trigger DNA synthesis. On the basis of these findings, we re-examined the role of androgen/AR axis in fibroblasts and human fibrosarcoma HT1080 cells. Recently, we obtained two original and integrated findings on the decision of mesenchymal cells to undergo reversible quiescence and migrate upon stimulation with 10 nM androgens (Castoria et al. 2011 and 2014). This decision is dependent upon the interaction of AR with FLNa. Once assembled, the bipartite AR/FLNa complex recruits a1-integrin and triggers Rac1 activation, thereby enhancing on the one hand cell motility. On the other, Rac 1 activation triggers its downstream effector DYRK 1B, which phosphorylates Ser10 of p27. Stabilization of p27 and cell quiescence then follow. These results strengthen and extend our studies, adding a new and exciting piece to the complex puzzle of signaling networks activated by androgens in target cells. Our findings might have implications for current approaches to AR-related diseases.

Sebaceous carcinoma: clinicopathologic features and diagnostic role of immunohistochemistry (including androgen receptor)

ObjectiveTo explicate the clinicopathologic features of periocular sebaceous carcinoma (SC) and emphasize the importance of immunohistochemical staining that includes androgen receptor (AR) in their diagnosis. DesignRetrospective study. MethodsClinical and histopathologic features in 56 patients with periocular SC were analyzed. Immunohistochemical staining for pan-cytokeratin (CK), epithelial membrane antigen (EMA), Ber-EP4, adipophilin (ADP), and AR was performed on all cases. Immunostaining in SC was compared with that of squamous cell carcinoma (SCC; n = 25) and basal cell carcinoma (BCC; n = 18) for all antibodies. ResultsSGC was more frequent in females, most commonly presenting as a discrete and nodular mass. Four patients had regional lymph node metastases, whereas in 2, the tumour metastasized to the liver. Eight patients had a locoregional recurrence between the 3rd and 45th months of follow-up. Fifty patients had a moderately differentiated tumour. In 3 patients, the resected margins displayed intraepithelial tumour spread, and 2 patients died of the disease. In general, the staining pattern in SGC was CK+, EMA+, BerEP4–, ADP+, and AR+. SCCs were CK+, EMA+, Ber-EP4–, ADP–/+, and AR–, whereas BCC were CK+, EMA–, Ber-EP4+, ADP–, and AR–/+. p53 expression and Ki-67 index were higher in SC than SCC or BCC. ConclusionsImmunostaining using a panel of antibodies comprising BerEP4, ADP, EMA, and AR is useful in diagnosing sebaceous carcinoma and differentiating it from SCC and BCC, which are common to the periocular location and sometimes are morphologically identical to SC.

Androgen receptor enhances kidney stone-CaOx crystal formation via modulation of oxalate biosynthesis & oxidative stress.

Males develop kidney stones far more frequently than females with a ratio of 2–3:1, suggesting that androgen receptor (AR) signaling might play a key role in the development of nephrolithiasis. Using the cre-loxP system to selectively knock out AR in glyoxylate-induced calcium oxalate (CaOx) crystal mouse models, we found that the mice lacking hepatic AR had less oxalate biosynthesis, which might lead to lower CaOx crystal formation, and that the mice lacking kidney proximal or distal epithelial AR also had lower CaOx crystal formation. We found that AR could directly up-regulate hepatic glycolate oxidase and kidney epithelial NADPH oxidase subunit p22-PHOX at the transcriptional level. This up-regulation might then increase oxalate biosynthesis and oxidative stress that resulted in induction of kidney tubular injury. Targeting AR with the AR degradation enhancer ASC-J9 led to suppression of CaOx crystal formation via modulation of oxalate biosynthesis and oxidative stress in both in vitro and in vivo studies. Taken together, these results established the roles of AR in CaOx crystal formation.

Finasteride treatment alters tissue specific androgen receptor expression in prostate tissues

Normal and pathologic growth of the prostate is dependent on the synthesis of dihydrotestosterone (DHT) from testosterone by 5α-reductase. Finasteride is a selective inhibitor of 5α-reductase 2, one isozyme of 5α-reductase found in abundance in the human prostate. The objective of this study was to investigate the effects of finasteride on androgen receptor expression and tissue morphology in human benign prostatic hyperplasia specimens. Patients undergoing transurethral resection of the prostate and either treated or not treated with finasteride between 2004 and 2010 at the University of Wisconsin-Hospital were retrospectively identified using an institutional database. Prostate specimens from each patient were triple-stained for androgen receptor, prostate-specific antigen, and basal marker cytokeratin 5. Morphometric analysis was performed using the multispectral imaging, and results were compared between groups of finasteride treated and non-treated patients. Epithelial androgen receptor but not stromal androgen receptor expression was significantly lower in patients treated with finasteride than in non-treated patients. Androgen receptor-regulated prostate-specific antigen was not significantly decreased in finasteride-treated patients. Significant luminal epithelial atrophy and basal cell hyperplasia were prevalent in finasteride treated patients. Epithelial androgen receptor expression was highly correlated to the level of luminal epithelial atrophy. In this study, finasteride decreased the expression of epithelial androgen receptor in a tissue specific manner. The correlation between epithelial androgen receptor and the extent of luminal epithelial atrophy suggests that epithelial androgen receptor may be directly regulating the atrophic effects observed with finasteride treatment.

Influence of insulin and testosterone on diabetic rat ventral prostate: Histological, morphometric and immunohistochemical study

Many studies have reported high prevalence of hypogonadism amongst the diabetic patients suggesting the therapeutic benefits of testosterone hormone administration to these patients. The present research was carried out to study the possible role of insulin, testosterone and combined insulin and testosterone therapy on the diabetic rat ventral prostate. Fifty-eight adult male albino rats were used and divided into 2 main groups; the control and the experimentally induced diabetic group that given a single intra-peritoneal injection of STZ (60mg/kg). The rats with confirmed DM were subdivided into: diabetic group that kept without treatment, diabetic–testosterone group (100mg/kg/4weeks, S.C.), diabetic–insulin group (5IU/day/rat, S.C.) and diabetic–insulin–testosterone group. After 8 weeks, the ventral prostatic lobes were dissected and processed for paraffin blocks to study the epithelial height, mast cell number, expression of AR, PCNA and active caspase-3. DM induced a significant decrease in mast cells number, epithelial height and androgen receptors (AR) with a significant increase in the apoptotic index. On the other hand, the epithelial cells showed a non-significant change in the proliferation rate compared to the control group. Treatment with testosterone induced a significant increase in the epithelial height, number of mast cells & AR positive epithelial cells and a non-significant increase in PCNA with a significant decrease in the active caspase-3. Treatment with insulin showed abundant mast cells in the stroma with a significant increase in epithelial height & AR with decrease in active caspase-3 positive cells and insignificant changes in PCNA. Combined treatment with both hormones produced similar outcomes to the group treated with insulin alone. It was concluded that diabetes leads to alterations in rat ventral prostate gland. Early insulin treatment attenuates these effects with preserving most of the measured parameters within the normal values. Furthermore, addition of testosterone to insulin did not seem to produce a significant therapeutic add.

A true epidermotropic apocrine neoplasm in the form of perianal Paget’s disease: a case report

IntroductionExtramammary Paget’s disease is an uncommon intraepithelial neoplasm that arises in areas rich in apocrine glands. Treatment includes wide surgical excision and nonsurgical modalities. We present the case of a patient with perianal Paget’s disease with no recurrent disease after wide surgical resection.Case presentationOur patient was a 46-year-old man of Macedonian ethnicity who presented with a pruritic perianal lesion measuring up to 6cm without pain or bleeding. Two biopsies and a perianal wide surgical excision were performed. The tissue specimens were formalin-fixed and the paraffin-embedded samples analyzed according to standard histochemical and immunohistochemical procedures.Surgical perianal skin excision revealed diffuse eczematoid, whitish plaques. Pathohistology showed Paget cells infiltrating his epidermis and adnexal epithelium, with ulceration. Immunohistochemical analysis revealed positive Paget cell expression for cytokeratin 7, epithelial membrane antigen, carcinoembryonic antigen, androgen receptor and human epidermal growth factor receptor 2, and negative expression for cytokeratin 20 and melan-A.ConclusionPaget’s disease is a rare disorder that should be considered in the differential diagnosis of perianal lesions. Reporting cases of extramammary Paget’s disease is crucial for diagnostic guidelines and different therapeutic options.

Abstract 4245: Silencing of the PMEPA1 gene, a key regulator of androgen receptor in prostate cancer.

Abstract Introduction and Objectives: PMEPA1 is an androgen inducible gene, abundantly expressed in prostate epithelial cells and controls androgen receptor (AR) protein levels by recruiting NEDD4-1 E3 ubiquitin ligase to degrade AR. PMEPA1 gene expression is either reduced or lost in two-thirds of prostate cancer (CaP). Methylation of the PMEPA1 promoter may contribute to PMEPA1 gene silencing. Previously we have shown that AR binds to an Androgen Responsive Element (ARE) in the proximal promoter of the PMEPA1 gene in a hormone-dependent manner. These results prompted us to investigate the methylation status of proximal ARE and intronic SP1 enhancer sites in cell lines and in laser capture micro-dissected (LCM) human prostate tumors. Methods: Methylation status of the PMEPA1 promoter was evaluated in AR positive LNCaP, VCaP and LAPC4 cell lines and in LCM-enriched tumor cells using a previously validated method combining precipitation of methylated-DNA and methylation-sensitive restriction enzyme digestion (COMPARE-MS). Expression of PMEPA1 at protein and transcript levels was assessed by Western blot and Q-PCR, respectively in response to DNA methyl transferase inhibitor 5-aza-2’-deoxycitidine. Results: In a panel of LCM derived genomic DNA (N=50), PMEPA1 promoter sequences were found to be hypermethylated in 44% of human prostate tumors (N=22). PMEPA1 methylation was consistent with loss of PMEPA1 gene expression. PMEPA1 methylation was also detected in androgen-sensitive VCaP, LNCaP and LAPC4 CaP cell lines. Western blot and Q-PCR analysis revealed that demethylation restores PMEPA1 expression in CaP cells. Evaluation of proximal promoter sequences of PMEPA1 suggested that DNA methylation may directly inhibit AR binding to the proximal ARE and to the downstream intronic SP1 enhancer site of PMEPA1 gene. PMEPA1 methylation status showed a trend towards association with race, with a very low (12%) incidence of PMEPA1 methylation in African Americans subjects. Conclusions: PMEPA1 promoter regulatory sequences are hypermethylated in prostate cancer cell lines and in primary tumors suggesting that DNA methylation may contribute to the silencing of PMEPA1 gene in a significant subset of prostate tumors. These data, along with our earlier observation showing elevation of AR levels in response to the inhibition of PMEPA1 expression suggest a role of DNA methylation in the silencing of PMEPA1 gene and AR regulation in human CaP. Overall, reduced or eliminated PMEPA1 expression is likely to interfere with AR signaling and/ or other pathways in CaP. Source of Funding: This study was supported by the NIH grant RO CA106653 to S.S. Citation Format: Shashwat E. Sharad, Hua Li, Micheal Haffner, Srinivasan Yegnasubramanian, Lakshmi Ravindranth, Yongmei Chen, Alagarsamy Srinivasan, Gyorgy Petrovics, Shiv Srivastava, Albert Dobi. Silencing of the PMEPA1 gene, a key regulator of androgen receptor in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4245. doi:10.1158/1538-7445.AM2013-4245

Targeting Thymic Epithelia AR Enhances T-Cell Reconstitution and Bone Marrow Transplant Grafting Efficacy

Although thymic involution has been linked to the increased testosterone in males after puberty, its detailed mechanism and clinical application related to T-cell reconstitution in bone marrow transplantation (BMT) remain unclear. By performing studies with reciprocal BMT and cell-specific androgen receptor (AR) knockout mice, we found that AR in thymic epithelial cells, but not thymocytes or fibroblasts, played a more critical role to determine thymic cellularity. Further dissecting the mechanism using cell-specific thymic epithelial cell-AR knockout mice bearing T-cell receptor transgene revealed that elevating thymocyte survival was due to the enhancement of positive selection resulting in increased positively selected T-cells in both male and female mice. Targeting AR, instead of androgens, either via genetic knockout of thymic epithelial AR or using an AR-degradation enhancer (ASC-J9®), led to increased BMT grafting efficacy, which may provide a new therapeutic approach to boost T-cell reconstitution in the future.

Chronic caffeine intake increases androgenic stimuli, epithelial cell proliferation and hyperplasia in rat ventral prostate.

Coffee intake has been associated with a low risk of developing cancer, including prostate cancer, which is one of the most commonly diagnosed cancer in men. However, few studies have evaluated the chronic effects of caffeine, which is the most abundant methylxanthine in coffee, on prostate morphology and physiology. In the present study, we investigated the effects of chronic, low-dose caffeine intake on rat prostate morphology from puberty to adulthood. Five-week-old male Wistar rats were randomized into two experimental groups: caffeine-treated (20ppm in drinking water, n=12) and control (n=12). The ventral and dorsolateral prostates were dissected, weighted and submitted to morphological, morphometrical and immunohistochemical analysis of cellular proliferation, apoptosis and androgen receptor (AR) tissue expression. The testosterone (T) and dihydrotestosterone (DHT) concentrations were measured in the plasma. Our results show that caffeine intake increased the concentrations of T and DHT, organ weight, epithelial cell proliferation and AR tissue expression in the ventral prostatic lobe. All the ventral prostates from the caffeine-treated animals presented various degrees of epithelial and stromal hyperplasia. Our results suggest that chronic caffeine intake from puberty increases androgenic signalling and cell proliferation in the rat prostate gland and can be related to the development of benign prostatic hyperplasia.

Targeting Androgen Receptor to Suppress Macrophage-induced EMT and Benign Prostatic Hyperplasia (BPH) Development

Early studies suggested macrophages might play roles in inflammation-associated benign prostatic hyperplasia (BPH) development, yet the underlying mechanisms remain unclear. Here we first showed that CD68(+) macrophages were identified in both epithelium and the stromal area of human BPH tissues. We then established an in vitro co-culture model with prostate epithelial and macrophage cell lines to study the potential impacts of infiltrating macrophages in the BPH development and found that co-culturing prostate epithelial cells with macrophages promoted migration of macrophages. In a three-dimensional culture system, the sphere diameter of BPH-1 prostate cells was significantly increased during coculture with THP-1 macrophage cells. Mechanism dissection suggested that expression levels of epithelial-mesenchymal transition (EMT) markers, such as N-cadherin, Snail, and TGF-β2, were increased, and administration of anti-TGF-β2 neutralizing antibody during co-culture suppressed the EMT and THP-1-mediated growth of BPH-1 cells, suggesting THP-1 might go through EMT to influence the BPH development and progression. Importantly, we found that modulation of androgen receptor (AR) in BPH-1 and mPrE cells significantly increased THP-1 and RAW264.7 cell migration, respectively, and enhanced expression levels of EMT markers, suggesting that AR in prostate epithelial cells might play a role in promoting macrophage-mediated EMT in prostate epithelial cells. Silencing AR function via an AR degradation enhancer, ASC-J9, decreased the macrophage migration to BPH-1 cells and suppressed EMT marker expression. Together, these results provide the first evidence to demonstrate that prostate epithelial AR function is important for macrophage-mediated EMT and proliferation of prostate epithelial cells, which represents a previously unrecognized role of AR in the cross-talk between macrophages and prostate epithelial cells. These results may provide new insights for a new therapeutic approach to battle BPH via targeting AR and AR-mediated inflammatory signaling pathways.

Androgen hormone action in prostatic carcinogenesis: stromal androgen receptors mediate prostate cancer progression, malignant transformation and metastasis

It has been postulated that prostatic carcinogenesis is androgen dependent and that androgens mediate their effects primarily through epithelial cells; however, definitive proof of androgen hormone action in prostate cancer (PRCA) progression is lacking. Here we demonstrate through genetic loss of function experiments that PRCA progression is androgen dependent and that androgen dependency occurs via prostatic stromal androgen receptors (AR) but not epithelial AR. Utilizing tissue recombination models of prostatic carcinogenesis, loss of AR function was evaluated by surgical castration or genetic deletion. Loss of AR function prevented prostatic carcinogenesis, malignant transformation and metastasis. Tissue-specific evaluation of androgen hormone action demonstrated that epithelial AR was not necessary for PRCA progression, whereas stromal AR was essential for PRCA progression, malignant transformation and metastasis. Stromal AR was not necessary for prostatic maintenance, suggesting that the lack of cancer progression due to stromal AR deletion was not related to altered prostatic homeostasis. Gene expression analysis identified numerous androgen-regulated stromal factors. Four candidate stromal AR-regulated genes were secreted growth factors: fibroblast growth factors-2, -7, -10 and hepatocyte growth factor which were significantly affected by androgens and anti-androgens in stromal cells grown in vitro. These data support the concept that androgens are necessary for PRCA progression and that the androgen-regulated stromal microenvironment is essential to carcinogenesis, malignant transformation and metastasis and may serve as a potential target in the prevention of PRCA.

Abstract SY09-02: Molecular mechanisms in prostate cancer in African Americans

Abstract African American men have long been known to have the highest rates of prostate cancer (PCa) in the world. In addition, age adjusted mortality rates from PCa patients are two times worse among African Americans (AA) than Caucasians (CA). The reasons for the disparity in PCa risk, incidence, clinical progression, and disease-specific death are still controversial. In addition to socioeconomic and environmental factors, such as diet, access to care, and screenings, a biologically more aggressive cancer is proposed as one important explanation for the racial disparity observed in AA men. Androgens and the androgen receptor pathway are well established key regulators of aggressive PCa behavior. Using a novel proteomic pathway analysis comparing the distinct signal pathway and network proteins different between AA and CA PCa, we identified a number of markers including stem/progenitor cell and EMT related proteins that are overexpressed in PCa among AA. Our ongoing study indicates a novel link among Notch, WNT and androgen receptor (AR) pathways. In contrast to increase in epithelial AR, our data revealed a decrease in the expression of the stromal AR in AA PCa compared to CA PCa. Androgen and its receptor inhibit proliferation of prostate stromal cells through transcriptional suppression of cyclin B1. Mechanistically, the negative regulation of cyclin B1 by AR is mediated through switching between E2F1 and E2F4 on the promoter of cyclin B1. E2F1 binds to cyclin B1 promoter and maintains its expression and subsequent cell cycle progression in AR negative stromal cells or AR positive stromal cells under androgen depleted condition. Upon stimulation with androgen in AR positive stromal cells, E2F1 is displaced from the binding site by AR and replaced with E2F4, leading to recruitment of the SMRT/HDAC3 co-repressor complex and repression of cyclin B1. Furthermore, the switch between E2F1 and E2F4 at the E2F binding site of the cyclin B1 promoter coincides with an androgen-dependent interaction between AR and E2F1 as well as cytoplasm to nucleus translocation of E2F4. In summary, our study revealed an association between distinct epithelial and stromal AR expression and PCa racial disparity as well as the molecular mechanism involved in regulating AR expression in epithelial and stromal prostate cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY09-02. doi:1538-7445.AM2012-SY09-02

What controls PTEN and what it controls (in prostate cancer)

What controls PTEN and what it controls (in prostate cancer)

Anterior prostate epithelial AR inactivation modifies estrogen receptor expression and increases estrogen sensitivity

Androgens influence prostate growth and development, so androgen withdrawal can control progression of prostate diseases. Although estrogen treatment was originally used to induce androgen withdrawal, more recently direct estrogen effects on the prostate have been recognized, but the nature of androgen-estrogen interactions within the prostate remain poorly understood. To characterize androgen effects on estrogen sensitivity in the mouse prostate, we contrasted models of castration-induced androgen withdrawal in the prostate stromal and epithelial compartments with a prostate epithelial androgen receptor (AR) knockout (PEARKO) mouse model of selective epithelial AR inactivation. Castration markedly increased prostate epithelial estrogen receptor (ER)α immunoreactivity compared with very low ERα expression in intact males. Similarly, strong basal and luminal ERα expression was detected in PEARKO prostate of intact males, suggesting that epithelial AR activity regulated epithelial ERα expression. ERβ was strongly expressed in intact, castrated, and PEARKO prostate. However, strong clusters of epithelial ERβ positivity coincided with epithelial stratification in PEARKO prostate. In vivo estrogen sensitivity was increased in PEARKO males, with greater estradiol-induced prostate growth and epithelial proliferation leading to squamous metaplasia, featuring markedly increased epithelial proliferation, thickening, and keratinization compared with littermate controls. Our results suggest that ERα expression in the prostate epithelial cells is regulated by local, epithelia-specific, androgen-dependent mechanisms, and this imbalance in the AR- and ER-mediated signaling sensitizes the mature prostate to exogenous estrogens.

Increased CK5/CK8-Positive Intermediate Cells with Stromal Smooth Muscle Cell Atrophy in the Mice Lacking Prostate Epithelial Androgen Receptor

Results from tissue recombination experiments documented well that stromal androgen receptor (AR) plays essential roles in prostate development, but epithelial AR has little roles in prostate development. Using cell specific knockout AR strategy, we generated pes-ARKO mouse with knock out of AR only in the prostate epithelial cells and demonstrated that epithelial AR might also play important roles in the development of prostate gland. We found mice lacking the prostate epithelial AR have increased apoptosis in epithelial CK8-positive luminal cells and increased proliferation in epithelial CK5-positive basal cells. The consequences of these two contrasting results could then lead to the expansion of CK5/CK8-positive intermediate cells, accompanied by stromal atrophy and impaired ductal morphogenesis. Molecular mechanism dissection found AR target gene, TGF-β1, might play important roles in this epithelial AR-to-stromal morphogenesis modulation. Collectively, these results provided novel information relevant to epithelial AR functions in epithelial-stromal interactions during the development of normal prostate, and suggested AR could also function as suppressor in selective cells within prostate.

Essential roles of androgen signaling in Wolffian duct stabilization and epididymal cell differentiation

The epididymis is a male accessory organ and functions for sperm maturation and storage under the control of androgen. The development of the epididymis is also androgen dependent. The Wolffian duct (WD), anlagen of the epididymis, is formed in both male and female embryos; however, it is stabilized only in male embryos by testicular androgen. Androgen drives subsequent differentiation of the WD into the epididymis. Although the essential roles of androgen in WD masculinization and epididymal function have been established, little is known about cellular events regulated precisely by androgen signaling during these processes. It is also unclear whether androgen signaling, especially in the epithelia, has further function for epididymal epithelial cell differentiation. In this study we examined the cellular death and proliferation controlled by androgen signaling via the androgen receptor (AR) in WD stabilization. Analyses using AR knockout mice revealed that androgen signaling inhibits epithelial cell death in this process. Analysis of AP2α-Cre;ARflox/Y mice, in which AR function is deleted in the WD epithelium, revealed that epithelial AR is not required for the WD stabilization but is required for epithelial cell differentiation in the epididymis. Specifically, loss of epithelial AR significantly reduced expression of p63 that is essential for differentiation of basal cells in the epididymal epithelium. We also interrogated the possibility of regulation of the p63 gene (Trp63) by AR in vitro and found that p63 is a likely direct target of AR regulation.

Essential Roles of Androgen Signaling in Wolffian Duct Stabilization and Epididymal Cell Differentiation

The epididymis is a male accessory organ and functions for sperm maturation and storage under the control of androgen. The development of the epididymis is also androgen dependent. The Wolffian duct (WD), anlagen of the epididymis, is formed in both male and female embryos; however, it is stabilized only in male embryos by testicular androgen. Androgen drives subsequent differentiation of the WD into the epididymis. Although the essential roles of androgen in WD masculinization and epididymal function have been established, little is known about cellular events regulated precisely by androgen signaling during these processes. It is also unclear whether androgen signaling, especially in the epithelia, has further function for epididymal epithelial cell differentiation. In this study we examined the cellular death and proliferation controlled by androgen signaling via the androgen receptor (AR) in WD stabilization. Analyses using AR knockout mice revealed that androgen signaling inhibits epithelial cell death in this process. Analysis of AP2α-Cre;AR(flox/Y) mice, in which AR function is deleted in the WD epithelium, revealed that epithelial AR is not required for the WD stabilization but is required for epithelial cell differentiation in the epididymis. Specifically, loss of epithelial AR significantly reduced expression of p63 that is essential for differentiation of basal cells in the epididymal epithelium. We also interrogated the possibility of regulation of the p63 gene (Trp63) by AR in vitro and found that p63 is a likely direct target of AR regulation.

Androgen Receptor Expression in the Caput Epididymal Epithelium Is Essential for Development of the Initial Segment and Epididymal Spermatozoa Transit

The epididymis plays an essential role in male fertility, and disruption of epididymal function can lead to obstructive azoospermia. Formation and function of the epididymis is androgen-dependent. The androgen receptor (AR) is expressed in both the stromal and epithelial compartments of the epididymis, and androgen action mediated via stromal cells is vital for its normal development and function. However the impact of epithelial specific AR-dependent signaling in the epididymis remains underexplored. To address this, we used conditional gene-targeting in mice to selectively ablate AR from the caput epididymal epithelium, and characterized the resulting phenotype at multiple postnatal ages. Caput epithelium androgen receptor knock-out (CEARKO) mice have normal serum testosterone concentrations at day (d) 21 and d100, but do not develop an epididymal initial segment. The remaining caput epithelium displays a significant decrease in epithelial cell height from d11 and lumen diameter from d21 and disruption of the smooth muscle layer of the caput epididymis at d100. From d21, CEARKO mice accumulate cell debris, proteinaceous material, and, at later ages, spermatozoa in their efferent ducts, which prevents normal passage of spermatozoa from the testis into the cauda epididymis resulting in infertility when tested at d100. This efferent duct obstruction leads to fluid back-pressure and disruption of the seminiferous epithelium of the adult testis. We conclude that epithelial AR signaling is essential for postnatal development and function of the epididymal epithelium and that disruption of this signaling can contribute to obstructive azoospermia.

Androgen Receptor Expression in the Caput Epididymal Epithelium Is Essential for Development of the Initial Segment and Epididymal Spermatozoa Transit

The epididymis plays an essential role in male fertility, and disruption of epididymal function can lead to obstructive azoospermia. Formation and function of the epididymis is androgen-dependent. The androgen receptor (AR) is expressed in both the stromal and epithelial compartments of the epididymis, and androgen action mediated via stromal cells is vital for its normal development and function. However the impact of epithelial specific AR-dependent signaling in the epididymis remains underexplored. To address this, we used conditional gene-targeting in mice to selectively ablate AR from the caput epididymal epithelium, and characterized the resulting phenotype at multiple postnatal ages. Caput epithelium androgen receptor knock-out mice have normal serum testosterone concentrations at day (d) 21 and d100, but do not develop an epididymal initial segment. The remaining caput epithelium displays a significant decrease in epithelial cell height from d11 and lumen diameter from d21 and disruption of the smooth muscle layer of the caput epididymis at d100. From d21, caput epithelium androgen receptor knock-out mice accumulate cell debris, proteinaceous material, and, at later ages, spermatozoa in their efferent ducts, which prevents normal passage of spermatozoa from the testis into the cauda epididymis resulting in infertility when tested at d100. This efferent duct obstruction leads to fluid back-pressure and disruption of the seminiferous epithelium of the adult testis. We conclude that epithelial AR signaling is essential for postnatal development and function of the epididymal epithelium and that disruption of this signaling can contribute to obstructive azoospermia.