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CONTEXT - The role of endogenous androgens and SHBG in the development of cardiovascular disease in young adult women is unclear.
OBJECTIVE - Our objective was to study the prospective association of serum androgens and SHBG with subclinical coronary and carotid disease among young to middle-aged women.
DESIGN AND SETTING - This was an ancillary study to the Coronary Artery Risk Development in Young Adults (CARDIA) study, a population-based multicenter cohort study with 20 yr of follow-up.
PARTICIPANTS - Participants included 1629 women with measurements of serum testosterone and SHBG from yr 2, 10, or 16 and subclinical disease assessment at yr 20 (ages 37-52 yr).
MAIN OUTCOME MEASURES - Coronary artery calcified plaques (CAC) and carotid artery intima-media thickness (IMT) were assessed at yr 20. The IMT measure incorporated the common carotid arteries, bifurcations, and internal carotid arteries.
RESULTS - SHBG (mean of yr 2, 10, and 16) was inversely associated with the presence of CAC (multivariable adjusted odds ratio for women with SHBG levels above the median = 0.59; 95% confidence interval = 0.40-0.87; P = 0.008). SHBG was also inversely associated with the highest quartile of carotid-IMT (odds ratio for women with SHBG levels in the highest quartile = 0.56; 95% confidence interval = 0.37-0.84; P for linear trend across quartiles = 0.005). No associations were observed for total or free testosterone with either CAC or IMT.
CONCLUSION - SHBG levels were inversely associated with subclinical cardiovascular disease in young to middle-aged women. The extent to which low SHBG is a risk marker or has its own independent effects on atherosclerosis is yet to be determined.
BACKGROUND - The androgen-regulated probasin (PB) promoter has been used extensively to target transgenes to the prostate in transgenic mice; however, limited data exist on the mechanism that dictates prostate-specific gene expression. Tissue-specific gene expression involves synergistic effects among transcription factors associated in a complex bound to cis-acting DNA elements.
METHODS - Using comprehensive linker scan mutagenesis, enzyme mobility shift and supershift assays, chromatin immunoprecipitation, and transgenic animal studies, we have extensively characterized the prostate-specific PB promoter.
RESULTS - We identified a series of nonreceptor transcription factors that are bound to the prostate-specific rat PB promoter. These factors include several ubiquitously distributed proteins known to participate in steroid receptor-mediated transcription. In addition, we identified two tissue-specific DNA elements that are crucial in directing prostate-specific PB expression, and confirmed the functional importance of both elements in transgenic animal studies. These two elements are functionally interchangeable and can be bound by multiple protein complexes, including the forkhead transcription factor FoxA1, a "pioneer factor" that has a restricted distribution to some cells type that are ectoderm and endoderm in origin. Using transgenic mice, we further demonstrate that the minimal PB promoter region (-244/-96 bp) that encompasses these tissue-specific elements results in prostate-specific gene expression in transgenic mice, contains androgen receptor and FoxA1-binding sites, as well as ubiquitous transcription factor binding sites.
CONCLUSION - We propose that these sequence-specific DNA-binding proteins, including tissue-restricted and ubiquitous factors, create the first level of transcriptional control, which responds to intracellular pathways that directs prostate-specific gene expression.
Benign prostatic hyperplasia (BPH) and prostate carcinoma (CaP) are linked to aging and the presence of androgens, suggesting that androgen regulated genes play a major role in these common diseases. Androgen regulation of prostate growth and development depends on the presence of intact epithelial-stromal interactions. Further, the prostatic stroma is implicated in BPH. This suggests that epithelial cell lines are inadequate to identify androgen regulated genes that could contribute to BPH and CaP and which could serve as potential clinical biomarkers. In this study, we used a human prostate xenograft model to define a profile of genes regulated in vivo by androgens, with an emphasis on identifying candidate biomarkers. Benign transition zone (TZ) human prostate tissue from radical prostatectomies was grafted to the sub-renal capsule site of intact or castrated male immunodeficient mice, followed by the removal or addition of androgens, respectively. Microarray analysis of RNA from these tissues was used to identify genes that were; 1) highly expressed in prostate, 2) had significant expression changes in response to androgens, and, 3) encode extracellular proteins. A total of 95 genes meeting these criteria were selected for analysis and validation of expression in patient prostate tissues using quantitative real-time PCR. Expression levels of these genes were measured in pooled RNAs from human prostate tissues with varying severity of BPH pathologic changes and CaP of varying Gleason score. A number of androgen regulated genes were identified. Additionally, a subset of these genes were over-expressed in RNA from clinical BPH tissues, and the levels of many were found to correlate with disease status. Our results demonstrate the feasibility, and some of the problems, of using a mouse xenograft model to characterize the androgen regulated expression profiles of intact human prostate tissues.
BACKGROUND/AIMS - Disrupting the enzyme Cyp4a14 in mice leads to hypertension, which is more severe in the male mice and appears to be due to androgen excess. Because the Cyp4a14 enzyme is located in the proximal tubule of the kidney, we hypothesized that there could be dysregulation of transport in this segment that could contribute to the hypertension.
METHODS - Wild-type (SV/129) mice and mice that had targeted disruption of the Cyp4a14 gene were studied. Proximal convoluted tubules (PCT) from knockout and wild-type mice were dissected and perfused in vitrofor measurement of volume absorption (J(V)). Expression of the sodium-hydrogen exchanger 3 (NHE3), the predominant transporter responsible for sodium transport in this segment, was measured by immunoblot. Renal vascular (afferent arteriole) responses to angiotensin and endothelin were also measured.
RESULTS - PCT volume absorption was elevated in tubules from the Cyp4a14 knockout mice as compared to the wild-type mice. Brush border membrane NHE3 expression was almost 2-fold higher in Cyp4a14 knockout mice than in wild-type mice. No difference was found in the afferent arteriolar response.
CONCLUSION - Thus, hypertension in the Cyp4a14 knockout mice appears to be driven by excessive fluid reabsorption in the proximal tubule, which is secondary to overexpression of NHE3.
2009 S. Karger AG, Basel.
BACKGROUND - The role of Wnt/beta-Catenin signaling in embryogenesis and carcinogenesis has been extensively studied in organs such as colon, lung and pancreas, but little is known about Wnt/beta-Catenin signaling in the prostate. Although stabilizing mutations in APC and beta-Catenin are rare in primary prostate tumors, recent studies suggest that cytoplasmic/nuclear beta-Catenin is associated with advanced, metastatic, hormone-refractory prostate carcinoma.
METHODS - To better understand the role of beta-Catenin in prostatic development and carcinogenesis, we studied Wnt expression during prostate development and activated Wnt/beta-Catenin signaling in the developing and adult prostate.
RESULTS - Our results demonstrated that during prostate development Wnt ligands display a dynamic expression pattern. Activation of beta-Catenin during prostate development caused epithelial hyperplasia followed by prostatic intraepithelial neoplasia (PIN) in prostate. In the adult prostate, activation of beta-Catenin resulted in high grade PIN (HGPIN) and continuous prostatic growth after castration. As a result of activation of beta-Catenin, AR was first up-regulated with the emergence of epithelial hyperplasia, but was later down-regulated when HGPIN developed. Furthermore, activation of beta-Catenin induced Foxa2 re-expression in adult prostate which normally is only expressed in the embryonic budding stage during prostate development.
CONCLUSIONS - The results from this study strongly suggest that Wnt/beta-Catenin signaling is involved in the regulation of prostate development and confirm that constitutive activation of this pathway enables the mouse prostate to grow after castration.
High-dose testosterone enanthate (TE) may prevent hypogonadism-induced osteopenia. For this study, 3-mo-old male and female Fisher SAS rats underwent sham surgery, gonadectomy (GX), or GX plus 28 days TE administration (7.0 mg/wk). GX reduced serum sex hormones (i.e., testosterone, dihydrotestosterone, and estradiol) (P < 0.05) in both sexes and bone concentrations of testosterone (males only), and estradiol (females only). GX also elevated urine deoxypyridinoline/creatinine in both sexes and serum osteocalcin (females only), findings that are consistent with high-turnover osteopenia. GX reduced cancellous bone volume (CBV) and increased osteoid surfaces in tibia of both sexes. GX males also experienced reduced trabecular number and width and increased trabecular separation, whereas GX females experienced increased osteoblast and osteoid surfaces. Bone biomechanical characteristics remained unaffected by GX, except that femoral stiffness was reduced in females. In contrast, TE administration to GX rats elevated serum and bone androgens to supraphysiological concentrations in both sexes but altered neither serum nor bone estradiol in males. Additionally, TE did not prevent GX-induced reductions in serum or bone estradiol in females. TE also reduced markers of high-turnover osteopenia in both sexes. In males, TE prevented GX-induced changes in trabecular number and separation, CBV, and osteoid surfaces while diminishing osteoblast and osteoclast surfaces; however, these changes were not fully prevented in females. In both sexes, TE increased femoral length and femoral maximal strength to above that of Sham and GX animals while preventing the loss of femoral stiffness in females. In conclusion, TE administration appears protective of cancellous bone in male rats and augments cortical bone strength in both sexes.
Typically, the initial response of a prostate cancer patient to androgen ablation therapy is regression of the disease. However, the tumor will progress to an "androgen-independent" stage that results in renewed growth and spread of the cancer. Both nuclear factor-kappaB (NF-kappaB) expression and neuroendocrine differentiation predict poor prognosis, but their precise contribution to prostate cancer progression is unknown. This report shows that secretory proteins from neuroendocrine cells will activate the NF-kappaB pathway in LNCaP cells, resulting in increased levels of active androgen receptor (AR). By blocking NF-kappaB signaling in vitro, AR activation is inhibited. In addition, the continuous activation of NF-kappaB signaling in vivo by the absence of the IkappaBalpha inhibitor prevents regression of the prostate after castration by sustaining high levels of nuclear AR and maintaining differentiated function and continued proliferation of the epithelium. Furthermore, the NF-kappaB pathway was activated in the ARR(2)PB-myc-PAI (Hi-myc) mouse prostate by cross-breeding into a IkappaBalpha(+/-) haploid insufficient line. After castration, the mouse prostate cancer continued to proliferate. These results indicate that activation of NF-kappaB is sufficient to maintain androgen-independent growth of prostate and prostate cancer by regulating AR action. Thus, the NF-kappaB pathway may be a potential target for therapy against androgen-independent prostate cancer.
Mechanisms of androgen dependence of the prostate are critical to understanding prostate cancer progression to androgen independence associated with disease mortality. Transient elevation of transforming growth factor-beta (TGF-beta) occurs after androgen ablation. To determine the role of TGF-beta on prostate response to androgen ablation, conditional TGF-beta type II receptor knockout mouse models of the epithelia (Tgfbr2(NKX3.1KO)) and stromal fibroblasts (Tgfbr2(fspKO)) were used. After castration, the prostates of Tgfbr2(NKX3.1KO) mice had apoptosis levels similar to those expected for control Tgfbr2(floxE2/floxE2) mice. Prostates of Tgfbr2(fspKO) mice, however, had reduced regression and high levels of proliferation associated with canonical Wnt activity throughout the glandular epithelia regardless of androgen status. In contrast, Tgfbr2(floxE2/floxE2) prostates had epithelial canonical Wnt activity only in the surviving proximal ducts after castration. In vitro studies showed that androgen antagonist, bicalutamide, transiently elevated both Tgfbr2(floxE2/floxE2) and Tgfbr2(fspKO) stromal expression of Wnt-2, Wnt-3a, and Wnt-5a. The neutralization of Wnt signaling by the expression of secreted frizzled related protein-2 (SFRP-2) resulted in decreased LNCaP prostate epithelial cell proliferation in stromal conditioned media transfer experiments. In vivo tissue recombination studies using Tgfbr2(fspKO) prostatic stromal cells in combination with wild-type or SV40 large T antigen expressing epithelia resulted in prostates that were refractile to androgen ablation. The expression of SFRP-2 restored the Tgfbr2(fspKO)-associated prostate responsiveness to androgen ablation. These studies reveal a novel TGF-beta, androgen, and Wnt paracrine signaling axis that enables prostatic regression of the distal ducts after androgen ablation while supporting proximal duct survival.
BACKGROUND - The androgen receptor (AR) plays critical roles in both androgen-dependent and castrate-resistant prostate cancer (PCa). However, little is known about AR target genes that mediate the receptor's roles in disease progression.
RESULTS - Using Chromatin Immunoprecipitation (ChIP) Display, we discovered 19 novel loci occupied by the AR in castrate resistant C4-2B PCa cells. Only four of the 19 AR-occupied regions were within 10-kb 5'-flanking regulatory sequences. Three were located up to 4-kb 3' of the nearest gene, eight were intragenic and four were in gene deserts. Whereas the AR occupied the same loci in C4-2B (castrate resistant) and LNCaP (androgen-dependent) PCa cells, differences between the two cell lines were observed in the response of nearby genes to androgens. Among the genes strongly stimulated by DHT in C4-2B cells--D-dopachrome tautomerase (DDT), Protein kinase C delta (PRKCD), Glutathione S- transferase theta 2 (GSTT2), Transient receptor potential cation channel subfamily V member 3 (TRPV3), and Pyrroline-5-carboxylate reductase 1 (PYCR1)--most were less strongly or hardly stimulated in LNCaP cells. Another AR target gene, ornithine aminotransferase (OAT), was AR-stimulated in a ligand-independent manner, since it was repressed by AR siRNA knockdown, but not stimulated by DHT. We also present evidence for in vivo AR-mediated regulation of several genes identified by ChIP Display. For example, PRKCD and PYCR1, which may contribute to PCa cell growth and survival, are expressed in PCa biopsies from primary tumors before and after ablation and in metastatic lesions in a manner consistent with AR-mediated stimulation.
CONCLUSION - AR genomic occupancy is similar between LNCaP and C4-2B cells and is not biased towards 5' gene flanking sequences. The AR transcriptionally regulates less than half the genes nearby AR-occupied regions, usually but not always, in a ligand-dependent manner. Most are stimulated and a few are repressed. In general, response is stronger in C4-2B compared to LNCaP cells. Some of the genes near AR-occupied regions appear to be regulated by the AR in vivo as evidenced by their expression levels in prostate cancer tumors of various stages. Several AR target genes discovered in the present study, for example PRKCD and PYCR1, may open avenues in PCa research and aid the development of new approaches for disease management.
Cell cultures representing different stages of prostatic carcinoma will be a useful tool allowing a more complete understanding of the role of individual genes in tumorigenesis. We used the androgen-regulated probasin promoter linked to the neomycin phosphotransferase (Neo) gene, to generate the ARR(2)PBneo transgenic mouse model. Development was normal and all six ARR(2)PBneo transgenic founder lines expressed the Neo gene in a prostate-specific manner. Line C, which expressed high levels of neo, was crossbred to LPB-Tag 12T-7f transgenic mice (in which the SV40 large T antigen (Tag) was targeted to the prostate by the large probasin (LPB) promoter). Three bigenic males (carrying both Neo and Tag transgenes) were identified. Prostatic lesions developed in these mice in a predictable and heritable manner, indicating that Neo did not alter Tag-induced prostate tumor development and progression. Three separate NeoTag epithelial cell strains were established from three bigenic mice. G418 selection was used to obtain immortalized epithelial cells in culture. Selected cells expressed the Neo and Tag transgenes, cytokeratins 8 and 18, and were androgen responsive for growth. To determine if these NeoTag cells maintained a similar in vivo phenotype to the 12T-7f transgenic line, tissue recombinations were made with rat urogenital sinus mesenchyme (rUGM) and grafted under the renal capsule of male nude mouse hosts. In recombinants, the three NeoTag strains developed PIN lesions and/or more extensive adenocarcinoma than seen in the 12T-7f mouse. Androgen ablation demonstrated that the grafts were androgen responsive. NeoTag cells grafted without rUGM developed undifferentiated adenocarcinoma demonstrating that prostatic stroma dictates the glandular architecture seen in the well-differentiated adenocarcinoma.