Human obesity leads to an increase in respiratory demands. As obesity becomes more pronounced some individuals are unable to compensate, leading to elevated arterial carbon dioxide levels (PaCO2), alveolar hypoventilation, and increased cardiorespiratory morbidity and mortality (Pickwickian syndrome). The mechanisms that link obesity and hypoventilation are unknown, but thought to involve depression of central respiratory control mechanisms. Here we report that obese C57BL/6J-Lepob mice, which lack circulating leptin, also exhibit respiratory depression and elevated PaCO2 >> 10 mm Hg; p < 0. 0001). A role for leptin in restoring ventilation in these obese, mutant mice was investigated. Three days of leptin infusion (30 microg/d) markedly increased minute ventilation (V E) across all sleep/wake states, but particularly during rapid eye movement (REM) sleep when respiration was otherwise profoundly depressed. The effect of leptin was independent of food intake, weight, and CO2 production, indicating a reversal of hypoventilation by stimulation of central respiratory control centers. Furthermore, leptin replacement in mutant mice increased CO2 chemosensitivity during non-rapid eye movement (NREM) (4.0 +/- 0.5 to 5.6 +/- 0.4 ml/min/%CO2; p < 0.01) and REM (-0.1 +/- 0.5 to 3.0 +/- 0.8 ml/min/%CO2; p < 0.01) sleep. We also demonstrate in wild-type mice that ventilation is appropriately compensated when obesity is diet-induced and endogenous leptin levels are raised more than tenfold. These results suggest that leptin can prevent respiratory depression in obesity, but a deficiency in central nervous system (CNS) leptin levels or activity may induce hypoventilation and the Pickwickian syndrome in some obese subjects. O'Donnell CP, Schaub CD, Haines AS, Berkowitz DE, Tankersley CG, Schwartz AR, Smith PL. Leptin prevents respiratory depression in obesity.

Recently, a unique fusion between the prostate-specific, androgen-regulated TMPRSS2 gene and the ETS genes ERG, ETV1, or ETV4 has been described in clinical prostate cancer. We investigated mechanisms of expression of four ETS genes, ERG, ETV1, ETV4, and FLI1, in 11 xenografts representing different stages of prostate cancer. All five androgen-dependent xenografts showed as major transcript overexpression of two splice variants of TMPRSS2:ERG, linking TMPRSS2 exon 1 or 2 sequences to ERG exon 4. In one of two androgen-sensitive xenografts, fusion transcripts of TMPRSS2 and ETV1 were detected. Array-based comparative genomic hybridization and interphase fluorescence in situ hybridization indicated both interstitial deletions and translocations as mechanisms of TMPRSS2:ERG gene fusion. Importantly, TMPRSS2 to ERG fusions were also observed in three of four androgen-independent, androgen receptor (AR)-negative xenografts and in two AR-negative clinical prostate cancer specimens; however, the fusion gene was not expressed. In almost all AR-negative tumor samples, overexpression of wild-type ETV4 or FLI1 was detected. Combined, our observations indicate a key role of fusion of TMPRSS2 and ETS genes in most androgen-regulated prostate cancers, which might be bypassed by androgen-independent expression of wild-type ETS factors in late-stage disease.

The calmodulin-binding properties of the rabbit skeletal muscle Ca2+ release channel (ryanodine receptor) and the channel's regulation by calmodulin were determined at < or = 0.1 microM and micromolar to millimolar Ca2+ concentrations. [125I]Calmodulin and [3H]ryanodine binding to sarcoplasmic reticulum (SR) vesicles and purified Ca2+ release channel preparations indicated that the large (2200 kDa) Ca2+ release channel complex binds with high affinity (KD = 5-25 nM) 16 calmodulins at < or = 0.1 microM Ca2+ and 4 calmodulins at 100 microM Ca2+. Calmodulin-binding affinity to the channel showed a broad maximum at pH 6.8 and was highest at 0.15 M KCl at both < or = 0.1 MicroM and 100 microM Ca2+. Under condition closely related to those during muscle contraction and relaxation, the half-times of calmodulin dissociation and binding were 50 +/- 20 s and 30 +/- 10 min, respectively. SR vesicle-45Ca2+ flux, single-channel, and [3H]ryanodine bind measurements showed that, at < or = 0.2 microM Ca2+, calmodulin activated the Ca2+ release channel severalfold. Ar micromolar to millimolar Ca2+ concentrations, calmodulin inhibited the Ca(2+)-activated channel severalfold. Hill coefficients of approximately 1.3 suggested no or only weak cooperative activation and inhibition of Ca2+ release channel activity by calmodulin. These results suggest a role for calmodulin in modulating SR Ca2+ release in skeletal muscle at both resting and elevated Ca2+ concentrations.

HOXB13 is a member of the homeodomain family of sequence-specific transcription factors and, together with the androgen receptor (AR), plays a critical role in the normal development of the prostate gland. We demonstrate here that, in prostate cancer cells, HOXB13 is a key determinant of the response to androgens. Specifically, it was determined that HOXB13 interacts with the DNA-binding domain of AR and inhibits the transcription of genes that contain an androgen-response element (ARE). In contrast, the AR:HOXB13 complex confers androgen responsiveness to promoters that contain a specific HOXB13-response element. Further, HOXB13 and AR synergize to enhance the transcription of genes that contain a HOX element juxtaposed to an ARE. The profound effects of HOXB13 knockdown on androgen-regulated proliferation, migration, and lipogenesis in prostate cancer cells highlight the importance of the observed changes in gene expression.

Androgen receptor (AR) gene aberrations are rare in prostate cancer before primary hormone treatment but emerge with castration resistance. To determine AR gene status using a minimally invasive assay that could have broad clinical utility, we developed a targeted next-generation sequencing approach amenable to plasma DNA, covering all AR coding bases and genomic regions that are highly informative in prostate cancer. We sequenced 274 plasma samples from 97 castration-resistant prostate cancer patients treated with abiraterone at two institutions. We controlled for normal DNA in patients' circulation and detected a sufficiently high tumor DNA fraction to quantify AR copy number state in 217 samples (80 patients). Detection of AR copy number gain and point mutations in plasma were inversely correlated, supported further by the enrichment of nonsynonymous versus synonymous mutations in AR copy number normal as opposed to AR gain samples. Whereas AR copy number was unchanged from before treatment to progression and no mutant AR alleles showed signal for acquired gain, we observed emergence of T878A or L702H AR amino acid changes in 13% of tumors at progression on abiraterone. Patients with AR gain or T878A or L702H before abiraterone (45%) were 4.9 and 7.8 times less likely to have a ≥50 or ≥90% decline in prostate-specific antigen (PSA), respectively, and had a significantly worse overall [hazard ratio (HR), 7.33; 95% confidence interval (CI), 3.51 to 15.34; P = 1.3 × 10(-9)) and progression-free (HR, 3.73; 95% CI, 2.17 to 6.41; P = 5.6 × 10(-7)) survival. Evaluation of plasma AR by next-generation sequencing could identify cancers with primary resistance to abiraterone.

Mutations in the androgen receptor (AR) that enable activation by antiandrogens occur in hormone-refractory prostate cancer, suggesting that mutant ARs are selected by treatment. To validate this hypothesis, we compared AR variants in metastases obtained by rapid autopsy of patients treated with flutamide or bicalutamide, or by excision of lymph node metastases from hormone-naïve patients. AR mutations occurred at low levels in all specimens, reflecting genetic heterogeneity of prostate cancer. Base changes recurring in multiple samples or multiple times per sample were considered putative selected mutations. Of 26 recurring missense mutations, most in the NH(2)-terminal domain (NTD) occurred in multiple tumors, whereas those in the ligand binding domain (LBD) were case specific. Hormone-naïve tumors had few recurring mutations and none in the LBD. Several AR variants were assessed for mechanisms that might underlie treatment resistance. Selection was evident for the promiscuous receptor AR-V716M, which dominated three metastases from one flutamide-treated patient. For the inactive cytoplasmically restricted splice variant ARAR enhanced ligand response, supporting a decoy function. A novel NTD mutation, W435L, in a motif involved in intramolecular interaction influenced promoter-selective, cell-dependent transactivation. AR-E255K, mutated in a domain that interacts with an E3 ubiquitin ligase, led to increased protein stability and nuclear localization in the absence of ligand. Thus, treatment with antiandrogens selects for gain-of-function AR mutations with altered stability, promoter preference, or ligand specificity. These processes reveal multiple targets for effective therapies regardless of AR mutation.

Prostate cancer is an androgen-dependent disease; metastatic prostate cancer is typically treated by androgen receptor (AR) blockade. Recurrence after androgen ablation and evidence that AR continues to play a role in many prostate cancers has led to an examination of other factors that potentiate AR activity. AR is a ligand-activated transcription factor whose activity is regulated not only by hormone but also by the levels of coactivators recruited by AR to facilitate transcription. We sought to assess the consequences of reducing expression of the transcription intermediary factor 2 (TIF2) coactivator on prostate cancer cell growth and AR action in cell lines to examine TIF2 expression in prostate cancer and to correlate expression with clinical outcome. Depletion of TIF2 reduced expression of AR-induced target genes and slowed proliferation of AR-dependent and AR-independent prostate cancer cells. Remarkably, we found that TIF2 expression is directly repressed by high levels of androgens in multiple AR-expressing cell lines. Expression of a reporter containing 5'-flanking region of the TIF2 was repressed both by androgens and by the antagonist, Casodex. Expression of TIF2 correlates with biochemical (prostate-specific antigen) recurrence (P = 0.0136). In agreement with our in vitro findings, the highest expression of TIF2 was found in patients whose cancer relapsed after androgen ablation therapy, supporting the idea that AR blockade might activate pathways that lead to stimulation of AR-dependent and AR-independent proliferation of prostate epithelium. The elevated expression of TIF2 at low hormone levels likely aids in inducing AR activity under these conditions; treatment with Casodex has the potential to counteract this induction.

The X-linked androgen receptor (AR) gene contains two polymorphic trinucleotide repeat segments that code for polyglutamine and polyglycine tracts in the N-terminal trans-activation domain of the AR protein. Changes in the lengths of these polymorphic repeat segments have been associated with increased risk of prostate cancer, an androgen-dependent tumor. Expansion of the polyglutamine tract causes a rare neuromuscular disease, spinal bulbar muscular atrophy, that is associated with low virilization, reduced sperm production, testicular atrophy, and infertility. As spermatogenesis is exquisitely androgen dependent, it is plausible that changes in these two repeat segments could have a role in some cases of male infertility associated with impaired spermatogenesis. To test this hypothesis, we examined the lengths of the polyglutamine and polyglycine repeats in 153 patients with defective sperm production and compared them to 72 normal controls of proven fertility. There was no significant association between the polyglycine tract and infertility. However, patients with 28 or more glutamines (Gln) in their AR had more than 4-fold (95% confidence interval, 4.9-3.2) increased risk of impaired spermatogenesis, and the more severe the spermatogenic defect, the higher the proportion of patients with a longer Gln repeat. Concordantly, the risk of defective spermatogenesis was halved when the polyglutamine tract was short (< or = 23 Gln). Whole cell transfection experiments using AR constructs harboring 15, 20, and 31 Gln repeats and a luciferase reporter gene with an androgen response element promoter confirmed an inverse relationship between Gln number and trans-regulatory activity. Immunoblot analyses indicated that the reduced androgenicity of the AR was unlikely to be due to a change in AR protein content. The data indicate a direct relation between length of the AR polyglutamine tract and the risk of defective spermatogenesis that is attributable to the decreased functional competence of AR with longer glutamine tracts.

Spinobulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by expansion of a polyglutamine tract in the androgen receptor (AR). This mutation confers toxic function to AR through unknown mechanisms. Mutant AR toxicity requires binding of its hormone ligand, suggesting that pathogenesis involves ligand-induced changes in AR. However, whether toxicity is mediated by native AR function or a novel AR function is unknown. We systematically investigated events downstream of ligand-dependent AR activation in a Drosophila model of SBMA. We show that nuclear translocation of AR is necessary, but not sufficient, for toxicity and that DNA binding by AR is necessary for toxicity. Mutagenesis studies demonstrated that a functional AF-2 domain is essential for toxicity, a finding corroborated by a genetic screen that identified AF-2 interactors as dominant modifiers of degeneration. These findings indicate that SBMA pathogenesis is mediated by misappropriation of native protein function, a mechanism that may apply broadly to polyglutamine diseases.

We sought to determine the prevalence of androgen receptor (AR) expression in a predominantly estrogen receptor (ER)-negative subset of breast cancers and delineate the immunohistochemical and clinical associations, including whether AR expression has prognostic significance in ER-negative tumors. We identified 69 ER-negative and 19 ER-positive breast cancer cases with concurrent immunohistochemical prognostic panels (ER, PR, HER-2/neu, Ki-67, and p53); immunohistochemical analysis was performed for AR using standard techniques. Clinical data were extracted from medical records. chi 2 tests were used to assess associations between variables. AR was found in 49% (34/69) of ER-negative and 89% (17/19) of ER-positive cases. In ER-negative tumors, AR was associated with increased age (P = .02), postmenopausal status (P < .001), tumor grade (P = .03), tumor size (P = .03), and HER-2/neu overexpression (P = .003). In ER-positive tumors, AR was associated with progesterone receptor expression (P < .03). In univariate analysis of ER-negative tumors, patients with AR-positive tumors had significantly better disease-free survival (P = .049). AR is expressed in a significant number of ER-negative cases and shows significant associations with important clinical and pathologic prognostic factors.

BACKGROUND

Chronic beta-adrenergic receptor (beta-AR) blockade improves cardiac contractility and prolongs survival in patients with heart failure; however, the mechanisms underlying these favorable responses are poorly understood. Stress-induced activation of the sympathetic nervous system results in protein kinase A (PKA)-mediated phosphorylation of the calcium (Ca2+) release channel/cardiac ryanodine receptor (RyR2), required for cardiac excitation-contraction (EC) coupling, activating the RyR2 channel, and increasing cardiac contractility. The hyperadrenergic state of heart failure results in leaky RyR2 channels attributable to PKA hyperphosphorylation and depletion of the stabilizing FK506 binding protein, FKBP12.6. We tested the hypothesis that improved cardiac muscle function attributable to beta-AR blockade is associated with restoration of normal RyR2 channel function in patients with heart failure.

RESULTS

We assessed the effects of beta-AR blockade on left ventricular volume using isolated perfused hearts and beta-agonist responsiveness using muscle strips from patients undergoing transplantation. Twenty-four human hearts were examined, 10 from patients with heart failure treated with beta-AR blockers (carvedilol, metoprolol, or atenolol), 9 from patients with heart failure without beta-AR blocker treatment, and 5 normal hearts. RyR2 PKA phosphorylation was determined by back-phosphorylation, FKBP12.6 in the RyR2 macromolecular complex was determined by coimmunoprecipitation, and channel function was assayed using planar lipid bilayers. beta-AR blockers reduced left ventricular volume (reverse remodeling) and restored beta-agonist response in cardiac muscle from patients with heart failure. Improved cardiac muscle function was associated with restoration of normal FKBP12.6 levels in the RyR2 macromolecular complex and RyR2 channel function.

CONCLUSIONS

Improved cardiac muscle function during beta-AR blockade is associated with improved cardiac Ca2+ release channel function in patients with heart failure.

Prostate cancer is dependent on androgen stimulation mediated by the androgen receptor (AR), a member of the steroid hormone receptor family of ligand-dependent nuclear receptors. Most patients respond to standard androgen ablation therapies, but virtually all patients eventually relapse with disease that has been termed hormone-refractory or androgen-independent disease. Efforts to use AR antagonists, such as flutamide or bicalutamide, to enhance responses to primary androgen ablation therapy or to treat androgen-independent prostate cancer have been disappointing, which has diminished enthusiasm for more aggressive or alternative methods to block AR function. However, many lines of evidence indicate that AR function contributes to tumor cell survival after androgen ablation and to growth of androgen-independent prostate cancer. This article outlines a number of mechanisms that may contribute to AR activity in androgen-independent prostate cancer, including AR amplification, AR mutation, altered expression of AR coactivator and corepressor proteins, and activation of other pathways that can enhance AR function. Understanding the mechanisms responsible for AR function in androgen-independent prostate cancer should allow the more rational development of antagonists that can enhance the efficacy of androgen ablation therapies.

Androgens are functionally required for the normal growth of the prostate gland and in prostate tumor development and progression. Epithelial-mesenchymal-transition (EMT) is an important process during normal development and in cancer cell metastasis induced by factors within the microenvironment, such as transforming growth factor-beta (TGF-beta). This study examined the ability of androgens to influence EMT of prostate cancer epithelial cells. The EMT pattern was evaluated on the basis of expression of the epithelial markers E-cadherin/beta-catenin, and the mesenchymal markers N-cadherin, as well as cytoskeleton reorganization in response to 5alpha-dihydrotestosterone (DHT; 1 nM) and/or TGF-beta (5 ng/ml). Overexpressing and silencing approaches to regulate androgen receptor (AR) expression were conducted to determine the involvement of AR in EMT in the presence or absence of an AR antagonist. Our results demonstrate that androgens induce the EMT pattern in prostate tumor epithelial cell with Snail activation and lead to significant changes in prostate cancer cell migration and invasion potential. Expression levels of AR inversely correlated with androgen-mediated EMT in prostate tumor epithelial cells, pointing to a low AR content required for the EMT phenotype. These findings indicate the ability of androgens to induce EMT by potentially bypassing the functional involvement of TGF-beta, thus contributing to metastatic behavior of prostate cancer cells.-Zhum, M.-L., Kyprianou, N. Role of androgens and the androgen receptor in epithelial-mesenchymal transition and invasion of prostate cancer cells.

The genetic dissection of various human infectious diseases has led to the definition of inborn errors of human STAT1 immunity of four types, including (i) autosomal recessive (AR) complete STAT1 deficiency, (ii) AR partial STAT1 deficiency, (iii) autosomal dominant (AD) STAT1 deficiency, and (iv) AD gain of STAT1 activity. The two types of AR STAT1 defect give rise to a broad infectious phenotype with susceptibility to intramacrophagic bacteria (mostly mycobacteria) and viruses (herpes viruses at least), due principally to the impairment of IFN-γ-mediated and IFN-α/β-mediated immunity, respectively. Clinical outcome depends on the extent to which the STAT1 defect decreases responsiveness to these cytokines. AD STAT1 deficiency selectively predisposes individuals to mycobacterial disease, owing to the impairment of IFN-γ-mediated immunity, as IFN-α/β-mediated immunity is maintained. Finally, AD gain of STAT1 activity is associated with autoimmunity, probably owing to an enhancement of IFN-α/β-mediated immunity. More surprisingly, it is also associated with chronic mucocutaneous candidiasis, through as yet undetermined mechanisms involving an inhibition of the development of IL-17-producing T cells. Thus, germline mutations in human STAT1 define four distinct clinical disorders. Various combinations of viral, mycobacterial and fungal infections are therefore allelic at the human STAT1 locus. These experiments of Nature neatly highlight the clinical and immunological impact of the human genetic dissection of infectious phenotypes.

Resistance of prostate cancer cells to the next-generation antiandrogen enzalutamide may be mediated by a multitude of survival signaling pathways. In this study, we tested whether increased expression of NF-κB2/p52 induces prostate cancer cell resistance to enzalutamide and whether this response is mediated by aberrant androgen receptor (AR) activation and AR splice variant production. LNCaP cells stably expressing NF-κB2/p52 exhibited higher survival rates than controls when treated with enzalutamide. C4-2B and CWR22Rv1 cells chronically treated with enzalutamide were found to express higher levels of NF-κB2/p52. Downregulation of NF-κB2/p52 in CWR22Rv1 cells chronically treated with enzalutamide rendered them more sensitive to cell growth inhibition by enzalutamide. Analysis of the expression levels of AR splice variants by quantitative reverse transcription PCR and Western blotting revealed that LNCaP cells expressing p52 exhibit higher expression of AR splice variants. Downregulation of expression of NF-κB2/p52 in VCaP and CWR22Rv1 cells by short hairpin RNA abolished expression of splice variants. Downregulation of expression of either full-length AR or the splice variant AR-V7 led to an increase in sensitivity of prostate cancer cells to enzalutamide. These results collectively demonstrate that resistance to enzalutamide may be mediated by NF-κB2/p52 via activation of AR and its splice variants.

SUMO is a novel ubiquitin-like protein that can covalently modify a large number of nuclear proteins. SUMO modification has emerged as an important regulatory mechanism for protein function and localization. Sumoylation is a dynamic process that is mediated by activating (E1), conjugating (E2), and ligating (E3) enzymes and is readily reversed by a family of SUMO-specific proteases (SENPs). Since SUMO was discovered 10 years ago, the biologic contribution of this posttranslational modification has remained unclear. In this review, we report that SENP1, a member of the SENP family, is overexpressed in human prostate cancer specimens. The induction of SENP1 is observed with the chronic exposure of prostate cancer cells to androgen and/or interleukin (IL) 6. SENP1 upregulation modulates the transcriptional activity of androgen receptors (ARs) and c-Jun, as well as cyclin D1 expression. Initial in vivo data from transgenic mice indicate that overexpression of SENP1 in the prostate leads to the development of prostatic intraepithelial neoplasia at an early age. Collectively, these studies indicate that overexpression of SENP1 is associated with prostate cancer development.

The multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) targets a number of Ca(2+) homeostatic proteins and regulates gene transcription. Many of the substrates phosphorylated by CaMKII are also substrates for protein kinase A (PKA), the best known downstream effector of beta-adrenergic receptor (beta-AR) signaling. While PKA and CaMKII are conventionally considered to transduce signals through separate pathways, there is a body of evidence suggesting that CaMKII is activated in response to beta-AR stimulation and that some of the downstream effects of beta-AR stimulation are actually mediated by CaMKII. The signaling pathway through which beta-AR stimulation activates CaMKII, in parallel with or downstream of PKA, is not well-defined. This review considers the evidence for and mechanisms by which CaMKII is activated in response to beta-AR stimulation. In addition the potential role of CaMKII in beta-AR regulation of cardiac function is considered. Notably, although many CaMKII targets (e.g., phospholamban or the ryanodine receptor) are central to the regulation of Ca(2+) handling, and effects of CaMKII on Ca(2+) handling are detectable, inhibition or gene deletion of CaMKII has relatively little effect on the acute physiological contractile response to beta-AR. On the other hand CaMKII expression and activity are increased in heart failure, a pathophysiological condition characterized by chronic stimulation of cardiac beta-ARs. Blockade of beta-ARs is an accepted therapy for treatment of chronic heart failure although the rationale for its beneficial effects in cardiomyocytes is uncertain. There is growing evidence that inhibition or gene deletion of CaMKII also has a significant beneficial impact on the development of heart failure. The possibility that excessive beta-AR stimulation is detrimental because of its effects on CaMKII mediated Ca(2+) handling disturbances (e.g., ryanodine receptor phosphorylation and diastolic SR Ca(2+) leak) is an intriguing hypothesis that merits future consideration.

BACKGROUND

GPCRs regulate a remarkable diversity of biological functions, and are thus often targeted for drug therapies. Stimulation of a GPCR by an extracellular ligand triggers receptor signaling via G proteins, and this process is highly regulated. Receptor activation is typically accompanied by desensitization of receptor signaling, a complex feedback regulatory process of which receptor internalization is postulated as a key event. The in vivo significance of GPCR internalization is poorly understood. In fact, the majority of studies have been performed in transfected cell systems, which do not adequately model physiological environments and the complexity of integrated responses observed in the whole animal.

RESULTS

In this study, we used knock-in mice expressing functional fluorescent delta opioid receptors (DOR-eGFP) in place of the native receptor to correlate receptor localization in neurons with behavioral responses. We analyzed the pain-relieving effects of two delta receptor agonists with similar signaling potencies and efficacies, but distinct internalizing properties. An initial treatment with the high (SNC80) or low (AR-M100390) internalizing agonist equally reduced CFA-induced inflammatory pain. However, subsequent drug treatment produced highly distinct responses. Animals initially treated with SNC80 showed no analgesic response to a second dose of either delta receptor agonist. Concomitant receptor internalization and G-protein uncoupling were observed throughout the nervous system. This loss of function was temporary, since full DOR-eGFP receptor responses were restored 24 hours after SNC80 administration. In contrast, treatment with AR-M100390 resulted in retained analgesic response to a subsequent agonist injection, and ex vivo analysis showed that DOR-eGFP receptor remained G protein-coupled on the cell surface. Finally SNC80 but not AR-M100390 produced DOR-eGFP phosphorylation, suggesting that the two agonists produce distinct active receptor conformations in vivo which likely lead to differential receptor trafficking.

CONCLUSIONS

Together our data show that delta agonists retain full analgesic efficacy when receptors remain on the cell surface. In contrast, delta agonist-induced analgesia is abolished following receptor internalization, and complete behavioral desensitization is observed. Overall these results establish that, in the context of pain control, receptor localization fully controls receptor function in vivo. This finding has both fundamental and therapeutic implications for slow-recycling GPCRs.

Men have a higher incidence of hepatocellular carcinoma (HCC) than women. Epidemiologic and animal studies have suggested that it might be due to the stimulatory effects of androgen and the protective effects of estrogen. Recently, increasing molecular mechanisms underlying the carcinogenic effect of both sex hormones were reported. Knockout of androgen receptor (AR) expression in hepatocytes delayed the development of N',N'-diethylnitrosamine (DEN)-induced HCC, suggesting the active AR pathway in augmenting the HCC risk. Moreover, an intriguing interaction between the viral protein of hepatitis B virus X protein (HBx) and the androgen pathway was established. HBx can enhance the transcriptional activity of AR in a ligand concentration-dependent manner, mainly through its effects on the c-Src and GSK-3beta kinase pathways. The studies from the DEN-induced HCC mouse model further provided a mechanism for the protective role of estrogen in female HCC. Estrogen can protect hepatocytes from malignant transformation via downregulation of IL-6 release from Kupffer cells, a critical process in this mouse model. Intriguingly, suppression of the ERalpha protein by overexpression of miR-18a, which occurs preferentially in female HCC, was identified as a novel mechanism to block the tumor-protective function of estrogen in female HCC. In conclusion, the current studies demonstrated that the gender disparity of HCC is attributed by both androgen and estrogen sex hormone pathways, with distinct roles in each gender. Therefore, the ligand and the receptor factors of both sex hormones need to be included for assessing the relative risk of HCC patients of each gender.

Despite encouraging clinical results with next generation drugs (MDV3100 and abiraterone) that inhibit androgen receptor (AR) signaling in patients with castration-resistant prostate cancer (CRPC), responses are variable and short-lived. There is an urgent need to understand the basis of resistance to optimize their future use. We reasoned that a radiopharmaceutical that measures intratumoral changes in AR signaling could substantially improve our understanding of AR pathway directed therapies. Expanding on previous observations, we first show that prostate-specific membrane antigen (PSMA) is repressed by androgen treatment in multiple models of AR-positive prostate cancer in an AR-dependent manner. Conversely, antiandrogens up-regulate PSMA expression. These expression changes, including increased PSMA expression in response to treatment with the antiandrogen MDV3100, can be quantitatively measured in vivo in human prostate cancer xenograft models through PET imaging with a fully humanized, radiolabeled antibody to PSMA, (64)Cu-J591. Collectively, these results establish that relative changes in PSMA expression levels can be quantitatively measured using a human-ready imaging reagent and could serve as a biomarker of AR signaling to noninvasively evaluate AR activity in patients with CRPC.

It has long been recognized that activation of sympathetic beta-adrenoceptors (beta-ARs) increases the spontaneous beating rate of sinoatrial nodal cells (SANCs); however, the specific links between stimulation of beta-ARs and the resultant increase in firing rate remain an enigma. In the present study, we show that the positive chronotropic effect of beta-AR stimulation is critically dependent on localized subsarcolemmal ryanodine receptor (RyR) Ca(2+) releases during diastolic depolarization (CRDD). Specifically, isoproterenol (ISO; 0.1 micromol/L) induces a 3-fold increase in the number of CRDDs per cycle; a shift to higher CRDD amplitudes (from 2.00+/-0.04 to 2.17+/-0.03 F/F(0); P<0.05 [F and F(0) refer to peak and minimal fluorescence]); and an increase in spatial width (from 3.80+/-0.44 to 5.45+/-0.47 microm; P<0.05). The net effect results in an augmentation of the amplitude of the local preaction potential subsarcolemmal Ca(2+) transient that, in turn, accelerates the diastolic depolarization rate, leading to an increase in SANC firing rate. When RyRs are disabled by ryanodine, beta-AR stimulation fails to amplify subsarcolemmal Ca(2+) releases, fails to augment the diastolic depolarization rate, and fails to increase the SANC firing rate, despite preserved beta-AR stimulation-induced augmentation of L-type Ca(2+) current amplitude. Thus, the RyR Ca(2+) release acts as a switchboard to link beta-AR stimulation to an increase in SANC firing rate: recruitment of additional localized CRDDs and partial synchronization of their occurrence by beta-AR stimulation lead to an increase in the heart rate.

The stability and activity of hypoxia-inducible factor (HIF) are regulated by the post-translational hydroxylation of specific prolyl and asparaginyl residues. We show that the HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also catalyzes hydroxylation of highly conserved asparaginyl residues within ankyrin repeat (AR) domains (ARDs) of endogenous Notch receptors. AR hydroxylation decreases the extent of ARD binding to FIH while not affecting signaling through the canonical Notch pathway. ARD proteins were found to efficiently compete with HIF for FIH-dependent hydroxylation. Crystallographic analyses of the hydroxylated Notch ARD (2.35A) and of Notch peptides bound to FIH (2.4-2.6A) reveal the stereochemistry of hydroxylation on the AR and imply that significant conformational changes are required in the ARD fold in order to enable hydroxylation at the FIH active site. We propose that ARD proteins function as natural inhibitors of FIH and that the hydroxylation status of these proteins provides another oxygen-dependent interface that modulates HIF signaling.

The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors and plays a critical role in regulating the expression of genes involved in androgen-dependent and -independent tumour formation. Regulation of the AR is achieved by alternate binding of either histone acetyltransferase (HAT)-containing co-activator proteins, or histone deacetylase 1 (HDAC1). Factors that control AR stability may also constitute an important regulatory mechanism, a notion that has been confirmed with the finding that the AR is a direct target for Mdm2-mediated ubiquitylation and proteolysis. Using chromatin immunoprecipitation (ChIP) and re-ChIP analyses, we show that Mdm2 associates with AR and HDAC1 at the active androgen-responsive PSA promoter in LNCaP prostate cancer cells. Furthermore, we demonstrate that Mdm2-mediated modification of AR and HDAC1 catalyses protein destabilization and attenuates AR sactivity, suggesting that ubiquitylation of the AR and HDAC1 may constitute an additional mechanism for regulating AR function. We also show that HDAC1 and Mdm2 function co-operatively to reduce AR-mediated transcription that is attenuated by the HAT activity of the AR co-activator Tip60, suggesting interplay between acetylation status and receptor ubiquitylation in AR regulation. In all, our data indicates a novel role for Mdm2 in regulating components of the AR transcriptosome.

The physiological significance of androgens in female reproduction was unclear until female mice with global knockout of androgen receptor (AR) expression were found to have reduced fertility with abnormal ovarian function. However, because ARs are expressed in a myriad of reproductive tissues, including the hypothalamus, pituitary, and various ovarian cells, the role of tissue-specific ARs in regulating female fertility remained unknown. To examine the importance of ovarian ARs in female reproduction, we generated granulosa cell (GC)- and oocyte-specific AR-knockout (ARKO) mice by crossing AR-flox mice with MisRIIcre (GC-specific) or growth differentiation factor growth differentiation factor-9cre (oocyte-specific) mice. Relative to heterozygous and wild-type mice, GC-specific ARKO mice had premature ovarian failure and were subfertile, with longer estrous cycles and fewer ovulated oocytes. In addition, ovaries from GC-specific knockout mice contained more preantral and atretic follicles, with fewer antral follicles and corpus lutea. Finally, in vitro growth of follicles from GC-specific AR-null mice was slower than follicles from wild-type animals. In contrast to GC-specific AR-null mice, fertility, estrous cycles, and ovarian morphology of oocyte-specific ARKO mice were normal, although androgens no longer promoted oocyte maturation in these animals. Together, our data indicate that nearly all reproductive phenotypes observed in global ARKO mice can be explained by the lack of AR expression in GCs. These GC-specific ARs appear to promote preantral follicle growth and prevent follicular atresia; thus they are essential for normal follicular development and fertility.