Androgens regulate ovarian follicular development by increasing follicle stimulating hormone receptor and microRNA-125b expression.

Although androgen excess is considered detrimental to women's health and fertility, global and ovarian granulosa cell-specific androgen-receptor (AR) knockout mouse models have been used to show that androgen actions through ARs are actually necessary for normal ovarian function and female fertility. Here we describe two AR-mediated pathways in granulosa cells that regulate ovarian follicular development and therefore female fertility. First, we show that androgens attenuate follicular atresia through nuclear and extranuclear signaling pathways by enhancing expression of the microRNA (miR) miR-125b, which in turn suppresses proapoptotic protein expression. Second, we demonstrate that, independent of transcription, androgens enhance follicle-stimulating hormone (FSH) receptor expression, which then augments FSH-mediated follicle growth and development. Interestingly, we find that the scaffold molecule paxillin regulates both processes, making it a critical regulator of AR actions in the ovary. Finally, we report that low doses of exogenous androgens enhance gonadotropin-induced ovulation in mice, further demonstrating the critical role that androgens play in follicular development and fertility. These data may explain reported positive effects of androgens on ovulation rates in women with diminished ovarian reserve. Furthermore, this study demonstrates mechanisms that might contribute to the unregulated follicle growth seen in diseases of excess androgens such as polycystic ovary syndrome.

A participatory model of the paradox of primary care.

PURPOSE: The paradox of primary care is the observation that primary care is associated with apparently low levels of evidence-based care for individual diseases, but systems based on primary care have healthier populations, use fewer resources, and have less health inequality. The purpose of this article is to explore, from a complex systems perspective, mechanisms that might account for the effects of primary care beyond disease-specific care. METHODS: In an 8-session, participatory group model-building process, patient, caregiver, and primary care clinician community stakeholders worked with academic investigators to develop and refine an agent-based computer simulation model to test hypotheses about mechanisms by which features of primary care could affect health and health equity. RESULTS: In the resulting model, patients are at risk for acute illness, acute life-changing illness, chronic illness, and mental illness. Patients have changeable health behaviors and care-seeking tendencies that relate to their living in advantaged or disadvantaged neighborhoods. There are 2 types of care available to patients: primary and specialty. Primary care in the model is less effective than specialty care in treating single diseases, but it has the ability to treat multiple diseases at once. Primary care also can provide disease prevention visits, help patients improve their health behaviors, refer to specialty care, and develop relationships with patients that cause them to lower their threshold for seeking care. In a model run with primary care features turned off, primary care patients have poorer health. In a model run with all primary care features turned on, their conjoint effect leads to better population health for patients who seek primary care, with the primary care effect being particularly pronounced for patients who are disadvantaged and patients with multiple chronic conditions. Primary care leads to more total health care visits that are due to more disease prevention visits, but there are reduced illness visits among people in disadvantaged neighborhoods. Supplemental appendices provide a working version of the model and worksheets that allow readers to run their own experiments that vary model parameters. CONCLUSION: This simulation model provides insights into possible mechanisms for the paradox of primary care and shows how participatory group model building can be used to evaluate hypotheses about the behavior of such complex systems as primary health care and population health.

Structural basis for leucine-rich nuclear export signal recognition by CRM1.

CRM1 (also known as XPO1 and exportin 1) mediates nuclear export of hundreds of proteins through the recognition of the leucine-rich nuclear export signal (LR-NES). Here we present the 2.9 A structure of CRM1 bound to snurportin 1 (SNUPN). Snurportin 1 binds CRM1 in a bipartite manner by means of an amino-terminal LR-NES and its nucleotide-binding domain. The LR-NES is a combined alpha-helical-extended structure that occupies a hydrophobic groove between two CRM1 outer helices. The LR-NES interface explains the consensus hydrophobic pattern, preference for intervening electronegative residues and inhibition by leptomycin B. The second nuclear export signal epitope is a basic surface on the snurportin 1 nucleotide-binding domain, which binds an acidic patch on CRM1 adjacent to the LR-NES site. Multipartite recognition of individually weak nuclear export signal epitopes may be common to CRM1 substrates, enhancing CRM1 binding beyond the generally low affinity LR-NES. Similar energetic construction is also used in multipartite nuclear localization signals to provide broad substrate specificity and rapid evolution in nuclear transport.

NF-kappaB mediates lipid-induced fetuin-A expression in hepatocytes that impairs adipocyte function effecting insulin resistance.

Fetuin-A, a hepatic secretory protein, has recently been implicated in insulin resistance and Type 2 diabetes. It is an endogenous inhibitor of insulin receptor tyrosine kinase. However, regulation of fetuin-A synthesis in relation to insulin resistance is unclear. In the present paper, we report that both non-esterified ('free') fatty acids and fetuin-A coexist at high levels in the serum of db/db mice, indicating an association between them. For an in-depth study, we incubated palmitate with HepG2 cells and rat primary hepatocytes, and found enhanced fetuin-A secretion to more than 4-fold over the control. Interestingly, cell lysates from these incubations showed overexpression and activity of NF-kappaB (nuclear factor kappaB). In NF-kappaB-knockout HepG2 cells, palmitate failed to increase fetuin-A secretion, whereas forced expression of NF-kappaB released fetuin-A massively in the absence of palmitate. Moreover, palmitate stimulated NF-kappaB binding to the fetuin-A promoter resulting in increased reporter activity. These results suggest NF-kappaB to be the mediator of the palmitate effect. Palmitate-induced robust expression of fetuin-A indicates the occurrence of additional targets, and we found that fetuin-A severely impaired adipocyte function leading to insulin resistance. Our results reveal a new dimension of lipid-induced insulin resistance and open another contemporary target for therapeutic intervention in Type 2 diabetes.

Lipid induced overexpression of NF-kappaB in skeletal muscle cells is linked to insulin resistance.

Lipid induced NF-kappaB activation is known to be associated with insulin resistance and type2 diabetes. Here we show that incubation of L6 skeletal muscle cells with palmitate significantly increased NF-kappaB p65 and NF-kappaB p50 expression along with their phosphorylation. NF-kappaB p65 siRNA inhibited palmitate induced overexpression of NF-kappaB p65 indicating palmitate effect on transcriptional activation. RT-PCR and real time PCR experiments also showed a significant increase in NF-kappaB p65 gene expression due to palmitate. Overexpression of NF-kappaB p65 by palmitate was linked to impairment of insulin activity. Palmitate effect on NF-kappaB gene and protein expression was found to be mediated by phospho-PKCepsilon as calphostin C (an inhibitor of PKC) and epsilonV1 (PKCepsilon translocation inhibitor) significantly reduced NF-kappaB expression. To understand the underlying mechanism, we purified NF-kappaB and pPKCepsilon from palmitate incubated skeletal muscle cells and their interaction in cell free system demonstrated the transfer of phosphate from PKCepsilon to NF-kappaB. This prompted us to transduct pPKCepsilon to the skeletal muscle cells. These cells showed increased amount of pNF-kappaB and NF-kappaB. Excess of NF-kappaB p65 pool thus created in the cells made them insulin resistant. Addition of NF-kappaB p65 siRNA and SN50 inhibited palmitate induced NF-kappaB p65 expression indicating NF-kappaB regulation of its gene expression. Increase of NF-kappaB did not affect the activation of IKK/IkappaB indicating NF-kappaB p65 expression to be a distinct effect of palmitate. Since NF-kappaB p65 is linked to several diseases, including type2 diabetes, this report may be important in understanding the pathogenicity of these diseases.

Insulin resistance due to lipid-induced signaling defects could be prevented by mahanine.

It is well known that free fatty acids (FFAs) play a key role in implementing insulin resistance and type 2 diabetes. Resources of chemical compounds that intervene the derogatory effect of FFAs are indeed very limited. We have isolated mahanine, a carbazole alkaloid, from the leaves of Murraya koenegii that prevented palmitate-induced inhibition of insulin-stimulated phosphorylation of IRbeta, PI3K, PDK1, and Akt in L6 myotubes. This was also reflected in the palmitate-induced inhibition of insulin-stimulated [(3)H] 2-DOG uptake by L6 myotubes, where palmitate adverse effect was significantly blocked by mahanine. Previous reports indicated that one of the major targets of lipid-induced damage in insulin signaling pathway resulting impairment of insulin sensitivity is insulin receptor (IR). Here, we have observed that palmitate significantly increased pPKCepsilon in both cytosol and nuclear region of L6 myotubes in comparison to control. Translocation of pPKCepsilon to the nucleus was associated with the impairment of HMGA1, the architectural transcription factor of IR gene and all these were reversed by mahanine. Palmitate-induced activation of IKK/IkappaBeta/NF-kappaBeta pathway was also attenuated by mahanine. Taken together, mahanine showed encouraging possibility to deal with lipid induced insulin resistance. In order to examine it further, mahanine was administered on nutritionally induced type 2 diabetic golden hamsters; it significantly improved hyperglycemia in all the treated animals. Our results, therefore, suggest that mahanine acts on two important sites of lipid induced insulin resistance (i) impairment of IR gene expression and (ii) activation of NF-kappaBeta pathway, thus, showing promise for its therapeutic choice for type 2 diabetes.

Paxillin regulated genomic networks in prostate cancer.

Paxillin is extensively involved in focal adhesion signaling and kinase signaling throughout the plasma membrane and cytoplasm. However, recent studies in prostate cancer suggest that paxillin also plays a critical role in regulating gene expression within the nucleus, serving as a liaison between cytoplasmic and nuclear MAPK and Androgen Receptor (AR) signaling. Here we used RNA-seq to examine the paxillin-regulated transcriptome in several human prostate cancer cell lines. First, we examined paxillin effects on androgen-mediated transcription in control or paxillin-depleted AR-positive LNCaP and C4-2 human prostate cancer cells. In androgen-dependent LNCaP cells, we found over 1000 paxillin-dependent androgen-responsive genes, some of which are involved in endocrine therapy resistance. Most paxillin-dependent AR-mediated genes in LNCaP cells were no longer paxillin-dependent in androgen-sensitive, castration-resistant C4-2 cells, suggesting that castration-resistance may markedly alter paxillin effects on genomic AR signaling. To examine the paxillin-regulated transcriptome in the absence of androgen signaling, we performed RNA-seq in AR-negative PC3 human prostate cancer cells. Paxillin enhanced several pro-proliferative pathways, including the CyclinD/Rb/E2F and DNA replication/repair pathways. Additionally, paxillin suppressed pro-apoptotic genes, including CASP1 and TNFSF10. Quantitative PCR confirmed that these pathways are similarly regulated by paxillin in LNCaP and C4-2 cells. Functional studies showed that, while paxillin stimulated cell proliferation, it had minimum effect on apoptosis. Thus, paxillin appears to be an important transcriptional regulator in prostate cancer, and analysis of its transcriptome might lead to novel approaches toward the diagnosis and treatment of this important disease.

Gestational Diabetes Epigenetically Reprograms the Cart Promoter in Fetal Ovary, Causing Subfertility in Adult Life.

Intrauterine exposure to various adverse conditions during fetal development can lead to epigenetic changes in fetal tissues, predisposing those tissues to disease conditions later in life. An example is gestational diabetes (GD), where the offspring has a higher risk of developing obesity, metabolic disorders, or cardiovascular disease in adult life. In this study, using two well-established GD (streptozotocin- and high-fat and high-sugar-induced) mouse models, we report that female offspring from GD dams are predisposed toward fertility problems later in life. This predisposition to fertility problems is due to altered ovarian expression of a peptide called cocaine- and amphetamine-regulated transcript (CART), which is known to negatively affect folliculogenesis and is induced by elevated leptin levels. Results show that the underlying cause of this altered expression is due to fetal epigenetic modifications involving glucose- and insulin-induced miRNA, miR-101, and the phosphatidylinositol 3-kinase/Akt pathway. These signaling events regulate Ezh2, a histone methyltransferase that promotes H3K27me3, a gene-repressive mark, and CBP/p300, a histone acetyltransferase that promotes H3K27ac, a transcription activation mark, in the fetal ovary. Moreover, the CART promoter has depleted 5-methylcytosine (5mC) and enriched 5-hydroxymethylcytosine (5hmC) levels. The depletion of H3K27me3 and 5mC repressive marks and subsequent increase in H3K27ac and 5hmC gene-activating marks convert the Cartpt promoter to a "superpromoter." This makes the Cartpt promoter more sensitive to leptin levels that predispose the GD offspring to fertility problems. Therefore, this study provides a mechanistic insight about fetal epigenome reprogramming that manifests to ovarian dysfunction and subfertility later in adult life.

Cajanus cajan Linn. (Leguminosae) prevents alcohol-induced rat liver damage and augments cytoprotective function.

AIM OF THE STUDY: Cajanus cajan Linn. (Leguminosae) is a nontoxic edible herb, widely used in Indian folk medicine for the prevention of various liver disorders. In the present study we have demonstrated that methanol-aqueous fraction (MAF2) of Cajanus cajan leaf extract could prevent the chronically treated alcohol induced rat liver damage. MATERIALS AND METHODS: Chronic doses of alcohol (3.7 g/ kg) orally administered to rats for 28 days and liver function marker enzymes such as GPT, GOT, ALP and anti-oxidant enzyme activities were determined. Effect of MAF2 at a dose of 50mg/kg body weight on alcohol treated rats was noted. RESULTS: Alcohol effected significant increase in liver marker enzyme activities and reduced the activities of anti-oxidant enzymes. Co-administration of MAF2 reversed the liver damage due to alcohol; it decreased the activities of liver marker enzymes and augmented antioxidant enzyme activities. We also demonstrate significant decrease of the phase II detoxifying enzyme, UDP-glucuronosyl transferase (UGT) activity along with a three- and two-fold decrease of UGT2B gene and protein expression respectively. MAF2 co-administration normalized UGT activity and revived the expression of UGT2B with a concomitant expression and nuclear translocation of Nrf2, a transcription factor that regulates the expression of many cytoprotective genes. CONCLUSION: Cajanus cajan extract therefore shows a promise in therapeutic use in alcohol induced liver dysfunction.

Bilateral Pheochromocytomas in a Patient with Y175C Von Hippel-Lindau Mutation.

Von Hippel-Lindau (VHL) disease, caused by germline mutations in the VHL gene, is characterized by metachronously occurring tumors including pheochromocytoma, renal cell carcinoma (RCC), and hemangioblastoma. Although VHL disease leads to reduced life expectancy, its diagnosis is often missed and tumor screening guidelines are sparse. VHL protein acts as a tumor suppressor by targeting hypoxia-inducible factors (HIFs) for degradation through an oxygen-dependent mechanism. VHL mutants with more severely reduced HIF degrading function carry a high risk of RCC, while mutants with preserved HIF degrading capacity do not cause RCC but still lead to other tumors. VHL disease is classified into clinical types (1 and 2A-2C) based on this genotype-phenotype relationship. We report a case of bilateral pheochromocytomas and no other VHL-related tumors in a patient with Y175C VHL and show that this mutant preserves the ability to degrade HIF in normal oxygen conditions but, similar to the wild-type VHL protein, loses its ability to degrade HIF under hypoxic conditions. This study adds to the current understanding of the structure-function relationship of VHL mutations, which is important for risk stratification of future tumor development in the patients.

Oocyte-Derived Factors (GDF9 and BMP15) and FSH Regulate AMH Expression Via Modulation of H3K27AC in Granulosa Cells.

Anti-Müllerian hormone (AMH) produced by ovarian granulosa cells (GCs) plays a crucial role in ovarian function. It is used as a diagnostic and/or prognostic marker of fertility as well as for pathophysiological conditions in women. In this study, we investigated the underlying mechanism for regulation of AMH expression in GCs using primary mouse GCs and a human GC tumor-derived KGN cell line. We find that growth differentiation factor 9 (GDF9) and bone morphogenetic factor 15 (BMP15) together (GDF9 + BMP15), but not when tested separately, significantly induce AMH expression in vitro and in vivo (serum AMH). Our results show that GDF9 + BMP15 through the PI3K/Akt and Smad2/3 pathways synergistically recruit the coactivator p300 on the AMH promoter region that promotes acetylation of histone 3 lysine 27 (H3K27ac), facilitating AMH/Amh expression. Intriguingly, we also find that FSH inhibits GDF9 + BMP15-induced increase of AMH/Amh expression. This inhibition occurs through FSH-induced protein kinase A/SF1-mediated expression of gonadotropin inducible ovarian transcription factor 1, a transcriptional repressor, that recruits histone deacetylase 2 to deacetylate H3K27ac, resulting in the suppression of AMH/Amh expression. Furthermore, we report that ovarian Amh mRNA levels are significantly higher in Fshß-null mice (Fshß-/-) compared with those in wild-type (WT) mice. In addition, ovarian Amh mRNA levels are restored in Fshß-null mice expressing a human WT FSHß transgene (FSHß-/-hFSHßWT). Our study provides a mechanistic insight into the regulation of AMH expression that has many implications in female reproduction/fertility.

Androgens Regulate Ovarian Gene Expression Through Modulation of Ezh2 Expression and Activity.

A substantial amount of evidence suggests that androgen signaling through classical androgen receptors is critical for both normal and pathologic ovarian physiology. Specifically, we and others have shown that, in mouse granulosa cells, androgen actions through both extranuclear and nuclear androgen receptor signaling are critical for normal follicle development and ovulation. Here, we show that androgens through the PI3K/Akt pathway rapidly (within minutes) phosphorylate and inhibit activity of the Polycomb group protein enhancer of zeste homolog 2 (Ezh2). Over the course of 24 to 48 hours, androgens then induce expression of the microRNA miR-101, which targets Ezh2 messenger RNA (mRNA), leading to a nearly complete loss of Ezh2 protein expression. This long-term androgen-induced loss of Ezh2 actions ultimately results in sustained reduction of the H3K27me3-repressive mark in the promoter region of the Runt-related transcription factor-1 (Runx1) gene, a luteinizing hormone (LH)-induced transcription factor essential for ovulation, leading to increased Runx1 mRNA expression. Accordingly, blocking androgen-induced inhibition of Ezh2 in vivo adversely affects LH-induced Runx1 mRNA expression and subsequent ovulation. Importantly, although estrogen treatment of granulosa cells similarly causes rapid activation of the PI3K/Akt pathway and short-term phosphorylation of Ezh2, it does not induce miR-101 expression and thereby does not reduce overall Ezh2 expression, demonstrating the androgen specificity of long-term Ezh2 suppression. Thus, this study provides insight regarding how androgen-induced extranuclear kinase signaling and intranuclear transcription through Ezh2 modifications may influence the expression pattern of genes, ultimately affecting various downstream physiological processes.

Intra-cellular mechanism of Anti-Müllerian hormone (AMH) in regulation of follicular development.

Anti-Müllerian hormone (AMH) is a member of the transforming growth factor-ß superfamily and plays a crucial role in testicular and ovarian functions. In clinical practice, AMH is used as a diagnostic and/or prognostic marker in women in association with ovulation induction and in various pathophysiological conditions. Despite widespread clinical use of AMH, our mechanistic understanding of AMH actions in regulating follicular development is limited. Using a mouse model, we in this study report that in vivo AMH treatment while stalls follicular development and inhibits ovulation, also prevents follicular atresia. We further show that these AMH actions are mediated through induction of two miRNAs, miR-181a and miR-181b, which regulate various aspects of FSH signaling and follicular growth, ultimately affecting downstream gene expression and folliculogenesis. We also report that in this mouse model AMH pre-treatment prior to superovulation improves oocyte yield. These studies, therefore, offer new mechanistic insight into AMH actions in folliculogenesis and point toward potential utilization of AMH as a therapeutic agent.

GPCR/EGFR cross talk is conserved in gonadal and adrenal steroidogenesis but is uniquely regulated by matrix metalloproteinases 2 and 9 in the ovary.

Previous work has demonstrated that cross talk between G protein-coupled LH receptors and epidermal growth factor receptors (EGFR) is essential for LH-induced steroid production in ovarian follicles and testicular Leydig cells. Here we demonstrate that G protein-coupled receptor (GPCR)/EGFR cross talk is also required for ACTH-induced steroidogenesis in Y1 adrenal cells. Moreover, we confirm that the signaling pathway from GPCR to Erk activation is conserved in all three steroidogenic tissues. ACTH or LH induces Gα(s), resulting in elevated cAMP and protein kinase A activation. cAMP/protein kinase A then triggers EGFR trans-activation, which promotes Erk signaling and subsequent steroidogenesis. Interestingly, although EGFR trans-activation is conserved in all three tissues, the specific mechanisms regulating this receptor cross talk differ. ACTH and LH trigger matrix metalloproteinase (MMP)-mediated release of EGFR ligands in adrenal and gonadal cells, respectively. However, this extracellular, ligand-dependent EGFR transactivation is required only for LH-induced steroidogenesis in ovarian follicles, reflecting the unique requirement of cell-cell cross talk for ovarian steroid production. Furthermore, MMP2 and MMP9 appear to regulate LH-induced steroidogenesis in mouse ovarian follicles, because a specific MMP2/9 inhibitor as well as the MMP2/9 inhibitor doxycycline suppress LH-induced follicular steroid production in vitro. Notably, although EGFR or MMP inhibition minimally affects estrous cycling in female mice, they attenuate ovarian steroidogenesis in response to LHR overstimulation in vivo. These results may have implications with regard to EGFR inhibitor use in various cancers as well as in polycystic ovarian syndrome, where excess LH-driven ovarian androgen production might be controlled by MMP2/9 inhibition.

Carlinoside reduces hepatic bilirubin accumulation by stimulating bilirubin-UGT activity through Nrf2 gene expression.

Accumulation of bilirubin, primarily because of its insolubility, has been found to be associated with liver diseases including jaundice. Free bilirubin is insoluble; its glucuronidation by bilirubin-UGT enzyme (UGT1A1) makes it soluble and eliminates it through urine and faeces. Taking CCl(4) induced rat liver dysfunction model, we demonstrated that suppression of UGT1A1 activity in rat liver increased serum bilirubin level which could be reversed by carlinoside (Cln), a flavone glycoside. Although Cln is a flavone compound, it escaped self-glucuronidation in the intestine and readily absorbed. Kinetic study of microsomal UGT1A1 from HepG2 cells suggested that Cln enhanced enzyme activity by increasing V(max) without altering K(m). This altered V(max) was found to be due to UGT1A1 overexpression by Cln which was observed in both HepG2 and rat primary hepatocytes. Since Nrf2 is the transcription factor of UGT1A1, we examined whether Cln effect on UGT1A1 overexpression is mediated through Nrf2. In Nrf2 knock-out cells, Cln could not elevate UGT1A1 activity indicating Nrf2 to be its target. Cln significantly increased Nrf2 gene expression in HepG2 cells which was subsequently localized in nuclear region. Results from ChIP assay showed that Cln markedly augmented Nrf2 binding to UGT1A1 promoter that consequently enhanced reporter activity. Our findings therefore show that Cln upregulated Nrf2 gene expression, increased its nuclear translocation and stimulated UGT1A1 promoter activity. Total outcome of these events brought about a significant increase of bilirubin glucuronidation. Cln therefore could be a worthy choice to intervene hyperbilirubinemia due to liver dysfunction.

Structural basis for assembly and disassembly of the CRM1 nuclear export complex.

CRM1 (or exportin 1, Xpo1) transports proteins out of the cell nucleus through the nuclear pore complex. In the cytoplasm, GTP hydrolysis and consequent dissociation of Ran from CRM1 releases low-affinity substrates, while additional factors facilitate release of high-affinity substrates. Here we provide a model for human CRM1 export complex assembly and disassembly through structural and biochemical analyses of CRM1 bound to the substrate snurportin 1 (SNUPN, also called snuportin 1).

How hair cells hear: the molecular basis of hair-cell mechanotransduction.

PURPOSE OF REVIEW: This review aims to summarize our current knowledge regarding mechanotransduction by hair cells and to highlight unresolved questions. RECENT FINDINGS: Despite over a quarter of a century of electrophysiological data describing hair-cell mechanotransduction, the molecular basis of this process is just now being revealed. Recent work has begun to identify candidate transduction complex molecules, and current work is aimed at confirming these hypotheses and identifying other proteins important for hair-cell function. SUMMARY: Our senses of hearing and balance rely on the exquisite sensitivity of the hair cell and its transduction complex. Understanding the molecular basis for hair-cell mechanotransduction may provide us with the foundation for understanding the causes of, and perhaps the treatments for, auditory and vestibular deficits resulting from hair-cell dysfunction.