Molecular Endocrinology Current Issue

Minireview: The Contribution of Different Androgen Receptor Domains to Receptor Dimerization and Signaling

Centenera, M. M., Harris, J. M., Tilley, W. D., Butler, L. M. — 2008-10-27

The androgen receptor (AR) is a ligand-activated transcription factor of the nuclear receptor superfamily that plays a critical role in male physiology and pathology. Activated by binding of the native androgens testosterone and 5-dihydrotestosterone, the AR regulates transcription of genes involved in the development and maintenance of male phenotype and male reproductive function as well as other tissues such as bone and muscle. Deregulation of AR signaling can cause a diverse range of clinical conditions, including the X-linked androgen insensitivity syndrome, a form of motor neuron disease known as Kennedy’s disease, and male infertility. In addition, there is now compelling evidence that the AR is involved in all stages of prostate tumorigenesis including initiation, progression, and treatment resistance. To better understand the role of AR signaling in the pathogenesis of these conditions, it is important to have a comprehensive understanding of the key determinants of AR structure and function. Binding of androgens to the AR induces receptor dimerization, facilitating DNA binding and the recruitment of cofactors and transcriptional machinery to regulate expression of target genes. Various models of dimerization have been described for the AR, the most well characterized interaction being DNA-binding domain- mediated dimerization, which is essential for the AR to bind DNA and regulate transcription. Additional AR interactions with potential to contribute to receptor dimerization include the intermolecular interaction between the AR amino terminal domain and ligand-binding domain known as the N-terminal/C-terminal interaction, and ligand-binding domain dimerization. In this review, we discuss each form of dimerization utilized by the AR to achieve transcriptional competence and highlight that dimerization through multiple domains is necessary for optimal AR signaling.

Minireview: The Wnt Signaling Pathway Effector TCF7L2 and Type 2 Diabetes Mellitus

Jin, T., Liu, L. — 2008-10-27

Since the relationship between TCF7L2 (also known as TCF-4) polymorphisms and type 2 diabetes mellitus was identified in 2006, extensive genome-wide association examinations in different ethnic groups have further confirmed this relationship. As a component of the bipartite transcription factor β-catenin/TCF, TCF7L2 is important in conveying Wnt signaling during embryonic development and in regulating gene expression during adulthood. Although we still do not know mechanistically how the polymorphisms within the intron regions of TCF7L2 affect the risk of type 2 diabetes, this transcriptional regulator was shown to be involved in stimulating the proliferation of pancreatic β-cells and the production of the incretin hormone glucagon-like peptide-1 in intestinal endocrine L cells. In this review, we introduce background knowledge of TCF7L2 as a component of the Wnt signaling pathway, summarize recent findings demonstrating the association between TCF7L2 polymorphisms and the risk of type 2 diabetes, outline experimental evidence of the potential function of TCF7L2 in pancreatic and intestinal endocrine cells, and present our perspective views.

Unique ER{alpha} Cistromes Control Cell Type-Specific Gene Regulation

2008-10-27

Estrogens play an important role in normal physiology and in a variety of pathological states involving diverse tissues including breast and bone. The mechanism by which estrogens exert cell type- and disease-specific effects, however, remains to be explained. We have compared the gene expression profile of the MCF7 breast cancer cell line with that of the osteoblast-like cell line U2OS-ER by expression microarrays. We find that fewer than 10% of the 17β-estradiol (E2)-regulated genes are common to both cell types. We have validated this in primary calvarial osteoblasts. To dissect the mechanism underlying the cell type-specific E2 regulation of gene expression in MCF7 and U2OS-ER cells, we compared the ER binding sites on DNA in the two cell types by performing chromatin immunoprecipitation (ChIP) on genomic tiling arrays (ChIP-on-chip). Consistent with the distinct patterns of E2-regulated gene expression in these two cell lines, we find that the vast majority of ER binding sites are also cell type specific and correlate both in position and number with cell type-specific gene regulation. Interestingly, although the forkhead factor FoxA1 plays a critical role in defining the ER cistrome in MCF7 cells, it is not expressed in U2OS-ER cells, and forkhead motifs are not enriched in the ER cistrome in these cells. Finally, the ER cistromes are correlated with cell type-specific epigenetic histone modifications. These results support a model for the cell type-specific action of E2 being driven primarily through specific ER occupancy of epigenetically marked cis-regulatory regions of target genes.

Liver X Receptor Is a Therapeutic Target for Photoaging and Chronological Skin Aging

2008-10-27

Liver X receptors (LXR and -β) are liposensors that exert their metabolic effects by orchestrating the expression of macrophage genes involved in lipid metabolism and inflammation. LXRs are also expressed in other tissues, including skin, where their natural oxysterol ligands induce keratinocyte differentiation and improve epidermal barrier function. To extend the potential use of LXR ligands to dermatological indications, we explored the possibility of using LXR as a target for skin aging. We demonstrate that LXR signaling is down-regulated in cell-based models of photoaging, i.e. UV-activated keratinocytes and TNF-activated dermal fibroblasts. We show that a synthetic LXR ligand inhibits the expression of cytokines and metalloproteinases in these in vitro models, thus indicating its potential in decreasing cutaneous inflammation associated with the etiology of photoaging. Furthermore, a synthetic LXR ligand induces the expression of differentiation markers, ceramide biosynthesis enzymes, and lipid synthesis and transport genes in keratinocytes. Remarkably, LXRβ-null mouse skin showed some of the molecular defects that are observed in chronologically aged human skin. Finally, we demonstrate that a synthetic LXR agonist inhibits UV-induced photodamage and skin wrinkle formation in a murine model of photoaging. Therefore, the ability of an LXR ligand to modulate multiple pathways underlying the etiology of skin aging suggests that LXR is a novel target for developing potential therapeutics for photoaging and chronological skin aging indications.

Target Gene-Specific Regulation of Androgen Receptor Activity by p42/p44 Mitogen-Activated Protein Kinase

2008-10-27

Evidence that the androgen receptor (AR) is not only important in androgen-dependent prostate cancer, but also continues to play a role in tumors that become resistant to androgen deprivation therapies, highlights the need to find alternate means to block AR activity. AR, a hormone-activated transcription factor, and its coactivators are phosphoproteins. Thus, we sought to determine whether inhibition of specific cell signaling pathways would reduce AR function. We found that short-term inhibition of p42/p44 MAPK activity either by a MAPK kinase inhibitor, U0126, or by depletion of kinase with small interfering RNA caused target gene-specific reductions in AR activity. AR enhances histone H3 acetylation of target genes that are sensitive to U0126 including prostate-specific antigen and TMPRSS2, but does not increase histone H3 acetylation of the U0126-resistant PMEPA1 gene. Thus, although AR induces transcription of many target genes, the molecular changes induced by AR at the chromatin level are target gene specific. Long-term treatment (24–48 h) with U0126 causes a G₁ cell cycle arrest and reduces AR expression both through a decrease in AR mRNA and a reduction in AR protein stability. Thus, treatments that reduce p42/p44 MAPK activity in prostate cancer have the potential to reduce AR activity through a reduction in expression levels as well as by target gene-selective inhibition of AR function.

Phosphorylation of Farnesoid X Receptor by Protein Kinase C Promotes Its Transcriptional Activity

2008-10-27

The farnesoid X receptor (FXR, NR1H4) belongs to the nuclear receptor superfamily and is activated by bile acids such as chenodeoxycholic acid, or synthetic ligands such as GW4064. FXR is implicated in the regulation of bile acid, lipid, and carbohydrate metabolism. Posttranslational modifications regulating its activity have not been investigated yet. Here, we demonstrate that calcium-dependent protein kinase C (PKC) inhibition impairs ligand-mediated regulation of FXR target genes. Moreover, in a transactivation assay, we show that FXR transcriptional activity is modulated by PKC. Furthermore, phorbol 12-myristate 13-acetate , a PKC activator, induces the phosphorylation of endogenous FXR in HepG2 cells and PKC phosphorylates in vitro FXR in its DNA-binding domain on S135 and S154. Mutation of S135 and S154 to alanine residues reduces in cell FXR phosphorylation. In contrast to wild-type FXR, mutant FXRS135AS154A displays an impaired PKC-induced transactivation and a decreased ligand-dependent FXR transactivation. Finally, phosphorylation of FXR by PKC promotes the recruitment of peroxisomal proliferator-activated receptor coactivator 1. In conclusion, these findings show that the phosphorylation of FXR induced by PKC directly modulates the ability of agonists to activate FXR.

Steroidal Androgens and Nonsteroidal, Tissue-Selective Androgen Receptor Modulator, S-22, Regulate Androgen Receptor Function through Distinct Genomic and Nongenomic Signaling Pathways

2008-10-27

Androgen receptor (AR) ligands are important for the development and function of several tissues and organs. However, the poor oral bioavailability, pharmacokinetic properties, and receptor cross-reactivity of testosterone, coupled with side effects, place limits on its clinical use. Selective AR modulators (SARMs) elicit anabolic effects in muscle and bone, sparing reproductive organs like the prostate. However, molecular mechanisms underlying the tissue selectivity remain ambiguous. We performed a variety of in vitro studies to compare and define the molecular mechanisms of an aryl propionamide SARM, S-22, as compared with dihydrotestosterone (DHT). Studies indicated that S-22 increased levator ani muscle weight but decreased the size of prostate in rats. Analysis of the upstream intracellular signaling events indicated that S-22 and DHT mediated their actions through distinct pathways. Modulation of these pathways altered the recruitment of AR and its cofactors to the PSA enhancer in a ligand-dependent fashion. In addition, S-22 induced Xenopus laevis oocyte maturation and rapid phosphorylation of several kinases, through pathways distinct from steroids. These studies reveal novel differences in the molecular mechanisms by which S-22, a nonsteroidal SARM, and DHT mediate their pharmacological effects.

Thyroid Hormone-Mediated Activation of the ERK/Dual Specificity Phosphatase 1 Pathway Augments the Apoptosis of GH4C1 Cells by Down-Regulating Nuclear Factor-{kappa}B Activity

Chiloeches, A., Sanchez-Pacheco, A., Gil-Araujo, B., Aranda, A., Lasa, M. — 2008-10-27

Thyroid hormone (T₃) plays a crucial role in processes such as cell proliferation and differentiation, whereas its implication on cellular apoptosis has not been well documented. Here we examined the effect of T₃ on the apoptosis of GH4C1 pituitary cells and the mechanisms underlying this effect. We show that T₃ produced a significant increase in apoptosis in serum-depleted conditions. This effect was accompanied by a decrease in nuclear factor-B (NF-B)-dependent transcription, IB phosphorylation, translocation of p65/NF-B to the nucleus, phosphorylation, and transactivation. Moreover, these effects were correlated with a T3-induced decrease in the expression of antiapoptotic gene products, such as members of the inhibitor of apoptosis protein and Bcl-2 families. On the other hand, ERK but not c-Jun N-terminal kinase or MAPK p38, was activated upon exposure to T₃, and inhibition of ERK alone abrogated T₃-mediated apoptosis. In addition, T₃ increased the expression of the MAPK phosphatase, dual specificity phosphatase 1 (DUSP1), in an ERK-dependent manner. Interestingly, the suppression of DUSP1 expression abrogated T₃-induced inhibition of NF-B-dependent transcription and p65/NF-B translocation to the nucleus, as well as T₃-mediated apoptosis. Overall, our results indicate that T₃ induces apoptosis in rat pituitary tumor cells by down-regulating NF-B activity through a mechanism dependent on the ERK/DUSP1 pathway.

Axl and Tyro3 Modulate Female Reproduction by Influencing Gonadotropin-Releasing Hormone Neuron Survival and Migration

2008-10-27

GnRH neurons must undergo a complex and precise pattern of neuronal migration to appropriately target their projections to the median eminence to trigger gonadotropin secretion and thereby control reproduction. Using NLT GnRH cells as a model of early GnRH neuronal development, we identified the potential importance of Axl and Tyro3, members of the TAM (Tyro3, Axl, and Mer) family of receptor tyrosine kinases in GnRH neuronal cell survival and migration. Silencing studies evaluated the role of Tyro3 and Axl in NLT GnRH neuronal cells and suggest that both play a role in Gas6 stimulation of GnRH neuronal survival and migration. Analysis of mice null for both Axl and Tyro3 showed normal onset of vaginal opening but delayed first estrus and persistently abnormal estrous cyclicity compared with wild-type controls. Analysis of GnRH neuronal numbers and positioning in the adult revealed a total loss of 24% of the neuronal network that was more striking (34%) when considered within specific anatomical compartments, with the largest deficit surrounding the organum vasculosum of the lamina terminalis. Analysis of GnRH neurons during embryogenesis identified a striking loss of immunoreactive cells within the context of the ventral forebrain compartment (36%) and not more rostrally. Studies using caspase 3 cleavage as a marker of apoptosis showed that Axl^–/–, Tyro3^–/– double-knockout mice had increased cell death in the nose and dorsal forebrain, supporting the underlying mechanism of cell loss. Together these data suggest that Axl and Tyro3 mediate the survival and appropriate targeting of GnRH neurons to the ventral forebrain, thereby contributing to normal reproductive function and cyclicity in the female.

The Expression of GPIHBP1, an Endothelial Cell Binding Site for Lipoprotein Lipase and Chylomicrons, Is Induced by Peroxisome Proliferator-Activated Receptor-{gamma}

2008-10-27

Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1), a protein in the lymphocyte antigen 6 (Ly-6) family, plays a key role in the lipolytic processing of triglyceride-rich lipoproteins. GPIHBP1 binds lipoprotein lipase and chylomicrons and is expressed along the luminal surface of microvascular endothelial cells. Lipolysis is known to be regulated by metabolic factors and is controlled at multiple levels, including the number of LPL binding sites on capillaries. Here, we tested the possibility that GPIHBP1 expression could be regulated by dietary perturbations and by peroxisome proliferator-activated receptors (PPARs). Gpihbp1 transcript levels in the heart and in brown and white adipose tissue increased with fasting and returned toward baseline after refeeding. A PPAR agonist increased Gpihbp1 expression in adipose tissue, heart, and skeletal muscle, whereas PPAR and PPAR agonists had no effect. Gpihbp1 was expressed in endothelial cells of embryoid bodies generated from mouse embryonic stem cells, and Gpihbp1 expression in embryoid bodies was up-regulated by a PPAR agonist. Sequences upstream from exon 1 of Gpihbp1 contain a strong PPAR binding site, and that site exhibited activity in a luciferase reporter assay. Gpihbp1 transcript levels in brown and white adipose tissue were lower in endothelial cell PPAR knockout mice than in littermate control mice, suggesting that PPAR regulates Gpihbp1 expression in vivo. We conclude that GPIHBP1 is regulated by dietary factors and by PPAR.

Structural Determinants Critical for Localization and Signaling within the Seventh Transmembrane Domain of the Type 1 Corticotropin Releasing Hormone Receptor: Lessons from the Receptor Variant R1d

Markovic, D., Lehnert, H., Levine, M. A., Grammatopoulos, D. K. — 2008-10-27

The type 1 CRH receptor (CRH-R1) plays a fundamental role in homeostatic adaptation to stressful stimuli. CRH-R1 gene activity is regulated through alternative splicing and generation of various CRH-R1 mRNA variants. One such variant is the CRH-R1d, which has 14 amino acids missing from the putative seventh transmembrane domain due to exon 13 deletion, a splicing event common to other members of the B1 family of G protein-coupled receptors. In this study, using overexpression of recombinant receptors in human embryonic kidney 293 and myometrial cells, we showed by confocal microscopy that in contrast to CRH-R1, the R1d variant is primarily retained in the cytoplasm, although some cell membrane expression is also evident. Use of antibodies against the CRH-R1 C terminus in nonpermeabilized cells showed that membrane-expressed CRH-R1d contains an extracellular C terminus. Interestingly, treatment of CRH-R1d-expressing cells with CRH (100 nM) for 45–60 min elicited functional responses associated with a significant reduction of plasma membrane receptor expression, redistribution of intracellular receptors, and increased receptor degradation. Site-directed mutagenesis studies identified the cassette G³⁵⁶-F³⁵⁸ within transmembrane domain 7 as crucial for CRH-R1 stability to the plasma membrane because deletion of this cassette caused substantial intracellular localization of CRH-R1 . Most importantly, coexpression studies between CRH-R1d and CRH-R2β demonstrated that the CRH-R2β could partially rescue CRH-R1d membrane expression, and this was associated with a significant attenuation of urocotrin II-induced cAMP production and ERK1/2 and p38MAPK activation, suggesting that CRH-R1d might specifically induce heterologous impairment of CRH-R2 signaling responses. This mechanism appears to involve accelerated CRH-R2β endocytosis.

A Crucial Role for G{alpha}q/11, But Not G{alpha}i/o or G{alpha}s, in Gonadotropin-Releasing Hormone Receptor-Mediated Cell Growth Inhibition

White, C. D., Coetsee, M., Morgan, K., Flanagan, C. A., Millar, R. P., Lu, Z.-L. — 2008-10-27

GnRH acts on its cognate receptor in pituitary gonadotropes to regulate the biosynthesis and secretion of gonadotropins. It may also have direct extrapituitary actions, including inhibition of cell growth in reproductive malignancies, in which GnRH activation of the MAPK cascades is thought to play a pivotal role. In extrapituitary tissues, GnRH receptor signaling has been postulated to involve coupling of the receptor to different G proteins. We examined the ability of the GnRH receptor to couple directly to G_q/11, G_i/o, and G_s, their roles in the activation of the MAPK cascades, and the subsequent cellular effects. We show that in G_q/11-negative cells stably expressing the GnRH receptor, GnRH did not induce activation of ERK, jun-N-terminal kinase, or P38 MAPK. In contrast to G_i or chimeric G_qi5, transfection of G_q cDNA enabled GnRH to induce phosphorylation of ERK, jun-N-terminal kinase, and P38. Furthermore, no GnRH-mediated cAMP response or inhibition of isoproterenol-induced cAMP accumulation was observed. In another cellular background, [³⁵S]GTPS binding assays confirmed that the GnRH receptor was unable to directly couple to G_i but could directly interact with G_q/11. Interestingly, GnRH stimulated a marked reduction in cell growth only in cells expressing G_q, and this inhibition could be significantly rescued by blocking ERK activation. We therefore provide direct evidence, in multiple cellular backgrounds, that coupling of the GnRH receptor to G_q/11, but not to G_i/o or G_s, and consequent activation of ERK plays a crucial role in GnRH-mediated cell death.

Insulin-Like Growth Factor I Receptor Signaling Is Required for Exercise-Induced Cardiac Hypertrophy

2008-10-27

The receptors for IGF-I (IGF-IR) and insulin (IR) have been implicated in physiological cardiac growth, but it is unknown whether IGF-IR or IR signaling are critically required. We generated mice with cardiomyocyte-specific knockout of IGF-IR (CIGF1RKO) and compared them with cardiomyocyte-specific insulin receptor knockout (CIRKO) mice in response to 5 wk exercise swim training. Cardiac development was normal in CIGF1RKO mice, but the hypertrophic response to exercise was prevented. In contrast, despite reduced baseline heart size, the hypertrophic response of CIRKO hearts to exercise was preserved. Exercise increased IGF-IR content in control and CIRKO hearts. Akt phosphorylation increased in exercise-trained control and CIRKO hearts and, surprisingly, in CIGF1RKO hearts as well. In exercise-trained control and CIRKO mice, expression of peroxisome proliferator-activated receptor- coactivator-1 (PGC-1) and glycogen content were both increased but were unchanged in trained CIGF1RKO mice. Activation of AMP-activated protein kinase (AMPK) and its downstream target eukaryotic elongation factor-2 was increased in exercise-trained CIGF1RKO but not in CIRKO or control hearts. In cultured neonatal rat cardiomyocytes, activation of AMPK with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) prevented IGF-I/insulin-induced cardiomyocyte hypertrophy. These studies identify an essential role for IGF-IR in mediating physiological cardiomyocyte hypertrophy. IGF-IR deficiency promotes energetic stress in response to exercise, thereby activating AMPK, which leads to phosphorylation of eukaryotic elongation factor-2. These signaling events antagonize Akt signaling, which although necessary for mediating physiological cardiac hypertrophy, is insufficient to promote cardiac hypertrophy in the absence of myocardial IGF-I signaling.

Research Resource: Rapid Recruitment of Temporally Distinct Vascular Gene Sets by Estrogen

2008-10-27

Cardiovascular disease is the leading cause of mortality for both men and women in developed countries. The sex steroid hormone estrogen is required for normal vascular physiology. Estrogen functions by binding to intracellular estrogen receptors (ER), ER and ERβ, ligand-activated transcription factors that are expressed in both vascular endothelial and smooth muscle cells. We recently demonstrated that long-term (8 d) estrogen treatment in vivo in mice recruits distinct vascular gene sets mediated by ER and ERβ and that the promoters from these gene sets are enriched for binding sites of specific transcription factors, leading to the hypothesis that estrogen initiates a cascade of early transcriptional events that modulate gene expression in the vasculature. Here we test this hypothesis using gene expression profiling to examine initial transcriptional events (2–8 h) mediated by estrogen in blood vessels. Our data reveal that 1) estrogen regulates temporally distinct cascades of vascular gene expression, 2) initially, estrogen-mediated vascular gene repression predominates, 3) the earliest estrogen-recruited gene program is enriched in vascular transcription factors that can interact with binding sites present in estrogen-regulated vascular genes recruited subsequently, and 4) estrogen-regulated genes recruited next have specific functions, including lipid metabolism and cellular growth and proliferation that are potentially important for estrogen’s known vascular functions. In summary, estrogen directly and rapidly recruits specific transcriptional factors that then propagate distinct cascades of gene expression. These data define the temporal recruitment of specific vascular genes by estrogen and enable further analysis of the mechanisms by which estrogen directly regulates vascular function.

Research Resource: Gene Expression Profile for Ectopic Versus Eutopic Endometrium Provides New Insights into Endometriosis Oncogenic Potential

2008-10-27

Endometriosis is a common gynecological disorder characterized by pain and infertility, where the lesions disseminate everywhere in the body with a preference for the pelvis. In that, it could be regarded as a benign metastatic disease, because its issue is not fatal. However, the molecular bases of this intriguing clinical condition are not well known. The objective of this study is to characterize the transcriptome differences between eutopic vs. ectopic endometrium with a special interest in pathways involved in cancerogenesis. We performed two hybridizations in technical replicate on highly specific long oligonucleotides microarrays (NimbleGen), with cDNA prepared from six-patients pools, where the same patient provided both eutopic and ectopic endometrium (endometriomas). To confirm the expression microarrays data, quantitative RT-PCR validation was performed on 12 individuals for 20 genes. Over 8000 transcripts were significantly modified (more than twice) in the lesions corresponding to 5600 down- or up-regulated genes. These were clustered through DAVID Bioinformatics Resources into 55 functional groups. The data are presented in a detailed and visual way on 24 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways implemented with induction ratios for each differentially expressed gene. An outstanding control of the cell cycle and a very specific modulation of the HOX genes were observed and provide some new evidence on why endometriosis only very rarely degenerates into cancer. The study constitutes a noteworthy update of gene profiling in endometriosis, by delivering the most complete and reliable list of dysregulated genes to date.

Retraction

2008-10-27

Endocrine-Related Resources from the National Institutes of Health

2008-10-27

National Hormone & Peptide Program (NHPP): New Recombinant Hormones, Hypothalamic Peptides, Natural Hormones, New Antisera, & Expanded Hormone Assay Services Available

2008-10-27