Molecular Endocrinology Recent Issues

Signals from NURSA

DeFranco, D. B., McKenna, N. J. — Tue, 24 Nov 2009 10:02:24 PST

Minireview: Modulation of Hormone Receptor Signaling by Dietary Anticancer Indoles

Firestone, G. L., Sundar, S. N. — Tue, 24 Nov 2009 10:02:25 PST

Indole-3-carbinol and its diindole condensation product 3-3'-diindolylmethane are dietary phytochemicals that have striking anticarcinogenic properties in human cancer cells. Molecular, cellular, physiological, and clinical studies have documented that both indole-3-carbinol and 3-3'-diindolylmethane have potent endocrine modulating activities through a myriad of mechanisms. The focus of this review is to discuss the evidence that directly links the anticancer actions of these two indole compounds to the control of steroid receptor and growth factor receptor signaling.

Osmotic Stress Regulates Mineralocorticoid Receptor Expression in a Novel Aldosterone-Sensitive Cortical Collecting Duct Cell Line

Tue, 24 Nov 2009 10:02:25 PST

Aldosterone effects are mediated by the mineralocorticoid receptor (MR), a transcription factor highly expressed in the distal nephron. Given that MR expression level constitutes a key element controlling hormone responsiveness, there is much interest in elucidating the molecular mechanisms governing MR expression. To investigate whether hyper- or hypotonicity could affect MR abundance, we established by targeted oncogenesis a novel immortalized cortical collecting duct (CCD) cell line and examined the impact of osmotic stress on MR expression. KC3AC1 cells form domes, exhibit a high transepithelial resistance, express 11β-hydroxysteroid dehydrogenase 2 and functional endogenous MR, which mediates aldosterone-stimulated Na⁺ reabsorption through the epithelial sodium channel activation. MR expression is tightly regulated by osmotic stress. Hypertonic conditions induce expression of tonicity-responsive enhancer binding protein, an osmoregulatory transcription factor capable of binding tonicity-responsive enhancer response elements located in MR regulatory sequences. Surprisingly, hypertonicity leads to a severe reduction in MR transcript and protein levels. This is accompanied by a concomitant tonicity-induced expression of Tis11b, a mRNA-destabilizing protein that, by binding to the AU-rich sequences of the 3'-untranslated region of MR mRNA, may favor hypertonicity-dependent degradation of labile MR transcripts. In sharp contrast, hypotonicity causes a strong increase in MR transcript and protein levels. Collectively, we demonstrate for the first time that optimal adaptation of CCD cells to changes in extracellular fluid composition is accompanied by drastic modification in MR abundance via transcriptional and posttranscriptional mechanisms. Osmotic stress-regulated MR expression may represent an important molecular determinant for cell-specific MR action, most notably in renal failure, hypertension, or mineralocorticoid resistance.

HDAC6 Regulates Androgen Receptor Hypersensitivity and Nuclear Localization via Modulating Hsp90 Acetylation in Castration-Resistant Prostate Cancer

Ai, J., Wang, Y., Dar, J. A., Liu, J., Liu, L., Nelson, J. B., Wang, Z. — Tue, 24 Nov 2009 10:02:25 PST

The development of castration-resistant prostate cancer (PCa) requires that under castration conditions, the androgen receptor (AR) remains active and thus nuclear. Heat shock protein 90 (Hsp90) plays a key role in androgen-induced and -independent nuclear localization and activation of AR. Histone deacetylase 6 (HDAC6) is implicated, but has not been proven, in regulating AR activity via modulating Hsp90 acetylation. Here, we report that knockdown of HDAC6 in C4-2 cells using short hairpin RNA impaired ligand-independent nuclear localization of endogenous AR and inhibited PSA expression and cell growth in the absence or presence of dihydrotestosterone (DHT). The dose-response curve of DHT-stimulated C4-2 colony formation was shifted by shHDAC6 such that approximately 10-fold higher concentration of DHT is required, indicating a requirement for HDAC6 in AR hypersensitivity. HDAC6 knockdown also inhibited C4-2 xenograft tumor establishment in castrated, but not in testes-intact, nude mice. Studies using HDAC6-deficient mouse embryonic fibroblasts cells showed that inhibition of AR nuclear localization by HDAC6 knockdown can be largely alleviated by expressing a deacetylation mimic Hsp90 mutant. Taken together, our studies suggest that HDAC6 regulates AR hypersensitivity and nuclear localization, mainly via modulating HSP90 acetylation. Targeting HDAC6 alone or in combination with other therapeutic approaches is a promising new strategy for prevention and/or treatment of castration-resistant PCa.

Rapid Regulation of KATP Channel Activity by 17{beta}-Estradiol in Pancreatic {beta}-Cells Involves the Estrogen Receptor {beta} and the Atrial Natriuretic Peptide Receptor

Tue, 24 Nov 2009 10:02:25 PST

The ATP-sensitive potassium (K_ATP) channel is a key molecule involved in glucose-stimulated insulin secretion. The activity of this channel regulates β-cell membrane potential, glucose- induced [Ca²⁺]_i signals, and insulin release. In this study, the rapid effect of physiological concentrations of 17β-estradiol (E2) on K_ATP channel activity was studied in intact β-cells by use of the patch-clamp technique. When cells from wild-type (WT) mice were used, 1 nm E2 rapidly reduced K_ATP channel activity by 60%. The action of E2 on K_ATP channel was not modified in β-cells from ER–/– mice, yet it was significantly reduced in cells from ERβ–/– mice. The effect of E2 was mimicked by the ERβ agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN). Activation of ERβ by DPN enhanced glucose-induced Ca²⁺ signals and insulin release. Previous evidence indicated that the acute inhibitory effects of E2 on K_ATP channel activity involve cyclic GMP and cyclic GMP-dependent protein kinase. In this study, we used β-cells from mice with genetic ablation of the membrane guanylate cyclase A receptor for atrial natriuretic peptide (also called the atrial natriuretic peptide receptor) (GC-A KO mice) to demonstrate the involvement of this membrane receptor in the rapid E2 actions triggered in β-cells. E2 rapidly inhibited K_ATP channel activity and enhanced insulin release in islets from WT mice but not in islets from GC-A KO mice. In addition, DPN reduced K_ATP channel activity in β-cells from WT mice, but not in β-cells from GC-A KO mice. This work unveils a new role for ERβ as an insulinotropic molecule that may have important physiological and pharmacological implications.

Opposite Clinical Phenotypes of Glucokinase Disease: Description of a Novel Activating Mutation and Contiguous Inactivating Mutations in Human Glucokinase (GCK) Gene

Tue, 24 Nov 2009 10:02:25 PST

Glucokinase is essential for glucose-stimulated insulin release from the pancreatic β-cell, serving as glucose sensor in humans. Inactivating or activating mutations of glucokinase lead to different forms of glucokinase disease, i.e. GCK-monogenic diabetes of youth, permanent neonatal diabetes (inactivating mutations), and congenital hyperinsulinism, respectively. Here we present a novel glucokinase gene (GCK)-activating mutation (p.E442K) found in an infant with neonatal hypoglycemia (1.5 mmol/liter) and in two other family members suffering from recurrent hypoglycemic episodes in their childhood and adult life. In contrast to the severe clinical presentation in the index case, functional studies showed only a slight activation of the protein (relative activity index of 3.3). We also report on functional studies of two inactivating mutations of the GCK (p.E440G and p.S441W), contiguous to the activating one, that lead to monogenic diabetes of youth. Interestingly, adult family members carrying the GCK pE440G mutation show an unusually heterogeneous and progressive diabetic phenotype, a feature not typical of GCK-monogenic diabetes of youth. In summary, we identified a novel activating GCK mutation that although being associated with severe neonatal hypoglycemia is characterized by the mildest activation of the glucokinase enzyme of all previously reported.

Mice Deficient for Glucagon Gene-Derived Peptides Display Normoglycemia and Hyperplasia of Islet {alpha}-Cells But Not of Intestinal L-Cells

Tue, 24 Nov 2009 10:02:25 PST

Multiple bioactive peptides, including glucagon, glucagon-like peptide-1 (GLP-1), and GLP-2, are derived from the glucagon gene (Gcg). In the present study, we disrupted Gcg by introduction of GFP cDNA and established a knock-in mouse line. Gcg^gfp/gfp mice that lack most, if not all, of Gcg-derived peptides were born in an expected Mendelian ratio without gross abnormalities. Gcg^gfp/gfp mice showed lower blood glucose levels at 2 wk of age, but those in adult Gcg^gfp/gfp mice were not significantly different from those in Gcg^+/+ and Gcg^gfp/+ mice, even after starvation for 16 h. Serum insulin levels in Gcg^gfp/gfp mice were lower than in Gcg^+/+ and Gcg^gfp/+ on ad libitum feeding, but no significant differences were observed on starvation. Islet -cells and intestinal L-cells were readily visualized in Gcg^gfp/gfp and Gcg^gfp/+ mice under fluorescence. The Gcg^gfp/gfp postnatally developed hyperplasia of islet -cells, whereas the population of intestinal L-cells was not increased. In the Gcg^gfp/gfp, expression of Aristaless-related homeobox (Arx) was markedly increased in pancreas but not in intestine and suggested involvement of Arx in differential regulation of proliferation of Gcg-expressing cells. These results illustrated that Gcg-derived peptides are dispensable for survival and maintaining normoglycemia in adult mice and that Gcg-derived peptides differentially regulate proliferation/differentiation of -cells and L-cells. The present model is useful for analyzing glucose/energy metabolism in the absence of Gcg-derived peptides. It is useful also for analysis of the development, differentiation, and function of Gcg-expressing cells, because such cells are readily visualized by fluorescence in this model.

E2F1 Induces Pituitary Tumor Transforming Gene (PTTG1) Expression in Human Pituitary Tumors

Zhou, C., Wawrowsky, K., Bannykh, S., Gutman, S., Melmed, S. — Tue, 24 Nov 2009 10:02:25 PST

Rb/E2F is dysregulated in murine and human pituitary tumors. Pituitary tumor transforming gene (PTTG1), a securin protein, is required for pituitary tumorigenesis, and PTTG1 deletion attenuates pituitary tumor development in Rb^+/– mice. E2F1 and PTTG1 were concordantly overexpressed in 29 of 46 Rb^+/– murine pituitary tissues and also in 45 of 80 human pituitary tumors (P < 0.05). E2F1 specifically bound the hPTTG1 promoter as assessed by chromatin immunoprecipitation and biotin-streptavidin pull-down assay, indicating that hPTTG1 may act as a direct E2F1 target. Transfection of E2F1 and its partner DP1 dose-dependently activated hPTTG1 transcription up to 3-fold in p53-devoid H1299 cells but not in p53-replete HCT116 cells. E2F1 overexpression enhanced endogenous hPTTG1 mRNA and protein levels up to 3-fold in H1299 cells. The presence of endogenous p53/p21 constrained the induction, whereas knocking down either p53 or p21 in HCT116 cells restored E2F1-induced hPTTG1 transactivation and expression. Moreover, suppressing Rb by small interfering RNA concordantly elevated E2F1 and hPTTG1 protein levels. In contrast, transfection of E2F1 small interfering RNA lowered hPTTG1 levels 24 h later in HCT116 than in H1299 cells, indicating that p53 delays E2F1 action on hPTTG1. These results elucidate a mechanism for abundant tumor hPTTG1 expression, whereby Rb inactivation releases E2F1 to induce hPTTG1. This signaling pathway may underlie the requirement of PTTG1 for pituitary tumorigenesis.

The IGF-I Receptor Can Alter the Matrix Metalloproteinase Repertoire of Tumor Cells through Transcriptional Regulation of PKC-{alpha}

Tue, 24 Nov 2009 10:02:25 PST

The IGF-I receptor (IGF-IR) was identified as a tumor progression factor, but its role in invasion and metastasis has been the subject of some controversy. Previously we reported that in murine lung carcinoma M-27 cells, overexpression of IGF-IR increased the synthesis and activation of matrix metalloproteinase (MMP)-2 via Akt/phosphatidylinositol 3-kinase signaling. In contrast, we show here that in these and other cells, IGF-IR overexpression reduced the constitutive and phorbol 12-myristate 13-acetate (PMA)-inducible expression of three protein kinase C (PKC)-regulated metalloproteinases, MMP-3, MMP-9, and MMP-13, in cultured cells as well as in vivo in sc tumors. To elucidate the underlying mechanism, we analyzed the effect of IGF-IR on PKC expression and activity using wild-type and IGF-IR-overexpressing (M-27^IGFIR) tumor cells. Our results show that overexpression and activation of IGF-IR reduced PKC- expression, PKC activity, and downstream ERK1/2 signaling, and these effects were reversed in cells expressing kinase (Y_{1131,1135,1136}F) or C-terminal (Y_1250/51F) domain mutants of IGF-IR. This reduction was due to transcriptional down-regulation of PKC- as evidenced by reduced PKC- mRNA expression in a phosphatidylinositol 3-kinase-dependent manner and a blockade of PKC- promoter activation as revealed by a reporter gene assay. Finally, reconstitution of PKC- levels could restore MMP-9 expression levels in these cells. Collectively, these results show that IGF-IR can inhibit PKC- gene transcription and thereby block the synthesis of PMA-regulated MMPs, suggesting that within the same cells, IGF-IR can act as both a positive and negative regulator of MMP expression and function.

2010 FASEB Summer Research Conferences

Tue, 24 Nov 2009 10:02:25 PST

Growth Hormone-Activated STAT5 May Indirectly Stimulate IGF-I Gene Transcription through HNF-3{gamma}

Eleswarapu, S., Ge, X., Wang, Y., Yu, J., Jiang, H. — Tue, 24 Nov 2009 10:02:25 PST

IGF-I is abundantly expressed in the liver under the stimulation of GH. We showed previously that expression of hepatocyte nuclear factor (HNF)-3, a liver-enriched transcription factor, was strongly stimulated by GH in bovine liver. In this study, we determined whether GH-increased HNF-3 might contribute to GH stimulation of IGF-I gene expression in bovine liver and the underlying mechanism. A sequence analysis of the bovine IGF-I promoter revealed three putative HNF-3 binding sites, which all appear to be conserved in mammals. Chromatin immunoprecipitation assays showed that GH injection increased binding of HNF-3 to the IGF-I promoter in bovine liver. Gel-shift assays indicated that one of the three putative HNF-3 binding sites, HNF-3 binding site 1, bound to the HNF-3 protein from bovine liver with high affinity. Cotransfection analyses demonstrated that this HNF-3 binding site was essential for the transcriptional response of the IGF-I promoter to HNF-3 in CHO cells and to GH in primary mouse hepatocytes. Using similar approaches, we found that GH increased binding of the signal transducer and activator of transcription 5 (STAT5) to the HNF-3 promoter in bovine liver, that this binding occurred at a conserved STAT5 binding site, and that this STAT5 binding site was necessary for the HNF-3 promoter to respond to GH. Taken together, these results suggest that in addition to direct action, GH-activated STAT5 may also indirectly stimulate IGF-I gene transcription in the liver by directly enhancing the expression of the HNF-3 gene.

Upstream Stimulatory Factor 2, a Novel FoxA1-Interacting Protein, Is Involved in Prostate-Specific Gene Expression

Sun, Q., Yu, X., Degraff, D. J., Matusik, R. J. — Tue, 24 Nov 2009 10:02:25 PST

The forkhead protein A1 (FoxA1) is critical for the androgenic regulation of prostate-specific promoters. Prostate tissue rescued from FoxA1 knockout mice exhibits abnormal prostate development, typified by the absence of expression of differentiation markers and inability to engage in secretion. Chromatin immunoprecipitation and coimmunoprecipitation studies revealed that FoxA1 is one of the earliest transcription factors that binds to prostate-specific promoters, and that a direct protein-protein interaction occurs between FoxA1 and androgen receptor. Interestingly, evidence of the interaction of FoxA1 with other transcription factors is lacking. The upstream stimulatory factor 2 (USF2), an E-box-binding transcription factor of the basic-helix-loop-helix-leucine-zipper family, binds to a consensus DNA sequence similar to FoxA1. Our in vitro and in vivo studies demonstrate the binding of USF2 to prostate-specific gene promoters including the probasin promoter, spermine-binding protein promoter, and prostate-specific antigen core enhancer. Furthermore, we show a direct physical interaction between FoxA1 and USF2 through the use of immunoprecipitation and glutathione-S-transferase pull-down assays. This interaction is mediated via the forkhead DNA-binding domain of FoxA1 and the DNA-binding domain of USF2. In summary, these data indicate that USF2 is one of the components of the FoxA1/androgen receptor transcriptional protein complex that contributes to the expression of androgen-regulated and prostate-specific genes.

Role of Phospholipase D in Parathyroid Hormone Type 1 Receptor Signaling and Trafficking

Garrido, J. L., Wheeler, D., Vega, L. L., Friedman, P. A., Romero, G. — Tue, 24 Nov 2009 10:02:25 PST

The role of phospholipase D (PLD) in the regulation of the traffic of the PTH type 1 receptor (PTH1R) was studied in Chinese hamster ovary cells stably transfected with a human PTH1R (CHO-R3) and in rat osteosarcoma 17/2.8 (ROS) cells. PTH(1–34) increased total PLD activity by 3-fold in CHO-R3 cells and by 2-fold in ROS cells. Overexpression of wild-type (WT) PLD1 and WT-PLD2 increased basal PLD activity in CHO-R3 but not in ROS cells. Ligand-stimulated PLD activity greatly increased in CHO-R3 cells transfected with WT-PLD1 and WT-PLD2. However, only WT-PLD2 expression increased PTH-dependent PLD activity in ROS cells. Expression of the catalytically inactive mutants R898K-PLD1 (DN-PLD1) and R758K-PLD2 (DN-PLD2) inhibited ligand-dependent PLD activity in both cell lines. PTH(1–34) induced internalization of the PTH1R with a concomitant increase in the colocalization of the receptor with PLD1 in intracellular vesicles and in a perinuclear, ADP ribosylation factor-1-positive compartment. The distribution of PLD1 and PLD2 remained unaltered after PTH treatment. Expression of DN-PLD1 had a small effect on endocytosis of the PTH1R; however, DN-PLD1 prevented accumulation of the PTH1R in the perinuclear compartment. Expression of DN-PLD2 significantly retarded ligand-induced PTH1R internalization in both cell lines. The differential effects of PLD1 and PLD2 on receptor traffic were confirmed using isoform-specific short hairpin RNA constructs. We conclude that PLD1 and PLD2 play distinct roles in regulating PTH1R traffic; PLD2 primarily regulates endocytosis, whereas PLD1 regulates receptor internalization and intracellular receptor traffic.

Regulation of GPR54 Signaling by GRK2 and {beta}-Arrestin

Tue, 24 Nov 2009 10:02:25 PST

Kisspeptin and its receptor, GPR54, are major regulators of the hypothalamic-pituitary-gonadal axis as well as regulators of human placentation and tumor metastases. GPR54 is a G_q/11-coupled G protein-coupled receptor (GPCR), and activation by kisspeptin stimulates phosphatidy linositol 4, 5-biphosphate hydrolysis, Ca²⁺ mobilization, arachidonic acid release, and ERK1/2 MAPK phosphorylation. Physiological evidence suggests that GPR54 undergoes agonist-dependent desensitization, but underlying molecular mechanisms are unknown. Furthermore, very little has been reported on the early events that regulate GPR54 signaling. The lack of information in these important areas led to this study. Here we report for the first time on the role of GPCR serine/threonine kinase (GRK)2 and β-arrestin in regulating GPR54 signaling in human embryonic kidney (HEK) 293 cells, a model cell system for studying the molecular regulation of GPCRs, and genetically modified MDA MB-231 cells, an invasive breast cancer cell line expressing about 75% less β-arrestin-2 than the control cell line. Our study reveals that in HEK 293 cells, GPR54 is expressed both at the plasma membrane and intracellularly and also that plasma membrane expression is regulated by cytoplasmic tail sequences. We also demonstrate that GPR54 exhibits constitutive activity, internalization, and association with GRK2 and β- arrestins-1 and 2 through sequences in the second intracellular loop and cytoplasmic tail of the receptor. We also show that GRK2 stimulates the desensitization of GPR54 in HEK 293 cells and that β-arrestin-2 mediates GPR54 activation of ERK1/2 in MDA-MB-231 cells. The significance of these findings in developing molecular-based therapies for treating certain endocrine-related disorders is discussed.

HIC1 Regulates Tumor Cell Responses to Endocrine Therapies

Zhang, B., Faller, D. V., Wang, S. — Tue, 24 Nov 2009 10:02:25 PST

An intractable problem impeding breast cancer treatment by the most frequently prescribed endocrine therapy tamoxifen is the inevitable development of resistance, and the molecular mechanisms underlying this loss of responsiveness by breast cancers have been under intense investigation but are not yet fully elucidated. Our recent reports demonstrated that the tumor suppressor heavily methylated in cancers 1 (HIC1) plays an essential role in growth suppression mediated by external stimuli. We report here that novel tumor suppressor HIC1 is required for growth suppression by estrogen antagonists in breast cancer cells. We also find that HIC1 expression is dramatically induced by exposure to estrogen antagonists in sensitive cells, via a c-Jun N-terminal kinase 1 (JNK1) and prohibitin-mediated signaling pathway. This induction is lost in spontaneously antagonist-resistant breast cancer cells. Furthermore, reintroducing HIC1 into resistant breast cancer cells restored their sensitivity to the estrogen antagonists, indicating the existence of a novel regulatory mechanism for growth control of breast cancer cells.

Deficiency of Ferritin Heavy-Chain Nuclear Import in Triple A Syndrome Implies Nuclear Oxidative Damage as the Primary Disease Mechanism

Tue, 24 Nov 2009 10:02:25 PST

Triple A syndrome is a rare autosomal recessive disorder characterized by ACTH-resistant adrenal failure, alacrima, achalasia, and progressive neurological manifestations. The majority of cases are associated with mutations in the AAAS gene, which encodes a novel, 60-kDa WD-repeat nuclear pore protein, alacrima-achalasia-adrenal insufficiency neurological disorder (ALADIN) of unknown function. Our aim was to elucidate the functional role of ALADIN by determining its interacting protein partners using the bacterial two-hybrid (B2-H) technique. Nonidentical cDNA fragments were identified from both a HeLa S-3 cell and human cerebellar cDNA library that encoded the full-length ferritin heavy chain protein (FTH1). This interaction was confirmed by both co-immunoprecipitation and fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer studies. Immunoblotting showed that fibroblasts from triple A patients (with known AAAS mutations) lack nuclear FTH1, suggesting that the nuclear translocation of FTH1 is defective. Cells transfected with FTH1 and visualized by confocal microscopy had very little nuclear FTH1, but when cotransfected with AAAS, FTH1 is readily visible in the nuclei. Therefore, FTH1 nuclear translocation is enhanced when ALADIN is coexpressed in these cells. In addition to its well known iron storage role, FTH1 has been shown to protect the nucleus from oxidative damage. Apoptosis of neuronal cells induced by hydrogen peroxide was significantly reduced by transfection of AAAS or by FTH1 or maximally by both genes together. Taken together, this work offers a plausible mechanism for the progressive clinical features of triple A syndrome.

A Novel Distal Enhancer Mediates Cytokine Induction of Mouse Rankl Gene Expression

Bishop, K. A., Meyer, M. B., Pike, J. W. — Tue, 24 Nov 2009 10:02:25 PST

Chronic inflammatory states are associated with increased bone loss. This increase is often linked to an elevation in receptor activator of nuclear factor-B ligand (RANKL), a TNF-like factor essential to osteoclast formation. In this study, we document the ability of IL-6 in combination with IL-6 soluble receptor (IL-6/IL-6sR) and oncostatin M to induce Rankl expression in stromal cells via signal transducer and activator of transcription 3 (STAT3). We used chromatin immunoprecipitation-tiled DNA microarray analysis to determine sites of action of STAT3 at the Rankl locus and to assess the consequences of binding on histone H4 acetylation and RNA polymerase II recruitment. Both IL-6/IL-6 soluble receptor and oncostatin M stimulated STAT3 binding upstream of the Rankl transcriptional start site. Although previously identified enhancers bound STAT3, a more distal enhancer termed mRLD6 was a particular focus of STAT3 binding. When fused to a heterologous promoter, this enhancer was highly active, containing two functionally active STAT response elements. Importantly, small interfering RNA knockdown of Stat3 mRNA and protein, but not that of Stat1 or Stat5a, was effective in limiting Rankl mRNA up-regulation. Interestingly, although RNA polymerase II and histone H4 acetylation marked many of the enhancers under basal conditions, the levels of both were strongly increased after cytokine treatment, particularly at mRLD6. Finally, mRLD6 was also a target for forskolin-induced cellular response element-binding protein (CREB) recruitment, which potentiated cytokine activity. Our studies provide new insight into mechanisms by which glycoprotein 130 activating cytokines induce RANKL expression.

Selective Disruption of ER{alpha} DNA-Binding Activity Alters Uterine Responsiveness to Estradiol

Hewitt, S. C., O'Brien, J. E., Jameson, J. L., Kissling, G. E., Korach, K. S. — Tue, 24 Nov 2009 10:02:25 PST

In vitro models have been used to demonstrate that estrogen receptors (ERs) can regulate estrogen-responsive genes either by directly interacting with estrogen-responsive element (ERE) DNA motifs or by interacting with other transcription factors such as AP1. In this study, we evaluated estrogen (E₂)-dependent uterine gene profiles by microarray in the KIKO mouse, an in vivo knock-in mouse model that lacks the DNA-binding function of ER and is consequently restricted to non-ERE-mediated responses. The 2- or 24-h E₂-mediated uterine gene responses were distinct in wild-type (WT), KIKO, and ERKO genotypes, indicating that unique sets of genes are regulated by ERE and non-ERE pathways. After 2 h E₂ treatment, 38% of the WT transcripts were also regulated in the KIKO, demonstrating that the tethered mechanism does operate in this in vivo model. Surprisingly, 1438 E₂-regulated transcripts were unique in the KIKO mouse and were not seen in either WT or ERKO. Pathway analyses revealed that some canonical pathways, such as the Jak/Stat pathway, were affected in a similar manner by E₂ in WT and KIKO. In other cases, however, the WT and KIKO differed. One example is the Wnt/β-catenin pathway; this pathway was impacted, but different members of the pathway were regulated by E₂ or were regulated in a different manner, consistent with differences in biological responses. In summary, this study provides a comprehensive analysis of uterine genes regulated by E₂ via ERE and non-ERE pathways.

Erratum

Tue, 24 Nov 2009 10:02:25 PST

The Glucocorticoid-Induced Leucine Zipper Gene (GILZ) Expression Decreases after Successful Treatment of Patients with Endogenous Cushing's Syndrome and May Play a Role in Glucocorticoid-Induced Osteoporosis

Lekva, T., Bollerslev, J., Kristo, C., Olstad, O. K., Ueland, T., Jemtland, R. — Tue, 24 Nov 2009 10:02:25 PST

Cytostatic Activity of Adenosine Triphosphate-Competitive Kinase Inhibitors in BRAF Mutant Thyroid Carcinoma Cells

Tue, 24 Nov 2009 10:02:25 PST

Differential Ligand-Mediated Pituitary Somatostatin Receptor Subtype Signaling: Implications for Corticotroph Tumor Therapy

Tue, 24 Nov 2009 10:02:25 PST

Medullary Thyroid Carcinoma Cell Lines Contain a Self-Renewing CD133+ Population that Is Dependent on RET Activity

Zhu, W., Hai, T., Ye, L., Cote, G. J. — Tue, 24 Nov 2009 10:02:25 PST

Frequency of Impaired Fibroblast Growth Factor Receptor 1 Signaling as a Cause of Normosmic Idiopathic Hypogonadotropic Hypogonadism

Tue, 24 Nov 2009 10:02:25 PST

Peptide YY (PYY) Gene Polymorphisms in the 3'-Untranslated Region and Proximal Promoter Regions Regulate Cellular Gene Expression and PYY Secretion and Metabolic Syndrome Traits in Vivo

Tue, 24 Nov 2009 10:02:26 PST

Minireview: Pref-1: Role in Adipogenesis and Mesenchymal Cell Fate

Sul, H. S. — Tue, 27 Oct 2009 10:02:17 PDT

Preadipocyte factor-1 [Pref-1; also called Dlk1 (Delta-like protein 1)] is made as an epidermal growth factor-repeat containing transmembrane protein that produces a biologically active soluble form by TNF--converting enzyme (TACE)-mediated cleavage. Soluble Pref-1 activates the MAPK kinase/ERK pathway. In adipose tissue, Pref-1 is specifically expressed in preadipocytes but not in adipocytes and thus is used as a preadipocyte marker. Inhibition of adipogenesis by Pref-1 has been well established in vitro as well as in vivo by ablation and overexpression of Pref-1. SRY (sex determining region Y)-box 9 (Sox9), a transcription factor expressed in preadipocytes to suppress CCAAT enhancer binding protein β and (C/EBP) expression, is required to be down-regulated before adipocyte differentiation can proceed. By activating MAPK kinase/ERK, Pref-1 prevents down-regulation of Sox9, resulting in inhibition of adipogenesis. Furthermore, by inducing Sox9, Pref-1 promotes chondrogenic induction of mesenchymal cells but prevents chondrocyte maturation as well as osteoblast differentiation. Thus, Pref-1 directs multipotent mesenchymal cells toward the chondrogenic lineage but inhibits differentiation into adipocytes as well as osteoblasts and chondrocytes. Pref-1, encoded by an imprinted gene, has also been detected in progenitor cells in various tissues during regeneration and therefore may have a more general role in maintaining cells in an undifferentiated state.

Genomic and Nongenomic Cross Talk between the Gonadotropin-Releasing Hormone Receptor and Glucocorticoid Receptor Signaling Pathways

Kotitschke, A., Sadie-Van Gijsen, H., Avenant, C., Fernandes, S., Hapgood, J. P. — Tue, 27 Oct 2009 10:02:17 PDT

The GnRH receptor (GnRHR), a member of the G protein-coupled receptor family, is a central regulator of reproductive function in all vertebrates. The peptide hormone GnRH exerts its effects via binding to the GnRHR in pituitary gonadotropes. We investigated the mechanisms of regulation of transcription of the mGnRHR gene in the mouse pituitary gonadotrope LβT2 cell line by GnRH and dexamethasone (dex). Reporter assays with transfected mGnRHR promoter show that both dex and GnRH increase transcription of the mGnRHR gene via an activating protein-1 (AP-1) site. Real-time PCR confirmed this on the endogenous mGnRHR gene, and small interfering RNA experiments revealed a requirement for the glucocorticoid receptor (GR) for both the dex and GnRH response. Chromatin immunoprecipitation (ChIP) and immunofluorescence assays provide evidence that both GnRH and dex up-regulate the GnRHR gene via nuclear translocation and interaction of the GR with the AP-1 region on the mGnRHR promoter. We show that GnRH activates the unliganded GR by rapid phosphorylation of the GR at Ser-234 in a GnRHR-dependent fashion to transactivate a GRE reporter gene in LβT2 and COS-1 cells. Using kinase inhibitors, we established a direct link between GnRH-induced protein kinase C and MAPK activation, leading to unliganded GR phosphorylation at Ser-234 and transactivation of the glucocorticoid response element. Furthermore, we show that GnRH and dex synergistically activate the endogenous GnRHR promoter in LβT2 cells, via a mechanism involving steroid receptor coactivator-1 recruitment to the GnRHR AP-1 region. Our results suggest a novel mechanism of rapid nongenomic cross talk between the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes via GnRHR-dependent phosphorylation and activation of the unliganded GR in response to GnRH.

A Novel Isoform of Human LZIP Negatively Regulates the Transactivation of the Glucocorticoid Receptor

Kang, H., Kim, Y. S., Ko, J. — Tue, 27 Oct 2009 10:02:17 PDT

The human leucine zipper protein (LZIP) is a basic leucine zipper transcription factor that is involved in leukocyte migration, tumor suppression, and endoplasmic reticulum stress-associated protein degradation. Although evidence suggests a diversity of roles for LZIP, its function is not fully understood, and the subcellular localization of LZIP is still controversial. We identified a novel isoform of LZIP and characterized its function in ligand-induced transactivation of the glucocorticoid receptor (GR) in COS-7 and HeLa cells. A novel isoform of human LZIP designated as "sLZIP" contains a deleted putative transmembrane domain (amino acids 229–245) of LZIP and consists of 345 amino acids. LZIP and sLZIP were ubiquitously expressed in a variety of cell lines and tissues, with LZIP being much more common. sLZIP was mainly localized in the nucleus, whereas LZIP was located in the cytoplasm. Unlike LZIP, sLZIP was not involved in the chemokine-mediated signal pathway. sLZIP recruited histone deacetylases (HDACs) to the promoter region of the mouse mammary tumor virus luciferase reporter gene and enhanced the activities of HDACs, resulting in suppression of expression of the GR target genes. Our findings suggest that sLZIP functions as a negative regulator in glucocorticoid-induced transcriptional activation of GR by recruitment and activation of HDACs.

Isoform-Specific Transcriptional Activity of Overlapping Target Genes that Respond to Thyroid Hormone Receptors {alpha}1 and {beta}1

Chan, I. H., Privalsky, M. L. — Tue, 27 Oct 2009 10:02:17 PDT

Thyroid hormone receptors (TRs) are hormone-regulated transcription factors that control multiple aspects of physiology and development. TRs are expressed in vertebrates as a series of distinct isoforms that exert distinct biological roles. We wished to determine whether the two most widely expressed isoforms, TR1 and TRβ1, exert their different biological effects by regulating different sets of target genes. Using stably transformed HepG2 cells and a microarray analysis, we were able to demonstrate that TR1 and TRβ1 regulate a largely overlapping repertoire of target genes in response to T₃ hormone. However, these two isoforms display very different transcriptional properties on each individual target gene, ranging from a much greater T₃-mediated regulation by TR1 than by TRβ1, to near equal regulation by both isoforms. We also identified TR1 and TRβ1 target genes that were regulated by these receptors in a hormone-independent fashion. We suggest that it is this gene-specific, isoform-specific amplitude of transcriptional regulation that is the likely basis for the appearance and maintenance of TR1 and TRβ1 over evolutionary time. In essence, TR1 and TRβ1 adjust the magnitude of the transcriptional response at different target genes to different levels; by altering the ratio of these isoforms in different tissues or at different developmental times, the intensity of T₃ response can be individually tailored to different physiological and developmental requirements.

Functional Screening of FxxLF-Like Peptide Motifs Identifies SMARCD1/BAF60a as an Androgen Receptor Cofactor that Modulates TMPRSS2 Expression

Tue, 27 Oct 2009 10:02:17 PDT

Androgen receptor (AR) transcriptional activity is tightly regulated by interacting cofactors and cofactor complexes. The best described cofactor interaction site in the AR is the hormone-induced coactivator binding groove in the ligand-binding domain, which serves as a high-affinity docking site for FxxLF-like motifs. This study aimed at identifying novel AR cofactors by in silico selection and functional screening of FxxLF-like peptide motifs. Candidate interacting motifs were selected from a proteome-wide screening and from a supervised screening focusing on components of protein complexes involved in transcriptional regulation. Of the 104 peptides tested, 12 displayed moderate to strong in vivo hormone-dependent interactions with AR. For three of these, ZBTB16/PLZF, SMARCA4/BRG1, and SMARCD1/BAF60a, the full-length protein was tested for interaction with AR. Of these, BAF60a, a subunit of the SWI/SNF chromatin remodeling complex, displayed hormone-dependent interactions with AR through its FxxFF motif. Vice versa, recruitment of BAF60a by the AR required an intact coactivator groove. BAF60a depletion by small interfering RNA in LNCaP cells demonstrated differential effects on expression of endogenous AR target genes. AR-driven expression of TMPRSS2 was almost completely blocked by BAF60a small interfering RNA. In summary, our data demonstrate that BAF60a directly interacts with the coactivator groove in the AR ligand-binding domain via its FxxFF motif, thereby selectively activating specific AR-driven promoters.

Decreased PPAR{gamma} Expression Compromises Perigonadal-Specific Fat Deposition and Insulin Sensitivity

Tue, 27 Oct 2009 10:02:17 PDT

Mutations and polymorphisms in PPARG have been linked to adiposity and partial lipodystrophy in humans. However, how disturbances in PPARG lead to depot-specific effects on adipose tissue, as shown by the characteristic aberrant fat distribution in patients, remains unclear. By manipulating the 3'-untranslated region of the Pparg gene, we have generated mice with peroxisome proliferator-activated receptor (PPAR) gene expression ranging from 25% to 100% normal. Basal levels of PPAR transcripts between 50% and approximately 100% had no significant effect on body weight, fat mass, and insulin sensitivity. In contrast, mice with 25% normal PPAR expression exhibited reduced body weight and total fat mass, insulin resistance, and dyslipidemia. Interestingly, fat mass was selectively reduced in perigonadal depot without significant changes in inguinal and other depots. Expression of adipogenic factor CCAAT enhancer binding protein- and some other metabolic genes containing peroxisome proliferator response element were reduced in a perigonadal depot-specific fashion. This was further associated with depot-specific reduction in the expression of adipokines, increased expression of TNF, and increased ectopic lipid deposition in muscles. Together, these results underscore the differential sensitivity of the individual fat depots on PPAR availability as an underlying mechanism of partial lipodystrophy.

Proteomic Analysis of Phosphorylated Nuclear Proteins Underscores Novel Roles for Rapid Actions of Retinoic Acid in the Regulation of mRNA Splicing and Translation

Tue, 27 Oct 2009 10:02:17 PDT

Retinoic acid (RA) signaling is mediated by the retinoic acid receptor (RAR), belonging to the nuclear hormone receptor superfamily. In addition to its classical transcriptional actions, RAR also mediates rapid transcription-independent (nongenomic) actions, consisting in the activation of signal transduction pathways, as the phosphatidyl-inositol-3-kinase or the ERK MAPK-signaling pathways. RA-induced rapid transcription-independent actions play a role in different physiological contexts. As an effort toward understanding the functions of those rapid actions on signaling elicited by RA, we have identified nuclear proteins the phosphorylation state of which is rapidly modified by RA treatment in neuroblastoma cells, using a proteomic approach. Our results show that RA treatment led to changes in the phosphorylation patterns in two families of proteins: 1) those related to chromatin dynamics in relation to transcriptional activation, and 2) those related to mRNA processing and, in particular, mRNA splicing. We show that treatment of neuroblastoma cells with RA leads to alteration of the regulation of pre-mRNA splicing and mRNA translation. Thus, our results underscore novel functions for the rapid signaling elicited by RAR in the regulation of mRNA processing. We conclude that RA activation of signaling pathways can indeed regulate mRNA processing as part of a cellular response orchestrated by the nuclear receptor RAR.

G Protein-Coupled Receptor 30 Expression Is Up-Regulated by EGF and TGF{alpha} in Estrogen Receptor {alpha}-Positive Cancer Cells

Tue, 27 Oct 2009 10:02:17 PDT

In the present study, we evaluated the regulation of G protein-coupled receptor (GPR)30 expression in estrogen receptor (ER)-positive endometrial, ovarian, and estrogen-sensitive, as well as tamoxifen-resistant breast cancer cells. We demonstrate that epidermal growth factor (EGF) and TGF transactivate the GPR30 promoter and accordingly up-regulate GPR30 mRNA and protein levels only in endometrial and tamoxifen-resistant breast cancer cells. These effects exerted by EGF and TGF were dependent on EGF receptor (EGFR) expression and activation and involved phosphorylation of the Tyr¹⁰⁴⁵ and Tyr¹¹⁷³ EGFR sites. Using gene-silencing experiments and specific pharmacological inhibitors, we have ascertained that EGF and TGF induce GPR30 expression through the EGFR/ERK transduction pathway, and the recruitment of c-fos to the activator protein-1 site located within GPR30 promoter sequence. Interestingly, we show that functional cross talk of GPR30 with both activated EGFR and ER relies on a physical interaction among these receptors, further extending the potential of estrogen to trigger a complex stimulatory signaling network in hormone-sensitive tumors. Given that EGFR/HER2 overexpression is associated with tamoxifen resistance, our data may suggest that ligand-activated EGFR could contribute to the failure of tamoxifen therapy also by up-regulating GPR30, which in turn could facilitates the action of estrogen. In addition, important for resistance is the ability of tamoxifen to bind to and activate GPR30, the expression of which is up-regulated by EGFR activation. Our results emphasize the need for new endocrine agents able to block widespread actions of estrogen without exerting any stimulatory activity on transduction pathways shared by the steroid and growth factor-signaling networks.

BMP-4 Induction of Arrest and Differentiation of Osteoblast-Like Cells via p21CIP1 and p27KIP1 Regulation

Tue, 27 Oct 2009 10:02:17 PDT

Cell cycle regulation by differentiation signals is critical for eukaryote development. We investigated the roles of bone morphogenetic protein (BMP)-4, an important stimulator of osteoblast differentiation and bone formation, in regulating cell cycle distribution in four osteoblast-like cell lines and mouse primary osteoblasts, and the underlying mechanisms. In all cells used, BMP-4 induced G₀/G₁ arrest. The molecular basis of the BMP-4 effect was analyzed, and the presentation on molecular mechanism is focused on human MG63 cells. BMP-4 induced p21^CIP1 and p27^KIP1 expressions and hence cell differentiation but had no effects on the expressions of cyclins A, B1, D1, and E, cyclin-dependent protein kinase-2, -4, and -6. Using specific small interfering RNA (siRNA), we found that BMP-4-induced G₀/G₁ arrest, and p21^CIP1 and p27^KIP1 expressions were mediated by BMP receptor type IA (BMPRIA)-specific Sma- and Mad-related protein (Smad)1/5. BMP-4 induced transient phosphorylations of ERK; transfection of MG63 cells with ERK2, but not ERK1, -specific siRNA inhibited the BMP-4-induced responses in MG63 cells. Pretreatment of MG63 cells with Arg-Gly-Asp-Ser, which blocks the cell-extracellular matrix interaction, or transfection with β₃ integrin-specific siRNA inhibited BMP-4-induced ERK and Smad1/5 phosphorylations. BMP-4 induced transient increases in associations of β₃-integrin with focal adhesion kinase and Shc, the dominant-negative mutants of which inhibited BMP-4-induced ERK and Smad1/5 phosphorylations. Our results indicate that BMP-4 induces G₀/G₁ arrest and hence differentiation in osteoblast-like cells through increased expressions of p21^CIP1 and p27^KIP1, which are mediated by BMPRIA-specific Smad1/5. The extracellular matrix/β₃ integrin/ focal adhesion kinase/Shc/ERK2 signaling pathway is involved in these BMP-4-induced responses in osteoblast-like cells.

Paradoxical Stimulation of Cyclooxygenase-2 Expression by Glucocorticoids via a Cyclic AMP Response Element in Human Amnion Fibroblasts

Tue, 27 Oct 2009 10:02:17 PDT

Human amnion fibroblasts produce abundant prostaglandins toward the end of gestation, which is one of the major events leading to parturition. In marked contrast to its well-described antiinflammatory effect, glucocorticoids have been shown to up-regulate cyclooxygenase-2 (COX-2) expression in human amnion fibroblasts. The mechanisms underlying this paradoxical induction of COX-2 by glucocorticoids have not been resolved. Using cultured human amnion fibroblasts, we found that the induction of COX-2 mRNA expression by cortisol was a glucocorticoid receptor (GR)-dependent process requiring ongoing transcription. Upon transfection of a COX-2 promoter-driven reporter gene into the amnion fibroblasts, cortisol stimulated the COX-2 promoter activity. This was abolished by mutagenesis of a cAMP response element (CRE) at –53 to approximately –59bp as well as by cotransfection of a plasmid expressing dominant-negative CRE-binding protein (CREB). The phosphorylation level of CREB-1 was significantly increased by cortisol treatment of the amnion fibroblasts, whereas the effect was attenuated either by the protein kinase A inhibitor H89 or the p38 -MAPK inhibitor SB203580. The induction of the COX-2 promoter activity and the phosphorylation of CREB-1 were also blocked by the GR antagonist RU486. Chromatin immunoprecipitation (ChIP) assay revealed that the binding of CREB-1 to the CRE of the COX-2 promoter was increased by cortisol treatment of the amnion fibroblasts. In conclusion, cortisol, via binding to GR, stimulated COX-2 expression by increasing phosphorylated CREB-1 binding to the CRE of the COX-2 gene. Cortisol may phosphorylate CREB-1 by activating either protein kinase A or p38-MAPK in the amnion fibroblasts.

A Preformed Signaling Complex Mediates GnRH-Activated ERK Phosphorylation of Paxillin and FAK at Focal Adhesions in L{beta}T2 Gonadotrope Cells

Tue, 27 Oct 2009 10:02:18 PDT

Most receptor tyrosine kinases and G protein-coupled receptors (GPCRs) operate via a limited number of MAPK cascades but still exert diverse functions, and therefore signal specificity remains an enigma. Also, most GPCR ligands utilize families of receptors for mediation of diverse biological actions; however, the mammalian type I GnRH receptor (GnRHR) seems to be the sole receptor mediating GnRH-induced gonadotropin synthesis and release. Signaling complexes associated with GPCRs may thus provide the means for signal specificity. Here we describe a signaling complex associated with the GnRHR, which is a unique GPCR lacking a C-terminal tail. Unlike other GPCRs, this signaling complex is preformed, and exposure of LβT2 gonadotropes to GnRH induces its dynamic rearrangement. The signaling complex includes c-Src, protein kinase C, -, and -, Ras, MAPK kinase 1/2, ERK1/2, tubulin, focal adhesion kinase (FAK), paxillin, vinculin, caveolin-1, kinase suppressor of Ras-1, and the GnRHR. Exposure to GnRH (5 min) causes MAPK kinase 1/2, ERK1/2, tubulin, vinculin, and the GnRHR to detach from c-Src, but they reassociate within 30 min. On the other hand, FAK, paxillin, the protein kinase Cs, and caveolin-1 stay bound to c-Src, whereas kinase suppressor of Ras-1 appears in the complex only 30 min after GnRH stimulation. GnRH was found to activate ERK1/2 in the complex in a c-Src-dependent manner, and the activated ERK1/2 subsequently phosphorylates FAK and paxillin. In parallel, caveolin-1, FAK, vinculin, and paxillin are phosphorylated on Tyr residues apparently by GnRH-activated c-Src. Receptor tyrosine kinases and GPCRs translocate ERK1/2 to the nucleus to phosphorylate and activate transcription factors. We therefore propose that the role of the multiprotein signaling complex is to sequester a cytosolic pool of activated ERK1/2 to phosphorylate FAK and paxillin at focal adhesions.

Cyclin D2 Protein Stability Is Regulated in Pancreatic {beta}-Cells

Tue, 27 Oct 2009 10:02:18 PDT

The molecular determinants of β-cell mass expansion remain poorly understood. Cyclin D2 is the major D-type cyclin expressed in β-cells, essential for adult β-cell growth. We hypothesized that cyclin D2 could be actively regulated in β-cells, which could allow mitogenic stimuli to influence β-cell expansion. Cyclin D2 protein was sharply increased after partial pancreatectomy, but cyclin D2 mRNA was unchanged, suggesting posttranscriptional regulatory mechanisms influence cyclin D2 expression in β-cells. Consistent with this hypothesis, cyclin D2 protein stability is powerfully regulated in fibroblasts. Threonine 280 of cyclin D2 is phosphorylated, and this residue critically limits D2 stability. We derived transgenic (tg) mice with threonine 280 of cyclin D2 mutated to alanine (T280A) or wild-type cyclin D2 under the control of the insulin promoter. Cyclin D2 T280A protein was expressed at much higher levels than wild-type cyclin D2 protein in β-cells, despite equivalent expression of tg mRNAs. Cyclin D2 T280A tg mice exhibited a constitutively nuclear cyclin D2 localization in β-cells, and increased cyclin D2 stability in islets. Interestingly, threonine 280-mutant cyclin D2 tg mice had greatly reduced β-cell apoptosis, with suppressed expression of proapoptotic genes. Suppressed β-cell apoptosis in threonine 280-mutant cyclin D2 tg mice resulted in greatly increased β-cell area in aged mice. Taken together, these data indicate that cyclin D2 is regulated by protein stability in pancreatic β-cells, that signals that act upon threonine 280 limit cyclin D2 stability in β-cells, and that threonine 280-mutant cyclin D2 overexpression prolongs β-cell survival and augments β-cell mass expansion.

MicroRNA 132 Regulates Nutritional Stress-Induced Chemokine Production through Repression of SirT1

Tue, 27 Oct 2009 10:02:18 PDT

Human adipose tissue secretes a number of proinflammatory mediators that may contribute to the pathophysiology of obesity-related disorders. Understanding the regulatory pathways that control their production is paramount to developing effective therapeutics to treat these diseases. Using primary human adipose-derived stem cells as a source of preadipocytes and in vitro differentiated adipocytes, we found IL-8 and monocyte chemoattractant protein-1 (MCP-1) are constitutively secreted by both cell types and induced in response to serum deprivation. MicroRNA profiling revealed the rapid induction of microRNA 132 (miR-132) in these cells when switched to serum-free medium. Furthermore, miR-132 overexpression was sufficient to induce nuclear factor-B translocation, acetylation of p65, and production of IL-8 and MCP-1. Inhibitors of miR-132 decreased acetylated p65 and partially inhibited the production of IL-8 and MCP-1 induced by serum deprivation. MiR-132 was shown to inhibit silent information regulator 1 (SirT1) expression through a miR-132 binding site in the 3'-untranslated region of SirT1. Thus, in response to nutritional availability, induction of miR-132 decreases SirT1-mediated deacetylation of p65 leading to activation of nuclear factor-B and transcription of IL-8 and MCP-1 in primary human preadipocytes and in vitro differentiated adipocytes.

Genomic Priming of the Antisecretory Response to Estrogen in Rat Distal Colon throughout the Estrous Cycle

O'Mahony, F., Alzamora, R., Chung, H.-L., Thomas, W., Harvey, B. J. — Tue, 27 Oct 2009 10:02:18 PDT

The secretion of Cl^– across distal colonic crypt cells provides the driving force for the movement of fluid into the luminal space. 17β-Estradiol (E2) produces a rapid and sustained reduction in secretion in females, which is dependent on the novel protein kinase C (PKC) isozyme and PKA isoform I targeting of KCNQ1 channels. This sexual dimorphism in the E2 response is associated with a higher expression level of PKC in female compared with the male tissue. The present study revealed the antisecretory response is regulated throughout the female reproductive (estrous) cycle and is primed by genomic regulation of the kinases. E2 (1–10 nm) decreased cAMP-dependent secretion in colonic epithelia during the estrus, metestrus, and diestrus stages. A weak inhibition of secretion was demonstrated in the proestrus stage. The expression levels of PKC and PKA fluctuated throughout the estrous cycle and correlated with the potency of the antisecretory effect of E2. The expression of PKC and PKA were up-regulated by estrogen at a transcriptional level via a PKC-MAPK-cAMP response element-binding protein-regulated pathway indicating a genomic priming of the antisecretory response. PKC was activated by the membrane-impermeant E2-BSA, and this response was inhibited by the estrogen receptor antagonist ICI 182,780. The 66-kDa estrogen receptor- isoform was present at the plasma membrane of female colonic crypt cells with a lower abundance found in male colonic crypts. The study demonstrates estrogen regulation of intestinal secretion both at a rapid and transcriptional level, demonstrating an interdependent relationship between both nongenomic and genomic hormone responses.

NIDDK Workshop on Circadian Rhythms and Metabolic Disease

Tue, 27 Oct 2009 10:02:18 PDT

Retinoblastoma Protein Plays Multiple Essential Roles in the Terminal Differentiation of Sertoli Cells

Nalam, R. L., Andreu-Vieyra, C., Braun, R. E., Akiyama, H., Matzuk, M. M. — Tue, 27 Oct 2009 10:02:18 PDT

Retinoblastoma protein (RB) plays crucial roles in cell cycle control and cellular differentiation. Specifically, RB impairs the G₁ to S phase transition by acting as a repressor of the E2F family of transcriptional activators while also contributing towards terminal differentiation by modulating the activity of tissue-specific transcription factors. To examine the role of RB in Sertoli cells, the androgen-dependant somatic support cell of the testis, we created a Sertoli cell-specific conditional knockout of Rb. Initially, loss of RB has no gross effect on Sertoli cell function because the mice are fertile with normal testis weights at 6 wk of age. However, by 10–14 wk of age, mutant mice demonstrate severe Sertoli cell dysfunction and infertility. We show that mutant mature Sertoli cells continue cycling with defective regulation of multiple E2F1- and androgen-regulated genes and concurrent activation of apoptotic and p53-regulated genes. The most striking defects in mature Sertoli cell function are increased permeability of the blood-testis barrier, impaired tissue remodeling, and defective germ cell-Sertoli cell interactions. Our results demonstrate that RB is essential for proper terminal differentiation of Sertoli cells.

Male-Specific Hepatic Bcl6: Growth Hormone-Induced Block of Transcription Elongation in Females and Binding to Target Genes Inversely Coordinated with STAT5

Meyer, R. D., Laz, E. V., Su, T., Waxman, D. J. — Tue, 27 Oct 2009 10:02:18 PDT

The transcriptional repressor Bcl6 is a male-specific rat liver gene product and one of 24 early GH-response genes encoding DNA-binding proteins. Presently, the sex specificity of Bcl6 was shown to emerge at puberty, when hepatic Bcl6 mRNA was induced in males and repressed in females by the female plasma GH profile. Hepatic Bcl6 mRNA was increased to near-normal male levels in hypophysectomized females and was extinguished in intact males given a continuous GH infusion (female-like GH pattern). Bcl6 was also repressed in adult male somatostatin-deficient mice, where plasma GH profiles are female like. Hepatic Bcl6 RNA was rapidly down-regulated by GH pulse treatment, both in hypophysectomized male rats and in primary rat hepatocytes. Bcl6 was substantially induced in female mice deficient in hepatic signal transducer and activator of transcription (STAT)5a/STAT5b, suggesting that these STAT transcriptional mediators of GH signaling repress Bcl6. Indeed, STAT5 was bound to Bcl6 STAT5-binding region-B, previously associated with Bcl6 repression, in both male and female liver chromatin. STAT5 also bound to Bcl6 region-A in male chromatin but only during a plasma GH pulse. Analysis of primary transcripts (heterogenous nuclear RNA) across the Bcl6 gene revealed a novel mechanism of GH-dependent sex specificity, with two apparent blocks in Bcl6 transcription elongation seen in female liver and in continuous GH-treated male liver, one early in intron 4 and one in exon 5, which together reduced transcription beyond exon 5 more than 300-fold. Finally, Bcl6 was bound to a subset of STAT5-binding sites in male liver chromatin, including a Socs2 STAT5-binding site where Bcl6 binding increased substantially between plasma GH pulses, i.e. when STAT5 binding was low. Bcl6 and STAT5 binding are thus inversely coordinated by the endogenous pulses of pituitary GH release, suggesting this male-specific transcriptional repressor modulates hepatic GH signaling to select STAT5 target genes.

Androgen-Responsive Gene Database: Integrated Knowledge on Androgen-Responsive Genes

Tue, 27 Oct 2009 10:02:18 PDT

Androgen signaling plays an important role in many biological processes. Androgen Responsive Gene Database (ARGDB) is devoted to providing integrated knowledge on androgen-controlled genes. Gene records were collected on the basis of PubMed literature collections. More than 6000 abstracts and 950 original publications were manually screened, leading to 1785 human genes, 993 mouse genes, and 583 rat genes finally included in the database. All the collected genes were experimentally proved to be regulated by androgen at the expression level or to contain androgen-responsive regions. For each gene important details of the androgen regulation experiments were collected from references, such as expression change, androgen-responsive sequence, response time, tissue/cell type, experimental method, ligand identity, and androgen amount, which will facilitate further evaluation by researchers. Furthermore, the database was integrated with multiple annotation resources, including National Center for Biotechnology Information, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathway, to reveal the biological characteristics and significance of androgen-regulated genes. The ARGDB web site is mainly composed of the Browse, Search, Element Scan, and Submission modules. It is user friendly and freely accessible at http://argdb.fudan.edu.cn. Preliminary analysis of the collected data was performed. Many disease pathways, such as prostate carcinogenesis, were found to be enriched in androgen-regulated genes. The discovered androgen-response motifs were similar to those in previous reports. The analysis results are displayed in the web site. In conclusion, ARGDB provides a unified gateway to storage, retrieval, and update of information on androgen-regulated genes.

Expression of Progesterone Receptor A form and Its Role in the Interaction of Progesterone with Cortisol on Cyclooxygenase-2 Expression in Amnionic Fibroblasts

Guo, C. M., Zhu, X. O., Ni, X. T., Yang, Z., Myatt, L., Sun, K. — Tue, 27 Oct 2009 10:02:18 PDT

11{beta}-Hydroxysteroid Dehydrogenase Type 1 and Its Role in the Hypothalamus-Pituitary-Adrenal Axis, Metabolic Syndrome, and Inflammation

Cooper, M. S., Stewart, P. M. — Tue, 27 Oct 2009 10:02:18 PDT

Correlation of Telomere Length and Telomerase Activity with Occult Ovarian Insufficiency

Tue, 27 Oct 2009 10:02:18 PDT

Programmed Death Ligand 1 (PD-L1) Gene Variants Contribute to Autoimmune Addison's Disease and Graves' Disease Susceptibility

Tue, 27 Oct 2009 10:02:18 PDT

Partial Primary Deficiency of Insulin-Like Growth Factor (IGF)-I Activity Associated with IGF1 Mutation Demonstrates Its Critical Role in Growth and Brain Development

Tue, 27 Oct 2009 10:02:18 PDT

Effects of Endogenous Growth Differentiation Factor 9 on Activin A-Induced Inhibin B Production in Human Granulosa-Lutein Cells

Shi, F.-T., Cheung, A. P., Huang, H.-F., Leung, P. C. K. — Tue, 27 Oct 2009 10:02:18 PDT

Dual Inhibition of Mitogen-Activated Protein Kinase Kinase and Mammalian Target of Rapamycin in Differentiated and Anaplastic Thyroid Cancer

Jin, N., Jiang, T., Rosen, D. M., Nelkin, B. D., Ball, D. W. — Tue, 27 Oct 2009 10:02:18 PDT

Minireview: Live and Let Die: Molecular Effects of Glucocorticoids on Bone Cells

Hofbauer, L. C., Rauner, M. — Mon, 28 Sep 2009 10:02:15 PDT

Glucocorticoids (GCs) are efficient drugs that are used to treat various immune-mediated diseases, but their long-term administration is associated with multiple metabolic side effects, including osteoporosis. Molecular analyses of the mechanisms exerted by the GC receptor have resulted in the development of GC receptor agonists that selectively repress or activate GC target genes. This review summarizes the cellular and molecular effects of GCs on bone cells and highlights the critical signaling pathways that may evolve into future therapeutic strategies.

Negative Regulation of Hedgehog Signaling by Liver X Receptors

Mon, 28 Sep 2009 10:02:16 PDT

Hedgehog (Hh) signaling is indispensable in embryonic development, and its dysregulated activity results in severe developmental disorders as shown by genetic models of naturally occurring mutations in animal and human pathologies. Hh signaling also functions in postembryonic development and adult tissue homeostasis, and its aberrant activity causes various human cancers. Better understanding of molecular regulators of Hh signaling is of fundamental importance in finding new strategies for pathway modulation. Here, we identify liver X receptors (LXRs), members of the nuclear hormone receptor family, as previously unrecognized negative regulators of Hh signaling. Activation of LXR by specific pharmacological ligands, TO901317 and GW3965, inhibited the responses of pluripotent bone marrow stromal cells and calvaria organ cultures to sonic Hh, resulting in the inhibition of expression of Hh-target genes, Gli1 and Patched1, and Gli-dependent transcriptional activity. Moreover, LXR ligands inhibited sonic Hh-induced differentiation of bone marrow stromal cells into osteoblasts. Elimination of LXRs by small interfering RNA inhibited ligand-induced inhibition of Hh target gene expression. Furthermore, LXR ligand did not inhibit Hh responsiveness in mouse embryonic fibroblasts that do not express LXRs, whereas introduction of LXR into these cells reestablished the inhibitory effects. Daily oral administration of TO901317 to mice after 3 d significantly inhibited baseline Hh target-gene expression in liver, lung, and spleen. Given the importance of modulating Hh signaling in various physiological and pathological settings, our findings suggest that pharmacological targeting of LXRs may be a novel strategy for Hh pathway modulation.

DNA Binding by Estrogen Receptor-{alpha} Is Essential for the Transcriptional Response to Estrogen in the Liver and the Uterus

Mon, 28 Sep 2009 10:02:16 PDT

The majority of the biological effects of estrogens in the reproductive tract are mediated by estrogen receptor (ER), which regulates transcription by several mechanisms. Because the tissue-specific effects of some ER ligands may be caused by tissue-specific transcriptional mechanisms of ER, we aimed to identify the contribution of DNA recognition to these mechanisms in two clinically important target organs, namely uterus and liver. We used a genetic mouse model that dissects DNA binding-dependent vs. independent transcriptional regulation elicited by ER. The EAAE mutant harbors amino acid exchanges at four positions of the DNA-binding domain (DBD) of ER. This construct was knocked in the ER gene locus to produce ER_(EAAE/EAAE) mice devoid of a functional ER DBD. The phenotype of the ER_(EAAE/EAAE) mice resembles the general loss-of-function phenotype of ER knockout mutant mice with hypoplastic uteri, hemorrhagic ovaries, and impaired mammary gland development. In agreement with this phenotype, the expression pattern of the ER_(EAAE/EAAE) mutant mice in liver obtained by genome-wide gene expression profiling supports the observation of a near-complete loss of estrogen-dependent gene regulation in comparison with the wild type. Further gene expression analyses to validate the results of the microarray data were performed by quantitative RT-PCR. The analyses indicate that both gene activation and repression by estrogen-bound ER rely on an intact DBD in vivo.

ASCOM Controls Farnesoid X Receptor Transactivation through Its Associated Histone H3 Lysine 4 Methyltransferase Activity

Kim, D.-H., Lee, J., Lee, B., Lee, J. W. — Mon, 28 Sep 2009 10:02:16 PDT

Activating signal cointegrator-2 (ASC-2), a coactivator of multiple nuclear receptors and transcription factors, belongs to a steady-state complex named ASCOM (for ASC-2 complex), which contains histone H3 lysine 4 (H3K4) methyltransferase MLL3 or its paralog MLL4. ASC-2 binds to many nuclear receptors in a ligand-dependent manner through its two LxxLL motifs. Here we show that the first LxxLL motif of ASC-2 shows relatively weak but specific interaction with the nuclear receptor farnesoid X receptor (FXR) and that ASCOM plays crucial roles in FXR transactivation. Our results reveal that ASC-2, MLL3, and MLL4 are recruited to FXR target genes in a ligand-dependent manner. We further show that the recruitment of MLL3 requires ASC-2 and that FXR ligand induces not only expression of FXR-target genes but also their H3K4 trimethylation in a manner dependent on the presence of ASC-2, MLL3, and MLL4. In addition, MLL3 and MLL4 function redundantly with FXR transactivation. Correspondingly, expression of FXR target genes is partially impaired in mice expressing an enzymatically inactivated mutant form of MLL3, and these mice show disrupted bile acid homeostasis. Overall, these results suggest that ASCOM-MLL3 and ASCOM-MLL4 play redundant but essential roles in FXR transactivation via their H3K4 trimethylation activity.

Expression of the Synaptotagmin I Gene Is Enhanced by Binding of the Pituitary-Specific Transcription Factor, POU1F1

Howard, P. W., Jue, S. F., Maurer, R. A. — Mon, 28 Sep 2009 10:02:16 PDT

The POU1F1 transcription factor (also known as Pit-1/GHF1) is required for development of pituitary cells that secrete prolactin, GH, and TSH. Presumably, POU1F1 regulates the expression of multiple genes required for expansion and differentiation of these pituitary cell lineages. However, only a few genes regulated by POU1F1 have been identified. In the present studies we have identified synaptotagmin I (Syt1) as a target gene for POU1F1 in GH₃ pituitary cells. Chromatin immunoprecipitation assays have provided evidence that POU1F1 binds close to the Syt1 exon that contains the initiation codon. Although this exon has previously been considered to be located far from the transcription initiation site, transcript mapping in GH₃ cells indicates that Syt1 mRNA synthesis is initiated close to the mapped POU1F1-binding site. POU1F1 knockdown studies using a short hairpin RNA vector have provided evidence that POU1F1 plays a role in stimulating expression of the endogenous Syt1 gene. Transfection studies with a Syt1-luciferase reporter gene are consistent with the presence of an internal, POU1F1-regulated promoter in the Syt1 gene. In vitro binding studies have provided further evidence for a POU1F1-binding site within this region of the Syt1 gene. Overall the studies provide evidence that Syt1 is a target gene regulated by POU1F1 in GH₃ pituitary cells. Because SYT1 has been extensively studied as an important transducer of Ca²⁺ signaling in regulated secretion, it seems likely that activation of Syt1 gene expression is part of a mechanism mediating POU1F-induced differentiation of pituitary cells.

Involvement of 4E-BP1 in the Protection Induced by HDLs on Pancreatic {beta}-Cells

Mon, 28 Sep 2009 10:02:16 PDT

High-density lipoproteins (HDLs) protect pancreatic β-cells against apoptosis. This property might relate to the increased risk to develop diabetes in patients with low HDL blood levels. However, the mechanisms by which HDLs protect β-cells are poorly characterized. Here we used a transcriptomic approach to identify genes differentially modulated by HDLs in β-cells subjected to apoptotic stimuli. The transcript encoding 4E-binding protein (4E-BP)1 was up-regulated by serum starvation, and HDLs blocked this increase. 4E-BP1 inhibits cap-dependent translation in its non- or hypophosphorylated state but it loses this ability when hyperphosphorylated. At the protein level, 4E-BP1 was also up-regulated in response to starvation and IL-1β, and this was blunted by HDLs. Whereas an ectopic increase of 4E-BP1 expression induced β-cell death, silencing 4E-BP1 increase with short hairpin RNAs inhibited the apoptotic-inducing capacities of starvation. HDLs can therefore protect β-cells by blocking 4E-BP1 protein expression, but this is not the sole protective mechanism activated by HDLs. Indeed, HDLs blocked apoptosis induced by endoplasmic reticulum stress with no associated decrease in total 4E-BP1 induction. Although, HDLs favored the phosphorylation, and hence the inactivation of 4E-BP1 in these conditions, this appeared not to be required for HDL protection. Our results indicate that HDLs can protect β-cells through modulation of 4E-BP1 depending on the type of stress stimuli.

Insulin Acts through FOXO3a to Activate Transcription of Plasminogen Activator Inhibitor Type 1

Jag, U. R., Zavadil, J., Stanley, F. M. — Mon, 28 Sep 2009 10:02:16 PDT

Plasminogen activator inhibitor-1 (PAI-1) is an important regulator of fibrinolysis. PAI-1 levels are elevated in type 2 diabetes, and this elevation correlates with macro- and microvascular complications of diabetes. However, the mechanistic link between insulin and up-regulation of PAI-1 is unclear. Here we demonstrate that overexpression of Forkhead-related transcription factor (Fox)O1, FoxO3a, and FoxC1 augment insulin’s ability to activate the PAI-1 promoter. In addition, insulin treatment promotes the phosphorylation of nuclear and cytoplasmic Fox03a and an increase of cytoplasmic Fox03a. In contrast, insulin treatment led to the accumulation of phospho-Fox01 only in the cytoplasm. Furthermore, insulin also increased the ability of chimeric LexA-FoxO1, LexA-FoxO3a, and LexA-FoxC1 proteins to increase the activity of a LexA reporter, suggesting that the effect of insulin on FoxO3a was direct. Using small interfering RNA to specifically deplete each of the Fox transcription factors tested, we demonstrate that only reduction of FoxO3a inhibits insulin-increased PAI-1-Luc expression and PAI-1 mRNA accumulation. Finally, chromatin immunoprecipitation assays confirm the presence of FoxO3a on the PAI-1 promoter. These results suggest that FoxO3a mediates insulin-increased PAI-1 gene expression.

Maternal Diabetes Causes Mitochondrial Dysfunction and Meiotic Defects in Murine Oocytes

Mon, 28 Sep 2009 10:02:16 PDT

The adverse effects of maternal diabetes on embryo development and pregnancy outcomes have recently been shown to occur as early as the one-cell zygote stage. The hypothesis of this study was that maternally inherited mitochondria in oocytes from diabetic mice are abnormal and thus responsible in part for this latency of developmental compromise. In ovulated oocytes from diabetic mice, transmission electron microscopy revealed an alteration in mitochondrial ultrastructure, and the quantitative analysis of mitochondrial DNA copy number demonstrated an increase. The levels of ATP and tricarboxylic acid cycle metabolites in diabetic oocytes were markedly reduced compared with controls, suggesting a mitochondrial metabolic dysfunction. Abnormal distribution of mitochondria within maturing oocytes also was seen in diabetic mice. Furthermore, oocytes from diabetic mice displayed a higher frequency of spindle defects and chromosome misalignment in meiosis, resulting in increased aneuploidy rates in ovulated oocytes. Collectively, our results suggest that maternal diabetes results in oocyte defects that are transmitted to the fetus by two routes: first, meiotic spindle and chromatin defects result in nondisjunction leading to embryonic aneuploidy; second, structural and functional abnormalities of oocyte mitochondria, through maternal transmission, provide the embryo with a dysfunctional complement of mitochondria that may be propagated during embryogenesis.

The Trafficking/Interaction of eNOS and Caveolin-1 Induced by Insulin Modulates Endothelial Nitric Oxide Production

Wang, H., Wang, A. X., Liu, Z., Chai, W., Barrett, E. J. — Mon, 28 Sep 2009 10:02:16 PDT

Endothelial nitric oxide synthase (eNOS) activity is tightly regulated by posttranscriptional modification and its subcellular localization. Here we examined whether insulin modulates nitric oxide (NO) production by regulating eNOS subcellular localization. We used confocal microscopy and immunoblots to examine the time course for 1) subcellular targeting/association of eNOS and caveolin-1 (CAV-1); 2) eNOS Ser¹¹⁷⁹ phosphorylation; and 3) NO production in cultured bovine aorta endothelial cells. Serum starvation increased eNOS/CAV-1 localization to the perinuclear region. Adding insulin provoked their prompt translocation to and association at the plasma membrane (PM). Specific monoclonal antibodies against either CAV-1 or eNOS coimmunoprecipitated the other from bovine aorta endothelial cell membrane extracts, and insulin increased this interaction. Insulin stimulated NO production transiently despite a persistent eNOS Ser¹¹⁷⁹ phosphorylation. The decline of NO production correlated temporally to insulin-induced translocation of eNOS and CAV-1 to PM. Knockdown of CAV-1 expression with a specific small interfering RNA duplex resulted in eNOS redistributing to the perinuclear region and nearly doubled insulin-induced NO production. Inhibition of phosphatidylinositol 3-kinase activity with wortmannin not only significantly inhibited insulin-induced translocation of eNOS and CAV-1 to PM but also blocked insulin-induced interaction of CAV-1 with eNOS at PM. Insulin increased incorporation of [³H]palmitic acid into eNOS immunoprecipitates by approximately 140%. Insulin-induced translocation of eNOS and CAV-1 to PM was palmitoylation dependent. Inhibiting eNOS and CAV-1 palmitoylation enhanced the NO production while blocking the translocation of eNOS and CAV-1 to PM induced by insulin. These data show that insulin acutely regulates eNOS and CAV-1 trafficking to PM of vascular endothelial cells where their interaction can regulate eNOS activity.

Enhancing the Apoptotic Potential of Insulin-Like Growth Factor-Binding Protein-3 in Prostate Cancer by Modulation of CK2 Phosphorylation

Cobb, L. J., Mehta, H., Cohen, P. — Mon, 28 Sep 2009 10:02:16 PDT

IGF-binding protein 3 (IGFBP-3) promotes apoptosis by both IGF-dependent and -independent mechanisms. We have previously reported that phosphorylation of IGFBP-3 (S156) by DNA-dependent protein kinase enhances its nuclear accumulation and is essential for its ability to interact with retinoid X receptor- and induce apoptosis in cultured prostate cancer cells. Using specific chemical inhibitors and small interfering RNA, we demonstrate that preventing casein kinase 2 (CK2) activation enhanced the apoptotic potential of IGFBP-3. We mapped potential CK2 phosphosphorylation sites in IGFBP-3 to S167 and S175 and identified that wild-type IGFBP-3- and IGFBP-3-S175A-induced apoptosis to a comparable extent. In contrast, IGFBP-3-S167A was far more potently apoptosis inducing due to inability to undergo CK2 phosphorylation. Pretreatment of 22RV1 cells with IGFBP-3 small interfering RNA also limits the ability of high doses of CK2 inhibitor to induce apoptosis. These effects can be reversed by the addition of exogenous IGFBP-3 protein, suggesting reciprocal regulation of cell survival and apoptosis by IGFBP-3 and CK2. These studies reveal multisite phosphorylation of IGFBP-3 that both positively and negatively regulate its apoptotic potential. Understanding such intrinsic regulation of IGFBP-3 action may enhance the development of potential cancer therapies.

Rapid Estradiol/ER{alpha} Signaling Enhances Aromatase Enzymatic Activity in Breast Cancer Cells

Mon, 28 Sep 2009 10:02:16 PDT

In situ estrogen production by aromatase conversion from androgens plays an important role in breast tumor promotion. Here, we show that 17β-estradiol (E₂) can rapidly enhance aromatase enzymatic activity through an increase of aromatase protein phosphorylation in breast cancer cell lines. In vivo labeling experiments and site-directed mutagenesis studies demonstrated that phosphorylation of the 361-tyrosine residue is crucial in the up-regulation of aromatase activity under E₂ exposure. Our results demonstrated a direct involvement of nonreceptor tyrosine-kinase c-Src in E₂-stimulated aromatase activity because inhibition of its signaling abrogated the up-regulatory effects induced by E₂ on aromatase activity as well as phosphorylation of aromatase protein. In addition, from our data it emerges that aromatase is a target of cross talk between growth factor receptors and estrogen receptor signaling. These findings show, for the first time, that tyrosine phosphorylation processes play a key role in the rapid changes induced by E₂ in aromatase enzymatic activity, revealing the existence of a short nongenomic autocrine loop between E₂ and aromatase in breast cancer cells.

Epidermal Growth Factor-Induced GnRH-II Synthesis Contributes to Ovarian Cancer Cell Invasion

Poon, S. L., Hammond, G. T., Leung, P. C. K. — Mon, 28 Sep 2009 10:02:16 PDT

GnRH-II modulates ovarian cancer cells invasion and is expressed in normal ovary and ovarian epithelial cancer cells; however, the upstream regulator(s) of GnRH-II expression in these cells remains unclear. We now demonstrate that epidermal growth factor (EGF) increases GnRH-II mRNA levels in several human ovarian carcinoma cell lines and up-regulates GnRH-II promoter activity in OVCAR-3 cells in a dose-dependent manner, whereas an EGF receptor inhibitor (AG148) abolishes EGF-induced increases in GnRH-II promoter activity and GnRH-II mRNA levels. EGF increases the phosphorylation of cAMP-responsive element-binding protein (p-CREB) and its association with the coregulator, CCAAT/enhancer binding protein β, whereas blocking the EGF-induced ERK1/2 phosphorylation with MAPK inhibitors (PD98059/U0126) markedly reduced these effects. Moreover, depletion of CREB using small interfering RNA attenuated EGF-induced GnRH-II promoter activity. Chromatin immunoprecipitation assays demonstrated that EGF induces p-CREB binding to a cAMP responsive-element within the GnRH-II promoter, likely in association with CCAAT/enhancer binding protein β, and mutagenesis of this cAMP responsive-element prevented EGF-induced GnRH-II promoter activity in OVCAR-3 cells. Importantly, GnRH-II acts additively with EGF to promote invasion of OVCAR-3 and CaOV-3 cells, but not SKOV-3 cells that express low levels of GnRH receptor (GnRHR). Treatment with GnRHR small interfering RNA also partially inhibited the EGF-induced invasion of OVCAR-3 and CaOV-3 cells. Furthermore, EGF treatment transiently increases GnRHR levels in OVCAR-3 and CaOV-3, which likely accentuates the effects of increase GnRH-II production on cell invasion. These results provide evidence that EGF is an upstream regulator of the autocrine actions of GnRH-II on the invasive properties of ovarian cancer cells.

Transgenic Expression of Ad4BP/SF-1 in Fetal Adrenal Progenitor Cells Leads to Ectopic Adrenal Formation

Zubair, M., Oka, S., Parker, K. L., Morohashi, K.-i. — Mon, 28 Sep 2009 10:02:16 PDT

Deficiency of adrenal 4 binding protein/steroidogenic factor 1 (Ad4BP/SF-1; NR5A1) impairs adrenal development in a dose-dependent manner, whereas overexpression of Ad4BP/SF-1 is associated with adrenocortical tumorigenesis. Despite its essential roles in adrenal development, the mechanism(s) by which Ad4BP/SF-1 regulates this process remain incompletely understood. We previously identified a fetal adrenal enhancer (FAdE) that stimulates Ad4BP/SF-1 expression in the fetal adrenal gland by a two-step mechanism in which homeobox proteins initiate Ad4BP/SF-1 expression, which then maintains FAdE activity in an autoregulatory loop. In the present study, we examined the effect of transgenic expression of Ad4BP/SF-1 controlled by FAdE on adrenal development. When Ad4BP/SF-1 was overexpressed using a FAdE-Ad4BP/SF-1 transgene, FAdE activity expanded outside of its normal field, resulting in increased adrenal size and the formation of ectopic adrenal tissue in the thorax. The increased size of the adrenal gland did not result from a corresponding increase in cell proliferation, suggesting rather that the increased levels of Ad4BP/SF-1 may divert uncommitted precursors to the steroidogenic lineage. The effects of FAdE-controlled Ad4BP/SF-1 overexpression in mice provide a novel model of ectopic adrenal formation that further supports the critical role of Ad4BP/SF-1 in the determination of steroidogenic cell fate in vivo.

The p62 P392L Mutation Linked to Paget's Disease Induces Activation of Human Osteoclasts

Chamoux, E., Couture, J., Bisson, M., Morissette, J., Brown, J. P., Roux, S. — Mon, 28 Sep 2009 10:02:16 PDT

Mutations of the gene encoding p62/SQSTM1 have been described in Paget’s disease of bone (PDB), identifying p62 as an important player in osteoclast signaling. We investigated the phenotype of osteoclasts differentiated from peripheral blood monocytes obtained from healthy donors or PDB patients, all genotyped for the presence of a mutation in the p62 ubiquitin-associated domain. The cohort included PDB patients carrying or not the p62 P392L mutation and healthy donors carrying or not this mutation. Osteoclasts from PDB patients were more numerous, contained more nuclei, were more resistant to apoptosis, and had a greater ability to resorb bone than their normal counterparts, regardless of whether the p62 mutation was present or not. A strong increase in p62 expression was observed in PDB osteoclasts. The presence of the p62^P392L gene in cells from healthy carriers conferred a unique, intermediate osteoclast phenotype. In addition, we report that two survival-promoting kinases, protein kinase C and phosphoinositide-dependent protein kinase 1, were associated with p62 in response to receptor activator of NF-B ligand (RANKL) stimulation in controls and before RANKL was added in PDB osteoclasts. In transfected osteoclasts derived from cord blood monocytes, the p62 P392L mutation contributed to increased activation of kinases protein kinase C/ and phosphoinositide-dependent protein kinase 1, along with basal activation of NF-B, independently of RANKL stimulation. These findings clearly indicate that the overexpression of p62 in PDB patients induces important shifts in the pathways activated by RANKL and up-regulates osteoclast functions. Moreover, the most-commonly reported p62 mutation, P392L, certainly contributes to the overactive state of osteoclasts in PDB.

Endogenous Parathyroid Hormone-Related Protein Regulates the Expression of PTH Type 1 Receptor and Proliferation of Vascular Smooth Muscle Cells

Song, G. J., Fiaschi-Taesch, N., Bisello, A. — Mon, 28 Sep 2009 10:02:16 PDT

The PTH type 1 receptor (PTH1R) and PTHrP are expressed in vessels, where they contribute to regulating vascular smooth muscle cell (VSMC) function. Elevated PTHrP levels in VSMC are often associated with hyperplasia. In contrast, exogenous PTHrP, acting through the PTH1R, inhibits VSMC proliferation. In this study, we investigated the regulation of PTH1R expression by endogenous PTHrP and the associated effects on VSMC proliferation. Blocking binding of secreted PTHrP fragments to the PTH1R by treatment with either an antagonist or an antibody against PTHrP, and inhibition of PTHrP expression by small interfering RNA significantly increased PTH1R expression. Interestingly, treatment of the cells with a PTHrP analog (Bpa¹-PTHrP) that activates the PTH1R without inducing its internalization had the same effect on receptor expression. To examine the association between receptor expression and the antiproliferative effect of N-terminal fragments of PTHrP, VSMC were treated with exogenous PTHrP (1–36) acutely and chronically to induce receptor down-regulation. Stimulation of VSMC with exogenous PTHrP (1–36) significantly reduced cell proliferation during the first 18 h of treatment but was no longer effective after 3 d, a time when PTH1R was down-regulated. In contrast, treatment with the noninternalizing agonist Bpa¹-PTHrP strongly inhibited cell proliferation at all time points. In conclusion, our study show that PTHrP, after its intracellular processing and secretion, promotes down-regulation of the PTH1R in VSMC, thereby regulating cell proliferation in an auto/paracrine fashion. This regulatory mechanism may have important implication during vascular remodeling, in particular in the development of neointima after arterial injury, where PTHrP overexpression occurs.

The Parathyroid Hormone 1 Receptor Directly Binds to the FERM Domain of Ezrin, an Interaction that Supports Apical Receptor Localization and Signaling in LLC-PK1 Cells

Mahon, M. J. — Mon, 28 Sep 2009 10:02:16 PDT

PTH 1 receptor (PTH1R) regulates mineral ion homeostasis. Both apical and basolateral PTH1R subpopulations exist within the renal proximal tubule. The purpose of this research was to examine determinants within the PTH1R that direct apical localization. When expressed in LLC-PK1 cells, a proximal tubule cell model, the PTH1R localizes to both apical and basolateral membranes. The C terminus of the PTH1R contains a psd-95, discs large, ZO-1 domain interaction motif that binds the sodium-hydrogen exchanger regulatory factor 1 (NHERF-1), a renal tubule scaffold protein. Receptors lacking the psd-95, discs large, ZO-1 domain interaction motif (PTH1R-C4) partly localize to apical membranes, suggesting that additional factors may be involved. Ezrin, a membrane-cytoskeleton linking protein, directly binds NHERF-1 and thus links assembled complexes to actin. In vitro, subdomain C of the ezrin band 4.1, ezrin, radixin, domain interacts with the C-terminal tail of the PTH1R on a site that is mutually exclusive from the NHERF-1 interaction domain, suggesting the presence of a ternary complex. Mutating the lysine-arginine-lysine motif within the juxtamembrane region of the PTH1R C-terminal tail to alanines markedly disrupts interactions with the band 4.1, ezrin, radixin, domain of ezrin both in vitro and within cells. Inclusion of these mutations in the context of the full-length PTH1R disrupts apical localization with no effect on basolateral expression. Expression of a dominant-negative ezrin selectively disrupts apical expression and signaling of the PTH1R. However, dominant-negative ezrin does not affect expression or signaling of the basolateral PTH1R subpopulation. These findings reveal that direct ezrin interactions promote PTH1R apical localization and signaling in LLC-PK1 cells.

The Transcriptional Response of the Islet to Pregnancy in Mice

Mon, 28 Sep 2009 10:02:16 PDT

The inability of the ß-cell to meet the demand for insulin brought about by insulin resistance leads to type 2 diabetes. In adults, ß-cell replication is one of the mechanisms thought to cause the expansion of ß-cell mass. Efforts to treat diabetes require knowledge of the pathways that drive facultative ß-cell proliferation in vivo. A robust physiological stimulus of ß-cell expansion is pregnancy and identifying the mechanisms underlying this stimulus may provide therapeutic leads for the treatment of type 2 diabetes. The peak in ß-cell proliferation during pregnancy occurs on d 14.5 of gestation in mice. Using advanced genomic approaches, we globally characterize the gene expression signature of pancreatic islets on d 14.5 of gestation during pregnancy. We identify a total of 1907 genes as differentially expressed in the islet during pregnancy. The islet’s ability to compensate for relative insulin deficiency during metabolic stress is associated with the induction of both proliferative and survival pathways. A comparison of the genes induced in three different models of islet expansion suggests that diverse mechanisms can be recruited to expand islet mass. The identification of many novel genes involved in islet expansion during pregnancy provides an important resource for diabetes researchers to further investigate how these factors contribute to the maintenance of not only islet mass, but ultimately ß-cell mass.

Dual Inhibition of Mitogen-Activated Protein Kinase and Mammalian Target of Rapamycin in Differentiated and Anaplastic Thyroid Cancer

Mon, 28 Sep 2009 10:02:16 PDT

8-Cl-Adenosine Inhibits Proliferation and Causes Apoptosis in B-Lymphocytes via Protein Kinase A-Dependent and Independent Effects: Implications for Treatment of Carney Complex-Associated Tumors

Mon, 28 Sep 2009 10:02:16 PDT

Differential Ligand-Mediated Pituitary Somatostatin Receptor Subtype Signaling: Implications for Corticotroph Tumor Therapy

Mon, 28 Sep 2009 10:02:16 PDT

The Glucocorticoid Receptor Is Overexpressed in Malignant Adrenocortical Tumors

Mon, 28 Sep 2009 10:02:16 PDT

Peptide YY (PYY) Gene Polymorphisms in the 3'-Untranslated Region and Proximal Promoter Regions Regulate Cellular Gene Expression and PYY Secretion and Metabolic Syndrome Traits in Vivo

Mon, 28 Sep 2009 10:02:16 PDT

RAS Mutations Are the Predominant Molecular Alteration in Poorly Differentiated Thyroid Carcinomas and Bear Prognostic Impact

Mon, 28 Sep 2009 10:02:16 PDT

Minireview: Role of Protein Methylation and Demethylation in Nuclear Hormone Signaling

Wu, S. C., Zhang, Y. — Thu, 27 Aug 2009 10:02:05 PDT

Nuclear hormone receptors (NRs) are transcription factors responsible for mediating the biological effects of hormones during development, metabolism, and homeostasis. Induction of NR target genes is accomplished through the assembly of hormone-bound NR complexes at target promoters and coincides with changes in histone modifications that promote transcription. Some coactivators and corepressors of NR can enhance or inhibit NR function by covalently modifying histones. One such modification is methylation, which plays important roles in transcriptional regulation. Histone methylation is catalyzed by histone methyltransferases and reversed by histone demethylases. Recent studies have uncovered the importance of these enzymes in the regulation of NR target genes. In addition to histones, these enzymes have nonhistone substrates and can methylate and demethylate NRs and coregulatory proteins in order to modulate their function. This review discusses recent progress in our understanding of the role of methylation and demethylation of histones, NRs, and their coregulators in NR-mediated transcription.

Resistance to Antiestrogen Arzoxifene Is Mediated by Overexpression of Cyclin D1

Thu, 27 Aug 2009 10:02:05 PDT

Resistance to tamoxifen treatment occurs in approximately 50% of the estrogen receptor (ER)-positive breast cancer patients. Resistant patients would benefit from treatment with other available antiestrogens. Arzoxifene is an effective growth inhibitor of ER-positive breast cancer cells, including tamoxifen-resistant tumors. In this study, we show that overexpression of a regular component of the ER transcription factor complex, cyclin D1, which occurs in approximately 40% of breast cancer patients, renders cells resistant to the new promising antiestrogen, arzoxifene. Overexpression of cyclin D1 alters the conformation of ER in the presence of arzoxifene. In this altered conformation, ER still recruits RNA polymerase II to an estrogen response element-containing promoter, inducing transcription of an ER-dependent reporter gene and of endogenous pS2, and promoting arzoxifene-stimulated growth of MCF-7 cells. Arzoxifene is then converted from an ER antagonist into an agonist. This can be explained by a stabilization of the ER/steroid receptor coactivator-1 complex in the presence of arzoxifene, only when cyclin D1 is overexpressed. These results indicate that subtle changes in the conformation of ER upon binding to antiestrogen are at the basis of resistance to antiestrogens.

Apurinic/Apyrimidinic Endonuclease 1 Alters Estrogen Receptor Activity and Estrogen-Responsive Gene Expression

Thu, 27 Aug 2009 10:02:05 PDT

Apurinic/apyrimidinic endonuclease 1 or redox factor-1 (Ape1/Ref-1) is a pleiotropic cellular protein involved in DNA repair and, through its redox activity, enhances the binding of a select group of transcription factors to their cognate recognition sequences in DNA. Thus, we were intrigued when we identified Ape1/Ref-1 and a number of DNA repair and oxidative stress proteins in a complex associated with the DNA-bound estrogen receptor (ER). Because Ape1/Ref-1 interacts with a number of transcription factors and influences their activity, we determined whether it might also influence ER activity. We found that endogenously expressed Ape1/Ref-1 and ER from MCF-7 human breast cancer cells interact and that Ape1/Ref-1 enhances the interaction of ER with estrogen-response elements (EREs) in DNA. More importantly, Ape1/Ref-1 alters expression of the endogenous, estrogen-responsive progesterone receptor and pS2 genes in MCF-7 cells and associates with ERE-containing regions of these genes in native chromatin. Interestingly, knocking down Ape1/Ref-1 expression or inhibiting its redox activity with the small molecule inhibitor E3330 enhances estrogen responsiveness of the progesterone receptor and pS2 genes but does not alter the expression of the constitutively active 36B4 gene. Additionally, the reduced form of Ape1/Ref-1 increases and E3330 limits ER-ERE complex formation in vitro and in native chromatin. Our studies demonstrate that Ape1/Ref-1 mediates its gene-specific effects, in part, by associating with endogenous, estrogen-responsive genes and that the redox activity of Ape1/Ref-1 is instrumental in altering estrogen-responsive gene expression.

Structural and Functional Characterization of the Interdomain Interaction in the Mineralocorticoid Receptor

Pippal, J. B., Yao, Y., Rogerson, F. M., Fuller, P. J. — Thu, 27 Aug 2009 10:02:05 PDT

The mineralocorticoid receptor (MR) plays a central role in electrolyte homeostasis and in cardiovascular disease. We have previously reported a ligand-dependent N/C-interaction in the MR. In the present study we sought to fully characterize the MR N/C-interaction. By using a range of natural and synthetic MR ligands in a mammalian two-hybrid assay we demonstrate that in contrast to aldosterone, which strongly induces the interaction, the physiological ligands deoxycorticosterone and cortisol weakly promote the interaction but predominantly inhibit the aldosterone-mediated N/C-interaction. Similarly, progesterone and dexamethasone antagonize the interaction. In contrast, the synthetic agonist 9-fludrocortisol robustly induces the interaction. The ability of the N/C interaction to discriminate between MR agonists suggests a subtle conformational difference in the ligand-binding domain induced by these agonists. We also demonstrate that the N/C interaction is not cell specific, consistent with the evidence from a glutathione-S-transferase pull-down assay, of a direct protein-protein interaction between the N- and C-terminal domains of the MR. Examination of a panel of deletions in the N terminus suggests that several regions may be critical to the N/C-interaction. These studies have identified functional differences between physiological MR ligands, which suggest that the ligand-specific dependence of the N/C-interaction may contribute to the differential activation of the MR that has been reported in vivo.

The 26-Amino Acid ss-Motif of the Pit-1ss Transcription Factor Is a Dominant and Independent Repressor Domain

Jonsen, M. D., Duval, D. L., Gutierrez-Hartmann, A. — Thu, 27 Aug 2009 10:02:05 PDT

The POU-homeodomain transcription factor Pit-1 governs the pituitary cell-specific expression of Pit-1, GH, prolactin (PRL), and TSHß genes. Alternative splicing generates Pit-1ß, which contains a 26-amino acid ß-domain inserted at amino acid 48, in the middle of the Pit-1 transcription activation domain (TAD). Pit-1ß represses GH, PRL, and TSHß promoters in a pituitary-specific manner, because Pit-1ß activates these same promoters in HeLa nonpituitary cells. Here we comprehensively analyze the role of ß-domain sequence, position, and context, to elucidate the mechanism of ß-dependent repression. Repositioning the ß-motif to the Pit-1 amino terminus, hinge, linker, and carboxyl terminus did not affect its ability to repress basal rat (r) PRL promoter activity in GH4 pituitary cells, but all lost the ability to repress Ras-induced rPRL promoter activity. To determine whether ß-domain repression is independent of Pit-1 protein and DNA binding sites, we generated Gal4-Pit-1TAD, Gal4-Pit-1ßTAD, and Gal4-ß-domain fusions and demonstrated that the ß-motif is sufficient to actively repress VP16-mediated transcription of a heterologous promoter. Moreover, ß-domain point mutants had the same effect whether fused to Gal4 or within the context of intact Pit-1ß. Surprisingly, Gal4-ß repression lost histone deacetylase sensitivity and pituitary specificity. Taken together, these results reveal that the ß-motif is a context-independent, modular, transferable, and dominant repressor domain, yet the ß-domain repressor activity within Pit-1ß contains cell type, promoter, and Pit-1 protein context dependence.

Induction of Kruppel-Like Factor 5 Expression by Androgens Results in Increased CXCR4-Dependent Migration of Prostate Cancer Cells in Vitro

Thu, 27 Aug 2009 10:02:05 PDT

Advanced prostate cancers preferentially metastasize to bone, suggesting that this tissue produces factors that provide a suitable microenvironment for prostate cancer cells. Recently, it has become clear that even in antiandrogen-resistant cancers, the androgen receptor (AR)-signaling axis is required for prostate cancer progression. Therefore, we hypothesized that AR may be involved in the regulation of pathways that are responsible for the homing of prostate cancer cells to select microenvironments. In support of this hypothesis, we have determined that chemokine (C-X-C motif) receptor 4 (CXCR4), the receptor for the chemokine CXCL12, is up-regulated in prostate cancer cells in response to androgens. Given that the levels of CXCL12 are elevated at sites of known prostate cancer metastases such as bone, these results suggest that androgens may influence prostate cancer metastasis. Specifically, we demonstrate that androgens increase the levels of both CXCR4 mRNA and functional protein in LNCaP prostate cancer cells. Importantly, androgens enhanced the migration of LNCaP cells toward a CXCL12 gradient, an effect that could be blocked by the specific CXCR4 antagonist AMD3100. Interestingly, CXCR4 is not directly regulated by androgens but rather is positively up-regulated by Krüppel-like factor 5 (KLF5), a transcription factor that we have shown to be an early, direct target of AR. Further, KLF5 is both required and sufficient for androgen-mediated CXCR4 expression and migration toward CXCL12. Taken together, these findings demonstrate that AR can utilize the CXCL12/CXCR4 axis through induction of KLF5 expression to promote prostate cancer progression and highlight the potential utility of CXCR4 antagonists as prostate cancer therapeutics.

Histone-Acetylated Control of Fibroblast Growth Factor Receptor 2 Intron 2 Polymorphisms and Isoform Splicing in Breast Cancer

Zhu, X., Asa, S. L., Ezzat, S. — Thu, 27 Aug 2009 10:02:05 PDT

Recent genome-wide association studies have identified fibroblast growth factor receptor (FGFR)2 as one of a few candidate genes linked with breast cancer susceptibility. In particular, the disease-predisposing allele of FGFR2 is inherited as a 7.5-kb region within intron 2 that harbors eight single nucleotide polymorphisms. The relationship between these single nucleotide polymorphisms and FGFR2 gene expression remains unclear. Here we show the common occurrence of polymorphisms within the intron 2 region in a panel of 10 breast cancer cell lines. High FGFR2-expressing cell lines such as MCF-7 cells displayed polymorphic sequences with constitutive histone acetylation at multiple intron 2 sequences harboring putative transcription binding sites. Knockdown of Runx2 or CCAAT enhancer binding protein β in these cells resulted in diminished endogenous FGFR2 gene expression. In contrast FGFR2-negative MDA-231 cells were wild type and showed evidence of histone 3/4 deacetylation at the rs2981578, rs10736303, and rs7895676 disease-associated alleles that harbor binding sites for Runx2, estrogen receptor, and CCAAT enhancer binding protein β, respectively. Histone deacetylation inhibition with trichostatin A resulted in enhanced acetylation at these intron 2 sites, an effect associated with robust FGFR2 reexpression. Isoform analysis proved reexpression of the FGFR2-IIIc variant the splicing of which was positively influenced by trichostatin A-mediated recruitment of the Fas-activated serine/threonine phosphoprotein survival protein. Our findings highlight the potential role of histone acetylation in modulating access to selected polymorphic sites within intron 2 as well as downstream splicing sites in generating variable FGFR2 levels and isoforms in breast cancer.

Dual Ablation of Grb10 and Grb14 in Mice Reveals Their Combined Role in Regulation of Insulin Signaling and Glucose Homeostasis

Thu, 27 Aug 2009 10:02:05 PDT

Growth factor receptor bound (Grb)10 and Grb14 are closely related adaptor proteins that bind directly to the insulin receptor (IR) and regulate insulin-induced IR tyrosine phosphorylation and signaling to IRS-1 and Akt. Grb10- and Grb14-deficient mice both exhibit improved whole-body glucose homeostasis as a consequence of enhanced insulin signaling and, in the case of the former, altered body composition. However, the combined physiological role of these adaptors has remained undefined. In this study we utilize compound gene knockout mice to demonstrate that although deficiency in one adaptor can enhance insulin-induced IRS-1 phosphorylation and Akt activation, insulin signaling is not increased further upon dual ablation of Grb10 and Grb14. Context-dependent limiting mechanisms appear to include IR hypophosphorylation and decreased IRS-1 expression. In addition, the compound knockouts exhibit an increase in lean mass comparable to Grb10-deficient mice, indicating that this reflects a regulatory function specific to Grb10. However, despite the absence of additive effects on insulin signaling and body composition, the double-knockout mice are protected from the impaired glucose tolerance that results from high-fat feeding, whereas protection is not observed with animals deficient for individual adaptors. These results indicate that, in addition to their described effects on IRS-1/Akt, Grb10 and Grb14 may regulate whole-body glucose homeostasis by additional mechanisms and highlight these adaptors as potential therapeutic targets for amelioration of the insulin resistance associated with type 2 diabetes.

Characterization of Anandamide-Stimulated Cannabinoid Receptor Signaling in Human ULTR Myometrial Smooth Muscle Cells

Thu, 27 Aug 2009 10:02:05 PDT

Accumulating evidence highlights the importance of the endocannabinoid anandamide (AEA) as a key mediator in reproductive physiology. Current data suggest potential roles for AEA in gametogenesis, fertilization, and parturition. AEA exerts its actions through two G protein-coupled receptors, termed cannabinoid receptor 1 (CB1), and 2 (CB2), and the ligand-gated transient receptor potential vanilloid receptor type 1 (TRPV1) ion channel. At present, the cellular mechanism(s) and consequences of AEA signaling in reproductive tissues, especially the myometrium, are poorly understood. Here, we examine the expression of CB1, CB2, and TRPV1 in the human myometrial smooth muscle cell-line (ULTR) and characterize intracellular signaling after stimulation with AEA. Radioligand binding analysis revealed a total CB receptor expression of 76 ± 24 fmol/mg protein, with both quantitative PCR and competition binding studies indicating a negligible CB2 component. AEA caused G_i/o-dependent inhibition of adenylate cyclase to reduce intracellular cAMP levels. In addition, AEA caused a 2.5- to 3.5-fold increase in ERK activation, which was ablated by inhibition of G_i/o, phosphoinositide-3-kinase and Src-kinase activities, but not by inhibition of Ca²⁺/calmodulin-dependent protein kinase or protein kinase C activities. TRPV1 channel activation with capsaicin failed to activate ERK. Consistent with these findings, the selective agonists, arachidonyl-2-chloroethylamide (CB1) and L759656 (CB2), and selective antagonists AM251 (CB1) and JTE907 (CB2), provided pharmacological evidence that the ERK signaling pathway is activated through endogenously expressed CB1. These findings provide an insight into myometrial AEA signaling, highlighting a potential role for endocannabinoids in the regulation of gene expression in myometrial smooth muscle cells.

Compartmentalizing VEGF-Induced ERK2/1 Signaling in Placental Artery Endothelial Cell Caveolae: A Paradoxical Role of Caveolin-1 in Placental Angiogenesis in Vitro

Liao, W.-x., Feng, L., Zhang, H., Zheng, J., Moore, T. R., Chen, D.-b. — Thu, 27 Aug 2009 10:02:06 PDT

On vascular endothelial growth factor (VEGF) stimulation, both VEGF R1 and R2 receptors were phosphorylated in ovine fetoplacental artery endothelial (oFPAE) cells. Treatment with VEGF stimulated both time- and dose-dependent activation of ERK2/1 in oFPAE cells. VEGF-induced ERK2/1 activation was mediated by VEGFR2, but not VEGFR1, and was linked to intracellular calcium, protein kinase C, and Raf-1. VEGF stimulated oFPAE cell proliferation, migration, and tube formation in vitro. Blockade of ERK2/1 pathway attenuated VEGF-induced cell proliferation and tube formation but failed to inhibit migration in oFPAE cells. Disruption of caveolae by cholesterol depletion with methyl-β-cyclodextrin or by down-regulation of its structural protein caveolin-1 blunted VEGF-induced ERK2/1 activation, proliferation, and tube formation in oFPAE cells, indicating an essential role of integral caveolae in these VEGF-induced responses. Adenoviral overexpression of caveolin-1 and addition of a caveolin scaffolding domain peptide also inhibited VEGF-stimulated ERK2/1 activation, cell proliferation, and tube formation in oFPAE cells. Furthermore, molecules comprising the ERK2/1 signaling module, including VEGFR2, protein kinase C, Raf-1, MAPK kinase 1/2, and ERK2/1, resided with caveolin-1 in caveolae. VEGF transiently stimulated ERK2/1 activation in the caveolae similarly as in intact cells. Caveolae disruption greatly diminished ERK2/1 activation by VEGF in oFPAE cell caveolae. We conclude that caveolae function as a platform for compartmentalizing the VEGF-induced ERK2/1 signaling module. Caveolin-1 and caveolae play a paradoxical role in regulating VEGF-induced ERK2/1 activation and in vitro angiogenesis as evidenced by the similar inhibitory effects of down-regulation and overexpression of caveolin-1 and disruption of caveolae in oFPAE cells.

FGF-2 Stimulation of RANK Ligand Expression in Paget's Disease of Bone

Sundaram, K., Senn, J., Yuvaraj, S., Rao, D. S., Reddy, S. V. — Thu, 27 Aug 2009 10:02:06 PDT

Receptor activator for nuclear factor-B ligand (RANKL), a critical osteoclastogenic factor expressed in marrow stromal/preosteoblast cells is up-regulated in Paget’s disease of bone (PDB). We previously demonstrated that heat-shock factor-2 (HSF-2) is a downstream target of fibroblast growth factor-2 (FGF-2) signaling to induce RANKL expression in bone marrow stromal/preosteoblast cells. In this study, we identified a 2.5-fold increase in serum FGF-2 levels in patients (n = 8) with PDB compared with normal subjects (n = 10). We showed that HSF-2 co-immunoprecipitates with heat-shock protein-27 (HSP-27) and that FGF-2 stimulation significantly increased phospho-HSP-27 levels in marrow stromal cells. Confocal microscopy revealed HSF-2 colocalization with HSP-27 in unstimulated cells and HSF-2 nuclear translocation upon FGF-2 stimulation. We further show that FGF-2 stimulation significantly increased the levels of phosphorylated signal transducers and activators of the transcription (p-STAT-1) in these cells. Western blot analysis confirmed that small interfering RNA suppression of STAT-1 significantly decreased (3.2-fold) RANKL expression and promoter activity in FGF-2-stimulated cells. Chromatin immunoprecipitation assay revealed STAT-1 binding to a putative motif located far upstream (–8 kb) in the hRANKL gene promoter region. These results suggest STAT-1 is a downstream effector of FGF-2 signaling and that elevated levels of FGF-2 stimulates RANKL expression in PDB.

Inhibition of WNT Signaling by G Protein-Coupled Receptor (GPCR) Kinase 2 (GRK2)

Wang, L., Gesty-Palmer, D., Fields, T. A., Spurney, R. F. — Thu, 27 Aug 2009 10:02:06 PDT

Activation of Wnt signaling pathways causes release and stabilization of the transcription regulator β-catenin from a destruction complex composed of axin and the adenomatous polyposis coli (APC) protein (canonical signaling pathway). Assembly of this complex is facilitated by a protein-protein interaction between APC and a regulator of G protein signaling (RGS) domain in axin. Because G protein-coupled receptor kinase 2 (GRK2) has a RGS domain that is closely related to the RGS domain in axin, we determined whether GRK2 regulated canonical signaling. We found that GRK2 inhibited Wnt1-induced activation of a reporter construct as well as reduced Wnt3a-dependent stabilization and nuclear translocation of β-catenin. GRK2 enzymatic activity was required for this negative regulatory effect, and depletion of endogenous GRK2 using small interfering RNA enhanced canonical signaling. GRK2-dependent inhibition of canonical signaling is relevant to osteoblast (OB) biology because overexpression of GRK2 attenuated Wnt/β-catenin signaling in calvarial OBs. Coimmunoprecipitation studies found that: 1) GRK2 bound APC; 2) The GRK2-APC interaction was promoted by GRK2 enzymatic activity; and 3) Deletion of the RGS domain in GRK2 prevented both the GRK2-APC interaction and GRK2-dependent inhibition of canonical signaling. These data suggest that: 1) GRK2 negatively regulates Wnt signaling; 2) GRK2-dependent inhibition of canonical signaling requires a protein-protein interaction between the RGS domain in GRK2 and APC; and 3) Enzymatic activity promotes the GRK2-APC interaction and is required for the negative regulatory effect on canonical signaling. We speculate that inhibiting GRK2 activity in bone-forming OBs might be a useful therapeutic strategy for increasing bone mass.

Subcellular Trafficking of the TRH Receptor: Effect of Phosphorylation

Jones, B. W., Hinkle, P. M. — Thu, 27 Aug 2009 10:02:06 PDT

Activation of the G protein-coupled TRH receptor leads to its phosphorylation and internalization. These studies addressed the fundamental question of whether phosphorylation regulates receptor trafficking or endosomal localization regulates the phosphorylation state of the receptor. Trafficking of phosphorylated and dephosphorylated TRH receptors was characterized using phosphosite-specific antibody after labeling surface receptors with antibody to an extracellular epitope tag. Rab5 and phosphoreceptor did not colocalize at the plasma membrane immediately after TRH addition but overlapped extensively by 15 min. Dominant-negative Rab5-S34N inhibited receptor internalization. Later, phosphoreceptor was in endosomes containing Rab5 and Rab4. Dephosphorylated receptor colocalized with Rab4 but not with Rab5. Dominant-negative Rab4, -5, or -11 did not affect receptor phosphorylation or dephosphorylation, showing that phosphorylation determines localization in Rab4⁺/Rab5^– vesicles and not vice versa. No receptor colocalized with Rab7; a small amount of phosphoreceptor colocalized with Rab11. To characterize recycling, surface receptors were tagged with antibody, or surface receptors containing an N-terminal biotin ligase acceptor sequence were labeled with biotin. Most recycling receptors did not return to the plasma membrane for more than 2 h after TRH was removed, whereas the total cell surface receptor density was largely restored in less than 1 h, indicating that recruited receptors contribute heavily to early repopulation of the plasma membrane.

Increased Connexin 43 Expression as a Potential Mediator of the Neuroprotective Activity of the Corticotropin-Releasing Hormone

Hanstein, R., Trotter, J., Behl, C., Clement, A. B. — Thu, 27 Aug 2009 10:02:06 PDT

CRH is a major central stress mediator, but also a potent neuroprotective effector. The mechanisms by which CRH mediates its neuroprotective actions are largely unknown. Here, we describe that the gap junction molecule connexin43 (Cx43) mediates neuroprotective effects of CRH toward experimentally induced oxidative stress. An enhanced gap junction communication has been reported to contribute to neuroprotection after neurotoxic insults. We show that CRH treatment up-regulates Cx43 expression and gap junctional communication in a CRH receptor-dependent manner in IMR32 neuroblastoma cells, primary astrocytes, and organotypic hippocampal slice cultures. MAPKs and protein kinase A-cAMP response element binding protein -coupled pathways are involved in the signaling cascade from CRH to enhanced Cx43 function. Inhibition of CRH-promoted gap junction communication by the gap junction inhibitor carbenoxolone could prevent neuroprotective actions of CRH in cell and tissue culture models suggesting that gap junction molecules are involved in the neuroprotective effects of CRH. The extent of oxidative stress-induced protein carbonylation and cell death inversely correlated with Cx43 protein levels as shown by Cx43 small interfering RNA knockdown experiments. Coculture studies of primary neurons and astrocytes revealed that astrocytic Cx43 likely contributes to the neuroprotective effects of CRH. To our knowledge this is the first description of Cx43 as a potential mediator of the neuroprotective actions of CRH.

Thyroid Hormone (T3)-Induced Up-Regulation of Voltage-Activated Sodium Current in Cultured Postnatal Hippocampal Neurons Requires Secretion of Soluble Factors from Glial Cells

Niederkinkhaus, V., Marx, R., Hoffmann, G., Dietzel, I. D. — Thu, 27 Aug 2009 10:02:06 PDT

We have previously shown that treatment with the thyroid hormone T₃ increases the voltage-gated Na⁺current density (Nav-D) in hippocampal neurons from postnatal rats, leading to accelerated action potential upstrokes and increased firing frequencies. Here we show that the Na⁺ current regulation depends on the presence of glial cells, which secrete a heat-instable soluble factor upon stimulation with T₃. The effect of conditioned medium from T₃-treated glial cells was mimicked by basic fibroblast growth factor (bFGF), known to be released from cerebellar glial cells after T₃ treatment. Neutralization assays of astrocyte-conditioned media with anti-bFGF antibody inhibited the regulation of the Nav-D by T₃. This suggests that the up-regulation of the neuronal sodium current density by T₃ is not a direct effect but involves bFGF release and satellite cells. Thus glial cells can modulate neuronal excitability via secretion of paracrinely acting factors.

Novel Regulators of Fgf23 Expression and Mineralization in Hyp Bone

Liu, S., Tang, W., Fang, J., Ren, J., Li, H., Xiao, Z., Quarles, L. D. — Thu, 27 Aug 2009 10:02:06 PDT

We used gene array analysis of cortical bone to identify Phex-dependent gene transcripts associated with abnormal Fgf23 production and mineralization in Hyp mice. We found evidence that elevation of Fgf23 expression in osteocytes is associated with increments in Fgf1, Fgf7, and Egr2 and decrements in Sost, an inhibitor in the Wnt-signaling pathway, were observed in Hyp bone. β-Catenin levels were increased in Hyp cortical bone, and TOPflash luciferase reporter assay showed increased transcriptional activity in Hyp-derived osteoblasts, consistent with Wnt activation. Moreover, activation of Fgf and Wnt-signaling stimulated Fgf23 promoter activity in osteoblasts. We also observed reductions in Bmp1, a metalloproteinase that metabolizes the extracellular matrix protein Dmp1. Alterations were also found in enzymes regulating the posttranslational processing and stability of Fgf23, including decrements in the glycosyltransferase Galnt3 and the proprotein convertase Pcsk5. In addition, we found that the Pcsk5 and the glycosyltransferase Galnt3 were decreased in Hyp bone, suggesting that reduced posttranslational processing of FGF23 may also contribute to increased Fgf23 levels in Hyp mice. With regard to mineralization, we identified additional candidates to explain the intrinsic mineralization defect in Hyp osteoblasts, including increases in the mineralization inhibitors Mgp and Thbs4, as well as increases in local pH-altering factors, carbonic anhydrase 12 (Car12) and 3 (Car3) and the sodium-dependent citrate transporter (Slc13a5). These studies demonstrate the complexity of gene expression alterations in bone that accompanies inactivating Phex mutations and identify novel pathways that may coordinate Fgf23 expression and mineralization of extracellular matrix in Hyp bone.

Uncoupling Protein-1 and Related Messenger Ribonucleic Acids in Human Epicardial and Other Adipose Tissues: Epicardial Fat Functioning as Brown Fat

Thu, 27 Aug 2009 10:02:06 PDT

c-Jun N-Terminal Kinase 1/2 Activation by Tumor Necrosis Factor-{alpha} Induces Insulin Resistance In Human Visceral But Not Subcutaneous Adipocytes: Reversal by Liver X Receptor Agonists

Fernandez-Veledo, S., Vila-Bedmar, R., Nieto-Vazquez, I., Lorenzo, M. — Thu, 27 Aug 2009 10:02:06 PDT

Prostaglandin E2 as a Regulator of Germ Cells during Ovarian Development

Thu, 27 Aug 2009 10:02:06 PDT

Estimated Glucose Disposal Rate in Assessment of the Metabolic Syndrome and Microvascular Complications in Patients with Type 1 Diabetes

Thu, 27 Aug 2009 10:02:06 PDT

Adipocyte Differentiation-Related Protein and OXPAT in Rat and Human Skeletal Muscle: Involvement in Lipid Accumulation and Type 2 Diabetes Mellitus

Thu, 27 Aug 2009 10:02:06 PDT

Corrigenda

Thu, 27 Aug 2009 10:02:06 PDT