Abnormal Paraventricular Nucleus of Hypothalamus and Growth Retardation Associated with Loss of Nuclear Receptor Gene COUP-TFII.
ABSTRACT: The paraventricular nucleus of hypothalamus plays important roles in the regulation of energy balance and fetal growth. However, the molecular mechanisms underlying its formation and function have not been clearly elucidated. Various mutations in the human COUP-TFII gene, which encodes a nuclear receptor, result in growth retardation, congenital diaphragmatic hernia and congenital heart defects. Here, we show that COUP-TFII gene is expressed in the developing hypothalamus in mouse. The ventral forebrain-specific RXCre/+; COUP-TFII F/F mutant mice display growth retardation. The development of the paraventricular nucleus of hypothalamus is compromised in the COUP-TFII mutant mainly because of increased apoptosis and mis-migration of the Brn2+ neurons. Moreover, hypoplastic anterior pituitary with blood cell clusters and shrunken posterior pituitary lacking AVP/OT neuron innervations are observed in the mutant, indicating the failure of formation of the hypothalamic-pituitary axis. Mechanistic studies show that the expression of Bdnf and Nrp1 genes is reduced in the mutant embryo, and that Bdnf is a direct downstream target of the COUP-TFII protein. Thus, our findings provide a novel functional validation that COUP-TFII gene promotes the expression of Bdnf and Nrp1 genes to ensure the appropriate morphogenesis of the hypothalamic-pituitary axis, especially the paraventricular nucleus of hypothalamus, and to prevent growth retardation.
Project description:The nuclear receptor Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) is an important coordinator of glucose homeostasis through its function in different organs such as the endocrine pancreas, adipose tissue, skeletal muscle, and liver. Recently we have demonstrated that COUP-TFII expression in the hypothalamus is restricted to a subpopulation of neurons expressing the steroidogenic factor 1 transcription factor, known to play a crucial role in glucose homeostasis. To understand the functional significance of COUP-TFII expression in the steroidogenic factor 1 neurons, we generated hypothalamic ventromedial nucleus-specific COUP-TFII KO mice using the cyclization recombination/locus of X-overP1 technology. The heterozygous mutant mice display insulin hypersensitivity and a leaner phenotype associated with increased energy expenditure and similar food intake. These mutant mice also present a defective counterregulation to hypoglycemia with altered glucagon secretion. Moreover, the mutant mice are more likely to develop hypoglycemia-associated autonomic failure in response to recurrent hypoglycemic or glucopenic events. Therefore, COUP-TFII expression levels in the ventromedial nucleus are keys in the ability to resist the onset of hypoglycemia-associated autonomic failure.
Project description:COUP-TFII (also known as Nr2f2), a member of the nuclear orphan receptor superfamily, is expressed in several regions of the central nervous system (CNS), including the ventral thalamus, hypothalamus, midbrain, pons, and spinal cord. To address the function of COUP-TFII in the CNS, we generated conditional COUP-TFII knockout mice using a tissue-specific NSE-Cre recombinase. Ablation of COUP-TFII in the brain resulted in malformation of the lobule VI in the cerebellum and a decrease in differentiation of cerebellar neurons and cerebellar growth. The decrease in cerebellar growth in NSE(Cre/+)/CII(F/F) mice is due to reduced proliferation and increased apoptosis in granule cell precursors (GCPs). Additional studies demonstrated that insulin like growth factor 1 (IGF-1) expression was reduced in the cerebellum of NSE(Cre/+)/CII(F/F) mice, thereby leading to decreased Akt1 and GSK-3beta activities, and the reduced expression of mTOR. Using ChIP assays, we demonstrated that COUP-TFII was recruited to the promoter region of IGF-1 in a Sp1-dependent manner. In addition, dendritic branching of Purkinje cells was decreased in the mutant mice. Thus, our results indicate that COUP-TFII regulates growth and maturation of the mouse postnatal cerebellum through modulation of IGF-1 expression.
Project description:The development of the progenitor zones in the pallium, lateral ganglionic eminence (LGE) and medial ganglionic eminence (MGE) in the subpallium has been well studied; however, so far the role of the caudal ganglionic eminence (CGE), a posterior subpallial domain, in telencephalon patterning remains poorly understood. COUP-TFII, an orphan nuclear receptor, is preferentially expressed in the CGE. We generated COUP-TFII mouse mutants, using Rx-Cre (RxCre;COUP-TFII(F/F)), to study its function in telencephalon development. In these mutants, we found severe defects in the formation of the amygdala complex, including the lateral (LA), basolateral (BLA) and basomedial (BMA) amygdala nuclei. Molecular analysis provided evidence that the migration of CGE-derived Pax6(+) cells failed to settle into the BMA nucleus, owing to reduced expression of neuropilin 1 (Nrp1) and Nrp2, two semaphorin receptors that regulate neuronal cell migration and axon guidance. Our ChIP assays revealed that Nrp1 and Nrp2 genes are the direct targets of COUP-TFII in the telencephalon in vivo. Furthermore, our results showed that the coordinated development between the CGE originated subpallial population (Pax6(+) cells) and pallial populations (Tbr1(+) and Lhx2(+) cells) was essential for patterning the amygdala assembly. Our study presented novel genetic evidence that the caudal ganglionic eminence, a distinct subpallial progenitor zone, contributes cells to the basal telencephalon, such as the BMA nucleus.
Project description:This study aimed to investigate the possible involvement of the orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) in the regulation of renin gene expression. COUP-TFII colocalized with renin in the juxtaglomerular cells of the kidney, which are the main source of renin in vivo. Protein-DNA binding studies demonstrated that COUP-TFII binds to an imperfect direct repeat COUP-TFII recognition sequence (termed hereafter proxDR) in the proximal renin promoter. Because cAMP signaling plays a central role in the control of the renin gene expression, we suggested that COUP-TFII may modulate this cAMP effect. Accordingly, knockdown of COUP-TFII in the clonal renin-producing cell lines As4.1 and Calu-6 diminished the stimulation of the renin mRNA expression by cAMP agonists. In addition, the mutation of the proxDR element in renin promoter reporter gene constructs abrogated the inducibility by cAMP. The proxDR sequence was found to be necessary for the function of a proximal renin promoter cAMP-response element (CRE). Knockdown of COUP-TFII or cAMP-binding protein (CREB), which is the archetypal transcription factor binding to CRE, decreased the basal renin gene expression. However, the deficiency of COUP-TFII did not further diminish the renin expression when CREB was knocked down. In agreement with the cell culture studies, mutant mice deficient in COUP-TFII have lower renin expression than their control strain. Altogether our data show that COUP-TFII is involved in the control of renin gene expression.
Project description:Mutations in phosphatase and tensin homologue (PTEN) or genomic alterations in the phosphatidylinositol-3-OH kinase-signalling pathway are the most common genetic alterations reported in human prostate cancer. However, the precise mechanism underlying how indolent tumours with PTEN alterations acquire metastatic potential remains poorly understood. Recent studies suggest that upregulation of transforming growth factor (TGF)-? signalling triggered by PTEN loss will form a growth barrier as a defence mechanism to constrain prostate cancer progression, underscoring that TGF-? signalling might represent a pre-invasive checkpoint to prevent PTEN-mediated prostate tumorigenesis. Here we show that COUP transcription factor II (COUP-TFII, also known as NR2F2), a member of the nuclear receptor superfamily, serves as a key regulator to inhibit SMAD4-dependent transcription, and consequently overrides the TGF-?-dependent checkpoint for PTEN-null indolent tumours. Overexpression of COUP-TFII in the mouse prostate epithelium cooperates with PTEN deletion to augment malignant progression and produce an aggressive metastasis-prone tumour. The functional counteraction between COUP-TFII and SMAD4 is reinforced by genetically engineered mouse models in which conditional loss of SMAD4 diminishes the inhibitory effects elicited by COUP-TFII ablation. The biological significance of COUP-TFII in prostate carcinogenesis is substantiated by patient sample analysis, in which COUP-TFII expression or activity is tightly correlated with tumour recurrence and disease progression, whereas it is inversely associated with TGF-? signalling. These findings reveal that the destruction of the TGF-?-dependent barrier by COUP-TFII is crucial for the progression of PTEN-mutant prostate cancer into a life-threatening disease, and supports COUP-TFII as a potential drug target for the intervention of metastatic human prostate cancer.
Project description:Adipose tissue development and function play a central role in the pathogenesis and pathophysiology of metabolic syndromes. Here, we show that chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) plays a pivotal role in adipogenesis and energy homeostasis. COUP-TFII is expressed in the early stages of white adipocyte development. COUP-TFII heterozygous mice (COUP-TFII(+/-)) have much less white adipose tissue (WAT) than wild-type mice (COUP-TFII(+/+)). COUP-TFII(+/-) mice display a decreased expression of key regulators for WAT development. Knockdown COUP-TFII in 3T3-L1 cells resulted in an increased expression of Wnt10b, while chromatin immunoprecipitation analysis revealed that Wnt10b is a direct target of COUP-TFII. Moreover, COUP-TFII(+/-) mice have increased mitochondrial biogenesis in WAT, and COUP-TFII(+/-) mice have improved glucose homeostasis and increased energy expenditure. Thus, COUP-TFII regulates adipogenesis by regulating the key molecules in adipocyte development and can serve as a target for regulating energy metabolism.
Project description:BACKGROUND:The control of the functional pancreatic ?-cell mass serves the key homeostatic function of releasing the right amount of insulin to keep blood sugar in the normal range. It is not fully understood though how ?-cell mass is determined. METHODOLOGY/PRINCIPAL FINDINGS:Conditional chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-deficient mice were generated and crossed with mice expressing Cre under the control of pancreatic duodenal homeobox 1 (pdx1) gene promoter. Ablation of COUP-TFII in pancreas resulted in glucose intolerance. Beta-cell number was reduced at 1 day and 3 weeks postnatal. Together with a reduced number of insulin-containing cells in the ductal epithelium and normal ?-cell proliferation and apoptosis, this suggests decreased ?-cell differentiation in the neonatal period. By testing islets isolated from these mice and cultured ?-cells with loss and gain of COUP-TFII function, we found that COUP-TFII induces the expression of the ?-catenin gene and its target genes such as cyclin D1 and axin 2. Moreover, induction of these genes by glucagon-like peptide 1 (GLP-1) via ?-catenin was impaired in absence of COUP-TFII. The expression of two other target genes of GLP-1 signaling, GLP-1R and PDX-1 was significantly lower in mutant islets compared to control islets, possibly contributing to reduced ?-cell mass. Finally, we demonstrated that COUP-TFII expression was activated by the Wnt signaling-associated transcription factor TCF7L2 (T-cell factor 7-like 2) in human islets and rat ?-cells providing a feedback loop. CONCLUSIONS/SIGNIFICANCE:Our findings show that COUP-TFII is a novel component of the GLP-1 signaling cascade that increases ?-cell number during the neonatal period. COUP-TFII is required for GLP-1 activation of the ?-catenin-dependent pathway and its expression is under the control of TCF7L2.
Project description:The nuclear receptor chicken ovalbumin upstream promoter-transcription factor type II (COUP-TFII)/NR2F2 is expressed in adult Leydig cells, and conditional deletion of the Coup-tfii/Nr2f2 gene impedes their differentiation. Steroid production is also reduced in COUP-TFII-depleted Leydig cells, supporting an additional role in steroidogenesis for this transcription factor. COUP-TFII action in Leydig cells remains to be fully characterized. In the present work, we report that COUP-TFII is an essential regulator of the gene encoding the anti-Müllerian hormone receptor type 2 (Amhr2), which participates in Leydig cell differentiation and steroidogenesis. We found that Amhr2 mRNA levels are reduced in COUP-TFII-depleted MA-10 Leydig cells. Consistent with this, COUP-TFII directly activates a -1486 bp fragment of the mouse Amhr2 promoter in transient transfection assays. The COUP-TFII responsive region was localized between -67 and -34 bp. Chromatin immunoprecipitation assay confirmed COUP-TFII recruitment to the proximal Amhr2 promoter whereas DNA precipitation assay revealed that COUP-TFII associates with the -67/-34 bp region in vitro. Even though the -67/-34 bp region contains an imperfect nuclear receptor element, COUP-TFII-mediated activation of the Amhr2 promoter requires a GC-rich sequence at -39 bp known to bind the specificity protein (SP)1 transcription factor. COUP-TFII transcriptionally cooperates with SP1 on the Amhr2 promoter. Mutations that altered the GCGGGGCGG sequence at -39 bp abolished COUP-TFII-mediated activation, COUP-TFII/SP1 cooperation, and reduced COUP-TFII binding to the proximal Amhr2 promoter. Our data provide a better understanding of the mechanism of COUP-TFII action in Leydig cells through the identification and regulation of the Amhr2 promoter as a novel target.
Project description:The orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine-resistant versus sensitive breast cancer cell lines. The identification of COUP-TFII-interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer.FLAG-affinity purification and multidimensional protein identification technology (MudPIT) identified nucleolin among the proteins interacting with COUP-TFII in MCF-7 tamoxifen-sensitive breast cancer cells. Interaction of COUP-TFII and nucleolin was confirmed by coimmunoprecipitation of endogenous proteins in MCF-7 and T47D breast cancer cells. In vitro studies revealed that COUP-TFII interacts with the C-terminal arginine-glycine repeat (RGG) domain of nucleolin. Functional interaction between COUP-TFII and nucleolin was indicated by studies showing that siRNA knockdown of nucleolin and an oligonucleotide aptamer that targets nucleolin, AS1411, inhibited endogenous COUP-TFII-stimulated RARB2 expression in MCF-7 and T47D cells. Chromatin immunoprecipitation revealed COUP-TFII occupancy of the RARB2 promoter was increased by all-trans retinoic acid (atRA). RAR?2 regulated gene RRIG1 was increased by atRA and COUP-TFII transfection and inhibited by siCOUP-TFII. Immunohistochemical staining of breast tumor microarrays showed nuclear COUP-TFII and nucleolin staining was correlated in invasive ductal carcinomas. COUP-TFII staining correlated with ER?, SRC-1, AIB1, Pea3, MMP2, and phospho-Src and was reduced with increased tumor grade.Our data indicate that nucleolin plays a coregulatory role in transcriptional regulation of the tumor suppressor RARB2 by COUP-TFII.
Project description:COUP-TFII, a member of the nuclear receptor superfamily plays a critical role in angiogenesis and organogenesis during embryonic development. Our results indicate that COUP-TFII expression is profoundly upregulated in prostate cancer patients and might serves as biomarker for recurrence prediction. Thus we conduct transcriptome comparison of control and COUP-TFII depleted PC3 cells to gain genomic insights on the biological processes that COUP-TFII is involved in prostate cancer cells. Ingenuity Pathway Analysis (IPA) shows that the most prominent altered pathways in the COUP-TFII depleted cells are related to cell growth; cell cycle progression and DNA damage response. Indeed many growth related genes including E2F1, p21, CDC25A, Cyclin A and Cyclin B are changed in COUP-TFII knockdown cells, suggesting that COUP-TFII might be an important regulator for prostate cancer cell growth. Further functional assays from cells and mice genetic studies confirm the hypothesis that COUP-TFII serve as the major regulator to control prostrate cancer growth. Together, results provide insight into the role of COUP-TFII in prostate tumorigenesis. PC3 Cells were transfected with siRNA (Control or COUP-TFII siRNA) duplexes (40 nM) and total RNA was isolated 48 hours later.