Chicken Ovalbumin Upstream Promoter-transcription Factor II Research Articles

The COUP-TFII variant lacking a DNA-binding domain inhibits the activation of the Cyp7a1 promoter through physical interaction with COUP-TFII

The COUP-TFII (chicken ovalbumin upstream promoter-transcription factor II) nuclear receptor, which is composed of a DNA-binding domain and a ligand-binding domain, exerts pleiotropic effects on development and cell differentiation by regulating the transcription of its target genes, including Cyp7a1 (cytochrome P450, family 7, subfamily a, polypeptide 1), which plays important roles in catabolism of cholesterol in the liver. Although multiple variants of COUP-TFII exist, their roles in the regulation of Cyp7a1 expression have not been elucidated. In the present study, we investigated the roles of COUP-TFII-V2 (variant 2), which lacks a DNA-binding domain, in the regulation of the transcriptional control of the Cyp7a1 gene by COUP-TFII in hepatocellular carcinoma cells. We found that COUP-TFII-V2 was significantly expressed in Huh7 cells, in which Cyp7a1 was not expressed. Furthermore, knockdown of COUP-TFII-V2 enhanced endogenous Cyp7a1 expression in Huh7 cells. Although COUP-TFII activates the Cyp7a1 promoter through direct binding to DNA, this activation was affected by COUP-TFII-V2, which physically interacted with COUP-TFII and inhibited its DNA-binding ability. Chromatin immunoprecipitation assays showed that COUP-TFII-V2 inhibited the binding of endogenous COUP-TFII to the intact Cyp7a1 promoter. The results of the present study suggest that COUP-TFII-V2 negatively regulates the function of COUP-TFII by inhibiting its binding to DNA to decrease Cyp7a1 expression.

Hypothalamic ventromedial COUP-TFII protects against hypoglycemia-associated autonomic failure

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.

Multiple roles of COUP-TFII in cancer initiation and progression

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an orphan nuclear receptor that acts as a transcriptional activator or repressor in a cell type-dependent manner. Best characterized for its role in the regulation of angiogenesis during mouse development, COUP-TFII also plays important roles in glucose metabolism and cancer. Expression of COUP-TFII is altered in various endocrine conditions. Cell type-specific functions and the regulation of COUP-TFII expression result in its varying physiological and pathological actions in diverse systems. Evidence will be reviewed for oncogenic and tumor-suppressive functions of COUP-TFII, with roles in angiogenesis, metastasis, steroidogenesis, and endocrine sensitivity of breast cancer described. The applicability of current data to our understanding of the role of COUP-TFII in cancer will be discussed.

Editorial: Molecular Endocrinology Articles in the Spotlight for August 2012

We are pleased to feature the following articles in this month’s issue of Molecular Endocrinology. In “COUP-TFII Is a Major Regulator of Cell Cycle and Notch Signaling Pathways,” Chen et al. investigate underlying mechanisms by which chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) controls specification of arterial-venous identity and tumor angiogenesis within endothelial cells. Their data indicate that COUP-TFII regulates the transcription of E2F1 and the activity of the Notch signaling pathway as well as repressing the expression of Hey2, Foxc1, and Np-1 at the transcriptional level. These results raise the possibility of screening for COUP-TFII antagonists to identify novel therapeutic agents that could be used to inhibit tumor angiogenesis. “Secretin Receptor Promotes the Proliferation of Endocrine Tumor Cells Via the PI3K/AKT Pathway” by Lee et al. demonstrates that the secretin receptor is overexpressed in various endocrine tumor cells and plays a proproliferative role in pheochromocytoma and adrenocortical carcinoma cells. Tumor cells expressing high levels of secretin receptor are sensitive to treatment with inhibitors of the phosphatidylinositol 3 kinase (PI3K)/serine-threonine protein kinase (AKT) pathway, offering a novel treatment option for patients presenting with tumors exhibiting high receptor expression. During pregnancy, progesterone signaling through its receptor (PR) maintains myometrial quiescence by repressing a group of contraction-associated (CAP) genes, such as connexin43 and the oxytocin receptor. Xie et al.’s “Expression and Function of Myometrial PSF Suggest a Role in Progesterone Withdrawal and the Initiation of Labor” demonstrates that PR and its associated coregulators [e.g., protein-associated splicing factor (PSF), switch independent 3 homolog A (Sin3A)/histone deacetylase (HDAC)] are recruited to CAP gene promoters, triggering repression of their transcriptional activity. Furthermore, expression of PSF was found to be negatively regulated by both estradiol and mechanical stretch of the myometrium by the growing fetus, providing a mechanism that could contribute to the functional withdrawal of progesterone action at term labor. Congratulations to the authors of these papers and to all authors published in the August issue. Donald B. DeFranco, Ph.D. Editor-in-Chief, Molecular Endocrinology E D I T O R I A L

Chicken Ovalbumin Upstream Promoter Transcription Factor II Regulates Renin Gene Expression

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.

Orphan Nuclear Receptor Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) Protein Negatively Regulates Bone Morphogenetic Protein 2-induced Osteoblast Differentiation through Suppressing Runt-related Gene 2 (Runx2) Activity

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is an orphan nuclear receptor of the steroid-thyroid hormone receptor superfamily. COUP-TFII is widely expressed in multiple tissues and organs throughout embryonic development and has been shown to regulate cellular growth, differentiation, and organ development. However, the role of COUP-TFII in osteoblast differentiation has not been systematically evaluated. In the present study, COUP-TFII was strongly expressed in multipotential mesenchymal cells, and the endogenous expression level decreased during osteoblast differentiation. Overexpression of COUP-TFII inhibited bone morphogenetic protein 2 (BMP2)-induced osteoblastic gene expression. The results of alkaline phosphatase, Alizarin Red staining, and osteocalcin production assay showed that COUP-TFII overexpression blocks BMP2-induced osteoblast differentiation. In contrast, the down-regulation of COUP-TFII synergistically induced the expression of BMP2-induced osteoblastic genes and osteoblast differentiation. Furthermore, the immunoprecipitation assay showed that COUP-TFII and Runx2 physically interacted and COUP-TFII significantly impaired the Runx2-dependent activation of the osteocalcin promoter. From the ChIP assay, we found that COUP-TFII repressed DNA binding of Runx2 to the osteocalcin gene, whereas Runx2 inhibited COUP-TFII expression via direct binding to the COUP-TFII promoter. Taken together, these findings demonstrate that COUP-TFII negatively regulates osteoblast differentiation via interaction with Runx2, and during the differentiation state, BMP2-induced Runx2 represses COUP-TFII expression and promotes osteoblast differentiation.

Proposed Role for COUP-TFII in Regulating Fetal Leydig Cell Steroidogenesis, Perturbation of Which Leads to Masculinization Disorders in Rodents

Reproductive disorders that are common/increasing in prevalence in human males may arise because of deficient androgen production/action during a fetal ‘masculinization programming window’. We identify a potentially important role for Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) in Leydig cell (LC) steroidogenesis that may partly explain this. In rats, fetal LC size and intratesticular testosterone (ITT) increased ∼3-fold between e15.5-e21.5 which associated with a progressive decrease in the percentage of LC expressing COUP-TFII. Exposure of fetuses to dibutyl phthalate (DBP), which induces masculinization disorders, dose-dependently prevented the age-related decrease in LC COUP-TFII expression and the normal increases in LC size and ITT. We show that nuclear COUP-TFII expression in fetal rat LC relates inversely to LC expression of steroidogenic factor-1 (SF-1)-dependent genes (StAR, Cyp11a1, Cyp17a1) with overlapping binding sites for SF-1 and COUP-TFII in their promoter regions, but does not affect an SF-1 dependent LC gene (3β-HSD) without overlapping sites. We also show that once COUP-TFII expression in LC has switched off, it is re-induced by DBP exposure, coincident with suppression of ITT. Furthermore, other treatments that reduce fetal ITT in rats (dexamethasone, diethylstilbestrol (DES)) also maintain/induce LC nuclear expression of COUP-TFII. In contrast to rats, in mice DBP neither causes persistence of fetal LC COUP-TFII nor reduces ITT, whereas DES-exposure of mice maintains COUP-TFII expression in fetal LC and decreases ITT, as in rats. These findings suggest that lifting of repression by COUP-TFII may be an important mechanism that promotes increased testosterone production by fetal LC to drive masculinization. As we also show an age-related decline in expression of COUP-TFII in human fetal LC, this mechanism may also be functional in humans, and its susceptibility to disruption by environmental chemicals, stress and pregnancy hormones could explain the origin of some human male reproductive disorders.

Nuclear receptor chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) modulates mesenchymal cell commitment and differentiation

The mesenchymal cell is a multipotent stem cell with the capacity to give rise to multiple cell types such as adipocytes, osteoblasts, chondrocytes, and myocytes. However, the molecular events responsible for their lineage specification and differentiation remain obscure. Here we show that inactivation of chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), a member of the nuclear receptor superfamily, in mesenchymal progenitors favors osteoblast and myoblast development while simultaneously impairing adipogenic and chondrogenic programs. During mouse embryogenesis, COUP-TFII protein is highly detected in the mesenchymal compartment and is involved in mesoderm tissue formation. Ablation of COUP-TFII in mice led to higher bone density, increased muscle mass, and suppression of cartilage and fat formation. We further demonstrate that COUP-TFII directs the plasticity of mesenchymal precursors primarily through the combined modulation of Wnt signaling, Runx2 activity, as well as PPARγ and Sox9 expression. Together, our results provide insight into the mechanisms whereby a single nuclear receptor can fine-tune the lineage-specific differentiation of a progenitor cell.

COUP‐TFII switches responses of venous endothelium to atherosclerotic factors through controlling the profile of various inherent genes expression

Endothelial cells of arteries (AEC) have a strikingly greater responsiveness to atherosclerosis factors than venous endothelial cells (VEC). However, the reasons for this phenomenon remain unclear. Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) plays an important role in regulating embryonic arterial-venous differentiation. We therefore investigate whether COUP-TFII is related to this different susceptibility between AEC and VEC. It is first confirmed that COUP-TFII is expressed in VEC but not in AEC in the adult. Using a siRNA strategy, we identified the expression of Jagged1 and Notch1 in cultured human VEC, which usually exist only in AEC, after knocking down of COUP-TFII. To further elucidate the role of COUP-TFII, we performed DNA microarrays in VEC transfected with the siRNA of COUP-TFII and subsequently stimulated with angiotensin II (AngII) and compared the expression profiles of 112 genes involved in various atherosclerosis-related pathways. The results indicated that expression of atherogenic genes was significantly upregulated after AngII stimulation in VEC transfected with COUP-TFII siRNA. Moreover, in vitro cell functional assay showed that knockdown of COUP-TFII in VEC increased not only basal but also AngII-induced cell adhesions. These results demonstrate that COUP-TFII suppresses the susceptibility of VEC to atherosclerosis through controlling the expression of various atherosclerosis-related molecules.

Deletion of steroid receptor coactivator-3 gene ameliorates hepatic steatosis

Excess dietary fat can cause hepatic steatosis, which can progress into severe liver disorders including steatohepatitis and cirrhosis. Steroid receptor coactivator-3 (SRC-3), a member of the p160 coactivator family, is reported as a key regulator of adipogenesis and energy homeostasis. We sought to determine the influence of SRC-3 on hepatic steatosis and the mechanism beneath. The influence of siRNA-mediated SRC-3 silencing on hepatic lipid accumulation was assessed in HepG2 cells. The molecular mechanism of SRC-3 regulation of hepatic lipid metabolism was also studied. Moreover, the effect of SRC-3 ablation on hepatic steatosis was examined in SRC-3 deficient mice. In this study, we report that SRC-3 ablation reduces palmitic acid-induced lipid accumulation in HepG2 cells. Moreover, deletion of SRC-3 ameliorates hepatic steatosis and inflammation response in mice fed a high fat diet (HFD). These metabolic improvements can presumably be explained by the reduction in chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) expression and the subsequent elevation in peroxisome proliferator-activated receptor α (PPARα) level. At the molecular level, SRC-3 interacts with retinoic receptor α (RARα) to activate COUP-TFII expression under all-trans retinoic acid (ARTA) treatment. These findings indicate a crucial role for SRC-3 in regulating hepatic lipid metabolism and provide the possible novel inner mechanisms.

Chicken ovalbumin upstream promoter-transcription factor II regulates nuclear receptor, myogenic, and metabolic gene expression in skeletal muscle cells

We demonstrate that chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) mRNA is more abundantly expressed (than COUP-TFI mRNA) in skeletal muscle C2C12 cells and in (type I and II) skeletal muscle tissue from C57BL/10 mice. Consequently, we have utilized the ABI TaqMan Low Density Array (TLDA) platform to analyze gene expression changes specifically attributable to ectopic COUP-TFII (relative to vector only) expression in muscle cells. Utilizing a TLDA-based platform and 5 internal controls, we analyze the entire NR superfamily, 96 critical metabolic genes, and 48 important myogenic regulatory genes on the TLDA platform utilizing 5 internal controls. The low density arrays were analyzed by rigorous statistical analysis (with Genorm normalization, Bioconductor R, and the Empirical Bayes statistic) using the (integromics) statminer software. In addition, we validated the differentially expressed patho-physiologically relevant gene (identified on the TLDA platform) glucose transporter type 4 (Glut4). We demonstrated that COUP-TFII expression increased the steady state levels of Glut4 mRNA and protein, while ectopic expression of truncated COUP-TFII lacking helix 12 (COUP-TFΔH12) reduced Glut4 mRNA expression in C2C12 cells. Moreover, COUP-TFII expression trans-activated the Glut4 promoter (-997/+3), and ChIP analysis identified selective recruitment of COUP-TFII to a region encompassing a highly conserved SP1 binding site (in mouse, rat, and human) at nt positions -131/-118. Mutation of the SpI site ablated COUP-TFII mediated trans-activation of the Glut4 promoter. In conclusion, this study demonstrates that in skeletal muscle cells, COUP-TFII regulates several nuclear hormone receptors, and critical metabolic and muscle specific genes.

The Nutritional Induction of COUP-TFII Gene Expression in Ventromedial Hypothalamic Neurons Is Mediated by the Melanocortin Pathway

BackgroundThe nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an important coordinator of glucose homeostasis. We report, for the first time, a unique differential regulation of its expression by the nutritional status in the mouse hypothalamus compared to peripheral tissues.Methodology/Principal FindingsUsing hyperinsulinemic-euglycemic clamps and insulinopenic mice, we show that insulin upregulates its expression in the hypothalamus. Immunofluorescence studies demonstrate that COUP-TFII gene expression is restricted to a subpopulation of ventromedial hypothalamic neurons expressing the melanocortin receptor. In GT1-7 hypothalamic cells, the MC4-R agonist MTII leads to a dose dependant increase of COUP-TFII gene expression secondarily to a local increase in cAMP concentrations. Transfection experiments, using a COUP-TFII promoter containing a functional cAMP responsive element, suggest a direct transcriptional activation by cAMP. Finally, we show that the fed state or intracerebroventricular injections of MTII in mice induce an increased hypothalamic COUP-TFII expression associated with a decreased hepatic and pancreatic COUP-TFII expression.Conclusions/SignificanceThese observations strongly suggest that hypothalamic COUP-TFII gene expression could be a central integrator of insulin and melanocortin signaling pathway within the ventromedial hypothalamus. COUP-TFII could play a crucial role in brain integration of circulating signal of hunger and satiety involved in energy balance regulation.

Suppression of ERalpha activity by COUP-TFII is essential for successful implantation and decidualization.

Synchrony between embryo competency and uterine receptivity is essential for successful implantation. Mice with ablation of chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) in the uterus (PR(Cre/+);COUP-TFII(flox/flox)) exhibit implantation defects and increased estrogen receptor (ER)alpha activity in the luminal epithelium, suggesting high ERalpha activity may disrupt the window of uterine receptivity. To determine whether increased ERalpha activity in the PR(Cre/+);COUP-TFII(flox/flox) uterus is the cause of defective implantation, we assessed whether inhibition of ERalpha activity could rescue the PR(Cre/+);COUP-TFII(flox/flox) uterine implantation defect. ICI 182,780 (ICI), a pure ERalpha antagonist, was administered to PR(Cre/+);COUP-TFII(flox/flox) mutant and COUP-TFII(flox/flox) control mice during the receptive period, and the number of implantation sites was examined. COUP-TFII(flox/flox) control mice treated with oil or ICI showed the normal number of implantation sites. As expected, no implantation sites were observed in PR(Cre/+);COUP-TFII(flox/flox) mutant mice treated with oil, consistent with previous observations. In contrast, implantation sites were greatly increased in ICI-treated PR(Cre/+);COUP-TFII(flox/flox) mutant mice, albeit at a reduced number in comparison with the control mice. ICI treatment was also able to restore the expression of Wnt4 and bone morphogenetic protein 2, important for endometrial decidualization in the PR(Cre/+);COUP-TFII(flox/flox) mutant mice. To confirm that the rescue of embryo attachment and decidualization is a consequence of a reduced ERalpha activity upon ICI treatment, we showed a reduction of the expression of ERalpha target genes in PR(Cre/+);COUP-TFII(flox/flox) mutant mice. Because COUP-TFII was also shown in our laboratory to be important for placentation during pregnancy, we asked whether ICI treatment could also rescue the placentation defect to allow full-term pregnancy in these mice. We found that whereas mice were born in COUP-TFII(flox/flox) control mice given ICI, no pups were born in the PR(Cre/+);COUP-TFII(flox/flox) mutant mice, suggesting that the increased ERalpha activity is not the reason for placentation defects. These results demonstrate that during the periimplantation period, COUP-TFII regulates embryo attachment and decidualization through controlling ERalpha activity. However, COUP-TFII expression is still required in the postimplantation period to facilitate placentation.

COUP-TFII regulates tumor growth and metastasis by modulating tumor angiogenesis

Tumor growth depends on nutrients and oxygen supplied by the vasculature through angiogenesis. Here, we show that the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), a member of the nuclear receptor family, is a major angiogenesis regulator within the tumor microenvironment. Conditional ablation of COUP-TFII in adults severely compromised neoangiogenesis and suppressed tumor growth in xenograft mouse models. In addition, tumor growth and tumor metastasis were also impaired in a spontaneous mammary-gland tumor model in the absence of COUP-TFII. We showed that COUP-TFII directly regulates the transcription of Angiopoietin-1 in pericytes to enhance neoangiogenesis. Importantly, provision of Angiopoietin-1 partially restores the angiogenic defects exhibited by the COUP-TFII-deficient mice, which supports the notion that COUP-TFII controls Angiopoietin-1/Tie2 signaling to regulate tumor angiogenesis. Because COUP-TFII has little impact on normal adult physiological function, our results raise an interesting possibility that inhibition of COUP-TFII may offer a therapeutic approach for anticancer intervention.

COUP-TFII is Regulated by High Glucose in Endothelial Cells

Diabetes mellitus is an important risk factor for cardiovascular diseases. Clinical evidence supports a link between hyperglycemia, endothelial dysfunction, and vascular disorders. However, the precise molecular mechanisms causing endothelial dysfunction in diabetic patients remain unclear. An interesting novel mediator could be chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), which plays an essential role in glucose metabolism. COUP-TFII is known to be expressed in venous endothelial cells. In this study, we show COUP-TFII expression in human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells. HUVECs express glucose transporters 1, 3, 6, and 10, and the insulin receptor. Insulin in combination with glucose activates protein kinase B (PKB or Akt) phosphorylation via phosphoinositide 3-kinase (PI3-kinase). Short-term (60-240 min) stimulation of HUVECs with high glucose increased COUP-TFII expression independent of insulin. Long-term (48 h) stimulation of HUVECs with high glucose augmented expression of the insulin receptor and E-selectin, but downregulated COUP-TFII protein expression. Downregulation of COUP-TFII by shRNA leads to downregulation of E-selectin and upregulation of eNOS and glucose transporters. Our data suggest that COUP-TFII is regulated by glucose in a time- and dose-dependent manner in endothelial cells. COUP-TFII might affect endothelial function in a diabetic background.

Prenatal retinoic acid up-regulates pulmonary gene expression of COUP-TFII, FOG2, and GATA4 in pulmonary hypoplasia

Purpose Retinoids play an important role in lung development. Recently, prenatal treatment with retinoic acid (RA) has been reported to stimulate alveologenesis in hypoplastic lungs in the nitrofen model of congenital diaphragmatic hernia (CDH). Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is a transcription factor in the steroid/thyroid hormone receptor superfamily, and targeted ablation of COUP-TFII causes CDH and associated lung hypoplasia in mice. Friend of GATA 2 (FOG2) is a zinc finger-containing protein that modulates the transcriptional activity of GATA proteins. GATA4 is a member of a family of DNA-binding proteins, which is found in the promoter regions of many genes. The COUP-TFII, FOG2, and GATA4 genes, regulated by the retinoid signaling pathway, are located on chromosomes 15q26, 8q23, and 8p23.1 respectively, regions reported to be deleted in individuals with CDH. The aim of this study was to examine the pulmonary gene expression of COUP-TFII, FOG2, and GATA4 in the nitrofen model of CDH. Materials and Methods Pregnant rats were exposed to either olive oil or 100 mg nitrofen on day 9 of gestation (D9). 5 mg/kg of RA was given intraperitoneally on days D18, D19, and D20. The fetuses were recovered by caesarean section on D21, and the diaphragm was carefully examined for the presence of a hernia under a microscope. Left lungs were obtained from CDH fetuses and controls and divided into four groups: control (n = 9), control + RA (n = 9), CDH (n = 9), and CDH + RA (n = 9). The relative mRNA expression levels of COUP-TFII, FOG2, and GATA4 were analyzed in each lung by real-time reverse transcriptase–polymerase chain reaction from cDNA generated by mRNA from pulmonary total RNA. Results The relative mRNA expression levels of COUP-TFII, FOG2, and GATA4 were significantly increased in CDH + RA lungs compared to control, control + RA, and CDH ( P < .05). Conclusions Up-regulation of pulmonary gene expression of COUP-TFII, FOG2, and GATA4 after prenatal treatment with retinoic acid in the nitrofen model of CDH suggests that RA may have a therapeutic potential in modulating lung growth. Furthermore, these results support the concept that these proteins work together to regulate downstream target genes that play an important role in the development of lung.

DLL1-mediated Notch activation regulates endothelial identity in mouse fetal arteries

Notch signaling has been shown to regulate various aspects of vascular development. However, a specific role of the ligand Delta-like 1 (DLL1) has not been shown thus far. Here, we demonstrate that during fetal development, DLL1 is an essential Notch ligand in the vascular endothelium of large arteries to activate Notch1 and maintain arterial identity. DLL1 was detected in fetal arterial endothelial cells beginning at embryonic day 13.5. While DLL4-mediated activation has been shown to suppress vascular endothelial growth factor (VEGF) pathway components in growing capillary beds, DLL1-Notch signaling was required for VEGF receptor expression in fetal arteries. In the absence of DLL1 function, VEGF receptor 2 (VEGFR2) and its coreceptor, neuropilin-1 (NRP1), were down-regulatedin mutant arteries, which was followed by up-regulation of chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), a repressor of arterial differentiation and Nrp1 expression in veins. Consistent with a positive modulation of the VEGF pathway by DLL1, the Nrp1 promoter contains several recombinant signal-binding protein 1 for J kappa (RBPJkappa)-binding sites and was responsive to Notch activity in cell culture. Our results establish DLL1 as a critical endothelial Notch ligand required for maintaining arterial identity during mouse fetal development and suggest context-dependent interrelations of the VEGFA and Notch signaling pathways.

The Nuclear Orphan Receptor COUP-TFII Plays an Essential Role in Adipogenesis, Glucose Homeostasis, and Energy Metabolism

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.

Molecular Cloning and Characterization of the Human PED/PEA-15 Gene Promoter Reveal Antagonistic Regulation by Hepatocyte Nuclear Factor 4α and Chicken Ovalbumin Upstream Promoter Transcription Factor II

Overexpression of the ped/pea-15 gene in mice impairs glucose tolerance and leads to diabetes in conjunction with high fat diet treatment. PED/PEA-15 is also overexpressed in type 2 diabetics as well as in euglycemic offspring from these subjects. The cause(s) of this abnormality remains unclear. In the present work we have cloned and localized the promoter region of the human PED/PEA-15 gene within the first 230 bp of the 5(R)-flanking region. A cis-acting regulatory element located between -320 and -335 bps upstream the PED/PEA-15 gene transcriptional start site (+1) is recognized by both the hepatocyte nuclear factor 4alpha (HNF-4alpha) and the chicken ovalbumin upstream promoter transcription factor II (COUP-TFII), two members of the steroid/thyroid superfamily of transcription factors, both of which are involved in the control of lipid and glucose homeostasis. HNF-4alpha represses PED/PEA-15 expression in HeLa cells, whereas COUP-TFII activates its expression. In hepatocytes, the activation of PED/PEA-15 gene transcription is paralleled by the establishment of a partially dedifferentiated phenotype accompanied by a reduction in mRNA levels encoded by genes normally expressed during liver development. Cotransfection of HeLa cells with a reporter construct containing the PED/PEA-15 response element and various combinations of HNF-4alpha and COUP-TFII expression vectors indicated that COUP-TFII antagonizes the repression of the PED/PEA-15 gene by HNF-4alpha. Thus, at least in part, transcription of the PED/PEA-15 gene in vivo is dependent upon the intracellular balance of these positive and negative regulatory factors. Abnormalities in HNF-4alpha and COUP-TFII balance might have important consequences on glucose tolerance in humans.

The MODY1 Gene for Hepatocyte Nuclear Factor 4α and a Feedback Loop Control COUP-TFII Expression in Pancreatic Beta Cells

Pancreatic islet beta cell differentiation and function are dependent upon a group of transcription factors that maintain the expression of key genes and suppress others. Knockout mice with the heterozygous deletion of the gene for chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) or the complete disruption of the gene for hepatocyte nuclear factor 4alpha (HNF4alpha) in pancreatic beta cells have similar insulin secretion defects, leading us to hypothesize that there is transcriptional cross talk between these two nuclear receptors. Here, we demonstrate specific HNF4alpha activation of a reporter plasmid containing the COUP-TFII gene promoter region in transfected pancreatic beta cells. The stable association of the endogenous HNF4alpha with a region of the COUP-TFII gene promoter that contains a direct repeat 1 (DR-1) binding site was revealed by chromatin immunoprecipitation. Mutation experiments showed that this DR-1 site is essential for HNF4alpha transactivation of COUP-TFII. The dominant negative suppression of HNF4alpha function decreased endogenous COUP-TFII expression, and the specific inactivation of COUP-TFII by small interfering RNA caused HNF4alpha mRNA levels in 832/13 INS-1 cells to decrease. This positive regulation of HNF4alpha by COUP-TFII was confirmed by the adenovirus-mediated overexpression of human COUP-TFII (hCOUP-TFII), which increased HNF4alpha mRNA levels in 832/13 INS-1 cells and in mouse pancreatic islets. Finally, hCOUP-TFII overexpression showed that there is direct COUP-TFII autorepression, as COUP-TFII occupies the proximal DR-1 binding site of its own gene in vivo. Therefore, COUP-TFII may contribute to the control of insulin secretion through the complex HNF4alpha/maturity-onset diabetes of the young 1 (MODY1) transcription factor network operating in beta cells.