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1.  Regulatory potential of COUP-TFs in development: stem/progenitor cells 
Seminars in cell & developmental biology  2013;24(0):10.1016/j.semcdb.2013.08.005.
The formation of complex organisms is highly dependent on the differentiation of specialized mature cells from common stem/progenitor cells. The orphan nuclear receptors chicken ovalbumin upstream promoter transcription factors (COUP-TFs) are broadly, but not ubiquitously, expressed in multiple tissues throughout embryonic development and COUP-TFs are indispensible for proper organogenesis. Recently, growing evidence suggests a critical role of COUP-TFs in multiple aspects of stem/progenitor cell biology. In this review, we highlight the progress of COUP-TFs function and its underlying mechanism in driving stem/progenitor cell self-renewal, lineage specification, differentiation, maintenance, and cell identity in diverse tissue types. These studies provide novel insights into future clinical utilities of COUP-TFs in stem cell based therapies and in the management of diseases.
doi:10.1016/j.semcdb.2013.08.005
PMCID: PMC3849206  PMID: 23978678
COUP-TFs; stem/progenitor cell
2.  Atrial Identity Is Determined by A COUP-TFII Regulatory Network 
Developmental cell  2013;25(4):417-426.
SUMMARY
Atria and ventricles exhibit distinct molecular profiles that produce structural and functional differences between the two cardiac compartments. However, factors that determine these differences remain largely undefined. Cardiomyocyte-specific COUP-TFII ablation produces ventricularized atria that exhibit ventricle-like action potentials, increased cardiomyocyte size, and development of extensive T-tubules. Changes in atrial characteristics are accompanied by alterations of 2584 genes, in which 81% of them were differentially expressed between atria and ventricles, suggesting that a major function of myocardial COUP-TFII is to determine the atrial identity. Chromatin immunoprecipitation assays using E13.5 atria identified classic atrial-ventricular identity genes Tbx5, Hey2, Irx4, MLC2v, MLC2a and MLC1a, among many other cardiac genes, as potential COUP-TFII direct targets. Collectively, our results reveal that COUP-TFII confers the atrial identity through direct binding and modulating expression of a broad spectrum of genes that have an impact on atrial development and function.
doi:10.1016/j.devcel.2013.04.017
PMCID: PMC3687546  PMID: 23725765
3.  Nuclear Receptor COUP-TFII Controls Pancreatic Islet Tumor Angiogenesis by Regulating VEGF/VEGFR-2 Signaling 
Cancer research  2010;70(21):8812-8821.
The significance of angiogenesis in cancer biology and therapy is well established. In this study, we utilized the prototypical RIP-Tag model of multistage pancreatic islet tumorigenesis to show that the nuclear receptor COUP-TFII is essential to regulate the balance between pro- and anti-angiogenic molecules that influence the angiogenic switch in cancer. Conditional ablation of COUP-TFII in the tumor microenvironment severely compromised neoangiogenesis and lymphangiogenesis during pancreatic tumor progression and metastasis. We found that COUP-TFII plays a cell autonomous role in endothelial cells to control blood vessel sprouting by regulating cell proliferation and migration. Mechanistic investigations revealed that COUP-TFII suppressed VEGF/VEGFR-2 signaling by transcriptionally repressing expression of VEGFR-1, thereby curtailing a central angiogenic driver for vascular growth. Together, our results implicate COUP-TFII as a critical factor in tumor angiogenesis via regulation of VEGF/VEGFR-2 signaling, suggesting COUP-TFII as a candidate target for anti-angiogenic therapy.
doi:10.1158/0008-5472.CAN-10-0551
PMCID: PMC2970665  PMID: 20978203
Nuclear Receptor; Endothelial Sprouting; VEGFR-2 signaling
4.  Direct transcriptional regulation of neuropilin-2 by COUP-TFII modulates multiple steps in murine lymphatic vessel development 
The Journal of Clinical Investigation  2010;120(5):1694-1707.
The lymphatic system plays a key role in tissue fluid homeostasis. Lymphatic dysfunction contributes to the pathogenesis of many human diseases, including lymphedema and tumor metastasis. However, the mechanisms regulating lymphangiogenesis remain largely unknown. Here, we show that COUP-TFII (also known as Nr2f2), an orphan member of the nuclear receptor superfamily, mediates both developmental and pathological lymphangiogenesis in mice. Conditional ablation of COUP-TFII at an early embryonic stage resulted in failed formation of pre-lymphatic ECs (pre-LECs) and lymphatic vessels. COUP-TFII deficiency at a late developmental stage resulted in loss of LEC identity, gain of blood EC fate, and impaired lymphatic vessel sprouting. siRNA-mediated downregulation of COUP-TFII in cultured primary human LECs demonstrated that the maintenance of lymphatic identity and VEGF-C–induced lymphangiogenic activity, including cell proliferation and migration, are COUP-TFII–dependent and cell-autonomous processes. COUP-TFII enhanced the pro-lymphangiogenic actions of VEGF-C, at least in part by directly stimulating expression of neuropilin-2, a coreceptor for VEGF-C. In addition, COUP-TFII inactivation in a mammary gland mouse tumor model resulted in inhibition of tumor lymphangiogenesis, suggesting that COUP-TFII also regulates neo-lymphangiogenesis in the adult. Thus, COUP-TFII is a critical factor that controls lymphangiogenesis in embryonic development and tumorigenesis in adults.
doi:10.1172/JCI40101
PMCID: PMC2860940  PMID: 20364082
5.  Steroid Receptor Coactivator-3/AIB1 Promotes Cell Migration and Invasiveness through Focal Adhesion Turnover and Matrix Metalloproteinase Expression 
Cancer research  2008;68(13):5460-5468.
Steroid receptor coactivator-3 (SRC-3)/AIB1 is a member of the p160 nuclear receptor coactivator family involved in development and cell cycle progression. We previously showed that SRC-3/AIB1 is required for prostate cancer cell proliferation and survival. Here, we reported that the elevated SRC-3/AIB1 expression is significantly correlated with human prostate cancer seminal vesicle invasion and lymph node metastasis. Furthermore, SRC-3/AIB1 is associated with increased prostate cancer cell migration and invasion. SRC-3/AIB1 is required for focal adhesion turnover and focal adhesion kinase activation. In addition, SRC-3/AIB1 directly regulates transcription of matrix metalloproteinase (MMP)-2 and MMP-13 through its coactivation of AP-1 and PEA3. Taken together, these data suggest that SRC-3/AIB1 plays an essential role in prostate cancer cell invasion and metastasis.
doi:10.1158/0008-5472.CAN-08-0955
PMCID: PMC2826835  PMID: 18593949
6.  The Nuclear Orphan Receptor COUP-TFII Plays an Essential Role in Adipogenesis, Glucose Homeostasis, and Energy Metabolism 
Cell metabolism  2009;9(1):77-87.
Summary
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 (WAT) development. COUP-TFII heterozygous mice (COUP-TFII+/-) have much less WAT than wild type mice (COUP-TFII+/+). COUP-TFII+/- mice display a decreased expression of key regulators for WAT development. Knock down 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 new target for regulating energy metabolism.
doi:10.1016/j.cmet.2008.12.002
PMCID: PMC2630393  PMID: 19117548
7.  Haploinsufficiency of COUP-TFII in Female Reproduction 
The chicken ovalbumin upstream promoter transcription factor II, COUP-TFII, is a member of the Orphan nuclear receptor transcription factor family. Genetic ablation of COUP-TFII results in early embryonic lethality and demonstrates that this gene is required for cardiac and vascular development. Expression of COUP-TFII persists throughout postnatal life in various tissues including the female reproductive tract. However, the physiological function of COUP-TFII in female reproduction has not been extensively analyzed. Here, we provide phenotypic evidences that haploinsufficiency of COUP-TFII in mice demonstrates an important role of COUP-TFII for normal female reproduction. COUP-TFII +/− females show significantly reduced fecundity, irregular estrus cycles, delayed puberty and retarded postnatal growth. Analysis of the reduced fertility revealed that although ovarian function was normal with respect to ovulation, the ovaries have reduced ability to synthesize progesterone in response to exogenous gonadotropins. This reduction is due to the reduction of the expression of steroidogenic enzymes important for P4 synthesis and the reduction of vascularization in COUP-TFII heterozygotes. Analysis of uterine function demonstrated a reduced response to an experimentally induced decidual cell reaction indicating that the ability of the uterus to support embryo implantation was reduced. Taken together, our data shows global impact of gene dosage effects of COUP-TFII on female postnatal life and indicates requirement of COUP-TFII in normal female reproduction, in particular for uterine endometrial functions during the peri-implantation period.
doi:10.1210/me.2005-0019
PMCID: PMC1198323  PMID: 15890675
8.  Neuronatin, a Downstream Target of BETA2/NeuroD1 in the Pancreas, Is Involved in Glucose-Mediated Insulin Secretion 
Diabetes  2005;54(4):1064-1073.
BETA2 (NeuroD1) is a member of the basic helix-loop-helix transcription factor family. BETA2 plays an important role in the development of the pancreas and the nervous system. Using microarray technology, we identified neuronatin (Nnat) as differentially expressed between wild-type (WT) and knockout (KO) pancreatic RNA from embryonic day 14 (e14.5). NNAT is a member of the proteolipid family of amphipathic polypeptides and is believed to be involved in ion channel transport or channel modulation. Northern blot and in situ hybridization analysis of WT and KO samples confirmed the downregulation of Nnat in pancreas of mutant BETA2 embryos. Chromatin immunoprecipitation and gel shift assays were performed and demonstrated the presence of BETA2 on the Nnat promoter, thus confirming the direct transcriptional regulation of Nnat by BETA2. To assess NNAT potential function, we performed knockdown studies by siRNA in NIT cells and observed a reduction in the ability of the NIT cells to respond to glucose. These results suggest for the first time an important role for NNAT in insulin secretion and for proper β-cell function.
PMCID: PMC1197706  PMID: 15793245
CHIP, chromatin immunoprecipitation assay; NNAT, neuronatin; RIA, radioimmunoassay; SSC, sodium chloride–sodium citrate; TBS-X, Tris-buffered saline with 0.1%Triton
9.  Surprise in the Battle Field of Vein vs. Artery 
Organogenesis  2005;2(2):31-32.
Formation of arteries and veins is a complex process. It was shown that activation of the notch signaling pathway in the artery results on the activation of arterial markers and the suppression of vein markers. However, factor, which instructs endothelial cells to take on the vein or artery identity, has not been defined. It was assumed that VEGF, the key molecule which stimulates notch signaling pathway in the artery, is not available in the vein. Thus, endothelial cells, lacking notch signaling, acquire vein identity. Recently, Drs. Tsai and their colleague demonstrated that COUP-TFII, an orphan nuclear receptor, is an important factor that regulates vein identity through suppression of the notch signaling.
PMCID: PMC2634082  PMID: 19521563
artery; COUP-TFil; endothelial cell; notch signal; orphan nuclear receptor; vein
10.  Compensational regulation of bHLH transcription factors in the postnatal development of BETA2/NeuroD1-null retina 
Mechanisms of development  2007;124(0):543-550.
The bHLH transcriptional factor BETA2/NeuroD1 is essential for the survival of photoreceptor cells in the retina. Although this gene is expressed throughout the retina, BETA2/NeuroD1 knockout mice show photoreceptor cell degeneration only in the outer nuclear layer of the retina; other retinal neurons are not affected. Previous studies on retina explants lacking three bHLH genes revealed that retinal neurons in the inner nuclear layer require multiple bHLH genes for their differentiation and survival. However, single- or double-gene mutations show no or a lesser degree of abnormalities during eye development, likely because of compensational or cooperative regulation among those genes. Because not all null mice survive until the retina is fully organized, no direct evidence of this concept has been reported. To understand the regulatory mechanisms between bHLH factors in retinal development, we performed a detailed analysis of BETA2/NeuroD1 knockout mice. BETA2/NeuroD1 was expressed in all three layers of the mouse retina, including all major types of neurons. In addition, a null mutation of BETA2/NeuroD1 resulted in up-regulation of other bHLH genes, Mash1, Neurogenin2, and Math3, in the inner nuclear layer. Our data suggest that compensatory and cross regulatory mechanisms exist among the bHLH factors during retinal development.
doi:10.1016/j.mod.2007.06.001
PMCID: PMC4300853  PMID: 17629466
BETA2/NeuroD1; Mash1; Math3; Neurogenin2; bHLH; Retina; Retinogenesis
11.  E2/Estrogen Receptor/Sjogren Syndrome-Associated Autoantigen Relieves Coactivator Activator-Induced G1/S Arrest To Promote Breast Tumorigenicity 
Molecular and Cellular Biology  2014;34(9):1670-1681.
Coactivator activator (CoAA) is a dual-functional coregulator that regulates steroid receptor-mediated transcription and alternative splicing. Previously, we have shown that CoAA has tumor-suppressive potential in tumorigenic human kidney cells. Here, we uncover a molecular mechanism by which Sjogren syndrome-associated autoantigen (SSA), an estrogen receptor (ER) coactivator, induces MYC oncogene by removing repressive CoAA through E2-dependent degradation of CoAA and promotes G1/S transition of the cell cycle as well as anchorage-independent growth capability of breast cancer cells. We also show that E2 and ER enhance the E3 ligase activity of SSA to modulate CoAA through splicing isoform-selective ubiquitylation. We propose this as one potential molecular basis for the reduced tumor incidence in autoimmune disease patients and suggest SSA as a potential therapeutic target to treat breast cancer.
doi:10.1128/MCB.01564-13
PMCID: PMC3993604  PMID: 24567374
12.  The critical roles of COUP-TFII in tumor progression and metastasis 
Cell & Bioscience  2014;4(1):58.
Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) belongs to the steroid/thyroid hormone receptor superfamily. Extensive evidence has indicated that COUP-TFII plays a critical and indispensable role in cell-fate specification, organogenesis, angiogenesis, and metabolism as well as in a variety of diseases. Recent studies obtained from genetically engineered mouse models (GEM) and patient specimen analysis indicate that COUP-TFII is also important for tumor progression and metastasis. In this article, we will comprehensively review the oncogenic roles of COUP-TFII within the tumor microenvironment and tumor cells and delineate the mechanism by which COUP-TFII contributes to tumorigenesis. The applicability of current data to our understanding of the role of COUP-TFII in cancer and the potential therapeutic implications will also be discussed.
doi:10.1186/2045-3701-4-58
PMCID: PMC4201699  PMID: 25328664
13.  The Health Effects of a Forest Environment on Subclinical Cardiovascular Disease and Heath-Related Quality of Life 
PLoS ONE  2014;9(7):e103231.
Background
Assessment of health effects of a forest environment is an important emerging area of public health and environmental sciences.
Purpose
To demonstrate the long-term health effects of living in a forest environment on subclinical cardiovascular diseases (CVDs) and health-related quality of life (HRQOL) compared with that in an urban environment.
Materials and Methods
This study included the detailed health examination and questionnaire assessment of 107 forest staff members (FSM) and 114 urban staff members (USM) to investigate the long-term health effects of a forest environment. Air quality monitoring between the forest and urban environments was compared. In addition, work-related factors and HRQOL were evaluated.
Results
Levels of total cholesterol, low-density lipoprotein cholesterol, and fasting glucose in the USM group were significantly higher than those in the FSM group. Furthermore, a significantly higher intima-media thickness of the internal carotid artery was found in the USM group compared with that in the FSM group. Concentrations of air pollutants, such as NO, NO2, NOx, SO2, CO, PM2.5, and PM10 in the forest environment were significantly lower compared with those in the outdoor urban environment. Working hours were longer in the FSM group; however, the work stress evaluation as assessed by the job content questionnaire revealed no significant differences between FSM and USM. HRQOL evaluated by the World Health Organization Quality of Life-BREF questionnaire showed FSM had better HRQOL scores in the physical health domain.
Conclusions
This study provides evidence of the potential beneficial effects of forest environments on CVDs and HRQOL.
doi:10.1371/journal.pone.0103231
PMCID: PMC4113370  PMID: 25068265
14.  Proteomic Analysis of Coregulators Bound to ERα on DNA and Nucleosomes Reveals Coregulator Dynamics 
Molecular cell  2013;51(2):185-199.
Summary
Chromatin immunoprecipitation studies have mapped protein occupancies at many genomic loci. However, a detailed picture of the complexity of coregulators (CoRs) bound to a defined enhancer along with a transcription factor is missing. To address this, we used biotin-DNA pulldown assays coupled with mass spectrometry-immunoblotting to identify at least 17 CoRs from nuclear extracts bound to 17β-estradiol (E2)-liganded estrogen receptor-α on estrogen response elements (EREs). Unexpectedly, these complexes initially are biochemically stable and contain certain atypical corepressors. Addition of ATP dynamically converts these complexes to an ‘activated’ state by phosphorylation events, primarily mediated by DNA-dependent protein kinase. Importantly, a ‘natural’ ERE-containing enhancer and nucleosomal EREs recruit similar complexes. We further discovered the mechanism whereby H3K4me3 stimulates ERα-mediated transcription as compared with unmodified nucleosomes. H3K4me3 templates promote specific CoR dynamics in the presence of ATP and AcCoA, as manifested by CBP/p300 and SRC-3 dismissal and SAGA and TFIID stabilization/recruitment.
doi:10.1016/j.molcel.2013.06.007
PMCID: PMC3900250  PMID: 23850489
15.  Perturbing the Cellular Levels of Steroid Receptor Coactivator-2 Impairs Murine Endometrial Function 
PLoS ONE  2014;9(6):e98664.
As pleiotropic coregulators, members of the p160/steroid receptor coactivator (SRC) family control a broad spectrum of transcriptional responses that underpin a diverse array of physiological and pathophysiological processes. Because of their potent coregulator properties, strict controls on SRC expression levels are required to maintain normal tissue functionality. Accordingly, an unwarranted increase in the cellular levels of SRC members has been causally linked to the initiation and/or progression of a number of clinical disorders. Although knockout mouse models have underscored the critical non-redundant roles for each SRC member in vivo, there are surprisingly few mouse models that have been engineered to overexpress SRCs. This deficiency is significant since SRC involvement in many of these disorders is based on unscheduled increases in the levels (rather than the absence) of SRC expression. To address this deficiency, we used recent mouse technology that allows for the targeted expression of human SRC-2 in cells which express the progesterone receptor. Through cre-loxP recombination driven by the endogenous progesterone receptor promoter, a marked elevation in expression levels of human SRC-2 was achieved in endometrial cells that are positive for the progesterone receptor. As a result of this increase in coregulator expression, female mice are severely subfertile due to a dysfunctional uterus, which exhibits a hypersensitivity to estrogen exposure. Our findings strongly support the proposal from clinical observations that increased levels of SRC-2 are causal for a number of endometrial disorders which compromise fertility. Future studies will use this mouse model to decipher the molecular mechanisms that underpin the endometrial defect. We believe such mechanistic insight may provide new molecular descriptors for diagnosis, prognosis, and/or therapy in the clinical management of female infertility.
doi:10.1371/journal.pone.0098664
PMCID: PMC4048228  PMID: 24905738
16.  COUP-TFII inhibits TGF-β-induced growth barrier to promote prostate tumorigenesis 
Nature  2012;493(7431):236-240.
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 cancer1–4. However, the precise mechanism underlying how indolent tumours with PTEN alterations acquire meta-static potential remaisns poorly understood. Recent studies suggest that upregulation of transforming growth factor (TGF)-β signalling triggered by PTEN loss will form a growth barrier as a defence mechanism to constrain prostate cancer progression5, underscoring that TGF-β 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)6–9, a member of the nuclear receptor superfamily, serves as a key regulator to inhibit SMAD4-dependent transcription, and consequently overrides the TGF-β-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-β signalling. These findings reveal that the destruction of the TGF-β-dependent barrier by COUP-TFII is crucial for the progression of PTEN-mutant prostate cancer into a life-threatening disease, and supports COUPTFII as a potential drug target for the intervention of metastatic human prostate cancer.
doi:10.1038/nature11674
PMCID: PMC4022346  PMID: 23201680
17.  Nuclear receptor LRH-1/NR5A2 is required and targetable for liver endoplasmic reticulum stress resolution 
eLife  2014;3:e01694.
Chronic endoplasmic reticulum (ER) stress results in toxicity that contributes to multiple human disorders. We report a stress resolution pathway initiated by the nuclear receptor LRH-1 that is independent of known unfolded protein response (UPR) pathways. Like mice lacking primary UPR components, hepatic Lrh-1-null mice cannot resolve ER stress, despite a functional UPR. In response to ER stress, LRH-1 induces expression of the kinase Plk3, which phosphorylates and activates the transcription factor ATF2. Plk3-null mice also cannot resolve ER stress, and restoring Plk3 expression in Lrh-1-null cells rescues ER stress resolution. Reduced or heightened ATF2 activity also sensitizes or desensitizes cells to ER stress, respectively. LRH-1 agonist treatment increases ER stress resistance and decreases cell death. We conclude that LRH-1 initiates a novel pathway of ER stress resolution that is independent of the UPR, yet equivalently required. Targeting LRH-1 may be beneficial in human disorders associated with chronic ER stress.
DOI: http://dx.doi.org/10.7554/eLife.01694.001
eLife digest
A protein can only work properly if it has been folded into the correct shape. However, it is estimated that about one third of new proteins have the wrong shape. This is a major challenge for cells because misfolded proteins are often toxic, and cause many neurodegenerative and metabolic disorders.
In eukaryotic cells, most protein folding takes place inside a part of the cell called the endoplasmic reticulum (ER). If an incorrectly folded protein is detected, it is prevented from leaving the ER until it is refolded correctly, or destroyed. If too many proteins are misfolded, a process called the unfolded protein response helps the cell to cope with this ‘ER stress’ by expanding the ER and producing more of the molecules that assist protein folding. If this does not relieve the ER stress, the cell self-destructs. Neighboring cells then have to increase protein production to compensate for what would have been produced by the dead cell, thereby increasing the chance that they will also experience ER stress.
Activation of a protein called LRH-1 (short for liver receptor homolog-1) that is produced in the liver, pancreas and intestine can relieve the symptoms of the various metabolic diseases that are associated with chronic ER stress, including type II diabetes and fatty liver disease. However, researchers have been puzzled by the fact that although LRH-1 performs many different roles, its molecular structure provides few clues as to how it can do this.
Mamrosh et al. now confirm the speculated link between LRH-1 and ER stress relief in mice. LRH-1 triggers a previously unknown pathway that can relieve ER stress and is completely independent of the unfolded protein response. Targeting LRH-1 with certain chemical compounds alters its activity, suggesting that drug treatments could be developed to relieve ER stress. As similar targets for drugs have not been found in the unfolded protein response, the discovery of the LRH-1 pathway could lead to new approaches to the treatment of the diseases that result from ER stress.
DOI: http://dx.doi.org/10.7554/eLife.01694.002
doi:10.7554/eLife.01694
PMCID: PMC3987120  PMID: 24737860
ER stress; nuclear receptors; liver metabolism; mouse
18.  Endocardial Cushion Morphogenesis and Coronary Vessel Development Require Chicken Ovalbumin Upstream Promoter-Transcription Factor II 
Objective
Septal defects and coronary vessel anomalies are common congenital heart defects, yet their ontogeny and the underlying genetic mechanisms are not well understood. Here, we investigated the role of chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII, NR2F2) in cardiac organogenesis.
Methods and Results
We analyzed embryos deficient in COUP-TFII and observed a spectrum of cardiac defects, including atrioventricular septal defect, thin-walled myocardium, and abnormal coronary morphogenesis. We show by expression analysis that COUP-TFII is expressed in the endocardium and the epicardium but not in the myocardium of the ventricle. Using endothelial-specific COUP-TFII mutants and molecular approaches, we show that COUP-TFII deficiency resulted in endocardial cushion hypoplasia. This was attributed to the reduced growth and survival of atrioventricular cushion mesenchymal cells and defective epithelial-mesenchymal transformation (EMT) in the underlying endocardium. In addition, the endocardial EMT defect was accompanied by downregulation of Snai1, one of the master regulators of EMT, and upregulation of vascular endothelial-cadherin. Furthermore, we show that although COUP-TFII does not play a major role in the formation of epicardial cell cysts, it is critically important for the formation of epicardium. Ablation of COUP-TFII impairs epicardial EMT and coronary plexus formation.
Conclusion
Our results reveal that COUP-TFII plays cell-autonomous roles in the endocardium and the epicardium for endocardial and epicardial EMT, which are required for proper valve and coronary vessel formation during heart development.
doi:10.1161/ATVBAHA.112.300255
PMCID: PMC3598627  PMID: 22962329
atrioventricular septal defect; cardiac morphogenesis; chicken ovalbumin upstream promoter-transcription factor II; epicardium; epithelial-mesenchymal transformation
19.  COUP-TFII, a prognostic marker and therapeutic target for prostate cancer 
Asian Journal of Andrology  2013;15(3):360-361.
doi:10.1038/aja.2013.12
PMCID: PMC3739656  PMID: 23435470
20.  ERK3 signals through SRC-3 coactivator to promote human lung cancer cell invasion 
The Journal of Clinical Investigation  2012;122(5):1869-1880.
In contrast to the well-studied classic MAPKs, such as ERK1/2, little is known concerning the regulation and substrates of the atypical MAPK ERK3 signaling cascade and its function in cancer progression. Here, we report that ERK3 interacted with and phosphorylated steroid receptor coactivator 3 (SRC-3), an oncogenic protein overexpressed in multiple human cancers at serine 857 (S857). This ERK3-mediated phosphorylation at S857 was essential for interaction of SRC-3 with the ETS transcription factor PEA3, which promotes upregulation of MMP gene expression and proinvasive activity in lung cancer cells. Importantly, knockdown of ERK3 or SRC-3 inhibited the ability of lung cancer cells to invade and form tumors in the lung in a xenograft mouse model. In addition, ERK3 was found to be highly upregulated in human lung carcinomas. Our study identifies a previously unknown role for ERK3 in promoting lung cancer cell invasiveness by phosphorylating SRC-3 and regulating SRC-3 proinvasive activity by site-specific phosphorylation. As such, ERK3 protein kinase may be an attractive target for therapeutic treatment of invasive lung cancer.
doi:10.1172/JCI61492
PMCID: PMC3336992  PMID: 22505454
21.  SRC-3Δ4 mediates the interaction of EGFR with FAK to promote cell migration 
Molecular cell  2010;37(3):321-332.
Summary
EGF induces signal transduction between EGFR and FAK, and FAK is required for EGF-induced cell migration. It is unknown, however, what factor mediates the interaction between EGFR and FAK and leads to EGF-induced FAK phosphorylation. Here we identify SRC-3Δ4, a splicing isoform of the SRC-3 oncogene, as a signaling adaptor that links EGFR and FAK and promotes EGF-induced phosphorylations of FAK and c-Src. We identify three PAK1-mediated phosphorylations in SRC-3Δ4 that promote the localization of SRC-3Δ4 to the plasma membrane and mediate the interactions with EGFR and FAK. Importantly, over-expression of SRC-3Δ4 promotes MDA-MB231-induced breast tumor metastasis. Our findings identify phosphorylated SRC-3Δ4 as a missing adaptor between EGFR and its downstream signaling molecule FAK, to coordinately regulate EGF-induced cell migration. Our study also reveals the new concept that a nuclear receptor coactivator can act in the periphery of a cell to directly mediate activation of an enzyme.
doi:10.1016/j.molcel.2010.01.004
PMCID: PMC2824333  PMID: 20159552
SRC-3Δ4; EGF; EGFR; FAK; PAK1; phosphorylation; cell migration; metastasis
22.  The spatial patterning of mouse cone opsin expression is regulated by BMP signaling through downstream effector COUP-TF nuclear receptors 
Cone photopigments, known as opsins, are pivotal elements and the first detection module employed in color vision. In mice, cone photoreceptors are distributed throughout the retina, and S- and M-opsins have unique expression patterns in the retina with a gradient along the dorsoventral axis; however, the mechanisms regulating the spatial patterning of cone opsin expression have not been well documented. The purpose of this study was to define the mechanisms regulating the spatial patterning of cone opsin expression. By analyzing knockouts for bone morphogenetic protein (BMP) signaling, we found an essential role for BMP in forming cone opsin expression patterns in the retina; however, BMP signaling is activated only transiently in the dorsal half of the retina during early retinal development. Thus, BMP is not likely to play a direct role in opsin gene expression, which starts at a later stage of retinal development. We identified the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) nuclear receptor as a link between BMP and opsin expression. BMP signaling is essential for the correct dorsoventral spatial expression of COUP-TFI and -TFII. Through gain- and loss-of-function analyses, we found that both COUP-TFI and -TFII are required to suppress S-opsin expression in the dorsal retina but that only COUP-TFI plays an essential role in suppressing M-opsin expression in the ventral retina. Based on these findings, we propose a new molecular cascade involving BMP and COUP-TFs that conveys dorsoventral information to direct the expression of cone opsins during retinal development.
doi:10.1523/JNEUROSCI.0951-09.2009
PMCID: PMC2791207  PMID: 19812316
BMP signal; mouse retina; nuclear receptor; S-opsin; M-opsin; photoreceptor
23.  Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) regulates growth and patterning of the postnatal mouse cerebellum 
Developmental biology  2008;326(2):378-391.
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 NSECre/+/CIIF/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 NSECre/+/CIIF/F mice, thereby leading to decreased Akt1 and GSK-3β 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.
doi:10.1016/j.ydbio.2008.11.001
PMCID: PMC2654226  PMID: 19041640
COUP-TFII; IGF-1; Akt1; mTOR; Cerebellum; Foliation; GSK-3β
24.  Expression of Disabled 1 suppresses astroglial differentiation in neural stem cells 
Disabled 1 (Dab1), a cytoplasmic adaptor protein expressed predominantly in the CNS, transduces a Reelin-initiated signaling that controls neuronal migration and positioning during brain development. To determine the role of Dab1 in neural stem cell (NSC) differentiation, we established a culture of neurospheres derived from the embryonic forebrain of the Dab1−/− mice, yotari. Differentiating Dab1−/− neurospheres exhibited a higher expression of GFAP, an astrocytic marker, at the expense of neuronal markers. Under Dab1-deficient condition, the expression of NeuroD, a transcription factor for neuronal differentiation, was decreased and the JAK-STAT pathway was evidently increased during differentiation of NSC, suggesting the possible involvement of Dab1 in astrocyte differentiation via JAK-STAT pathway. Notably, expression of neural and glial markers and the level of JAK-STAT signaling molecules were not changed in differentiating NSC by Reelin treatment, indicating that differentiation of NSC is Reelin-independent. Immunohistochemical analyses showed a decrease in the number of neurons and an increase in the number of GFAP-positive cells in developing yotari brains. Our results suggest that Dab1 participates in the differentiation of NSCs into a specific cell lineage, thereby maintaining a balance between neurogenesis and gliogenesis.
doi:10.1016/j.mcn.2008.08.012
PMCID: PMC2820303  PMID: 18848628
Disabled 1 Reelin; Astrocyte; Neuron; NeuroD; JAK-STAT; Neural stem cells
25.  Bortezomib-Mediated Inhibition of Steroid Receptor Coactivator-3 Degradation Leads to Activated Akt 
Purpose
To assess the safety of administering bortezomib to patients undergoing a radical prostatectomy, to assess pathologic changes induced by bortezomib in prostate cancer specimen, and to verify alterations by the drug in proteasome protein targets.
Experimental Design
Bortezomib is a proteasome inhibitor that has shown activity in vitro and in vivo in prostate cancer. We performed a neoadjuvant clinical trial of bortezomib in men with prostate cancer at high risk of recurrence. The primary endpoints were to evaluate safety and biological activity.
Results
Bortezomib is generally safe in the preoperative setting. Antitumor activity was manifested by tumor cytopathic effect, drops in serum prostate-specific antigen in some patients, and increases in tumor apoptosis. This was associated with cytoplasmic entrapment of nuclear factor-κB. We found an unexpected increase in proliferation in treated tissues and in vitro. Bortezomib also increased SRC-3 levels and phosphorylated Akt, both in vitro and in treated prostate cancer tissues. Knockdown of SRC-3 blocked the increase in activated Akt in vitro. Combined treatment with bortezomib and the Akt inhibitor perifosine was more effective than either agent alone in vitro.
Conclusion
These data suggest that combined therapies targeting the proteasome and the Akt pathway may have increased efficacy.
doi:10.1158/1078-0432.CCR-08-0839
PMCID: PMC2820291  PMID: 19010869

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