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1.  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.
PMCID: PMC1198323  PMID: 15890675
2.  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
3.  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
4.  Endocardial Cushion Morphogenesis and Coronary Vessel Development Require Chicken Ovalbumin Upstream Promoter-Transcription Factor II 
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.
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.
PMCID: PMC3598627  PMID: 22962329
atrioventricular septal defect; cardiac morphogenesis; chicken ovalbumin upstream promoter-transcription factor II; epicardium; epithelial-mesenchymal transformation
5.  COUP-TFII, a prognostic marker and therapeutic target for prostate cancer 
Asian Journal of Andrology  2013;15(3):360-361.
PMCID: PMC3739656  PMID: 23435470
6.  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.
PMCID: PMC3336992  PMID: 22505454
7.  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.
PMCID: PMC2970665  PMID: 20978203
Nuclear Receptor; Endothelial Sprouting; VEGFR-2 signaling
8.  SRC-3Δ4 mediates the interaction of EGFR with FAK to promote cell migration 
Molecular cell  2010;37(3):321-332.
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.
PMCID: PMC2824333  PMID: 20159552
SRC-3Δ4; EGF; EGFR; FAK; PAK1; phosphorylation; cell migration; metastasis
9.  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.
PMCID: PMC2791207  PMID: 19812316
BMP signal; mouse retina; nuclear receptor; S-opsin; M-opsin; photoreceptor
10.  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.
PMCID: PMC2860940  PMID: 20364082
11.  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.
PMCID: PMC2826835  PMID: 18593949
12.  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.
PMCID: PMC2654226  PMID: 19041640
COUP-TFII; IGF-1; Akt1; mTOR; Cerebellum; Foliation; GSK-3β
13.  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.
PMCID: PMC2820303  PMID: 18848628
Disabled 1 Reelin; Astrocyte; Neuron; NeuroD; JAK-STAT; Neural stem cells
14.  Bortezomib-Mediated Inhibition of Steroid Receptor Coactivator-3 Degradation Leads to Activated Akt 
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.
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.
These data suggest that combined therapies targeting the proteasome and the Akt pathway may have increased efficacy.
PMCID: PMC2820291  PMID: 19010869
15.  The Nuclear Orphan Receptor COUP-TFII Plays an Essential Role in Adipogenesis, Glucose Homeostasis, and Energy Metabolism 
Cell metabolism  2009;9(1):77-87.
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.
PMCID: PMC2630393  PMID: 19117548
16.  Dual roles for CoAA and its counterbalancing isoform CoAM in human kidney cell tumorigenesis 
Cancer research  2008;68(19):7887-7896.
Co-Activator Activator (CoAA) has been reported to be a coactivator that regulates steroid receptor-mediated transcription and alternative RNA splicing. Herein we show that CoAA is a dual-function coregulator that inhibits G1/S transition in human kidney cells and suppresses anchorage independent growth and xenograft tumor formation. Suppression occurs in part by downregulating c-myc and its downstream effectors ccnd1 and skp2, and causing accumulation of p27/Kip1 protein. In this cellular setting, CoAA directly represses the proto-oncogene, c-myc by recruiting HDAC3 protein and decreasing both the acetylation of histone H3 and the presence of RNA polymerase II on the c-myc promoter. Interestingly, a splicing isoform of CoAA, Coactivator Modulator (CoAM), antagonizes CoAA-induced G1/S transition and growth inhibition by negatively regulating the mRNA levels of the endogenous CoAA isoform. In addition, we found that expression of CoAA protein is significantly decreased in human renal cell carcinoma as compared to normal kidney. Our study presents evidence that CoAA is a potential tumor suppressor in renal carcinoma and that CoAM is a counterbalancing splice-isoform. This is so far the only example of a nuclear receptor coregulator involved in suppression of kidney cancer, and suggests potentially significant new roles for coregulators in renal cancer biology.
PMCID: PMC2597518  PMID: 18829545
17.  COUP-TFI Coordinates Cortical Patterning, Neurogenesis, and Laminar Fate and Modulates MAPK/ERK, AKT, and ß-Catenin Signaling 
Cerebral Cortex (New York, NY)  2007;18(9):2117-2131.
A major unsolved question in cortical development is how proliferation, neurogenesis, regional growth, regional identity, and laminar fate specification are coordinated. Here we provide evidence, using loss-of-function and gain-of-function manipulations, that the COUP-TFI orphan nuclear receptor promotes ventral cortical fate, promotes cell cycle exit and neural differentiation, regulates the balance of early- and late-born neurons, and regulates the balanced production of different types of layer V cortical projection neurons. We suggest that COUP-TFI controls these processes by repressing Mapk/Erk, Akt, and β-catenin signaling.
PMCID: PMC2733307  PMID: 18165280
β-catenin; COUP-TFI; Mapk/Erk; neurogenesis; PI3K/Akt; proliferation
18.  Atypical Protein Kinase C Regulates Dual Pathways for Degradation of the Oncogenic Coactivator SRC-3/AIB1 
Molecular cell  2008;29(4):465-476.
SRC-3/AIB1 is a steroid receptor coactivator with potent growth promoting activity and its overexpression is sufficient to induce tumorigenesis. Previous studies indicate that the cellular level of SRC-3 is tightly regulated by both ubiquitin-dependent and ubiquitin-independent proteasomal degradation pathways. Atypical protein kinase C (aPKC) is frequently overexpressed in cancers. In the present study, we show that aPKC phosphorylates and specifically stabilizes SRC-3 in a selective ER-dependent manner. We further demonstrate that an acidic residue rich region in SRC-3 is an important determinant for aPKC mediated phosphorylation and stabilization. The mechanism of the aPKC mediated stabilization appears due to a decreased interaction between SRC-3 and the C8 subunit of the 20S core proteasome, thus preventing SRC-3 degradation. Our results demonstrate a new and potent signaling mechanism for regulating SRC-3 levels in cells by coordinate enzymatic inhibition of both ubiquitin-dependent and ubiquitin-independent proteolytic pathways.
PMCID: PMC2293272  PMID: 18313384
19.  Essential Phosphatases and a Phospho-Degron Are Critical for Regulation of SRC-3/AIB1 Coactivator Function and Turnover 
Molecular cell  2008;31(6):835-849.
SRC-3/AIB1 is a master growth coactivator and oncogene, and phosphorylation activates it into a powerful coregulator. Dephosphorylation is a potential regulatory mechanism for SRC-3 function but the identity of such phosphatases remains unexplored. Herein, we report that using functional genomic screening of human Ser/Thr phosphatases targeting SRC-3’s known phosphorylation sites, the phosphatases PDXP, PP1 and PP2A were identified to be key negative regulators of SRC-3 transcriptional coregulatory activity in steroid receptor signalings. PDXP and PP2A dephosphorylate SRC-3 and inhibit its ligand-dependent association with estrogen receptor. PP1 stabilizes SRC-3 protein by blocking its proteasome-dependent turnover through dephosphorylation of two previously unidentified phosphorylation sites (Ser101 and S102) required for activity. These two sites are located within a degron of SRC-3, and are primary determinants of SRC-3 turnover. Moreover, PP1 regulates the oncogenic cell proliferation and invasion functions of SRC-3 in breast cancer cells.
PMCID: PMC2597059  PMID: 18922467
20.  Altered Traveling Wave Propagation and Reduced Endocochlear Potential Associated with Cochlear Dysplasia in the BETA2/NeuroD1 Null Mouse 
The BETA2/NeuroD1 null mouse has cochlear dysplasia. Its cochlear duct is shorter than normal, there is a lack of spiral ganglion neurons, and there is hair cell disorganization. We measured vertical movements of the tectorial membrane at acoustic frequencies in excised cochleae in response to mechanical stimulation of the stapes using laser doppler vibrometry. While tuning curve sharpness was similar between wild-type, heterozygotes, and null mice in the base, null mutants had broader tuning in the apex. At both the base and the apex, null mice had less phase lag accumulation with increasing stimulus frequency than wild-type or heterozygote mice. In vivo studies demonstrated that the null mouse lacked distortion product otoacoustic emissions, and the cochlear microphonic and endocochlear potential were found to be severely reduced. Electrically evoked otoacoustic emissions could be elicited, although the amplitudes were lower than those of wild-type mice. Cochlear cross-sections revealed an incomplete partition malformation, with fenestrations within the modiolus that connected the cochlear turns. Outer hair cells from null mice demonstrated the normal pattern of prestin expression within their lateral walls and normal FM 1-43 dye entry. Overall, these data demonstrate that while tonotopicity can exist with cochlear dysplasia, traveling wave propagation is abnormally fast. Additionally, the presence of electrically evoked otoacoustic emissions suggests that outer hair cell reverse transduction is present, although the acoustic response is shaped by the alterations in cochlear mechanics.
PMCID: PMC2538339  PMID: 17701252
cochlea; cochlear malformation; cochlear mechanics; incomplete partition; hearing loss; cochlear partition
21.  Essential Roles of COUP-TFII in Leydig Cell Differentiation and Male Fertility 
PLoS ONE  2008;3(9):e3285.
Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII; also known as NR2F2), is an orphan nuclear receptor of the steroid/thyroid hormone receptor superfamily. COUP-TFII-null mice die during the early embryonic development due to angiogenesis and cardiovascular defects. To circumvent the early embryonic lethality and investigate the physiological function of COUP-TFII, we knocked out COUP-TFII gene in a time-specific manner by using a tamoxifen inducible Cre recombinase. The ablation of COUP-TFII during pre-pubertal stages of male development results in infertility, hypogonadism and spermatogenetic arrest. Homozygous adult male mutants are defective in testosterone synthesis, and administration of testosterone could largely rescue the mutant defects. Notably, the rescued results also provide the evidence that the major function of adult Leydig cell is to synthesize testosterone. Further phenotypic analysis reveals that Leydig cell differentiation is arrested at the progenitor cell stage in the testes of null mice. The failure of testosterone to resumption of Leydig cell maturation in the null mice indicates that COUP-TFII itself is essential for this process. In addition, we identify that COUP-TFII plays roles in progenitor Leydig cell formation and early testis organogenesis, as demonstrated by the ablation of COUP-TFII at E18.5. On the other hand, when COUP-TFII is deleted in the adult stage after Leydig cells are well differentiated, there are no obvious defects in reproduction and Leydig cell function. Taken together, these results indicate that COUP-TFII plays a major role in differentiation, but not the maintenance of Leydig cells.
PMCID: PMC2553269  PMID: 18818749
22.  Identification of COUP-TFII Orphan Nuclear Receptor as a Retinoic Acid–Activated Receptor 
PLoS Biology  2008;6(9):e227.
The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 Å crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix α10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.
Author Summary
Unlike other classes of receptors, nuclear receptors can bind directly to DNA and act as transcription factors, playing key roles in embryonic development and cellular metabolism. Most nuclear receptors are activated by signal-triggering molecules (ligands) and can regulate their activity by recruiting coactivator proteins. However, the ligands are unknown for a subset of “orphan” nuclear receptors, including the chicken ovalbumin promoter-transcription factors (COUP-TFI and II, and EAR2). COUP-TFs are the most conserved nuclear receptors, with roles in angiogenesis, neuronal development, organogenesis, and metabolic homeostasis. Here we demonstrate that COUP-TFII is a ligand-regulated nuclear receptor that can be activated by unphysiological micromolar concentrations of retinoic acids. We determined the structure of the ligand-free ligand-binding domain of the human COUP-TFII, revealing the autorepressed conformation of the receptor, where helix α10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. These results suggest a mechanism where ligands activate COUP-TFII by releasing the receptor from the autorepressed conformation. The identification of COUP-TFII as a low-affinity retinoic acid receptor suggests ways of searching for the endogenous ligands that may ultimately link retinoic acid and COUP-TF signaling pathways.
Structural and functional studies reveal that the orphan nuclear receptor COUP-TFII is a low-affinity receptor for retinoic acids. paving the way to finding the endogenous ligands that may ultimately link retinoic acid and COUP-TF signaling pathways.
PMCID: PMC2535662  PMID: 18798693
23.  Targeted Disruption of NeuroD, a Proneural Basic Helix-Loop-Helix Factor, Impairs Distal Lung Formation and Neuroendocrine Morphology in the Neonatal Lung*S⃞ 
The Journal of Biological Chemistry  2008;283(30):21160-21169.
Despite the importance of airspace integrity in vertebrate gas exchange, the molecular pathways that instruct distal lung formation are poorly understood. Recently, we found that fibrillin-1 deficiency in mice impairs alveolar formation and recapitulates the pulmonary features of human Marfan syndrome. To further elucidate effectors involved in distal lung formation, we performed expression profiling analysis comparing the fibrillin-1-deficient and wild-type developing lung. NeuroD, a basic helix-loop-helix transcription factor, fulfilled the expression criteria for a candidate mediator of distal lung development. We investigated its role in murine lung development using genetically targeted NeuroD-deficient mice. We found that NeuroD deficiency results in both impaired alveolar septation and altered morphology of the pulmonary neuroendocrine cells. NeuroD-deficient mice had enlarged alveoli associated with reduced epithelial proliferation in the airway and airspace compartments during development. Additionally, the neuroendocrine compartment in these mice manifested an increased number of neuroepithelial bodies but a reduced number of solitary pulmonary neuroendocrine cells in the neonatal lung. Overexpression of NeuroD in a murine lung epithelial cell line conferred a neuroendocrine phenotype characterized by the induction of neuroendocrine markers as well as increased proliferation. These results support an unanticipated role for NeuroD in the regulation of pulmonary neuroendocrine and alveolar morphogenesis and suggest an intimate connection between the neuroendocrine compartment and distal lung development.
PMCID: PMC2475704  PMID: 18339630

Results 1-25 (39)