COUP-TFII Expression Research Articles

16p11.2 deletion accelerates subpallial maturation and increases variability in human iPSC-derived ventral telencephalic organoids.

Inhibitory interneurons regulate cortical circuit activity, and their dysfunction has been implicated in autism spectrum disorder (ASD). 16p11.2 microdeletions are genetically linked to 1% of ASD cases. However, few studies investigate the effects of this microdeletion on interneuron development. Using ventral telencephalic organoids derived from human induced pluripotent stem cells, we have investigated the effect of this microdeletion on organoid size, progenitor proliferation and organisation into neural rosettes, ganglionic eminence marker expression at early developmental timepoints, and expression of the neuronal marker NEUN at later stages. At early stages, deletion organoids exhibited greater variations in size with concomitant increases in relative neural rosette area and the expression of the ventral telencephalic marker COUPTFII, with increased variability in these properties. Cell cycle analysis revealed an increase in total cell cycle length caused primarily by an elongated G1 phase, the duration of which also varied more than normal. At later stages, deletion organoids increased their NEUN expression. We propose that 16p11.2 microdeletions increase developmental variability and may contribute to ASD aetiology by lengthening the cell cycle of ventral progenitors, promoting premature differentiation into interneurons.

COUP-TFII plays a role in cAMP-induced Schwann cell differentiation and in vitro myelination by up-regulating Krox20.

Schwann cells (SCs) are known to produce myelin for saltatory nerve conduction in the peripheral nervous system (PNS). Schwann cell differentiation and myelination processes are controlled by several transcription factors including Sox10, Oct6/Pou3f1, and Krox20/Egr2. Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII/NR2F2) is an orphan receptor that plays a role in the development and differentiation. However, the role of COUP-TFII in the transcriptional regulatory network of SC differentiation has not been fully identified yet. Thus, the objective of this study was to investigate the role and molecular hierarchy of COUP-TFII during cAMP-induced SC differentiation. Our results showed that dibutyryl-cAMP (db-cAMP) increased expression levels of COUP-TFII along with the expressions of Oct6, Krox20, and myelin-related genes known to be related to SC differentiation. Our mechanistic studies showed that COUP-TFII acted downstream of Hsp90/ErbB2/Gab1/ERK-AKT pathway during db-cAMP-induced SC differentiation. In addition, we found that COUP-TFII induced Krox20 expression by directly binding to Krox20-MSE8 as revealed by chromatin immunoprecipitation assay and promoter activity assay. In line with this, the expression of COUP-TFII was increased before up-regulation of Oct6, Krox20, and myelin-related genes in the sciatic nerves during early postnatal myelination period. Finally, COUP-TFII knockdown by COUP-TFII siRNA or via AAV-COUP-TFII shRNA in SCs inhibited db-cAMP-induced SC differentiation and in vitro myelination of sensory axons, respectively. Taken together, these findings indicate that COUP-TFII might be involved in postnatal myelination through induction of Krox20 in SCs. Our results present a new insight into the transcriptional regulatory mechanism in SC differentiation and myelination.

Orphan nuclear receptor COUP-TFII enhances myofibroblast glycolysis leading to kidney fibrosis.

Recent studies demonstrate that metabolic disturbance, such as augmented glycolysis, contributes to fibrosis. The molecular regulation of this metabolic perturbation in fibrosis, however, has been elusive. COUP-TFII (also known as NR2F2) is an important regulator of glucose and lipid metabolism. Its contribution to organ fibrosis is undefined. Here, we found increased COUP-TFII expression in myofibroblasts in human fibrotic kidneys, lungs, kidney organoids, and mouse kidneys after injury. Genetic ablation of COUP-TFII in mice resulted in attenuation of injury-induced kidney fibrosis. A non-biased proteomic study revealed the suppression of fatty acid oxidation and the enhancement of glycolysis pathways in COUP-TFII overexpressing fibroblasts. Overexpression of COUP-TFII in fibroblasts also induced production of alpha-smooth muscle actin (αSMA) and collagen 1. Knockout of COUP-TFII decreased glycolysis and collagen 1 levels in fibroblasts. Chip-qPCR revealed the binding of COUP-TFII on the promoter of PGC1α. Overexpression of COUP-TFII reduced the cellular level of PGC1α. Targeting COUP-TFII serves as a novel treatment approach for mitigating fibrosis in chronic kidney disease and potentially fibrosis in other organs.

Persistent COUP-TFII Expression Underlies the Myopathy and Impaired Muscle Regeneration Observed in Resistance to Thyroid Hormone-Alpha

Myopathic changes, including muscular dystrophy and weakness, are commonly described in hypothyroid and hyperthyroid patients. Thyroid hormone signaling, via activation of thyroid nuclear receptor alpha (THRA), plays an essential role in maintaining muscle mass, function, and regeneration. A mouse model of resistance to thyroid hormone carrying a frameshift mutation in the THRA gene (THRA-PV) is associated with accelerated skeletal muscle loss with aging and impaired regeneration after injury(1,2). We previously demonstrated that the expression of nuclear orphan receptor chicken ovalbumin upstream promoter-factor II (COUP-TFII, or Nr2f2) persists during myogenic differentiation in THRA-PV myoblasts and skeletal muscle of aged THRA- PV mice. COUP-TFII is known to regulate myogenesis negatively and has a role in Duchenne-like Muscular Dystrophies(3). COUP-TFII physically and functionally interacts with THRA in primary myoblasts isolated from WT and THRA-PV mice, as demonstrated via co-immunoprecipitation and chromatin-immunoprecipitation. We observed that satellite cells from THRA-PV mice display impaired myoblast proliferation and in vitro myogenic differentiation compared to WT cells. However, the silencing of COUP-TFII expression using siRNA probes restores in vitro myogenic potential of THRA-PV myoblasts and shifts the mRNA expression profile closer to WT myoblasts, with a higher proliferation of myoblasts and a higher number of fully differentiated myotubes after 5 days of myogenic induction. Moreover, RNAseq analysis on myoblasts from THRA-PV mice after COUP-TFII knockdown shows that COUP-TFII silencing reverses the transcriptomic profile of THRA-PV myoblasts and results in reactivation of pathways involved in muscle function and extracellular matrix remodeling/deposition. These findings indicate that the persistent COUP-TFII expression in THRA-PV mice is responsible for the abnormal muscle phenotype. In conclusion, COUP-TFII and THRA cooperate during murine post-natal myogenesis, and COUP-TFII is critical for the accelerated skeletal muscle loss with aging and impaired muscle regeneration after injury in THRA-PV mice. These studies can help increase our knowledge of the mechanisms involved in thyroid hormone signaling during skeletal muscle regeneration, ultimately increasing the possibility of designing more specific treatments for patients with thyroid hormone-induced myopathies.

Persistent COUP-TFII expression underlies the myopathy and impaired muscle regeneration observed in resistance to thyroid hormone-alpha

Thyroid hormone signaling plays an essential role in muscle development and function, in the maintenance of muscle mass, and in regeneration after injury, via activation of thyroid nuclear receptor alpha (THRA). A mouse model of resistance to thyroid hormone carrying a frame-shift mutation in the THRA gene (THRA-PV) is associated with accelerated skeletal muscle loss with aging and impaired regeneration after injury. The expression of nuclear orphan receptor chicken ovalbumin upstream promoter-factor II (COUP-TFII, or Nr2f2) persists during myogenic differentiation in THRA-PV myoblasts and skeletal muscle of aged THRA-PV mice and it is known to negatively regulate myogenesis. Here, we report that in murine myoblasts COUP-TFII interacts with THRA and modulates THRA binding to thyroid response elements (TREs). Silencing of COUP-TFII expression restores in vitro myogenic potential of THRA-PV myoblasts and shifts the mRNA expression profile closer to WT myoblasts. Moreover, COUP-TFII silencing reverses the transcriptomic profile of THRA-PV myoblasts and results in reactivation of pathways involved in muscle function and extracellular matrix remodeling/deposition. These findings indicate that the persistent COUP-TFII expression in THRA-PV mice is responsible for the abnormal muscle phenotype. In conclusion, COUP-TFII and THRA cooperate during post-natal myogenesis, and COUP-TFII is critical for the accelerated skeletal muscle loss with aging and impaired muscle regeneration after injury in THRA-PV mice.

Development of a putative adverse outcome pathway network for male rat reproductive tract abnormalities with specific considerations for the androgen sensitive window of development

Structured approaches like the adverse outcome pathway (AOP) framework offer great potential for depicting complex toxicological processes in a manner that can facilitate informed integration of mechanistic information in regulatory decisions. While this concept provides a structure for organizing evidence and facilitates consistency in evidence integration; the process, inputs, and manner in which AOPs and AOP networks are developed is still evolving. Following the OECD guiding principles of AOP development, we propose three AOPs for male reproductive tract abnormalities and derive a putative AOP network. The AOPs were developed using a fundamental understanding of the developmental biology of the organs of interest, paying close attention to the gestational timing of key events (KEs) to very specifically inform the domain of life stage applicability for the key event relationships (KERs). Chemical stressor data primarily from studies on low molecular weight phthalates (LMWPs) served to ‘bound’ the pathways of focus in this dynamic period of development and were integrated with the developmental biology data through an iterative process to define KEs and conclude on the extent of evidence in support of the KERs. The AOPs developed describe the linkage between 1) a decrease in Insl3 gene expression and cryptorchidism, 2) the sustained expression of Coup-tfII and hypospadias and 3) the sustained expression of Coup-tfII and altered Wolffian duct development/ epididymal agenesis. A putative AOP network linking AOP2 and AOP3 through decreased steroidogenic biosynthetic protein expression and converging of all AOPS at the population level impaired fertility adverse outcome is proposed. The network depiction specifies and displays the KEs aligned with their occurrence in gestational time. The pathways and network described herein are intended to catalyze collaborative initiatives for expansion into a larger network to enable effective data collection and inform alternative approaches for identifying stressors impacting this sensitive period of male reproductive tract development.

Synovial Mesenchymal Stem Cell-Derived EV-Packaged miR-31 Downregulates Histone Demethylase KDM2A to Prevent Knee Osteoarthritis

Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have emerged as important mediators of intercellular communication in response to cartilage damage. In this study, we sought to characterize the inhibitory role of microRNA (miR)-31 encapsulated in synovial MSC (SMSC)-derived EVs in knee osteoarthritis (OA). The expression of miR-31, lysine demethylase 2A (KDM2A), E2F transcription factor 1 (E2F1), and pituitary tumor transforming gene 1 (PTTG1) was validated in cartilage tissues of knee OA patients. Following SMSC-EV extraction and identification, chondrocytes with the miR-31 inhibitor were added with SMSC-EVs, whereupon the effects of miR-31 on proliferation and migration of chondrocytes were assessed. The interaction among miR-31, KDM2A, E2F1, and PTTG1 in chondrocyte activities was probed in vitro, along with an in vivo mouse knee OA model. We identified downregulated miR-31, E2F1, and PTTG1 and upregulated KDM2A in cartilage tissues of knee OA patients. SMSC-EV-packaged miR-31 potentiated chondrocyte proliferation and migration as well as cartilage formation by targeting KDM2A. Mechanistically, KDM2A bound to the transcription factor E2F1 and inhibited its transcriptional activity. Enrichment of E2F1 in the PTTG1 promoter region activated PTTG1 transcription, accelerating chondrocyte proliferation and migration. SMSC-EVs and EVs from miR-31-overexpressed SMSCs alleviated cartilage damage and inflammation in knee joints in vivo. SMSC-EV-encapsulated miR-31 ameliorates knee OA via the KDM2A/E2F1/PTTG1 axis.

Elevated COUP-TFII expression in dopaminergic neurons accelerates the progression of Parkinson's disease through mitochondrial dysfunction.

Parkinson’s disease (PD) is a neurodegenerative disorder featuring progressive loss of midbrain dopaminergic (DA) neurons that leads to motor symptoms. The etiology and pathogenesis of PD are not clear. We found that expression of COUP-TFII, an orphan nuclear receptor, in DA neurons is upregulated in PD patients through the analysis of public datasets. We show here that through epigenetic regulation, COUP-TFII contributes to oxidative stress, suggesting that COUP-TFII may play a role in PD pathogenesis. Elevated COUP-TFII expression specifically in DA neurons evokes DA neuronal loss in mice and accelerates the progression of phenotypes in a PD mouse model, MitoPark. Compared to control mice, those with elevated COUP-TFII expression displayed reduced cristae in mitochondria and enhanced cellular electron-dense vacuoles in the substantia nigra pars compacta. Mechanistically, we found that overexpression of COUP-TFII disturbs mitochondrial pathways, resulting in mitochondrial dysfunction. In particular, there is repressed expression of genes encoding cytosolic aldehyde dehydrogenases, which could enhance oxidative stress and interfere with mitochondrial function via 3,4-dihydroxyphenylacetaldehyde (DOPAL) buildup in DA neurons. Importantly, under-expression of COUP-TFII in DA neurons slowed the deterioration in motor functions of MitoPark mice. Taken together, our results suggest that COUP-TFII may be an important contributor to PD development and a potential therapeutic target.

OR01-03 Thyroid Receptor Alpha Interacts with COUP-TFII in the Regulation of Postnatal Skeletal Muscle Regeneration

Myopathic changes, including muscular dystrophy and weakness, are commonly described in hypothyroid and hyperthyroid patients. Thyroid hormone signaling, via activation of thyroid nuclear receptors (TRs), plays an essential role in the maintenance of muscle mass, function, and regeneration. TRs are ligand-inducible transcription factors expressed in almost all tissues, including skeletal muscle. In a mouse model of Resistance to Thyroid Hormone carrying a frame-shift mutation in the TRα gene (TRα1PV)1,2 we observed skeletal muscle loss with aging and impaired skeletal muscle regeneration after injury. We recently described that TRα interacts with the nuclear orphan receptor Chicken Ovalbumin Upstream Promoter-factor II (COUP-TFII, or NR2F2), which is known to regulate myogenesis negatively and has a role in Duchenne-like Muscular Dystrophies3. We showed that COUP-TFII expression declines with age in WT mice, while the skeletal muscle of TRα1PV mice shows a sustained significantly higher expression of COUP-TFII. Our findings suggest that the TRα/COUP-TFII interaction might mediate the impaired skeletal muscle phenotype observed in TRα1PV mice. To better characterize this interaction, we isolated SC from 10 months old WT and TRα1PV mice and cultured them in vitro using novel methods established within our lab. Using siRNA probes, we next silenced COUP-TFII and characterized the cells via RNA-seq analysis. In vitro, we assessed myoblast differentiation and proliferation using differentiation assays and EdU incorporation. We observed that satellite cells from TRα1PV mice display impaired myoblast proliferation and in vitro myogenic differentiation compared to WT SCs. However, when COUP-TFII was silenced, the myogenic potential of TRα1PV satellite cells was restored, with a higher proliferation of myoblasts and a higher number of fully differentiated myotubes after 4 days of myogenic induction. RNAseq analysis on satellite cells from TRα1PV mice after COUP-TFII knockdown showed upregulation of genes involved in the myogenic pathway, such as Myod1 and Pax7, and of genes in the thyroid hormone signaling, such as Dio2. Ingenuity Pathway Analysis further showed activation of pathways regarding cell growth, differentiation, matrix remodeling along with muscle function, muscle contractility, and muscle contraction. These in vitro results suggest that by silencing COUP-TFII we promote the myogenic pathway and may further rescue the impaired phenotype of TRα1PV mice. These studies can help increase our knowledge of the mechanisms involved in thyroid hormone signaling in skeletal muscle regeneration, which will ultimately increase the possibility of designing more specific treatments for patients with thyroid hormone-induced myopathies.

In Vitro Model of Coronary Angiogenesis.

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

COUP-TFII in Health and Disease.

The nuclear receptors (NRs) belong to a vast family of evolutionary conserved proteins acting as ligand-activated transcription factors. Functionally, NRs are essential in embryogenesis and organogenesis and in adulthood they are involved in almost every physiological and pathological process. Our knowledge of NRs action has greatly improved in recent years, demonstrating that both their expression and activity are tightly regulated by a network of signaling pathways, miRNA and reciprocal interactions. The Chicken Ovalbumin Upstream Promoter Transcription Factor II (COUP-TFII, NR2F2) is a NR classified as an orphan due to the lack of a known natural ligand. Although its expression peaks during development, and then decreases considerably, in adult tissues, COUP-TFII is an important regulator of differentiation and it is variably implicated in tissues homeostasis. As such, alterations of its expression or its transcriptional activity have been studied and linked to a spectrum of diseases in organs and tissues of different origins. Indeed, an altered COUP-TFII expression and activity may cause infertility, abnormality in the vascular system and metabolic diseases like diabetes. Moreover, COUP-TFII is actively investigated in cancer research but its role in tumor progression is yet to be fully understood. In this review, we summarize the current understanding of COUP-TFII in healthy and pathological conditions, proposing an updated and critical view of the many functions of this NR.

Dysregulation of FGFR signalling by a selective inhibitor reduces germ cell survival in human fetal gonads of both sexes and alters the somatic niche in fetal testes.

STUDY QUESTIONDoes experimental manipulation of fibroblast growth factor 9 (FGF9)-signalling in human fetal gonads alter sex-specific gonadal differentiation?SUMMARY ANSWERInhibition of FGFR signalling following SU5402 treatment impaired germ cell survival in both sexes and severely altered the developing somatic niche in testes, while stimulation of FGF9 signalling promoted Sertoli cell proliferation in testes and inhibited meiotic entry of germ cells in ovaries.WHAT IS KNOWN ALREADYSex-specific differentiation of bipotential gonads involves a complex signalling cascade that includes a combination of factors promoting either testicular or ovarian differentiation and inhibition of the opposing pathway. In mice, FGF9/FGFR2 signalling has been shown to promote testicular differentiation and antagonize the female developmental pathway through inhibition of WNT4.STUDY DESIGN, SIZE, DURATIONFGF signalling was manipulated in human fetal gonads in an established ex vivo culture model by treatments with recombinant FGF9 (25 ng/ml) and the tyrosine kinase inhibitor SU5402 (10 μM) that was used to inhibit FGFR signalling. Human fetal testis and ovary tissues were cultured for 14 days and effects on gonadal development and expression of cell lineage markers were determined.PARTICIPANTS/MATERIALS, SETTING, METHODSGonadal tissues from 44 male and 33 female embryos/fetuses from first trimester were used for ex vivo culture experiments. Tissues were analyzed by evaluation of histology and immunohistochemical analysis of markers for germ cells, somatic cells, proliferation and apoptosis. Culture media were collected throughout the experimental period and production of steroid hormone metabolites was analyzed in media from fetal testis cultures by liquid chromatography–tandem mass spectrometry (LC-MS/MS).MAIN RESULTS AND THE ROLE OF CHANCETreatment with SU5402 resulted in near complete loss of gonocytes (224 vs. 14 OCT4+ cells per mm2, P < 0.05) and oogonia (1456 vs. 28 OCT4+ cells per mm2, P < 0.001) in human fetal testes and ovaries, respectively. This was a result of both increased apoptosis and reduced proliferation in the germ cells. Addition of exogenous FGF9 to the culture media resulted in a reduced number of germ cells entering meiosis in fetal ovaries (102 vs. 60 γH2AX+ germ cells per mm2, P < 0.05), while in fetal testes FGF9 stimulation resulted in an increased number of Sertoli cells (2503 vs. 3872 SOX9+ cells per mm2, P < 0.05). In fetal testes, inhibition of FGFR signalling by SU5402 treatment altered seminiferous cord morphology and reduced the AMH expression as well as the number of SOX9-positive Sertoli cells (2503 vs. 1561 SOX9+ cells per mm2, P < 0.05). In interstitial cells, reduced expression of COUP-TFII and increased expression of CYP11A1 and CYP17A1 in fetal Leydig cells was observed, although there were no subsequent changes in steroidogenesis.LARGE SCALE DATAN/ALIMITATIONS, REASONS FOR CAUTION Ex vivo culture may not replicate all aspects of fetal gonadal development and function in vivo. Although the effects of FGF9 were studied in ex vivo culture experiments, there is no direct evidence that FGF9 acts in vivo during human fetal gonadogenesis. The FGFR inhibitor (SU5402) used in this study is not specific to FGFR2 but inhibits all FGF receptors and off-target effects on unrelated tyrosine kinases should be considered.WIDER IMPLICATIONS OF THE FINDINGSThe findings of this study suggest that dysregulation of FGFR-mediated signalling may affect both testicular and ovarian development, in particular impacting the fetal germ cell populations in both sexes.STUDY FUNDING/COMPETING INTEREST(S)This work was supported in part by an ESPE Research Fellowship, sponsored by Novo Nordisk A/S to A.JØ. Additional funding was obtained from the Erichsen Family Fund (A.JØ.), the Aase and Ejnar Danielsens Fund (A.JØ.), the Danish Government’s support for the EDMaRC programme (A.JU.) and a Wellcome Trust Intermediate Clinical Fellowship (R.T.M., Grant no. 098522). The Medical Research Council (MRC) Centre for Reproductive Health (R.T.M.) is supported by an MRC Centre Grant (MR/N022556/1). The authors have no conflict of interest to disclose.

SAT-560 Role of COUP-TFII and Thyroid Hormone Receptor α in Skeletal Muscle Loss

Myopathic changes including muscular dystrophy and weakness are commonly described in hypothyroid and hyperthyroid patients. The genomic actions of triiodothyronine (T3) are mediated by thyroid hormone nuclear receptors (TRs), which act as ligand-inducible transcription factors in almost all tissues, including skeletal muscle. Impaired skeletal muscle regeneration and sarcopenia with aging have been reported in a mouse model of Resistance to Thyroid Hormone (RTH) carrying a frame-shift mutation in the TRα gene (TRα1PV). One salient finding was a significantly smaller pool of PAX7-positive satellite cells (SCs) in the skeletal muscle of TRα1PV mice. Moreover, SC function during skeletal muscle injury was impaired four days after cardiotoxin (CTX)-induced skeletal muscle injury with a decreased activation of SC and a reduced proliferation of Myf5 expressing cells. TRα plays an important role in the maintenance of muscle mass and skeletal muscle regeneration after injury with aging, and loss of skeletal muscle mass with aging in TRα1PV mice was reported to be associated with loss of SC pool {1,2}. Interestingly, overexpression of the nuclear orphan receptor Chicken Ovalbumin Upstream Promoter-factor II (COUP-TFII or NR2F2) in murine satellite cells have been shown to induce a similar skeletal muscle phenotype, including skeletal muscle loss with aging, and inhibit myogenesis through modulation of Myf5 and MyoD expression {3}. We detected a higher expression of COUP-TFII in C2C12, a murine myoblast cell line, during proliferation and a decline during differentiation into myotube. Moreover, we analyzed the skeletal muscle of mice at different ages and we found a higher expression of COUP-TFII in the first 2 months of life followed by a decline with age. Thus, suggesting the important role of COUP-TFII in modulating post-natal myogenesis and onset of sarcopenia with aging. In proliferating C2C12 myoblasts and SCs from wild-type and TRα1PV mice, we demonstrated via co-immunoprecipitation that COUP-TFII and TRα interact. In addition, the skeletal muscle of TRα1PV mice showed significantly higher expression of COUP-TFII compared to their WT siblings, and in TRα1PV mice COUP-TFII expression remained higher with skeletal muscle aging. These results suggest a COUP-TFII role in skeletal muscle loss with aging and impaired skeletal muscle regeneration in TRαPV mice that may be mediated by COUP-TFII-TRα interaction. These new insights can provide a therapeutic target to prevent or treat myopathies, such as sarcopenia and Duchenne-like muscular dystrophy. 1. Milanesi A et al, Endocrinology 2015. 2. Milanesi A et al, Thyroid, 2017. 3. Lee HJ et al, Scientific Reports 2017. Sources of Research Support: NIH grants and VA MERIT GRANT.

Determination of critical shear stress for maturation of human pluripotent stem cell‐derived endothelial cells towards an arterial subtype

Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) present an attractive alternative to primary EC sources for vascular grafting. However, there is a need to mature them towards either an arterial or venous subtype. A vital environmental factor involved in the arteriovenous specification of ECs during early embryonic development is fluid shear stress; therefore, there have been attempts to employ adult arterial shear stress conditions to mature hPSC-ECs. However, hPSC-ECs are naïve to fluid shear stress, and their shear responses are still not well understood. Here, we used a multiplex microfluidic platform to systematically investigate the dose-time shear responses on hPSC-EC morphology and arterial-venous phenotypes over a range of magnitudes coincidental with physiological levels of embryonic and adult vasculatures. The device comprised of six parallel cell culture chambers that were individually linked to flow-setting resistance channels, allowing us to simultaneously apply shear stress ranging from 0.4 to 15 dyne/cm 2 . We found that hPSC-ECs required up to 40 hr of shear exposure to elicit a stable phenotypic change. Cell alignment was visible at shear stress <1 dyne/cm 2 , which was independent of shear stress magnitude and duration of exposure. We discovered that the arterial markers NOTCH1 and EphrinB2 exhibited a dose-dependent increase in a similar manner beyond a threshold level of 3.8 dyne/cm 2 , whereas the venous markers COUP-TFII and EphB4 expression remained relatively constant across different magnitudes. These findings indicated that hPSC-ECs were sensitive to relatively low magnitudes of shear stress, and a critical level of ~4 dyne/cm 2 was sufficient to preferentially enhance their maturation into an arterial phenotype for future vascular tissue engineering applications.

The microRNA miR-21 Is a Mediator of FGF8 Action on Cortical COUP-TFI Translation

SummaryThe morphogen FGF8 plays a pivotal role in neocortical area patterning through its inhibitory effect on COUP-TFI/Nr2f1 anterior expression, but its mechanism of action is poorly understood. We established an in vitro model of mouse embryonic stem cell corticogenesis in which COUP-TFI protein expression is inhibited by the activation of FGF8 in a time window corresponding to cortical area patterning. Interestingly, overexpression of the COUP-TFI 3′UTR reduces the inhibitory effect of FGF8 on COUP-TFI translation. FGF8 induces the expression of few miRNAs targeting COUP-TFI 3′UTR in silico. We found that the functional inhibition of miR-21 can effectively counteract the inhibitory effect of FGF8 in vitro and regulate COUP-TFI protein levels in vivo. Accordingly, miR-21 expression is complementary to COUP-TFI expression during corticogenesis. These data support a translational control of COUP-TFI gradient expression by FGF8 via miR-21 and contribute to our understanding of how regionalized expression is established during neocortical area mapping.

Abstract 334: Temporal Regulation of Retinoic Acid Delineates First Heart Field Ventricular and Atrial Cardiac Differentiation of Human Pluripotent Stem Cells

Retinoic acid plays a pivotal role during the heart development. The mammalian heart is formed from cardiac progenitors in the first and second heart fields (FHF and SHF). FHF progenitors populate the left ventricle and the atria; SHF progenitors populate the right ventricle, outflow tract and parts of the atria. Loss of retinoic acid signaling leads to depletion of FHF progenitors and compensatory increase in SHF progenitors. Here we hypothesized that mimicking the in vivo retinoic acid signaling during human pluripotent stem cell differentiation could lead to FHF cardiomyocytes.Human pluripotent stem cells were differentiated to cardiac lineage by treatment with Activin A, BMP4 and bFGF (control) for the first three days (d0-3). This lead to &gt;70% Brachyury+ mesodermal cells, and &gt;90% of Brachyury+ cells became cTnT+ cardiac myocytes. Retinoic acid signaling activated with exogenous RA (1μM) from d5-8 (RA protocol). This RA protocol was compared to another protocol in which d5-8 retinoic acid activation was followed by initial inhibition with a pan-retinoic acid (RiRA protocol). At d14, both RA and RiRA cardiomyocytes expressed higher transcript and protein levels of the FHF markers, NKX2.5, TBX5and HCN4, but lower levels of the SHF markers, ISL1, MEF2c, FGF8/10, compared to control. RiRA cardiomyocytes showed highest expression of ventricular markers, IRX4 and HRT2 , lowest expression of an atrial specific marker CoupTFII, which were mirrored in RA cardiomyocytes. In line with their atrial/ventricular-specific gene expression profile, RA cardiomyocytes demonstrated higher beating rates, higher maximum shortening velocity, faster conduction velocity, higher atrial specific ANP secretion and higher sensitivity to atrial-selective sodium blocker, ranolazine compared to control. In contrast, RiRA cardiomyocytes generated higher force but shorter action potential duration compared to control.Taken together, the data demonstrate temporal regulation of retinoic acid signaling can not only accurately recapitulate ventricular to atrial cardiomyocyte differentiation by delineating FHF vs. SHF lineages.

Neuron-Astroglia Cell Fate Decision in the Adult Mouse Hippocampal Neurogenic Niche Is Cell-Intrinsically Controlled by COUP-TFI In Vivo

In the dentate gyrus (DG) of the mouse hippocampus, neurogenesis and astrogliogenesis persist throughout life. Adult-born neurons and astrocytes originate from multipotent neural stem cells (NSCs) whose activity is tightly regulated within the neurogenic niche. However, the cell-intrinsic mechanisms controlling neuron-glia NSC fate choice are largely unknown. Here, we show COUP-TFI/NR2F1 expression in DG NSCs and its downregulation upon neuroinflammation. By using invivo inducible knockout lines, a retroviral-based loss-of-function approach and genetic fate mapping, we demonstrate that COUP-TFI inactivation in adult NSCs and/or mitotic progenitors reduces neurogenesis and increases astrocyte production without depleting the NSC pool. Moreover, forced COUP-TFI expression in adult NSCs/progenitors decreases DG astrogliogenesis and rescues the neuro-astrogliogenic imbalance under neuroinflammation. Thus, COUP-TFI is necessary and sufficient to promote neurogenesis by suppressing astrogliogenesis. Our data propose COUP-TFI as a central regulator of the neuron-astroglia cell fate decision and a key modulator during neuroinflammation in the adult hippocampus.

Role of COUP‐TFII in Glucose Homeostasis in Dahl Salt Sensitive Rats

The Interaction between genetic and environmental factors plays an important role in disease progression. One of the major environmental factor includes diets, which governs significant attention based on the broad spectrum of effect in cardiovascular and associated diseases. Consumptions of diets that are rich in calories and salts can significantly increases the risk for developing life threatening cardiovascular events including hypertension and metabolic syndrome. Through GWAS studies, a locus with significant association for hypertension was found in human chromosome 15 containing the gene Chicken Ovalbumin Upstream Promotor Transcription Factor II (COUP‐TFII). To validate this gene, a mutant rat was developed with a 15‐base pair deletion in Dahl Salt Sensitive (SS) rat background. Under high salt diet regimen, the Coup‐TfIImutant rat had an improved cardiac and renal function when compared to SS rats. Being a known transcriptional regulator, Coup‐TfII also plays a role in other physiological events including glucose homeostasis and energy metabolism. In H4IIE rat hepatoma cells, Coup‐TfII expression modulated in response to glucose levels; under high glucose condition Coup‐TfII expression was significantly down‐regulated (p&lt; 0.05); whereas in low glucose condition Coup‐TfII expression was significantly up‐regulated (p&lt; 0.05). In addition, Coup‐TfII protein expression is found to negatively correlate with insulin regardless on the amount of glucose present in H4IIE cells (p&lt; 0.05). Similar to our in‐vitro findings, Coup‐TfII gene expression was significantly up‐regulated in SS rat liver during fasting as compared to re‐fed condition (p&lt; 0.05). Coup‐TfIImutant rat have also showed a significant decrease in fasting blood glucose levels (p&lt; 0.05) compared to SS rat along with a significant decrease in their body weight (p&lt; 0.05). Furthermore, the Coup‐TfIImutant rat demonstrated a superior glucose response and insulin response as measured by glucose tolerance test (GTT) and Insulin tolerance test (ITT) (p&lt; 0.05). Further, under fasting condition the mRNA expression analysis in livers of SS and CoupTfIImutant rats revealed that the key enzymes involved in both beta oxidation and gluconeogenesis was significantly down regulated in Coup‐TfIImutant rats compared to SS rats (p&lt; 0.05). Overall, our preliminary findings suggest that Coup‐TfII is important for glucose homeostasis in SS rats. Further understanding of Coup‐TfII function will help to dissect the relationship between metabolic syndrome and blood pressure regulation in SS rats.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The expression of p-mTOR and COUP-TFII correlates with increased lymphangiogenesis and lymph node metastasis in prostate adenocarcinoma

BackgroundMammalian target of rapamycin (mTOR) is a central regulator of major cellular processes such as growth and proliferation. Deregulated mTOR signaling is implicated in a wide spectrum of human malignancies including prostate cancer. The aim of this study is to address the role of phosphorylated mTOR (p-mTOR) in prostate adenocarcinoma-induced lymphangiogenesis and lymph node metastasis as well as to investigate its relationship with chicken ovalbumin upstream promoter transcriptional factor 2 (COUP-TFII) and the vascular endothelial growth factors A/C (VEGF A/C). MethodsWe analyzed 92 paraffin embedded specimens from patients with prostate cancer who underwent radical prostatectomy with pelvic lymph node (LN) dissection. Twenty-four of these men were pathologically assessed to have regional LN metastasis (pN1 group) and 68 with negative lymph nodes (pN0 group). Lymph vessel density was measured using anti-D2-40 and anti-LYVE-1 antibodies. The expression of p-mTOR, COUP-TFII, and VEGF A/C was also evaluated by immunohistochemistry. ResultsSpecimens from pN1 group exhibited higher cytoplasmic p-mTOR expression compared to pN0 specimens. Mean vessel densities assessed by COUP-TFII and D2-40 were increased in pN1 tumors and positively associated with higher p-mTOR expression. Interestingly, increased expression of p-mTOR was positively associated with COUP-TFII expression in cancer cells and elevated immunoreactivity for both VEGF A and C, which in turn exhibited higher expression in pN1 group. ConclusionsOur findings suggest that increased p-mTOR and COUP-TFII expression are implicated in human prostate adenocarcinoma-induced lymphangiogenesis and LN metastasis.

Overexpression of COUP‑TFII suppresses proliferation and metastasis of human gastric cancer cells.

The abnormal expression of the chicken ovalbumin upstream promoter transcription factor 2 (COUP-TFII) is associated with numerous forms of cancer, including gastric, prostate, colon and lung cancer. However, previous studies investigating the association between COUP-TFII expression and the occurrence, recurrence, invasion and metastasis of gastric cancer are limited in number. In the present study, it was revealed that the expression of COUP-TFII is significantly reduced in gastric carcinoma tissues compared with normal gastric mucosa cells (GES-1). In addition, the expression of COUP-TFII was also reduced in gastric cancer cell lines compared with GES-1 cells. Furthermore, it was revealed that ectopic expression of COUP-TFII was able to suppress the proliferation, migration and invasion of gastric cells, as well as inhibit hepatic metastasis, in vivo. In addition, it was demonstrated that COUP-TFII knockdown was able to promote the proliferation, migration and invasion of GES-1 cells in vitro. Furthermore, database analysis suggested that COUP-TFII expression in patients with gastric cancer is correlated with clinical stage classification and increased expression levels of COUP-TFII improved overall survival rates in patients with gastric cancer. The results of the present study suggest that COUP-TFII functions as a significant regulatory suppressor of gastric cancer growth and metastasis, and suggests that COUP-TFII may serve as a novel diagnostic and prognostic biomarker for gastric cancer metastasis.