Nuclear Receptor Subfamily Research Articles

Role of Nuclear receptor subfamily 1 group D member 1 in the proliferation, migration of VSMC and vascular intimal hyperplasia.

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) causes neointimal hyperplasia after percutaneous vascular interventions. Nuclear receptor subfamily 1 group D member 1 (NR1D1), a crucial member of circadian clock, is involved in regulation on atherosclerosis and cellular proliferation. However, whether NR1D1 affects vascular neointimal hyperplasia remains unclear. In the current study, we found that activating NR1D1 reduced injury-induced vascular neointimal hyperplasia. Overexpression of NR1D1 reduced the number of Ki-67-positive VSMCs and migrated VSMCs after platelet-derived growth factor (PDGF)-BB treatment. Mechanistically, NR1D1 suppressed the phosphorylation of AKT and two main effectors of mammalian target of rapamycin complex 1 (mTORC1), S6 and 4EBP1 in PDGF-BB-challenged VSMCs. Both re-activation of mTORC1 by Tuberous sclerosis 1 siRNA (siTsc1) and re-activation of AKT by SC-79 abolished NR1D1-mediated inhibitory effects on proliferation and migration of VSMCs. Moreover, decreased mTORC1 activity induced by NR1D1 was also reversed by SC-79. Simultaneously, Tsc1 knockdown abolished the vascular protective effects of NR1D1 in vivo. In conclusion, NR1D1 reduces vascular neointimal hyperplasia by suppressing proliferation and migration of VSMCs in an AKT/mTORC1-dependent manner.

Reduced NR2F2 Expression in the Host Response to Infectious Bursal Disease Virus Infection Suppressed Viral Replication by Enhancing Type I Interferon Expression by Targeting SOCS5.

Nuclear receptors are ligand-activated transcription factors that play an important role in regulating innate antiviral immunity and other biological processes. However, the role of nuclear receptors in the host response to infectious bursal disease virus (IBDV) infection remains elusive. In this study, we show that IBDV infection or poly(I·C) treatment of DF-1 or HD11 cells markedly decreased nuclear receptor subfamily 2 group F member 2 (NR2F2) expression. Surprisingly, knockdown, knockout, or inhibition of NR2F2 expression in host cells remarkably inhibited IBDV replication and promoted IBDV/poly(I·C)-induced type I interferon and interferon-stimulated genes expression. Furthermore, our data show that NR2F2 negatively regulates the antiviral innate immune response by promoting the suppressor of cytokine signaling 5 (SOCS5) expression. Thus, reduced NR2F2 expression in the host response to IBDV infection inhibited viral replication by enhancing the expression of type I interferon by targeting SOCS5. These findings reveal that NR2F2 plays a crucial role in antiviral innate immunity, furthering our understanding of the mechanism underlying the host response to viral infection. IMPORTANCE Infectious bursal disease (IBD) is an immunosuppressive disease causing considerable economic losses to the poultry industry worldwide. Nuclear receptors play an important role in regulating innate antiviral immunity. However, the role of nuclear receptors in the host response to IBD virus (IBDV) infection remains elusive. Here, we report that NR2F2 expression decreased in IBDV-infected cells, which consequently reduced SOCS5 expression, promoted type I interferon expression, and suppressed IBDV infection. Thus, NR2F2 serves as a negative factor in the host response to IBDV infection by regulating SOCS5 expression, and intervention in the NR2F2-mediated host response by specific inhibitors might be employed as a strategy for prevention and treatment of IBD.

Development and pharmacological evaluation of a new chemical series of potent pan-ERR agonists, identification of SLU-PP-915

Estrogen-related receptors (ERR) are an orphan nuclear receptor sub-family that play a critical role in regulating gene transcription for several physiological processes including mitochondrial function, cellular energy utilization and homeostasis. They have also been implicated to play a role in several pathological conditions. Herein, we report the identification, synthesis, structure-activity relationships and pharmacological evaluation of a new chemical series of potent pan-ERR agonists. This template was designed for ERRγ starting from the known acyl hydrazide template and compounds such as agonist GSK-4716 employing a structure-based drug design approach. This led to the preparation of a series of 2,5-disubstituted thiophenes from which several were found to be potent agonists of ERRγ in cell-based co-transfection assays. Additionally, direct binding to ERRγ was established through 1H NMR protein-ligand binding experiments. Compound optimization revealed that the phenolic or aniline groups could be replaced with a boronic acid moiety, which was able to maintain activity and demonstrated improved metabolic stability in microsomal in vitro assays. Further pharmacological evaluation of these compounds showed that they had roughly equivalent agonist activity on ERR isoforms α and β representing an ERR pan-agonist profile. One potent agonist, SLU-PP-915 (10s), which contained a boronic acid moiety was profiled in gene expression assays and found to significantly upregulate the expression of ERR target genes such as peroxisome-proliferator activated receptor γ co-activators-1α, lactate dehydrogenase A, DNA damage inducible transcript 4 and pyruvate dehydrogenase kinase 4 both in vitro and in vivo.

Nuclear Receptor Subfamily 2 Group E Member 3 (NR2E3): Role in Retinal Development and Disease.

NR2E3 is a nuclear hormone receptor gene required for the correct development of the retinal rod photoreceptors. Expression of NR2E3 protein in rod cell precursors suppresses cone-specific gene expression and, in concert with other transcription factors including NRL, activates the expression of rod-specific genes. Pathogenic variants involving NR2E3 cause a spectrum of retinopathies, including enhanced S-cone syndrome, Goldmann-Favre syndrome, retinitis pigmentosa, and clumped pigmentary retinal degeneration, with limited evidence of genotype-phenotype correlations. A common feature of NR2E3-related disease is an abnormally high number of cone photoreceptors that are sensitive to short wavelength light, the S-cones. This characteristic has been supported by mouse studies, which have also revealed that loss of Nr2e3 function causes photoreceptors to develop as cells that are intermediate between rods and cones. While there is currently no available cure for NR2E3-related retinopathies, there are a number of emerging therapeutic strategies under investigation, including the use of viral gene therapy and gene editing, that have shown promise for the future treatment of patients with NR2E3 variants and other inherited retinal diseases. This review provides a detailed overview of the current understanding of the role of NR2E3 in normal development and disease, and the associated clinical phenotypes, animal models, and therapeutic studies.

Melatonin Alters Innate Immune Function in Infants with Neonatal Encephalopathy

Introduction: Melatonin has been suggested an adjunctive therapy in neonatal encephalopathy (NE). Melatonin reduces oxidative stress and neutrophil activation; however, the immunological effects in NE have not been studied. Methods: Infants with NE and neonatal controls were prospectively recruited. Whole blood was sampled in the first week of life. Following endotoxin and or melatonin treatment, diurnal variation was measured by RT PCR for circadian rhythm genes (brain and Muscle Arnt-Like protein [BMAL1], circadian locomotor output cycles kaput [CLOCK], Nuclear Receptor Subfamily 1 Group D Member 2 [REV Erβ], and cryptochrome circadian clock [CRY]). Neutrophil and monocyte cell surface markers of activation CD11b, reactive oxygen intermediates (ROIs), and Toll-like receptor (TLR)-4 were also examined by flow cytometry in matching samples. Results: Serum and RNA samples from forty infants were included (controls n = 20; NE n = 20) over the first week of life. Melatonin reduced neutrophil CD11b and TLR-4 expression in response to LPS in infants with NE compared to controls. There were no differences in ROIs. BMAL1 and CLOCK baseline gene expression levels were similar. BMAL1 was significantly decreased with LPS stimulation in NE. There was no significant diurnal variation in melatonin, neutrophil, and monocyte function or circadian genes. Conclusions: Melatonin alters immune function ex vivo in infants with NE. Infants with NE have altered immune circadian responses following LPS stimulation, which have potential for modulation.

Endothelial TIE1 Restricts Angiogenic Sprouting to Coordinate Vein Assembly in Synergy With Its Homologue TIE2

BACKGROUND: Vascular growth followed by vessel specification is crucial for the establishment of a hierarchical blood vascular network. We have shown that TIE2 is required for vein development while little is known about its homologue TIE1 (tyrosine kinase with immunoglobulin-like and EGF [epithelial growth factor]-like domains 1) in this process. METHODS: We analyzed functions of TIE1 as well as its synergy with TIE2 in the regulation of vein formation by employing genetic mouse models targeting Tie1 , Tek , and Nr2f2 , together with in vitro cultured endothelial cells to decipher the underlying mechanism. RESULTS: Cardinal vein growth appeared normal in TIE1-deficient mice, whereas TIE2 deficiency altered the identity of cardinal vein endothelial cells with the aberrant expression of DLL4 (delta-like canonical Notch ligand 4). Interestingly, the growth of cutaneous veins, which was initiated at approximately embryonic day 13.5, was retarded in mice lack of TIE1. TIE1 deficiency disrupted the venous integrity, displaying increased sprouting angiogenesis and vascular bleeding. Abnormal venous sprouts with defective arteriovenous alignment were also observed in the mesenteries of Tie1 -deleted mice. Mechanistically, TIE1 deficiency resulted in the decreased expression of venous regulators including TIE2 and COUP-TFII (chicken ovalbumin upstream promoter transcription factor, encoded by Nr2f2 , nuclear receptor subfamily 2 group F member 2) while angiogenic regulators were upregulated. The alteration of TIE2 level by TIE1 insufficiency was further confirmed by the siRNA-mediated knockdown of Tie1 in cultured endothelial cells. Interestingly, TIE2 insufficiency also reduced the expression of TIE1. Combining the endothelial deletion of Tie1 with 1 null allele of Tek resulted in a progressive increase of vein-associated angiogenesis leading to the formation of vascular tufts in retinas, whereas the loss of Tie1 alone produced a relatively mild venous defect. Furthermore, the induced deletion of endothelial Nr2f2 decreased both TIE1 and TIE2. CONCLUSIONS: Findings from this study imply that TIE1 and TIE2, together with COUP-TFII, act in a synergistic manner to restrict sprouting angiogenesis during the development of venous system.

Transcription Factor BCL11A Regulates Schwann Cell Behavior During Peripheral Nerve Regeneration.

Nerve injury-induced Schwann cell dedifferentiation helps to construct a favorable microenvironment for axon growth. Transcription factors regulate cell reprogramming and thus may be critical for Schwann cell phenotype switch during peripheral nerve regeneration. Here, we show that transcription factor B-cell lymphoma/leukemia 11A (BCL11A) is up-regulated in Schwann cells of injured peripheral nerves. Bcl11a silencing suppresses Schwann cell viability, decreases Schwann cell proliferation and migration rates, and impairs the debris clearance ability of Schwann cells. Reduced Bcl11a in injured peripheral nerves results in restricted axon elongation and myelin wrapping, leading to recovery failure. Mechanistically, we demonstrate that BCL11A may mediate Schwann cell activity through binding to the promoter of nuclear receptor subfamily 2 group F member 2 (Nr2f2) and regulating Nr2f2 expression. Collectively, we conclude that BCL11A is essential for Schwann cell activation and peripheral nerve regeneration, providing a potential therapeutic target for the treatment of peripheral nerve injury.

In Silico Analysis Predicts Nuclear Factors NR2F6 and YAP1 as Mesenchymal Subtype-Specific Therapeutic Targets for Ovarian Cancer Patients

Background: Tumour heterogeneity in high-grade serous ovarian cancer (HGSOC) is a proposed cause of acquired resistance to treatment and high rates of relapse. Among the four distinct molecular subtypes of HGSOC, the mesenchymal subtype (MES) has been observed with high frequency in several study cohorts. Moreover, it exhibits aggressive characteristics with poor prognosis. The failure to adequately exploit such subtypes for treatment results in high mortality rates, highlighting the need for effective targeted therapeutic strategies that follow the idea of personalized medicine (PM). Methods: As a proof-of-concept, bulk and single-cell RNA data were used to characterize the distinct composition of the tumour microenvironment (TME), as well as the cell–cell communication and its effects on downstream transcription of MES. Moreover, transcription factor activity contextualized with causal inference analysis identified novel therapeutic targets with potential causal impact on transcription factor dysregulation promoting the malignant phenotype. Findings: Fibroblast and macrophage phenotypes are of utmost importance for the complex intercellular crosstalk of MES. Specifically, tumour-associated macrophages were identified as the source of interleukin 1 beta (IL1B), a signalling molecule with significant impact on downstream transcription in tumour cells. Likewise, signalling molecules tumour necrosis factor (TNF), transforming growth factor beta (TGFB1), and C-X-C motif chemokine 12 (CXCL12) were prominent drivers of downstream gene expression associated with multiple cancer hallmarks. Furthermore, several consistently hyperactivated transcription factors were identified as potential sources for treatment opportunities. Finally, causal inference analysis identified Yes-associated protein 1 (YAP1) and Nuclear Receptor Subfamily 2 Group F Member 6 (NR2F6) as novel therapeutic targets in MES, verified in an independent dataset. Interpretation: By utilizing a sophisticated bioinformatics approach, several candidates for treatment opportunities, including YAP1 and NR2F6 were identified. These candidates represent signalling regulators within the cellular network of the MES. Hence, further studies to confirm these candidates as potential targeted therapies in PM are warranted.

Screening of novel biomarkers for breast cancer based on WGCNA and multiple machine learning algorithms.

Breast cancer (BC) ranks first in incidence among women, with approximately 2 million new cases per year. Therefore, it is essential to investigate emerging targets for BC patients' diagnosis and prognosis. We analyzed gene expression data from 99 normal and 1,081 BC tissues in The Cancer Genome Atlas (TCGA) database. Differentially expressed genes (DEGs) were identified using "limma" R package, and relevant modules were chosen through Weighted Gene Coexpression Network Analysis (WGCNA). Intersection genes were obtained by matching DEGs to WGCNA module genes. Functional enrichment studies were performed on these genes using Gene Ontology (GO), Disease Ontology (DO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Biomarkers were screened via Protein-Protein Interaction (PPI) networks and multiple machine-learning algorithms. The Gene Expression Profiling Interactive Analysis (GEPIA), The University of ALabama at Birmingham CANcer (UALCAN), and Human Protein Atlas (HPA) databases were employed to examine mRNA and protein expression of eight biomarkers. Kaplan-Meier mapper tool assessed their prognostic capabilities. Key biomarkers were analyzed via single-cell sequencing, and their relationship with immune infiltration was examined using Tumor Immune Estimation Resource (TIMER) database and "xCell" R package. Lastly, drug prediction was conducted based on the identified biomarkers. We identified 1,673 DEGs and 542 important genes through differential analysis and WGCNA, respectively. Intersection analysis revealed 76 genes, which play significant roles in immune-related viral infection and IL-17 signaling pathways. DIX domain containing 1 (DIXDC1), Dual specificity phosphatase 6 (DUSP6), Pyruvate dehydrogenase kinase 4 (PDK4), C-X-C motif chemokine ligand 12 (CXCL12), Interferon regulatory factor 7 (IRF7), Integrin subunit alpha 7 (ITGA7), NIMA related kinase 2 (NEK2), and Nuclear receptor subfamily 3 group C member 1 (NR3C1) were selected as BC biomarkers using machine-learning algorithms. NEK2 was the most critical gene for diagnosis. Prospective drugs targeting NEK2 include etoposide and lukasunone. Our study identified DIXDC1, DUSP6, PDK4, CXCL12, IRF7, ITGA7, NEK2, and NR3C1 as potential diagnostic biomarkers for BC, with NEK2 having the highest potential to aid in diagnosis and prognosis in clinical settings.

NR4A1 Aggravates Myocardial Ischaemia-Reperfusion Injury by Inhibiting OPA1-Mediated Mitochondrial Fusion.

Mitochondrial fusion is an important process that protects the myocardium. However, mitochondrial fusion is often inhibited in myocardial ischaemia-reperfusion injury (IR). The upstream mechanism of this effect is unclear. Nuclear receptor subfamily 4 group A member 1 (NR4A1) can aggravate myocardial IR and increase the level of oxidative stress, thereby affecting mitochondrial function and morphology. Inhibiting NR4A1 can improve oxidative stress levels and mitochondrial function and morphology, thereby reducing IR. Downregulating NR4A1 increases the expression level of the mitochondrial fusion-related protein optic atrophy 1 (OPA1), which is associated with these benefits. Inhibiting OPA1 expression with MYLS22 abrogates the effects of NR4A1 downregulation on IR. Furthermore, NR4A1 disrupts mitochondrial dynamics and activates the STING and NF-κB pathways. Insufficient mitochondrial fusion and increased apoptosis and inflammatory reactions worsen irreversible damage to cardiomyocytes. In conclusion, NR4A1 can exacerbate IR by inhibiting OPA1, causing mitochondrial damage.

NRPreTo: A Machine Learning-Based Nuclear Receptor and Subfamily Prediction Tool.

The nuclear receptor (NR) superfamily includes phylogenetically related ligand-activated proteins, which play a key role in various cellular activities. NR proteins are subdivided into seven subfamilies based on their function, mechanism, and nature of the interacting ligand. Developing robust tools to identify NR could give insights into their functional relationships and involvement in disease pathways. Existing NR prediction tools only use a few types of sequence-based features and are tested on relatively similar independent datasets; thus, they may suffer from overfitting when extended to new genera of sequences. To address this problem, we developed Nuclear Receptor Prediction Tool (NRPreTo), a two-level NR prediction tool with a unique training approach where in addition to the sequence-based features used by existing NR prediction tools, six additional feature groups depicting various physiochemical, structural, and evolutionary features of proteins were utilized. The first level of NRPreTo allows for the successful prediction of a query protein as NR or non-NR and further subclassifies the protein into one of the seven NR subfamilies in the second level. We developed Random Forest classifiers to test on benchmark datasets, as well as the entire human protein datasets from RefSeq and Human Protein Reference Database (HPRD). We observed that using additional feature groups improved the performance. We also observed that NRPreTo achieved high performance on the external datasets and predicted 59 novel NRs in the human proteome. The source code of NRPreTo is publicly available at https://github.com/bozdaglab/NRPreTo.

Impact of mineralocorticoid receptor gene NRC2 on the prediction of arrhythmia after acute myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: None. Introduction Patients with acute myocardial infarction (AMI) suffer from distress that causes a rise in cortisol (stress hormone) which acts through glucocorticoid receptors (GR) and mineralocorticoid receptors (MR). Both receptors are present in cardiomyocyte nuclei (1-4). The animal model study in 2017 showed that activated MR increases cardiomyocyte oxidative stress leading to adverse electrophysiological remodelling that causes arrhythmias. 1.4% of tachyarrhythmias which is the main cause of cardiac death occur within the first month in AMI patients. MR are encoded by the human gene NR3C2 (nuclear receptor subfamily 3 group C member (5-6). Thus, we hypothesized that NR3C2 rs2070950, rs4635799 and rs5522 gene polymorphisms might have an extensive impact on rhythm disorders that occur in a cohort of AMI patients. Aim The aim is to evaluate the impact of the NR3C2 rs2070950, rs4635799 and rs5522 genes polymorphism in patients that developed rhythm and conduction disorders after suffering AMI. Methods The study included 301 patients treated at our hospital after suffering from AMI; all study subjects underwent primary percutaneous coronary intervention (PCI) and guideline-directed medical treatment (1). Patients' blood samples that were collected upon arrival had cortisol and troponin I levels checked and SNPs of the NR3C2 gene (rs2070950, rs4635799 and rs5522) assessed. To avoid inaccuracies all patients with a previous history of coronary syndrome or PCI were excluded. Rhythm conduction disorders and NR3C2 gene polymorphism were analyzed using the exact Fisher’s test. All the statistical analyses were performed with SPSS 27.0 software. The value of p < 0.05 was considered statistically significant. Results Patients with ventricular and atrial events in the early phase of AMI had higher levels of serum cortisol than the patients that did not have rhythm disorders (p=0.001) (Table 1). Rhythm disorders such as ventricular tachycardia (VT), ventricular flutter (VF) and atrial flutter or atrial fibrillation (AF/AFL) that occurred during hospitalization for AMI were notably associated with rs5522 gene polymorphism. During a hospital stay for AMI higher rs5522 TT genotype frequency was noticed in patients with AF/AFL than in patients without. C allele recurrence was 19.6%. Moreover, the protective effect was detected for AF/AFL there is also a protective effect in VA. (Table 2). Despite this, the gene polymorphism and high-grade atrioventricular block (HAVB) were unrelated. Genes rs2070950 and rs4635799 were not found significant to rhythm and conduction disorders. Conclusions Patients with the rs5522 TT allele are more prone to experience a higher frequency of arrhythmias. However, if at least one rs5522 C is found it associates with a smaller risk of arrhythmias post-AMI, meaning that this gene possibly limits damaging effects on cardiomyocytes that are caused by cortisol.

Apple polyphenol extract alleviates DSS-induced ulcerative colitis and linked behavioral disorders via regulating the gut-brain axis

To explore the effects of apple polyphenol extract (APE) on dextran sulfate sodium (DSS) induced acute ulcerative colitis (UC) and linked behavioral disorders, C57BL/6 male mice aged 9–11 weeks were randomly divided into the following groups: (1) Control group (CON), (2) 3% DSS group (DSS), (3) 3% DSS + APE at 8 a.m. group (DA-ZT0), (4) 3% DSS + APE at 8 p.m. group (DA-ZT12). APE was given at a dosage of 500 mg/(kg·bw·d). APE treatment elevated protein expressions of Occludin, zonula occludins-1 (ZO-1) and mucoprotein-2 (MUC2), and inhibited inflammatory response by down-regulating the (NOD)-like receptor family and pyrin domain containing 3 (NLRP3)/apoptosis-associated speck-like protein (ASC)/cysteine aspartate-specific protease-1 (caspase-1) signaling pathway. Meanwhile, APE alleviated anxiety and depression-like behavior disorders by upregulating brain-derived neurotrophic factor (BDNF) and postsynaptic-density protein 95 (PSD-95) and downregulating allograft inflammatory factor 1 (AIF1). Additionally, APE reshaped the structure of the intestinal microbiota, with an increased Firmicutes/Bacterodetes ratio and reduced the relative abundances of Escherichia-Shigella, Bacteroides and Parasutterella. Finally, APE reset DSS-induced circadian rhythm disturbance of clock genes, with significant induction of Cryptochrome2 (Cry2), Period2 (Per2) and Nuclear receptor subfamily 1 group D member 1 (Rev-erbα) in hippocampus and Brain and muscle arnt-like protein 1 (Bmal1) and Circadian locomotor output cycles kaput (Clock) in cortex in DA-ZT0 group, but APE treatment at ZT12 induced longer colon length, lower serum IL-β concentration and proteins expression of NLRP3 and ASC in colon, and better recovery of behavioral disorder. Thus, APE might conserve the potential as a diet-derived nutraceutical for UC treatment.

Tongqiao Huoxue Decoction ameliorates traumatic brain injury-induced gastrointestinal dysfunction by regulating CD36/15-LO/NR4A1 signaling, which fails when CD36 and CX3CR1 are deficient.

Gastrointestinal (GI) dysfunction, as a common peripheral-organ complication after traumatic brain injury (TBI), is primarily characterized by gut inflammation and damage to the intestinal mucosal barrier (IMB). Previous studies have confirmed that TongQiao HuoXue Decoction (TQHXD) has strong anti-inflammatory properties and protects against gut injury. However, few have reported on the therapeutic effects of TQHXD in a TBI-induced GI dysfunction model. We aimed to explore the effects of TQHXD on TBI-induced GI dysfunction and the underlying mechanism thereof. We assessed the protective effects and possible mechanism of TQHXD in treating TBI-induced GI dysfunction via gene engineering, histological staining, immunofluorescence (IF), 16S ribosomal ribonucleic acid (rRNA) sequencing, real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), Western blot (WB), and flow cytometry (FCM). TQHXD administration ameliorated TBI-induced GI dysfunction by modulating the abundance and structure of bacteria; reconstructing the destroyed epithelial and chemical barriers of the IMB; and improving M1/M2 macrophage, T-regulatory cell (Treg)/T helper 1 cell (Th1 ), as well as Th17 /Treg ratios to preserve homeostasis of the intestinal immune barrier. Notably, Cluster of Differentiation 36 (CD36)/15-lipoxygenase (15-LO)/nuclear receptor subfamily 4 group A member 1 (NR4A1) signaling was markedly stimulated in colonic tissue of TQHXD-treated mice. However, insufficiency of both CD36 and (C-X3-C motif) chemokine receptor 1 (CX3CR1) worsened GI dysfunction induced by TBI, which could not be rescued by TQHXD. TQHXD exerted therapeutic effects on TBI-induced GI dysfunction by regulating the intestinal biological, chemical, epithelial, and immune barriers of the IMB, and this effect resulted from the stimulation of CD36/NR4A1/15-LO signaling; however, it could not do so when CX3CR1 and CD36 were deficient. TQHXD might therefore be a potential drug candidate for treating TBI-induced GI dysfunction.

Differentiation of human adipose tissue–derived mesenchymal stromal cells into steroidogenic cells by adenovirus-mediated overexpression of NR5A1 and implantation into adrenal insufficient mice

Background aimsCell therapy for adrenal insufficiency is a potential method for physiological glucocorticoid and mineralocorticoid replacement. We have previously shown that mouse mesenchymal stromal cells (MSCs) differentiated into steroidogenic cells by the viral vector–mediated overexpression of nuclear receptor subfamily 5 group A member 1 (NR5A1), an essential regulator of steroidogenesis, and their implantation extended the survival of bilateral adrenalectomized (bADX) mice. MethodsIn this study, we examined the capability of NR5A1-induced steroidogenic cells prepared from human adipose tissue-derived MSCs (MSC [AT]) and the therapeutic effect of the implantation of human NR5A1-induced steroidogenic cells into immunodeficient bADX mice. ResultsHuman NR5A1-induced steroidogenic cells secreted adrenal and gonadal steroids and exhibited responsiveness to adrenocorticotropic hormone and angiotensin II in vitro. In vivo, the survival time of bADX mice implanted with NR5A1-induced steroidogenic cells was significantly prolonged compared with that of bADX mice implanted with control MSC (AT). Serum cortisol levels, which indicate hormone secretion from the graft, were detected in bADX mice implanted with steroidogenic cells. ConclusionsThis is the first report to demonstrate steroid replacement by the implantation of steroid-producing cells derived from human MSC (AT). These results indicate the potential of human MSC (AT) to be a source of steroid hormone-producing cells.

The clock gene Bmal1 inhibits kidney expression of SGLT2 and glucose reabsorption via the NR1d1/NRF1 pathway

Background & Aims: The nuclear receptor subfamily 1, group D, member 1 (Nr1d1, also called Rev-erbα) is highly expressed in the kidney, and its transcriptional activity is increased by the clock gene Bmal1 (ARNTL or MOP3). Here we studied the roles and functional interactions of Bmal1 and Nr1d1 in mouse kidney, particularly in regulating the expression of the glucose transporter SGLT2 (SLC5A2) and glucose reabsorption. Methods: We performed histologic and biochemical analyses of kidney tissues from male C57BL/6 and Bmal1-knockout mice and performed gene expression analyses to identify genes regulated by Bmal1. The effects of fasting and refeeding on renal expression of Sglt2 was examined in male C57BL/6, Bmal1-knockout, and Nr1d1-knockout mice. Some mice were given hemin to stimulate Nr1d1 gene expression (daily for 1 week) at 12:00 with 100 μg/kg/day; renal glucose reabsorption, and glucose and fatty acid levels were measured. Renal proximal tubule epithelial cells (RPTECs) were generated from C57BL/6 and Bmal1-knockout mice and glucose uptake determined. Luciferase reporter assays were performed using HK2 cell. Results: We found that kidney SGLT2 mRNA and protein expression and glucose absorption were increased in Bmal1-knockout mice compared with C57BL/6 mice. Expression of SGLT2 was affected by fasting and refeeding in C57BL/6 mice, but not in Bmal1-knockout or Nr1d1-knockout mice. Bmal1-knockout mice also presented with higher levels of renal fatty acids compared with C57BL/6 mice. Hemin injection reduced expression of SGLT2, decreased glucose re-absorption in RPTECs, and increased levels of fatty acid in kidney; these changes were not observed in Bmal1-knockout mice. Nuclear respiratory factor 1 (NRF1), which regulates glucose, activated transcription of Sglt2. Fasting caused MAPK signing pathway to phosphorylate Bmal1, which activated NR1d1 and inhibited NRF1 activity and thereby reduced Sglt2 expression. Conclusions: The data indicate that fasting activates Bmal1 and Nr1d1, which inhibits NRF1 and leads to repression of renal SGLT2 expression and glucose reabsorption. This is the first study that mechanistically links food intake to the regulation of kidney glucose transport through a circadian clock gene. NIH National Heart, Lung, and Blood Institute grant R56 HL137912-01, and American Heart Association grant-in-aid 16GRNT30960027 to PX; NIH R01DK112042 to VV This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Proteolysis-targeting chimera against NR4A1 for cancer immunotherapy

Abstract Modulating tumor-infiltrating immune cells is the key mission for the development of cancer immunotherapy. Based on recent literature and our bioinformatic analysis, nuclear receptor subfamily 4 group A member 1 (NR4A1) is an ideal target for cancer immunotherapy due to its important role in T cell exhaustion, regulatory T (Treg) cell function. Here we targets NR4A1 via proteolysis-targeting chimeric (PROTAC) technology, expecting to achieve immune activation and cancer clearance. We designed and synthesized over 80 NR4A1 PROTACs based on several reported NR4A1 ligands, such as celastrol. We found that several celastrol-based PROTACs achieved effective degradation of target protein NR4A1. We studied the therapeutic efficiency of the lead NR-V04, a celastrol- and von Hippel-Lindau (VHL)-based PROTAC. As expected, we confirmed that the NR-V04-induced NR4A1 degradation is through the uniquitin proteasome system with a tenery complex formation among NR4A1, VHL and NR-V04. The lead NR-V04 exhibits outstanding pharmacokinetics and effectively inhibits tumor growth at a low dose, 1.8mg/kg twice a week, in B16F10, Yummer 1.7, and MC38 mouse tumor models. We proved that NR-V04 significantly enhances anti-tumor immunity with seemingly distinct mechanisms of immune activation in a tumor-dependent manner. For example, NR-V04 induces the expansion of B cells and the reduction in myeloid-derived suppressor cells (MDSC) in B16F10; the reduction of Tregs, and the induction of CD8 effector population in Yummer 1.7 and MC38 tumors. we did not find obvious toxicity at these effective doses for up to 2 months. Therefore, the lead NR-V04 or other better ones under test have the potential for translation as a novel immunotherapeutic strategy. BC200100 - “Developing a Novel PROTAC-Based NR4A1 Degrader for Breast Cancer Therapy” (DEPARTMENT OF THE ARMY)

Abstract 378: Loss Of Nuclear Receptor Subfamily 4 Group A 1 (NR4A1) Results In Impaired Post-thrombotic Vein Wall Remodeling By Promoting A Dysregulated Antifibrinolytic Phenotype In Monocytes

Objectives: Nuclear Receptor Subfamily 4 Group A1 (NR4A1) is a suppressor of monocyte/macrophage (MO/Mϕ) inflammatory output that antagonizes TGFβ-signaling. As NR4A1 inhibits epithelial-to-mesenchymal transition and tissue fibrosis, we sought to determine the role of NR4A1 in MO/Mϕ mediated post-thrombotic vein wall remodeling, which when exaggerated, can result in post-thrombotic syndrome (PTS). Methods: To assess the role of MO/Mϕ NR4A1 in post-thrombotic vein wall remodeling, we employed a knockout-rescue schema which featured whole mouse loss of function ( Nr4a1 -/- ) models and adoptive bone marrow derived MO/Mϕtransfer from Nr4a1 replete mice (WT-BMDMs). Stasis venous thrombosis was induced by the inferior vena cava (IVC) ligation. Characterization of vein wall remodeling/endothelialization was performed using Masson Trichrome staining and CD31 immunostaining, respectively. Quantitation of mRNA and protein levels of factors critical for MO/Mϕ-mediated vein wall remodeling was performed using qRT-PCR and ELISA, and matrix metalloproteinase (MMP) activity was measured using zymography. Results: We observed that Nr4a1 -/- mice exhibited impaired vein wall remodeling after 8- and 14-days post IVC ligation as indicated by thicker vein wall measurements and impaired luminal endothelialization. Adoptive transfer of WT-BMDMs into Nr4a1 -/- mice rescued this phenotype (Fig. 1A-C). Nr4a1 -/- mice did not exhibit any differences in thrombus burden (data not shown). Finally, although we observed induced mRNA levels of Mmp2 and Mmp9 in the vein walls of Nr4a1 -/- mice that underwent adoptive transfer, we noted suppressed MMP2 enzyme activity despite no compensatory changes in MMP9 activity or TGFβ/IL6 levels (Fig. 1D, data not shown). Conclusions: NR4A1 loss results in impaired post-thrombotic vein wall remodeling and agonism of NR4A1 may be a potential therapeutic strategy to combat PTS.

PIMT Controls Insulin Synthesis and Secretion through PDX1.

Pancreatic beta cell function is an important component of glucose homeostasis. Here, we investigated the function of PIMT (PRIP-interacting protein with methyl transferase domain), a transcriptional co-activator binding protein, in the pancreatic beta cells. We observed that the protein levels of PIMT, along with key beta cell markers such as PDX1 (pancreatic and duodenal homeobox 1) and MafA (MAF bZIP transcription factor A), were reduced in the beta cells exposed to hyperglycemic and hyperlipidemic conditions. Consistently, PIMT levels were reduced in the pancreatic islets isolated from high fat diet (HFD)-fed mice. The RNA sequencing analysis of PIMT knockdown beta cells identified that the expression of key genes involved in insulin secretory pathway, Ins1 (insulin 1), Ins2 (insulin 2), Kcnj11 (potassium inwardly-rectifying channel, subfamily J, member 11), Kcnn1 (potassium calcium-activated channel subfamily N member 1), Rab3a (member RAS oncogene family), Gnas (GNAS complex locus), Syt13 (synaptotagmin 13), Pax6 (paired box 6), Klf11 (Kruppel-Like Factor 11), and Nr4a1 (nuclear receptor subfamily 4, group A, member 1) was attenuated due to PIMT depletion. PIMT ablation in the pancreatic beta cells and in the rat pancreatic islets led to decreased protein levels of PDX1 and MafA, resulting in the reduction in glucose-stimulated insulin secretion (GSIS). The results from the immunoprecipitation and ChIP experiments revealed the interaction of PIMT with PDX1 and MafA, and its recruitment to the insulin promoter, respectively. Importantly, PIMT ablation in beta cells resulted in the nuclear translocation of insulin. Surprisingly, forced expression of PIMT in beta cells abrogated GSIS, while Ins1 and Ins2 transcript levels were subtly enhanced. On the other hand, the expression of genes, PRIP/Asc2/Ncoa6 (nuclear receptor coactivator 6), Pax6, Kcnj11, Syt13, Stxbp1 (syntaxin binding protein 1), and Snap25 (synaptosome associated protein 25) associated with insulin secretion, was significantly reduced, providing an explanation for the decreased GSIS upon PIMT overexpression. Our findings highlight the importance of PIMT in the regulation of insulin synthesis and secretion in beta cells.

In silico identification of molecular mechanisms for stroke risk caused by heavy metals and their mixtures: Sponges and drugs involved

This study used various approaches and databases to evaluate the molecular processes and identify miRNA sponges and drugs associated with the pathophysiology of stroke caused by heavy metals and their combinations. We found that the genes ALB (albumin), IL1B (Interleukin-1β), F2 (coagulation factor II), APOA1 (apolipoprotein A1), IL6 (Interleukin 6), and NOS2 (nitric oxide synthase 2) were linked to the development of strokes by 18 chemicals and a combination of cadmium, copper, and lead. These results may point to the significance of detoxification and neuroinflammation in stroke as well as the potential for targeting these genes in future stroke therapies. ALB and IL1B were the most common and significant genes. The "selenium micronutrient network," "vitamin B12 metabolism," and "folate metabolism" were shown to be the most significant pathways connected to the risk of stroke brought on by combined heavy metals. The two main cellular elements that may increase the risk of stroke caused by heavy metals were discovered to be "blood microparticle" and "endoplasmic reticulum lumen." We also observed an important chromosome (chr7p15.3), two transcription factors (NFKB2 [nuclear factor kappa B subunit 2] and NR1I2 [nuclear receptor subfamily 1 group, member 2]), and four microRNAs (hsa-miR-26a-5p, hsa-miR-9–5p, hsa-miR-124–3p, and hsa-miR-155–5p) associated with stroke caused by combined heavy metals. Additionally, for these miRNAs, we created and examined in silico microRNA sponge sequences. Triflusal and andrographolide have been identified as potential treatments for heavy metal-induced stroke. Taken together, heavy metals may be a significant contributor to the pathophysiology of stroke, but further investigation into the precise molecular pathways implicated in stroke pathophysiology is required to corroborate these findings.