NR1D1-transactivated lncRNA NUTM2A-AS1 promotes chemoresistance and immune evasion in neuroblastoma via inhibiting B7-H3 degradation.

Neuroblastoma (NB), a common solid tumour in young children originating from the sympathetic nervous system during embryonic development, poses challenges despite therapeutic advances like high-dose chemotherapy and immunotherapy. Some survivors still grapple with severe side effects and drug resistance. The role of lncRNA NUTM2A-AS1 has been explored in various cancers, but its function in drug-resistant NB progression is unclear. Our study found that NUTM2A-AS1 expression in cisplatin-resistant NB cells increased in a time- and dose-dependent manner. Knockdown of NUTM2A-AS1 significantly improved NB cell sensitivity to cisplatin and inhibited metastatic abilities. Additionally, we identified B7-H3, an immune checkpoint-related protein, as a NUTM2A-AS1-associated protein in NB cells. NUTM2A-AS1 was shown to inhibit the protein degradation of B7-H3. Moreover, NUTM2A-AS1 modulated immune evasion in cisplatin-resistant NB cells through B7-H3. Furthermore, NUTM2A-AS1 expression in cisplatin-resistant NB cells was transactivated by NR1D1. In summary, our results unveil the molecular or biological relationship within the NR1D1/NUTM2A-AS1/B7-H3 axis in NB cells under cisplatin treatment, providing an intriguing avenue for fundamental research into cisplatin-resistant NB.

Acute exercise modulates Trim63 and Bmal1 in the skeletal muscle of IL-10 knockout mice.

The anti-inflammatory role of physical exercise is mediated by interleukin 10 (IL-10), and their release is possibly upregulated in response to IL-6. Previous studies demonstrated that mice lacking IL-6 (IL-6 KO mice) exhibited diminished exercise tolerance, and reduced strength. Rev-erbα, a transcriptional suppressor involved in circadian rhythm, has been discovered to inhibit the expression of genes linked to bodily functions, encompassing inflammation and metabolism. It also plays a significant role in skeletal muscle and exercise performance capacity. Given the potential association between Rev-erbα and the immune system and the fact that both pathways are modulated following acute aerobic exercise, we examined the physical performance of IL-10 KO mice and analyzed the modulation of the atrophy and Rev-erbα pathways in the muscle of wild type (WT) and IL-10 KO mice following one session of acute exercise. For each phenotype, WT and IL-10 KO were divided into two subgroups (Control and Exercise). The acute exercise session started at 6 m/min, followed by 3 m/min increments every 3 min until animal exhaustion. Two hours after the end of the exercise protocol, the gastrocnemius muscle was removed and prepared for the reverse transcription-quantitative polymerase chain reaction (RT-q-PCR) and immunoblotting technique. In summary, compared to WT, the IL-10 KO animals showed lower body weight and grip strength in the baseline. The IL-10 control group presented a lower protein content of BMAL1. After the exercise protocol, the IL-10 KO group had higher mRNA levels of Trim63 (atrophy signaling pathway) and lower mRNA levels of Clock and Bmal1 (Rev-erbα signaling pathway). This is the first study showing the relationship between Rev-erbα and atrophy in IL-10 KO mice. Also, we accessed a public database that analyzed the gastrocnemius of MuRF KO mice submitted to two processes of muscle atrophy, a denervation surgery and dexamethasone (Dexa) injections. Independently of knockout, the denervation demonstrated lower Nr1d1 levels. In conclusion, IL-10 seems to be a determinant in the Rev-erbα pathway and atrophy after acute exercise, with no modulation in the baseline state.

NR1D1-rearranged soft tissue tumour: A clinicopathological and molecular analysis of four additional cases.

AIMS: Nuclear receptor subfamily 1 group D member 1 (NR1D1)-rearranged soft tissue tumour is a newly described entity with an epithelioid morphology and a potential for aggressive behaviour. Largely due to under-recognition, this tumour type has not yet been widely acknowledged. Herein, we report four additional cases to further expand its clinicopathological and molecular spectrum. METHODS AND RESULTS: Four mesenchymal tumours with NR1D1 rearrangement were identified from our consultation files. There were one male and three females with ages ranging from 19 to 47 years (median = 28.5 years). Tumour occurred in the tongue, neck, hip and index finger, respectively. Histologically, two tumours were composed predominantly of epithelioid cells; one tumour had admixed epithelioid-spindle cells and one tumour consisted of monomorphic small round to ovoid cells. By immunohistochemistry, none of the tumours expressed lineage-specific markers. Targeted RNA-sequencing identified NR1D1 fusions in all four tumours, the partner genes being MAML2, MAML3, KMT2A and NCOA2, respectively. The novel MAML3 and NCOA2 rearrangements were confirmed by fluorescence in-situ hybridisation analysis. On follow-up (2-23 months), one patient experienced local recurrence due to incomplete resection and one patient developed lung metastasis. The other two patients were alive without disease. CONCLUSIONS: This study adds more support for NR1D1-rearranged soft tissue tumour as an emerging entity. The occurrence of two additional tumours in the head and neck region, description of a small round cell variant and identification of novel MAML3, KMT2A and NCOA2 partners further expand its clinicopathological and molecular spectrum. More studies on larger series are necessary to validate the fully malignant potential of NR1D1-rearranged soft tissue tumour.

Light pollution during pregnancy influences the growth of offspring in rats.

OBJECTIVE: To investigate the effects of excessive light exposure during gestation on intrauterine development and early growth of neonates in rats. METHODS: Pregnant rats were randomly allocated to three groups: the constant light exposure group, non-light exposure group and control group. Blood samples were collected from the tail vein to analyze melatonin and cortisol levels. Weight, daily food and water consumption were recorded. Uterine weight, placental weight and placental diameter were measured on gestational day 19. Natural birth and neonate growth were also monitored. The expression of NR1D1(nuclear receptor subfamily 1 group D member 1) in offspring's SCN (suprachiasmatic nuclei), liver and adipose tissue was measured. Expression of NR1D1, MT1(melatonin 1 A receptor) and 11ß-HSD2 (placental 11ß-hydroxysteroid dehydrogenase type 2) in placenta was also measured. Finally, the expression of MT1 and 11ß-HSD2 in NR1D1 siRNA transfected JEG-3 cells was evaluated. RESULTS: There were no significant differences in maternal weight gain, pregnancy duration, uterine weight, placental body weight, placental diameter, fetal number among three groups. There were no significant differences in weights or lengths of offspring at birth. Compared to other two groups, constant light exposure group showed significantly more rapid growth of offspring in 21st day post-birth. The expression of NR1D1 in SCN, liver and adipose tissues of offspring was not significantly different among three groups. The maternal serum melatonin and cortisol levels of the constant light exposure group were lower and higher than other two groups, respectively. The expressions of NR1D1, MT1 and 11ß-HSD2 were all decreased in placenta of the constant light exposure group. The expression of MT1 and 11ß-HSD2 in JEG-3 cells were decreased after NR1D1 siRNA transfection. CONCLUSION: Excessive light exposure during pregnancy results in elevated cortisol and reduced melatonin exposure to fetuses in uterus, potentially contributing to an accelerated early growth of offspring in rats.

Mitophagy Regulates the Circadian Rhythms by Degrading NR1D1 in Simulated Microgravity and Isolation Environments.

Long-term spaceflight is known to induce disruptions in circadian rhythms, which are driven by a central pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus, but the underlying molecular mechanisms remain unclear. Here, we developed a rat model that simulated microgravity and isolation environments through tail suspension and isolation (TSI). We found that the TSI environment imposed circadian disruptions to the core body temperature, heart rate, and locomotor-activity rhythms of rats, especially in the amplitude of these rhythms. In TSI model rats' SCNs, the core circadian gene NR1D1 showed higher protein but not mRNA levels along with decreased BMAL1 levels, which indicated that NR1D1 could be regulated through post-translational regulation. The autophagosome marker LC3 could directly bind to NR1D1 via the LC3-interacting region (LIR) motifs and induce the degradation of NR1D1 in a mitophagy-dependent manner. Defects in mitophagy led to the reversal of NR1D1 degradation, thereby suppressing the expression of BMAL1. Mitophagy deficiency and subsequent mitochondrial dysfunction were observed in the SCN of TSI models. Urolithin A (UA), a mitophagy activator, demonstrated an ability to enhance the amplitude of core body temperature, heart rate, and locomotor-activity rhythms by prompting mitophagy induction to degrade NR1D1. Cumulatively, our results demonstrate that mitophagy exerts circadian control by regulating NR1D1 degradation, revealing mitophagy as a potential target for long-term spaceflight as well as diseases with SCN circadian disruption.

NR1D1::MAML1 epithelioid and spindle cell sarcoma mimicking pseudomyogenic hemangioendothelioma in core biopsy: A case report and review of the literature.

Epithelioid and spindle cell sarcomas with NR1D1::MAML1/2 gene fusions are rare and emerging entities. Only six cases of NR1D1-rearranged mesenchymal tumors have previously been reported in the literature; they are often characterized by an epithelioid morphology, at least focal pseudogland formation, prominent cytoplasmic vacuoles, and focal to diffuse immunohistochemical expression of keratin. We herein report the first case of an NR1D1::MAML1 epithelioid and spindle cell sarcoma with dual immunohistochemical expression of ERG and FOSB, mimicking a pseudomyogenic hemangioendothelioma (PHE) on core biopsy. The sarcoma arose in the left forearm of a 64-year-old man. Initial biopsy showed a mesenchymal neoplasm composed of epithelioid and spindle cells dispersed in myxoid stroma with scattered stromal neutrophils. The morphologic features, combined with the dual immunohistochemical expression of ERG and FOSB, initially mimicked PHE, representing an important potential diagnostic pitfall. The patient subsequently underwent a radical resection, which showed a much more diffuse epithelioid appearance with nested architecture and pseudogland formation. Next-generation sequencing was performed on the resection specimen, which revealed an NR1D1::MAML1 gene fusion, confirming the final diagnosis. Given the fully malignant potential of this tumor, knowledge and recognition of this rare entity are essential to ensure proper management, prevent misdiagnosis, and further characterize the clinical course of this emerging entity. Comprehensive molecular testing can help to identify these rare tumors and exclude the possibility of epithelioid mimics, including PHE.

Low expression of NR1D1 and NR2E3 is associated with advanced features of retinoblastoma.

PURPOSE: To investigate the expression of nuclear receptor subfamily 1 group D member 1 (NR1D1) and nuclear receptor subfamily 2 group E Member 3 (NR2E3) in retinoblastoma (RB) and their correlation with the clinical and pathological features of RB. METHODS: Immunohistochemical (IHC) assays were performed to detect and evaluate the expression levels of NR1D1 and NR2E3 in paraffin-embedded tissue samples. The relationship between the expression levels and clinicopathological characteristics of RB patients was analyzed using the χ2 test or Fisher exact test. RESULTS: A total of 51 RB patients were involved in this research. The expression levels of NR1D1 (P = 0.004) and NR2E3 (P = 0.024) were significantly lower in RB tumor tissues than in normal retina. The expression levels of NR1D1 and NR2E3 were less positive in RB patients with advanced stages (P = 0.007, P = 0.015), choroidal infiltration (P = 0.003, P = 0.029), and optic nerve infiltration (P = 0.036, P = 0.003). In addition, a low expression level of NR2E3 was associated with high-risk pathology (P = 0.025) and necrosis (P = 0.035) of RB tissues. CONCLUSION: The expression levels of NR1D1 and NR2E3 were decreased in RB and closely associated with the clinical stage and high invasion of the disease. These findings provide new insights into the mechanism of RB progression and suggest that NR1D1 and NR2E3 could be potential targets for treatment strategies.

Hypoxia activation attenuates progesterone synthesis in goat trophoblast cells via NR1D1 inhibition of StAR expression†.

Trophoblast plays a crucial role in gestation maintenance and embryo implantation, partly due to the synthesis of progesterone. It has been demonstrated that hypoxia regulates invasion, proliferation, and differentiation of trophoblast cells. Additionally, human trophoblasts display rhythmic expression of circadian clock genes. However, it remains unclear if the circadian clock system is present in goat trophoblast cells (GTCs), and its involvement in hypoxia regulation of steroid hormone synthesis remains elusive. In this study, immunofluorescence staining revealed that both BMAL1 and NR1D1 (two circadian clock components) were highly expressed in GTCs. Quantitative real-time PCR analysis showed that several circadian clock genes were rhythmically expressed in forskolin-synchronized GTCs. To mimic hypoxia, GTCs were treated with hypoxia-inducing reagents (CoCl2 or DMOG). Quantitative real-time PCR results demonstrated that hypoxia perturbed the mRNA expression of circadian clock genes and StAR. Notably, the increased expression of NR1D1 and the reduction of StAR expression in hypoxic GTCs were also detected by western blotting. In addition, progesterone secretion exhibited a notable decline in hypoxic GTCs. SR9009, an NR1D1 agonist, significantly decreased StAR expression at both the mRNA and protein levels and markedly inhibited progesterone secretion in GTCs. Moreover, SR8278, an NR1D1 antagonist, partially reversed the inhibitory effect of CoCl2 on mRNA and protein expression levels of StAR and progesterone synthesis in GTCs. Our results demonstrate that hypoxia reduces StAR expression via the activation of NR1D1 signaling in GTCs, thus inhibiting progesterone synthesis. These findings provide new insights into the NR1D1 regulation of progesterone synthesis in GTCs under hypoxic conditions.

NR1D1 downregulation in astrocytes induces a phenotype that is detrimental to cocultured motor neurons.

Nuclear receptor subfamily 1 group D member 1 (NR1D1, also known as Rev-erbα) is a nuclear transcription factor that is part of the molecular clock encoding circadian rhythms and may link daily rhythms with metabolism and inflammation. NR1D1, unlike most nuclear receptors, lacks a ligand-dependent activation function domain 2 and is a constitutive transcriptional repressor. Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease, caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Approximately 10%-20% of familial ALS is caused by a toxic gain-of-function induced by mutations of the Cu/Zn superoxide dismutase (SOD1). Dysregulated clock and clock-controlled gene expression occur in multiple tissues from mutant hSOD1-linked ALS mouse models. Here we explore NR1D1 dysregulation in the spinal cord of ALS mouse models and its consequences on astrocyte-motor neuron interaction. NR1D1 protein and mRNA expression are significantly downregulated in the spinal cord of symptomatic mice expressing mutant hSOD1, while no changes were observed in age-matched animals overexpressing wild-type hSOD1. In addition, NR1D1 downregulation in primary astrocyte cultures induces a pro-inflammatory phenotype and decreases the survival of cocultured motor neurons. NR1D1 orchestrates the cross talk between physiological pathways identified to be disrupted in ALS (e.g., metabolism, inflammation, redox homeostasis, and circadian rhythms) and we observed that downregulation of NR1D1 alters astrocyte-motor neuron interaction. Our results suggest that NR1D1 could be a potential therapeutic target to prevent astrocyte-mediated motor neuron toxicity in ALS.

m6A methylation-induced NR1D1 ablation disrupts the HSC circadian clock and promotes hepatic fibrosis.

The roles of nuclear receptor subfamily 1 group d member 1 (NR1D1) and the circadian clock in liver fibrosis remain unclear. Here, we showed that liver clock genes, especially NR1D1, were dysregulated in mice with carbon tetrachloride (CCl4)-induced liver fibrosis. In turn, disruption of the circadian clock exacerbated experimental liver fibrosis. NR1D1-deficient mice were more sensitive to CCl4-induced liver fibrosis, supporting a critical role of NR1D1 in liver fibrosis development. Validation at the tissue and cellular levels showed that NR1D1 was primarily degraded by N6-methyladenosine (m6A) methylation in a CCl4-induced liver fibrosis model, and this result was also validated in rhythm-disordered mouse models. In addition, the degradation of NR1D1 further inhibited the phosphorylation of dynein-related protein 1-serine site 616 (DRP1S616), resulting in weakened mitochondrial fission function and increased mitochondrial DNA (mtDNA) release in hepatic stellate cell (HSC), which in turn activated the cGMP-AMP synthase (cGAS) pathway. Activation of the cGAS pathway induced a local inflammatory microenvironment that further stimulated liver fibrosis progression. Interestingly, in the NR1D1 overexpression model, we observed that DRP1S616 phosphorylation was restored, and cGAS pathway was also inhibited in HSCs, resulting in improved liver fibrosis. Taken together, our results suggest that targeting NR1D1 may be an effective approach to liver fibrosis prevention and management.

Nuclear receptor subfamily 1 group D member 1 suppresses the proliferation, migration of adventitial fibroblasts, and vascular intimal hyperplasia via mammalian target of rapamycin complex 1/ß-catenin pathway.

BACKGROUND: In-stent restenosis hardly limits the therapeutic effect of the percutaneous vascular intervention. Although the restenosis is significantly ameliorated after the application of new drug-eluting stents, the incidence of restenosis remains at a high level. OBJECTIVE: Vascular adventitial fibroblasts (AFs) play an important role in intimal hyperplasia and subsequent restenosis. The current study was aimed to investigate the role of nuclear receptor subfamily 1, group D, member 1 (NR1D1) in the vascular intimal hyperplasia. METHODS AND RESULTS: We observed increased expression of NR1D1 after the transduction of adenovirus carrying Nr1d1 gene (Ad-Nr1d1) in AFs. Ad-Nr1d1 transduction significantly reduced the numbers of total AFs, Ki-67-positive AFs, and the migration rate of AFs. NR1D1 overexpression decreased the expression level of ß-catenin and attenuated the phosphorylation of the effectors of mammalian target of rapamycin complex 1 (mTORC1), including mammalian target of rapamycin (mTOR) and 4E binding protein 1 (4EBP1). Restoration of ß-catenin by SKL2001 abolished the inhibitory effects of NR1D1 overexpression on the proliferation and migration of AFs. Surprisingly, the restoration of mTORC1 activity by insulin could also reverse the decreased expression of ß-catenin, attenuated proliferation, and migration in AFs induced by NR1D1 overexpression. In vivo, we found that SR9009 (an agonist of NR1D1) ameliorated the intimal hyperplasia at days 28 after injury of carotid artery. We further observed that SR9009 attenuated the increased Ki-67-positive AFs, an essential part of vascular restenosis at days 7 after injury to the carotid artery. CONCLUSION: These data suggest that NR1D1 inhibits intimal hyperplasia by suppressing the proliferation and migration of AFs in a mTORC1/ß-catenin-dependent manner.

Nuclear receptor REVERBα is a state-dependent regulator of liver energy metabolism.

The nuclear receptor REVERBα is a core component of the circadian clock and proposed to be a dominant regulator of hepatic lipid metabolism. Using antibody-independent ChIP-sequencing of REVERBα in mouse liver, we reveal a high-confidence cistrome and define direct target genes. REVERBα-binding sites are highly enriched for consensus RORE or RevDR2 motifs and overlap with corepressor complex binding. We find no evidence for transcription factor tethering and DNA-binding domain-independent action. Moreover, hepatocyte-specific deletion of Reverbα drives only modest physiological and transcriptional dysregulation, with derepressed target gene enrichment limited to circadian processes. Thus, contrary to previous reports, hepatic REVERBα does not repress lipogenesis under basal conditions. REVERBα control of a more extensive transcriptional program is only revealed under conditions of metabolic perturbation (including mistimed feeding, which is a feature of the global Reverbα-/- mouse). Repressive action of REVERBα in the liver therefore serves to buffer against metabolic challenge, rather than drive basal rhythmicity in metabolic activity.

Daily rhythms of REV-ERBα and its role as transcriptional repressor of clock genes in fish hepatic oscillator.

The REV-ERBα nuclear receptor is a key component of the molecular machinery of circadian oscillators in mammals. While the rhythmic expression of this receptor has been described in teleosts, several critical aspects of its regulation remain unknown, such as which synchronizers entrain its rhythm, and whether it can modulate the expression of other clock genes. The objective of this study was to gain deeper understanding of the role of REV-ERBα in the fish circadian system. To this end, we first investigated the cues that entrain the rhythm of rev-erbα expression in the goldfish (Carassius auratus) liver and hypothalamus. A 12-h shift in feeding time induced a parallel shift in the hepatic rhythm of rev-erbα expression, confirming that this gene is food-entrainable in the goldfish liver. In contrast, light seems the main driver of rev-erbα rhythmic expression in the hypothalamus. Next, we examined the effects of REV-ERBα activation on locomotor activity and hepatic expression of clock genes. Subchronic treatment with the REV-ERBα agonist SR9009 slightly decreased locomotor activity anticipating light onset and food arrival, and downregulated hepatic bmal1a, clock1a, cry1a, per1a and pparα expression. This generalized repressing action of REV-ERBα on the expression of hepatic clock genes was confirmed in vitro by using agonists (SR9009 and GSK4112) and antagonist (SR8278) of this receptor. Overall, the present work reveals that REV-ERBα modulates the daily expression of the main genes of the teleostean liver clock, reinforcing its role in the liver temporal homeostasis, which seems highly conserved in both fish and mammals.

Polycyclic aromatic hydrocarbons (PAHs) in ambient air of Guangzhou city: Exposure levels, health effects and cytotoxicity.

Polycyclic aromatic hydrocarbons (PAHs) in PM2.5 pose potentially serious threats to human health. In this study, the distribution characteristics of 16 priority controlled, fine PM (PM2.5)-bound PAHs in the ambient air of Guangzhou city were analysed from 2016 to 2019. Four high-molecular-weight PAHs with the highest annual average concentrations were benzo[ghi]perylene (BghiP; 0.757 ng/m3), indeno(1,2,3-cd)pyrene (IcdP; 0.627 ng/m3), benzo[b]fluoranthene (BbF, 0.519 ng/m3) and 3,4-benzopyrene (BaP; 0.426 ng/m3). Increasing concentrations of BghiP, IcdP, BbF and BaP were associated with increasing numbers of outpatient visits for respiratory diseases, indicating that exposure to these PAHs potentially causes acute respiratory injury in residents. Acute exposure of the human bronchial epithelial cell line BEAS-2B cells to BghiP, IcdP, BbF and BaP in vitro resulted in acute inflammation, DNA damage and apoptosis. Further bioinformatic analysis indicated that nuclear receptor subfamily 1 group D member 1 (NR1D1) may be a key target gene involved in mediating the toxic effects of BghiP. Collectively, our results suggest that BghiP and the other PAHs represented by it can damage the respiratory system and induce lung cancer. This study provides valuable evidence regarding the potential health risks posed by local ambient PAHs pollution.

Uncovering the Novel Role of NR1D1 in Regulating BNIP3-Mediated Mitophagy in Ulcerative Colitis.

BACKGROUND: Ulcerative colitis (UC) is a chronic, incurable condition characterized by mucosal inflammation and intestinal epithelial cell (IEC) damage. The circadian clock gene NR1D1, implicated in UC and the critical mitophagy process for epithelial repair, needs further exploration regarding its role in mitophagy regulation in UC. METHODS: We created a jet lag mouse model and induced colitis with dextran sulfate sodium (DSS), investigating NR1D1's role. Intestinal-specific Nr1d1 knockout mice were also generated. RNA sequencing, chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays helped ascertain NR1D1's regulatory effect on BNIP3 expression. The mitochondrial state in IECs was assessed through transmission electron microscopy, while confocal microscopy evaluated mitophagy-associated protein expression in colon tissue and CCD841 cells. Cell apoptosis and reactive oxygen species (ROS) were measured via flow cytometry. RESULTS: We observed reduced NR1D1 expression in the IECs of UC patients, accentuated under jet lag and DSS exposure in mice. NR1D1 ablation led to disrupted immune homeostasis and declined mitophagy in IECs. NR1D1, usually a transcriptional repressor, was a positive regulator of BNIP3 expression, leading to impaired mitophagy, cellular inflammation, and apoptosis. Administering the NR1D1 agonist SR9009 ameliorated colitis symptoms, primarily by rectifying defective mitophagy. CONCLUSIONS: Our results suggest that NR1D1 bridges the circadian clock and UC, controlling BNIP3-mediated mitophagy and representing a potential therapeutic target. Its agonist, SR9009, shows promise in UC symptom alleviation.

Expanding the spectrum of mesenchymal neoplasms with NR1D1-rearrangement.

Undifferentiated mesenchymal neoplasms can be morphologically subclassified based on cell shape; epithelioid tumors may be diagnostically challenging, particularly since they can show morphologic and immunohistochemical overlap with epithelial neoplasms. Following the recent report of an NR1D1::MAML1 gene fusion in an undifferentiated pediatric neoplasm, we performed a retrospective archival review and identified four additional cases of undifferentiated mesenchymal neoplasms with NR1D1-rearrangement. All four tumors occurred in adult women. The tumors involved superficial and/or deep soft tissues of the extremities or abdomen. Morphologically, they showed a spectrum of overlapping features. In addition to epithelioid cells, two cases also had a prominent spindle cell component. Two cases also had admixed polygonal cells containing prominent cytoplasmic vacuoles with amorphous debris. The immunophenotype was nonspecific but all cases had at least focal keratin expression; this was extensive in two tumors. Targeted RNA-sequencing revealed two cases each with NR1D1::MAML1 and NR1D1::MAML2 gene fusions. One patient developed lung and liver metastases, and one patient required amputation due to multifocal disease and underlying bone involvement. This study confirms undifferentiated NR1D1-rearranged sarcoma represents a distinct mesenchymal neoplasm with an epithelioid morphology and potential for aggressive behavior. Further, we offer new insight into the spectrum of clinical, morphologic, immunohistochemical, and molecular findings possible in these rare neoplasms. An awareness of this entity is especially important given the potential for misclassification as a carcinoma.

Rev-erbα Knockout Reduces Ethanol Consumption and Preference in Male and Female Mice.

Alcohol use is a contributor in the premature deaths of approximately 3 million people annually. Among the risk factors for alcohol misuse is circadian rhythm disruption; however, this connection remains poorly understood. Inhibition of the circadian nuclear receptor REV-ERBα is known to disrupt molecular feedback loops integral to daily oscillations, and impact diurnal fluctuations in the expression of proteins required for reward-related neurotransmission. However, the role of REV-ERBα in alcohol and substance use-related phenotypes is unknown. Herein, we used a Rev-erbα knockout mouse line and ethanol two-bottle choice preference testing to show that disruption of Rev-erbα reduces ethanol preference in male and female mice. Rev-erbα null mice showed the lowest ethanol preference in a two-bottle choice test across all genotypes, whereas there were no ethanol preference differences between heterozygotes and wildtypes. In a separate experiment, alcohol-consuming wildtype C57Bl/6N mice were administered the REV-ERBα/ß inhibitor SR8278 (25 mg/kg or 50 mg/kg) for 7 days and alcohol preference was evaluated daily. No differences in alcohol preference were observed between the treatment and vehicle groups. Our data provides evidence that genetic variation in REV-ERBα may contribute to differences in alcohol drinking.

REV-ERBα Agonist SR9009 Promotes a Negative Energy Balance in Goldfish.

REV-ERBα (nr1d1, nuclear receptor subfamily 1 group D member 1) is a transcriptional repressor that in mammals regulates nutrient metabolism, and has effects on energy homeostasis, although its role in teleosts is poorly understood. To determine REV-ERBα's involvement in fish energy balance and metabolism, we studied the effects of acute and 7-day administration of its agonist SR9009 on food intake, weight and length gain, locomotor activity, feeding regulators, plasma and hepatic metabolites, and liver enzymatic activity. SR9009 inhibited feeding, lowering body weight and length gain. In addition, the abundance of ghrelin mRNA decreased in the intestine, and abundance of leptin-aI mRNA increased in the liver. Hypocretin, neuropeptide y (npy), and proopiomelanocortin (pomc) mRNA abundance was not modified after acute or subchronic SR9009 administration, while hypothalamic cocaine- and amphetamine-regulated transcript (cartpt-I) was induced in the subchronic treatment, being a possible mediator of the anorectic effects. Moreover, SR9009 decreased plasma glucose, coinciding with increased glycolysis and a decreased gluconeogenesis in the liver. Decreased triglyceride levels and activity of lipogenic enzymes suggest a lipogenesis reduction by SR9009. Energy expenditure by locomotor activity was not significantly affected by SR9009. Overall, this study shows for the first time in fish the effects of REV-ERBα activation via SR9009, promoting a negative energy balance by reducing energetic inputs and regulating lipid and glucose metabolism.

Regulation of Circadian Genes Nr1d1 and Nr1d2 in Sex-Different Manners during Liver Aging.

BACKGROUND: Circadian rhythm is associated with the aging process and sex differences; however, how age and sex can change circadian regulation systems remains unclear. Thus, we aimed to evaluate age- and sex-related changes in gene expression and identify sex-specific target molecules that can regulate aging. METHODS: Rat livers were categorized into four groups, namely, young male, old male, young female, and old female, and the expression of several genes involved in the regulation of the circadian rhythm was confirmed by in silico and in vitro studies. RESULTS: Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed that the expression of genes related to circadian rhythms changed more in males than in females during liver aging. In addition, differentially expressed gene analysis and quantitative real-time polymerase chain reaction/western blotting analysis revealed that Nr1d1 and Nr1d2 expression was upregulated in males during liver aging. Furthermore, the expression of other circadian genes, such as Arntl, Clock, Cry1/2, Per1/2, and Rora/c, decreased in males during liver aging; however, these genes showed various gene expression patterns in females during liver aging. CONCLUSIONS: Age-related elevation of Nr1d1/2 downregulates the expression of other circadian genes in males, but not females, during liver aging. Consequently, age-related upregulation of Nr1d1/2 may play a more crucial role in the change in circadian rhythms in males than in females during liver aging.

Nr1d1 Mediated Cell Senescence in Mouse Heart-Derived Sca-1+CD31- Cells.

AIM: Sca-1+CD31- cells are resident cardiac progenitor cells, found in many mammalian tissues including the heart, and able to differentiate into cardiomyocytes in vitro and in vivo. Our previous work indicated that heart-derived Sca-1+CD31- cells increased the Nr1d1 mRNA level of Nr1d1 with aging. However, how Nr1d1 affects the senescence of Sca-1+CD31- cells. METHODS: Overexpression and knockdown of Nr1d1 in Sca-1+CD31- cells and mouse cardiac myocyte (MCM) cell lines were performed by lentiviral transduction. The effects of Nr1d1 abundance on cell differentiation, proliferation, apoptosis, cell cycle, and transcriptomics were evaluated. Moreover, binding of Nr1d1 to the promoter region of Nr4a3 and Serpina3 was examined by a luciferase reporter assay. RESULTS AND CONCLUSIONS: Upregulation Nr1d1 in young Sca-1+CD31- cells inhibited cell proliferation and promoted apoptosis. However, depletion of Nr1d1 in aged Sca-1+CD31- cells promoted cell proliferation and inhibited apoptosis. Furthermore, Nr1d1 was negatively associated with cell proliferation, promoting apoptosis and senescence-associated beta-galactosidase production in MCMs. Our findings show that Nr1d1 stimulates Serpina3 expression through its interaction with Nr4a3. Nr1d1 may therefore act as a potent anti-aging receptor that can be a therapeutic target for aging-related diseases.