NPAS2, transcriptionally activated by ARRB1, promotes the malignant behaviours of lung adenocarcinoma cells and regulates the reprogramming of glucose metabolism.

Lung adenocarcinoma (LUAD) is a serious threat to public health and is accompanied by increased morbidity and mortality worldwide. Neuronal PAS domain protein2 (NPAS2) has been confirmed as an oncogene in LUAD; however, little is known about its molecular mechanism. Here, the expression level of NPAS2 was detected in LUAD cell lines and 16HBE cells. Gain- and loss-of-function experiments were performed. Cell Counting Kit-8, colony formation, flow cytometry, wound-healing and Transwell assays were conducted to assess cell proliferation, apoptosis, migration and invasion, respectively. Reprogramming of glucose metabolism was evaluated via oxygen consumption rate (OCR), complexes activities, lactic production and glucose consumption. The expression of critical proteins was examined by western blot. We demonstrated aberrant upregulation of NPAS2 and ß-arrestin-1 (ARRB1) in LUAD cell lines. ARRB1 was found to be a critical transcription factor of NPAS2 with binding sites within the promoter region of NPAS2, thereby causing its transcriptional activation. Functional experiments revealed that NPAS2 depletion significantly inhibited the malignant behaviours of A549 cells by suppressing cell proliferation, migration, invasion and epithelial-mesenchymal transition and promoting cell apoptosis. Meanwhile, NPAS2 depletion increased OCR and activities of complexes (I, II, III and V), and reduced lactic acid production and glucose uptake in A549 cells, indicating that NPAS2 depletion inhibited aerobic glycolysis, accompanied by reduced expression of glycolytic enzymes. However, the changes caused by NPAS2 knockdown were partly restored by ARRB1 overexpression. In conclusion, our study suggests that ARRB1 could transcriptionally activate NPAS2, facilitating malignant activities and glycolysis, and ultimately promoting the progression of LUAD, proving a novel therapeutic strategy for the treatment of LUAD.

MICALL2 participates in the regulation of epithelial-mesenchymal transition in alveolar epithelial cells - Potential roles in pulmonary fibrosis.

Epithelial-mesenchymal transition (EMT) of alveolar epithelial cells is a vital process in idiopathic pulmonary fibrosis (IPF), which results in the accumulation of fibroblasts and myofibroblasts and excessive extracellular matrix deposition. Based on RNA sequencing analysis and GEO dataset reanalysis, we screened out MICALL2, a gene upregulated in the lungs of IPF mice and alveolar epithelial type II (ATII) cells from IPF patients, and aimed to explore its role in IPF. We validated the expression of MICALL2 in bleomycin (BLM)-induced IPF mice and TGF-ß1-stimulated ATII cells (primary murine ATII cells and A549 cells), and explored the role of MICALL2 in IPF by knockdown of MICALL2 in BLM-induced mice and TGF-ß1-stimulated ATII cells. We found that MICALL2 was upregulated in the lungs of BLM-induced mice and TGF-ß1-stimulated ATII cells. MICALL2-deficient mice had reduced fibrogenesis and restrained EMT upon BLM challenge. Knockdown of MICALL2 restrained the EMT process, in vitro, through impeding ß-catenin nuclear translocation. Mechanistically, we demonstrated that NPAS2 is directly bound to the promoter of MICALL2. Altogether, our data revealed transactivation of MICALL2 induced by NPAS2, contributing to activation of the Wnt/ß-catenin pathway in ATII cells, thus leading to the EMT process and subsequent pulmonary fibrosis. Interfering with MICALL2 may represent an innovative therapeutic target to mitigate the extent of IPF.

NPAS2 promotes aerobic glycolysis and tumor growth in prostate cancer through HIF-1A signaling.

BACKGROUND: Prostate cancer (PCa), one of the common malignant tumors, is the second leading cause of cancer-related deaths in men. The circadian rhythm plays a critical role in disease. Circadian disturbances are often found in patients with tumors and enable to promote tumor development and accelerate its progression. Accumulating evidence suggests that the core clock gene NPAS2 (neuronal PAS domain-containing protein 2) has been implicated in tumors initiation and progression. However, there are few studies on the association between NPAS2 and prostate cancer. The purpose of this paper is to investigate the impact of NPAS2 on cell growth and glucose metabolism in prostate cancer. METHODS: Quantitative real-time PCR (qRT-PCR), immunohistochemical (IHC) staining, western blot, GEO (Gene Expression Omnibus) and CCLE (Cancer Cell Line Encyclopedia) databases were used to analyze the expression of NPAS2 in human PCa tissues and various PCa cell lines. Cell proliferation was assessed using MTS, clonogenic assays, apoptotic analyses, and subcutaneous tumor formation experiments in nude mice. Glucose uptake, lactate production, cellular oxygen consumption rate and medium pH were measured to examine the effect of NPAS2 on glucose metabolism. The relation of NPAS2 and glycolytic genes was analyzed based on TCGA (The Cancer Genome Atlas) database. RESULTS: Our data showed that NPAS2 expression in prostate cancer patient tissue was elevated compared with that in normal prostate tissue. NPAS2 knockdown inhibited cell proliferation and promoted cell apoptosis in vitro and suppressed tumor growth in a nude mouse model in vivo. NPAS2 knockdown led to glucose uptake and lactate production diminished, oxygen consumption rate and pH elevated. NPAS2 increased HIF-1A (hypoxia-inducible factor-1A) expression, leading to enhanced glycolytic metabolism. There was a positive correlation with the expression of NPAS2 and glycolytic genes, these genes were upregulated with overexpression of NPAS2 while knockdown of NPAS2 led to a lower level. CONCLUSION: NPAS2 is upregulated in prostate cancer and promotes cell survival by promoting glycolysis and inhibiting oxidative phosphorylation in PCa cells.

Candidate gene polymorphisms are linked to dispersive and migratory behaviour: Searching for a mechanism behind the "paradox of the great speciators".

The "paradox of the great speciators" has puzzled evolutionary biologists for over half a century. A great speciator requires excellent dispersal propensity to explain its occurrence on multiple islands, but reduced dispersal ability to explain its high number of subspecies. A rapid reduction in dispersal ability is often invoked to solve this apparent paradox, but a proximate mechanism has not been identified yet. Here, we explored the role of six genes linked to migration and animal personality differences (CREB1, CLOCK, ADCYAP1, NPAS2, DRD4, and SERT) in 20 South Pacific populations of silvereye (Zosterops lateralis) that range from highly sedentary to partially migratory, to determine if genetic variation is associated with dispersal propensity and migration. We detected genetic associations in three of the six genes: (i) in a partial migrant population, migrant individuals had longer microsatellite alleles at the CLOCK gene compared to resident individuals from the same population; (ii) CREB1 displayed longer average microsatellite allele lengths in recently colonized island populations (<200 years), compared to evolutionarily older populations. Bayesian broken stick regression models supported a reduction in CREB1 length with time since colonization; and (iii) like CREB1, DRD4 showed differences in polymorphisms between recent and old colonizations but a larger sample is needed to confirm. ADCYAP1, SERT, and NPAS2 were variable but that variation was not associated with dispersal propensity. The association of genetic variants at three genes with migration and dispersal ability in silvereyes provides the impetus for further exploration of genetic mechanisms underlying dispersal shifts, and the prospect of resolving a long-running evolutionary paradox through a genetic lens.

Circadian-Dependent and Sex-Dependent Increases in Intravenous Cocaine Self-Administration in Npas2 Mutant Mice.

Substance use disorder (SUD) is associated with disruptions in circadian rhythms. The circadian transcription factor neuronal PAS domain protein 2 (NPAS2) is enriched in reward-related brain regions and regulates reward, but its role in SU is unclear. To examine the role of NPAS2 in drug taking, we measured intravenous cocaine self-administration (acquisition, dose-response, progressive ratio, extinction, cue-induced reinstatement) in wild-type (WT) and Npas2 mutant mice at different times of day. In the light (inactive) phase, cocaine self-administration, reinforcement, motivation and extinction responding were increased in all Npas2 mutants. Sex differences emerged during the dark (active) phase with Npas2 mutation increasing self-administration, extinction responding, and reinstatement only in females as well as reinforcement and motivation in males and females. To determine whether circulating hormones are driving these sex differences, we ovariectomized WT and Npas2 mutant females and confirmed that unlike sham controls, ovariectomized mutant mice showed no increase in self-administration. To identify whether striatal brain regions are activated in Npas2 mutant females, we measured cocaine-induced ΔFosB expression. Relative to WT, ΔFosB expression was increased in D1+ neurons in the nucleus accumbens (NAc) core and dorsolateral (DLS) striatum in Npas2 mutant females after dark phase self-administration. We also identified potential target genes that may underlie the behavioral responses to cocaine in Npas2 mutant females. These results suggest NPAS2 regulates reward and activity in specific striatal regions in a sex and time of day (TOD)-specific manner. Striatal activation could be augmented by circulating sex hormones, leading to an increased effect of Npas2 mutation in females.SIGNIFICANCE STATEMENT Circadian disruptions are a common symptom of substance use disorders (SUDs) and chronic exposure to drugs of abuse alters circadian rhythms, which may contribute to subsequent SU. Diurnal rhythms are commonly found in behavioral responses to drugs of abuse with drug sensitivity and motivation peaking during the dark (active) phase in nocturnal rodents. Emerging evidence links disrupted circadian genes to SU vulnerability and drug-induced alterations to these genes may augment drug-seeking. The circadian transcription factor neuronal PAS domain protein 2 (NPAS2) is enriched in reward-related brain regions and regulates reward, but its role in SU is unclear. To examine the role of NPAS2 in drug taking, we measured intravenous cocaine self-administration in wild-type (WT) and Npas2 mutant mice at different times of day.

Circadian transcription factor NPAS2 and the NAD+ -dependent deacetylase SIRT1 interact in the mouse nucleus accumbens and regulate reward.

Substance use disorders are associated with disruptions to both circadian rhythms and cellular metabolic state. At the molecular level, the circadian molecular clock and cellular metabolic state may be interconnected through interactions with the nicotinamide adenine dinucleotide (NAD+ )-dependent deacetylase, sirtuin 1 (SIRT1). In the nucleus accumbens (NAc), a region important for reward, both SIRT1 and the circadian transcription factor neuronal PAS domain protein 2 (NPAS2) are highly enriched, and both are regulated by the metabolic cofactor NAD+ . Substances of abuse, like cocaine, greatly disrupt cellular metabolism and promote oxidative stress; however, their effects on NAD+ in the brain remain unclear. Interestingly, cocaine also induces NAc expression of both NPAS2 and SIRT1, and both have independently been shown to regulate cocaine reward in mice. However, whether NPAS2 and SIRT1 interact in the NAc and/or whether together they regulate reward is unknown. Here, we demonstrate diurnal expression of Npas2, Sirt1 and NAD+ in the NAc, which is altered by cocaine-induced upregulation. Additionally, co-immunoprecipitation reveals NPAS2 and SIRT1 interact in the NAc, and cross-analysis of NPAS2 and SIRT1 chromatin immunoprecipitation sequencing reveals several reward-relevant and metabolic-related pathways enriched among shared gene targets. Notably, NAc-specific Npas2 knock-down or a functional Npas2 mutation in mice attenuates SIRT1-mediated increases in cocaine preference. Together, our data reveal an interaction between NPAS2 and SIRT1 in the NAc, which may serve to integrate cocaine's effects on circadian and metabolic factors, leading to regulation of drug reward.

Genetic variants in NPAS2 gene and clinical outcomes of resectable non-small-cell lung cancer.

Background: A series of studies have demonstrated that NPAS2 plays a critical role in the development and progression of several cancers. However, the association between genetic variants in the NPAS2 gene and the clinical outcome of patients with non-small-cell lung cancer (NSCLC) has not been investigated. Methods: Six functional SNPs in NPAS2 were selected and genotyped using the Sequenom iPLEX genotyping system in a cohort of 484 Chinese NSCLC patients undergoing surgery. Multivariate Cox proportional hazards model were used for the prognosis analysis. Results: We found that SNP rs2305158 exhibited a significant association with overall survival of NSCLC patients in the dominant model (hazard ratio [HR]: 0.68; 95% CI: 0.49-0.95; p = 0.02). Lymph node metastasis was significantly associated with increased death risk (HR: 1.73; 95% CI: 1.24-2.40; p = 0.001) in patients with the homozygous wildtype (WW) genotype of rs2305158. However, no significant association was observed between them in patients carrying a heterozygous variant (WV) or homozygous variant (VV) genotype of rs2305158. Finally, in the joint and interaction analysis, the patients carrying homozygous wildtype (WW) genotype and lymph node metastasis from N1 to N3 conferred a significant increased effect on death (HR: 2.29; 95% CI: 1.40-3.76; p = 0.001). Conclusions: Our results suggest that NPAS2 polymorphisms may serve as an independent prognostic marker for NSCLC patients.

Roles of NPAS2 in circadian rhythm and disease.

NPAS2, a circadian rhythm gene encoding the neuronal PAS domain protein 2 (NPAS2), has received widespread attention because of its complex functions in cells and diverse roles in disease progression, especially tumorigenesis. NPAS2 binds with DNA at E-box sequences and forms heterodimers with another circadian protein, brain and muscle ARNT-like protein 1 (BMAL1). Nucleotide variations of the NPAS2 gene have been shown to influence the overall survival and risk of death of cancer patients, and differential expression of NPAS2 has been linked to patient outcomes in breast cancer, lung cancer, non-Hodgkin's lymphoma, and other diseases. Here, we review the latest advances in our understanding of NPAS2 with the aim of drawing attention to its potential clinical applications and prospects.

NPAS2 regulates proliferation of acute myeloid leukemia cells via CDC25A-mediated cell cycle progression and apoptosis.

Promoted proliferation and associated suppression of apoptosis at various stages of myeloid differentiation are well-known features of acute myeloid leukemia (AML), but understanding of the molecular processes involved remains limited. As a crucial circadian agent, neuronal PAS domain protein 2 (NPAS2) is widely recognized as a promising predictor of clinical outcome in various malignancies. Nevertheless, the understanding of its influence on AML is insufficient. Using KD cells and expression assays, we carried out detailed investigation of the role of NPAS2 in AML in vivo and in vitro. Firstly, we found that NPAS2 expression was elevated in AML cells both in vivo and in vitro. NPAS2 knockdown via lentiviral infection clearly suppressed proliferation of MV4-11 and MOLM-14 cells. Additionally, NPAS2 knockdown caused G1/S cell cycle arrest (CCA), which inhibited CDC25A expression. Moreover, NPAS2 knockdown promoted cell death, as evidenced by increased caspase-3 cleavage, and change in Bcl2/Bax production. Excessive CDC25A expression eliminated G1/S CCA triggered by NPAS2 knockdown and death of NPAS2 knocked down MOLM and MV4-11 cells. The expression of CDC25A was stabilized by NPAS2, which induced cell cycle progression and participated in suppression of cell death by modulating caspase-3 cleavage, and expression of Bcl2/Bax. We therefore indicated NPAS2 to be a crucial modulator of survival as well as proliferation. Our research sheds light on the etiology of the proliferation of promyelocytes modulated via NPAS2 with regard to AML.

Genetic variants in the circadian rhythm pathway as indicators of prostate cancer progression.

BACKGROUND: To determine the association between circadian pathway genetic variants and the risk of prostate cancer progression. METHODS: We systematically evaluated 79 germline variants in nine circadian pathway genes in a cohort of 458 patients with localized prostate cancer as the discovery phase. We then replicated the significant findings in another cohort of 324 men with more advanced disease. The association of each variant with prostate cancer progression was evaluated by a log-rank test and Cox regression. RESULTS: A single nucleotide polymorphism of the neuronal PAS domain protein 2 (NPAS2) gene (rs6542993 A>T) was found to be associated with a significantly higher risk of disease progression in both localized (P = 0.001) and advanced (P = 0.039) prostate cancer cases. In silico analysis revealed decreased expression levels of NPAS2 in carriers of the T allele of rs6542993 compared with those carrying the A allele. Consistently, downregulation of NPAS2 expression was associated with more aggressive prostate cancer and poor progression-free survival (log-rank P = 0.002). CONCLUSIONS: The NPAS2 rs6542993 polymorphism may be a promising biomarker, and may shed light on the pathways that govern prostate cancer progression.

Conditional postnatal deletion of the neonatal murine hepatic circadian gene, Npas2, alters the gut microbiome following restricted feeding.

BACKGROUND: We have recently shown in both non-human primates and in rodents that fetal and neonatal hepatic expression of the circadian transcription factor, Npas2, is modulated by a high fat maternal diet and plays a critical role in establishing life-long metabolic homeostasis. Similarly, we and others have also established the importance of the maternal and early postnatal diet on establishment of the early gut microbiome. OBJECTIVE: We hypothesized that altered circadian gene expression solely in the neonatal liver would result in gut microbiome dysbiosis, especially with diet-induced metabolic stress (ie, restricted feeding). Using a murine model in which we conditionally knock out Npas2 in the neonatal liver, we aimed to determine the role of the circadian machinery in gut dysbiosis with restricted feeding. STUDY DESIGN: We collected fecal samples from liver Npas2 conditional knockout (n = 11) and wild-type (n = 13) reproductive-aged mice before (study day 0) and after the restricted feeding study (study day 17). Extracted DNA was sequenced using the MiSeq Illumina platform using primers specific for the V4 region of the 16S ribosomal DNA gene. The resulting sequences were quality filtered, aligned, and assigned taxonomy. Principal coordinate analysis was performed on unweighted and weighted UniFrac distances between samples with a permutation analysis of variance to assess clustering significance between groups. Microbial taxa that significantly differ between groups of interest was determined using linear discriminate analysis effect size and randomForrest. RESULTS: Principal coordinate analysis performed on weighted UniFrac distances between male conditional knockout and wild-type cohorts revealed that the gut microbiome of the mice did not differ by genotype at the start of the restricted feeding study but did differ by virtue of genotype at the end of the study (P = .001). Moreover, these differences could be at least partially attributed to restricted feeding-associated alterations in relative abundance of the Bacteroides genus, which has been implicated as crucial to establishing a healthy gut microbiome early in development. CONCLUSION: Here we have provided an initial key insight into the interplay between neonatal establishment of the peripheral circadian clock in the liver and the ability of the gut microbiome to respond to dietary and metabolic stress. Because Npas2 expression in the liver is a target of maternal high-fat diet-induced metabolic perturbations during fetal development, we speculate that these findings have potential implications in the long-term metabolic health of their offspring.

DEC2 Blocks the Effect of the ARNTL2/NPAS2 Dimer on the Expression of PER3 and DBP.

The expression of clock genes ARNTL2, NPAS2 and DEC2 are disturbed in rheumatoid arthritis, an autoimmune disease with circadian variation of symptoms. We have shown that TNF is a potent inducer of these genes. We investigated the regulation of ARNTL2 and NPAS2 by TNF and elucidated their effect on other clock gene expressions. Additionally, we studied the effect of DEC1 and DEC2 on ARNTL, ARNTL2 and NPAS2. Cultured primary human fibroblasts were stimulated with TNF and the effects on ARNTL2 and NPAS2 were studied with RT-qPCR and immunofluorescence staining. The role of NF-κB was analyzed using IKK-2 inhibitor IMD-0354. TNF promoted ARNTL2 localization into the nuclei. Similar to DEC2, the effects of TNF on ARNTL2 and NPAS2 expressions were mediated via NF-κB. Cloned ARNTL, ARNTL2, NPAS2, DEC1 and DEC2 were transfected into HEK293. The ARNTL2/NPAS2 dimer was a weaker inducer of PER3 and DBP than ARNTL/NPAS2. ARNTL2 and NPAS2 are regulated by TNF via the same mechanism as DEC2. Compared to their paralogs they have unique effects on other circadian components. Our data suggest that these genes are responsible, at least in fibroblasts, for the accurate adaptation of circadian timekeeping in individual cells during inflammation.

Sex differences in circadian timing systems: implications for disease.

Virtually every eukaryotic cell has an endogenous circadian clock and a biological sex. These cell-based clocks have been conceptualized as oscillators whose phase can be reset by internal signals such as hormones, and external cues such as light. The present review highlights the inter-relationship between circadian clocks and sex differences. In mammals, the suprachiasmatic nucleus (SCN) serves as a master clock synchronizing the phase of clocks throughout the body. Gonadal steroid receptors are expressed in almost every site that receives direct SCN input. Here we review sex differences in the circadian timing system in the hypothalamic-pituitary-gonadal axis (HPG), the hypothalamic-adrenal-pituitary (HPA) axis, and sleep-arousal systems. We also point to ways in which disruption of circadian rhythms within these systems differs in the sexes and is associated with dysfunction and disease. Understanding sex differentiated circadian timing systems can lead to improved treatment strategies for these conditions.

Functional polymorphisms in the NPAS2 gene are associated with overall survival in transcatheter arterial chemoembolization-treated hepatocellular carcinoma patients.

The functional abnormality of circadian regulation genes is involved in the development and progression of hepatocellular carcinoma (HCC). However, the association between functional single nucleotide polymorphisms (SNPs) in circadian gene NPAS2 and the overall survival of HCC patients treated with transcatheter arterial chemoembolization (TACE) has never been investigated. Six functional SNPs in the NPAS2 gene were genotyped using the Sequenom iPLEX genotyping system in a cohort of 448 unresectable Chinese patients with HCC treated with TACE. Multivariate Cox proportional hazards model and Kaplan-Meier curves were used for the prognosis analysis. We found that two SNPs, rs1053096 and rs2305160, in the NPAS2 gene showed significant associations with overall death risk in HCC patients in the recessive model (hazard ratio [HR] = 1.48; 95% confidence interval [CI], 1.13-1.94; P = 0.004) and in the dominant model (HR = 1.63; 95% CI, 1.29-2.07; P < 0.001), respectively. Moreover, we observed a cumulative effect of these two SNPs on HCC overall survival, indicating a significant trend of increasing death risk with increasing number of unfavorable genotypes (P for trend < 0.001). Compared with the patients without any unfavorable genotypes, the HRs for patients with one and two unfavorable genotypes were 1.41 (95% CI, 1.10-1.82; P = 0.007) and 2.09 (95% CI, 1.46-2.97, P < 0.001), respectively. The haplotype and diplotype analyses further characterized the association between NPAS2 genotype and survival of HCC patients. Our results for the first time suggest that NPAS2 gene polymorphisms may serve as an independent prognostic marker for HCC patients treated with TACE.

Silencing NPAS2 promotes cell growth and invasion in DLD-1 cells and correlated with poor prognosis of colorectal cancer.

Emerging evidences show that circadian rhythm disorder is an important factor of tumor initiation and development. Neuronal PAS domain protein2 (NPAS2), which is the largest circadian gene, has been proved to be a novel prognostic biomarker in breast cancer and non-Hodgkin's lymphoma. However, the potential functions of NPAS2 in colorectal cancer are still unknown. In our present study, we detected the mRNA expressions of NPAS2 in 108 CRC patients by RT-PCR, and found that NPAS2 expression was significantly down-regulated in tumor tissues than that in NATs. Clinicopathologic analysis revealed that low expression of NPAS2 was associated with the tumor size, TNM stage and tumor distance metastasis in colorectal cancer (p<0.05). Furthermore, we effectively down-regulated NPAS2 mRNA expression by transfecting RNA interfere fragments into DLD-1 cells, and our results in vitro demonstrated that silencing NPAS2 expression could promote cell proliferation, cell invasion and increase the wound healing ability (p<0.05). However, down-regulating NPAS2 expression did not influence the apoptotic rate in DLD-1 cells (p>0.05). In conclusion, our study suggested that NPAS2, functioned as a potential tumor suppressor gene, could serve as a promising target and potential prognostic indicator for colorectal cancer.

Association between glucose metabolism, the circadian cycle and hypoxia: Evaluation of the NPAS2 and Rev-Erb-α protein serum levels in obstructive sleep apnea patients - a pilot study.

BACKGROUND: Obstructive sleep apnea (OSA) is one of the risk factors for diabetes mellitus type 2 (DM2). As OSA is associated with the disruption of the circadian rhythm, it affects circadian clock proteins, including neuronal PAS domain protein 2 (NPAS2) and nuclear receptor subfamily 1 group D member 1 (Rev-Erb-α). These proteins have been shown to be related to metabolic abnormalities, i.a., insulin resistance. OBJECTIVES: The present pilot study aimed to investigate the NPAS2 and Rev-Erb-α protein serum levels in the groups of patients with severe OSA and severe OSA+DM2 in comparison with healthy controls, taking into account correlations with polysomnography (PSG) parameters (e.g., oxygen saturation (SpO2) variables). MATERIAL AND METHODS: A total of 40 participants were included in the study. They were split into 3 groups as follows: the OSA group (n = 17; apnea-hypopnea index (AHI) >30, no DM2); the OSA+DM2 group (n = 7; AHI > 30 and DM2); and the control group (n = 16; AHI < 5, no DM2). All participants underwent a nocturnal PSG examination and had their blood collected the following morning. The serum levels of NPAS2 and Rev-Erb-α proteins were assessed using the enzyme-linked immunosorbent assay (ELISA). RESULTS: The mean NPAS2 protein level was significantly lower in the OSA group as compared to healthy individuals (p = 0.017). Additionally, the OSA group presented with lower NPAS2 protein levels as compared to the OSA+DM2 group, but only a tendency was observed (p = 0.094). No differences in the Rev-Erb-α protein concentration were noticed. Furthermore, a negative correlation between AHI during rapid eye movement (REM) sleep and the NPAS2 protein serum level was observed (r = -0.478; p = 0.002). CONCLUSIONS: Serum NPAS2 protein might be involved in metabolic dysregulation present among OSA patients, while the mechanism itself may be associated with REM sleep.

Effects of NAD(P)H and its derivatives on the DNA-binding activity of NPAS2, a mammalian circadian transcription factor.

NPAS2 is a transcription factor that regulates mammalian circadian rhythms. It has been suggested that NPAS2 DNA-binding activity is regulated by the intracellular redox state of NAD(P)H, although the mechanism remains unclear. To investigate the NAD(P)H interaction site of murine NPAS2, we performed electrophoretic mobility shift assays using several truncation mutants of the NPAS2 bHLH domain. Among the mutants, NPAS2 containing the N-terminal 61 residues formed a heterodimer with BMAL1 to bind DNA, and NAD(P)H enhanced the binding activity, while NAD(P)H inhibited the DNA-binding activity of the BMAL1 homodimer in a dose-dependent manner. NAD(P)H derivatives such as 2',5'-ADP, nicotinamide, nicotinic acid and nicotinic acid adenine dinucleotide (NAAD) did not affect the DNA-binding activity. Interestingly, NAD(P)(+), previously reported as an inhibitor, did not affect NPAS2 binding activity in the presence or absence of NAD(P)H in our system. These results suggest that NPAS2 DNA-binding activity is specifically enhanced by NAD(P)H independently of NAD(P)(+) and that the N-terminal 1-61 amino acids of NPAS2 are sufficient to sense NAD(P)H.

Role of the Circadian Gas-Responsive Hemeprotein NPAS2 in Physiology and Pathology.

Neuronal PAS domain protein 2 (NPAS2) is a hemeprotein comprising a basic helix-loop-helix domain (bHLH) and two heme-binding sites, the PAS-A and PAS-B domains. This protein acts as a pyridine nucleotide-dependent and gas-responsive CO-dependent transcription factor and is encoded by a gene whose expression fluctuates with circadian rhythmicity. NPAS2 is a core cog of the molecular clockwork and plays a regulatory role on metabolic pathways, is important for the function of the central nervous system in mammals, and is involved in carcinogenesis as well as in normal biological functions and processes, such as cardiovascular function and wound healing. We reviewed the scientific literature addressing the various facets of NPAS2 and framing this gene/protein in several and very different research and clinical fields.

Characterization of molecular subtypes based on chromatin regulators and identification of the role of NPAS2 in lung adenocarcinoma.

BACKGROUND: Chromatin regulators (CRs) are critical epigenetic modifiers and have been reported to play critical roles during the progression of various tumors, but their role in lung adenocarcinoma (LUAD) has not been comprehensively studied. METHODS: Differential expression and univariate Cox regression analyses were conducted to identify the prognostic CRs. Consensus clustering was applied to classify the subtypes of LUAD based on prognostic CRs. LASSO-multivariate Cox regression method was used for construction of a prognostic signature and development of chromatin regulator-related gene index (CRGI). The capacity of CRGI to distinguish survival was evaluated via Kaplan-Meier method in multiple datasets. Relationship between CRGI and tumor microenvironment (TME) was evaluated. Additionally, clinical variables and CRGI were incorporated to create a nomogram. The role of the prognostic gene NPAS2 in LUAD was elucidated via clinical samples validation and a series of in vitro and in vivo experiments. RESULTS: Two subtypes of LUAD were classified based on 46 prognostic CRs via consensus clustering which had significantly different survival and TME. A prognostic signature consisting of six CRs (MOCS, PBK, CBX3, A1CF, NPAS2, and CTCFL) was developed and proved to be an effective survival predictor in multiple independent datasets. The prognostic signature was also demonstrated to be an indicator of TME and sensitivity to immunotherapy and chemotherapy. The nomogram was suggested to be a simple tool that can predict survival accurately. Clinical samples show that NPAS2 is highly expressed in LUAD tissues, and in vitro and in vivo experiments demonstrated that inhibition of NPAS2 impeded malignant progression of LUAD cells. CONCLUSIONS: Our study comprehensively unveiled the functions of CRs in LUAD, developed a classifier to predict survival and response to treatments, and suggested that NPAS2 promoted LUAD progression for the first time.

High expression of the circadian clock gene NPAS2 is associated with progression and poor prognosis of gastric cancer: A single-center study.

BACKGROUND: The prognostic assessment of patients after surgical resection of gastric cancer (GC) patients is critical. However, the role of the circadian clock gene NPAS2 expression in GC remains unknown. AIM: To explore the relationship between NPAS2 and the survival prognosis of GC patients and clarify its role in evaluating GC prognosis. METHODS: The tumor tissues and clinical data of 101 patients with GC were collected retrospectively. Immunohistochemical staining (IHC) was used to detect the expression of NPAS2 protein in GC and adjacent tissues. Univariate and multivariate Cox regression analysis was used to determine the independent prognostic factors of GC, and a nomogram prediction model was established. The receiver operating characteristic (ROC) curve, the ROC area under the curve, the calibration curve, and C-index were used to evaluate the predictive effectiveness of the model. Kaplan Meier analysis was used to compare the risk stratification of subgroups according to the median score in the nomogram model of each patient. RESULTS: Microarray IHC analysis showed that the positive rate of NPAS2 protein expression in GC tissues was 65.35%, which was significantly higher than 30.69% in adjacent tissues. The high expression of NPAS2 was correlated with tumor-node-metastasis (TNM) stage (P < 0.05), pN stage (P < 0.05), metastasis (P < 0.05), venous invasion (P < 0.05), lymphatic invasion (P < 0.05), and lymph node positive (P < 0.05) of GC. Kaplan Meier survival analysis showed that the 3-year overall survival (OS) of patients with high NPAS2 expression was significantly shortened (P < 0.0001). Univariate and multivariate COX regression analysis showed that TNM stage (P = 0.009), metastasis (P = 0.009), and NPAS2 expression (P = 0.020) were independent prognostic factors of OS in GC patients for 3 years. The nomogram prediction model based on independent prognostic factors has a C-Index of 0.740 (95%CI: 0.713-0.767). Furthermore, subgroup analysis showed that the 3-year OS time of the high-risk group was significantly lower than that of the low-risk group (P < 0.0001). CONCLUSION: NPAS2 is highly expressed in GC tissues and is closely related to worse OS in patients. Therefore, the evaluation of NPAS2 expression may be a potential marker for GC prognosis evaluation. Notably, the nomogram model based on NPAS2 can improve the accuracy of GC prognosis prediction and assist clinicians in postoperative patient management and decision-making.