Npas2 Expression Research Articles

Folium crataegi boosts skin regeneration for burn injury in rats through multiple ways

Appropriate topical dressings for burn treatments are important to accelerate skin wound recovery and prevent external infections. This study aimed to evaluate the effect and investigate the mechanism of folium crataegi (Crataegus pinnatifida Bge.) for the treatment of burn wounds, as well as to compare the therapeutic effects of aqueous extracts (HLW) and alcoholic extracts (HLE) from folium crataegi. The results demonstrated that both HLW and HLE groups exhibited a higher wound contraction rate than the silver sulfadiazine (SSD) ointment group. Moreover, HLW showed more significant wound repair effects than HLE. HLW significantly increased levels of EGF and FGF-2 in wound tissue, as well as TGF-β1, VEGF, CAT and IL-10 in serum. Folium crataegi extract, especially aqueous extracts, exerted good anti-inflammatory, anti-oxidant and anti-bacterial effects by upregulating the expression of lag3, txn1 and slpi, respectively. Folium crataegi extract significantly inhibits the expression of npas2, a key gene in the circadian rhythm pathway. In conclusion, this research illustrated that the folium crataegi extract, especially aqueous extracts, had better therapeutic effects on skin burns through multiple ways, possibly including a novel mechanism related to circadian rhythm pathway. These findings suggest that folium crataegi could be a valuable source of compounds for enhancing skin regeneration through multiple ways.

In vitro assessment of Neuronal PAS domain 2 mitigating compounds for scarless wound healing.

The core circadian gene Neuronal PAS domain 2 (NPAS2) is expressed in dermal fibroblasts and has been shown to play a critical role in regulating collagen synthesis during wound healing. We have performed high throughput drug screening to identify genes responsible for downregulation of Npas2 while maintaining cell viability. From this, five FDA-approved hit compounds were shown to suppress Npas2 expression in fibroblasts. In this study, we hypothesize that the therapeutic suppression of Npas2 by hit compounds will have two effects: (1) attenuated excessive collagen deposition and (2) accelerated dermal wound healing without hypertrophic scarring. To test the effects of each hit compound (named Dwn1, 2, 3, 4, and 5), primary adult human dermal fibroblasts (HDFa) were treated with either 0, 0.1, 1, or 10 μM of a single hit compound. HDFa behaviors were assessed by picrosirius red staining and quantitative RT-PCR for in vitro collagen synthesis, cell viability assay, in vitro fibroblast-to-myofibroblast differentiation test, and cell migration assays. Dwn1 and Dwn2 were found to significantly affect collagen synthesis and cell migration without any cytotoxicity. Dwn3, Dwn4, and Dwn5 did not affect collagen synthesis and were thereby eliminated from further consideration for their role in mitigation of gene expression or myofibroblast differentiation. Dwn1 also attenuated myofibroblast differentiation on HDFa. Dwn1 and Dwn2 may serve as possible therapeutic agents for future studies related to skin wound healing.

Expression pattern of circadian rhythm-related genes and its potential relationship with miRNAs activity in endometrial cancer.

The circadian clock is an autonomous oscillator that controls key aspects of cell physiology, including metabolism, transcriptional state, and cell signaling. Disturbances of circadian rhythms lead to disruption of cell and tissue homeostasis, which promotes carcinogenesis. The aim of the study was to determine the expression of circadian rhythm-related genes in endometrial cancer and to select miRNAs involved in the regulation of their expression. 50 endometrial tissue samples were collected from patients who underwent hysterectomy: 40 diagnosed with endometrial cancer and 10 without cancer. Expression profile of circadian rhythm-related genes was evaluated using microarrays and validated with RT-qPCR. MicroRNA expression was assessed using microarrays. Then mirTAR tool was used to identify miRNAs involved in the expression regulation of circadian rhythm-related genes. CLOCK expression is disrupted in endometrial cancer, which may be due to miR-15b, miR-331-3p and miR-200a overexpression. Elevated NPAS2 and CSNK1D levels may be associated with miR-432 silencing. In addition, high miR-874 and miR-200a expression may be potentially responsible for the reduction of PER3 level. Change of CLOCK, CSNK1D, NPAS2 and PER3 expression may suggest that circadian rhythms are disrupted in endometrial cancer. A possible mechanism of the observed changes may be related to miRNAs activity.

Indirubin relieves inflammatory injury of chondrocytes in a mouse model of osteoarthritis

To investigate the effect of indirubin for relieving joint inflammation and injury in a rat model of osteoarthritis. Articular cartilage chondrocytes were isolated from adult rat knee joint and cultured in the presence of interleukin-1β (IL-1β) and 0.1, 0.5, 1.0, or 2.0 μmol/L indirubin. The cells were transfected with NPAS2 siRNA or a non-specific siRNA, and the cell proliferation and apoptosis were evaluated using tetramethylthiazole blue staining and flow cytometry. The protein expression levels of Bax, Bcl-2, ACAN, COL2A1, MMP-13 and NPAS2 were detected with Western blotting, and the levels of NO, PGE2 and TNF-α in the culture supernatant were determined with ELISA. The mRNA expression levels of NPAS2, ACAN, COL2A1 and MMP-13 were detected using fluorescence quantitative PCR. In a C57BL/6 mouse model of osteoarthritis, the effect of indirubin on BAX, Bcl-2, ACAN and MMP-13 protein expressions in the bone and joint tissues were evaluated with Western blotting. Treatment with 0.1 μmol/L indirubin produced no significant changes in chondrocyte proliferation, apoptosis, caspase-3 activity, or BAX and Bcl-2 protein expressions. At higher doses (0.5, 1.0 and 2.0 μmol/L), indirubin significantly promoted cell proliferation, increased Bcl-2 protein expression, and lowered cell apoptosis rate, caspase-3 activity and Bax protein expression (P < 0.05). Indirubin treatment at 0.5 μmol/L up-regulated the protein and mRNA expressions of NPAS2, ACAN and COL2A1, and down-regulated the expressions of MMP-13, NO, PGE2 and TNF-α (P < 0.05). Interference of NPAS2 expression significantly attenuated the protective effect of 0.5 μmol/L indirubin against IL-1β-induced chondrocyte injury. The mouse model of osteoarthritis showed obviously increased protein levels of BAX and MMP-13 (P < 0.01) and decreased levels of Bcl-2 (P < 0.05) and ACAN (P < 0.01) in the knee joint, and indirubin treatment of the mouse models significantly inhibited the increase of BAX and MMP-13 protein expressions (P < 0.01) and up-regulated the protein expressions of Bcl-2 and ACAN (P < 0.05). Indirubin has a protective effect on osteoarthritis tissue and alleviates inflammation and damage of osteoarthritis chondrocytes possibly through NPAS2.

A signature of circadian rhythm genes in driving anaplastic thyroid carcinoma malignant progression

BackgroundAnaplastic thyroid carcinoma (ATC) was a rare and extremely malignant endocrine cancer. Recently, dysregulation of circadian rhythm genes was demonstrated to play an essential role in tumor progression, while its exact role and mechanism in ATC remained poorly clear. Methods4 ATC-related datasets were integrated to screen for differentially expressed circadian rhythm genes (DE-CRGs). Thereafter, Multiscale Embedded Gene Co-expression Network Analysis (MEGENA) and network enrichment analysis were conducted to investigate the dynamic characteristics of circadian rhythm genes. Next, Lasso-logistic model and immunohistochemistry were applied for determining the candidates. Finally, cell biological experiments and gene set enrichment analysis (GSEA) were used to confirm the roles of NPAS2 in ATC. Results25 DE-CRGs were firstly identified in ATC. These DE-CRGs mainly regulated mitotic nuclear division, cytokinesis and DNA replication signals. Notably, NPAS2, CSNK1E, NAMPT, TYMS, SERPINE1, TOP2A, JUN, EGR3 and HEBP1 were identified as the dynamic signature in the malignant progression of ATC, which were confirmed by prognostic analysis. Furthermore, NPAS2 was found to be significantly up-regulated in ATC through clinical samples and cell experiments. Silencing NPAS2 effectively inhibited the proliferation, migration and invasion of ATC cells. GSEA showed that high expression of NPAS2 was mainly associated with cell cycle and focal adhesion, and silencing of NPAS2 suppressed these signals in our experiments. ConclusionsIn summary, we found a dynamic 9-DE-CRGs signature in ATC. And the aberrant expression of NPAS2 drove the malignant phenotypes of ATC, which facilitated to deepen our understanding of the roles of circadian rhythm genes in ATC.

Time of trauma prospectively affects PTSD symptom severity: The impact of circadian rhythms and cortisol

A key feature of posttraumatic stress disorder (PTSD) is a disruption of hypothalamic-pituitary-adrenal (HPA) axis feedback sensitivity and cortisol levels. Despite known diurnal rhythmicity of cortisol, there has been little exploration of the circadian timing of the index trauma and consequent cortisol release. Stress-related glucocorticoid pulses have been shown to shift clocks in peripheral organs but not the suprachiasmatic nucleus, uncoupling the central and peripheral clocks. A sample of 425 participants was recruited in the Emergency Department following a DSM-IV-TR Criterion A trauma. The Zeitgeber time of the trauma was indexed in minutes since sunrise, which was hypothesized to covary with circadian blood cortisol levels (high around sunrise and decreasing over the day). Blood samples were collected M(SD)= 4.0(4.0) hours post-trauma. PTSD symptoms six months post-trauma were found to be negatively correlated with trauma time since sunrise (r(233) = −0.15, p = 0.02). The effect remained when adjusting for sex, age, race, clinician-rated severity, education, pre-trauma PTSD symptoms, and time of the blood draw (β = -0.21, p = 0.00057). Cortisol levels did not correlate with blood draw time, consistent with a masking effect of the acute stress response obscuring the underlying circadian rhythm. Interactions between trauma time and expression of NPAS2 (punadjusted=0.042) and TIMELESS (punadjusted=0.029) predicted six-month PTSD symptoms. The interaction of trauma time and cortisol concentration was significantly correlated with the expression of PER1 (padjusted=0.029). The differential effect of time of day on future symptom severity suggests a role of circadian effects in PTSD development, potentially through peripheral clock disruption.

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.

Therapeutic downregulation of neuronal PAS domain 2 (Npas2) promotes surgical skin wound healing.

Attempts to minimize scarring remain among the most difficult challenges facing surgeons, despite the use of optimal wound closure techniques. Previously, we reported improved healing of dermal excisional wounds in circadian clock neuronal PAS domain 2 (Npas2)-null mice. In this study, we performed high-throughput drug screening to identify a compound that downregulates Npas2 activity. The hit compound (Dwn1) suppressed circadian Npas2 expression, increased murine dermal fibroblast cell migration, and decreased collagen synthesis in vitro. Based on the in vitro results, Dwn1 was topically applied to iatrogenic full-thickness dorsal cutaneous wounds in a murine model. The Dwn1-treated dermal wounds healed faster with favorable mechanical strength and developed less granulation tissue than the controls. The expression of type I collagen, Tgfβ1, and α-smooth muscle actin was significantly decreased in Dwn1-treated wounds, suggesting that hypertrophic scarring and myofibroblast differentiation are attenuated by Dwn1 treatment. NPAS2 may represent an important target for therapeutic approaches to optimal surgical wound management.

Involvement of Npas2 and Per2 modifications in zinc-induced acute diurnal toxicity in mice.

Zinc (Zn) is one of the most essential trace elements in the body and an integral part of many enzyme systems. Zn deficiency is characterized by growth retardation, loss of appetite, and impaired immune function. In contrast, Zn overdoses can be associated with liver, kidney, and stomach damage. We focused on the "chronotoxicity," or the relationship between injection time and severity of chemical toxicity. The aim of this study was to investigate the chronotoxicity of Zn and the in vivo factors involved. Seven-week-old male ICR mice were administered Zn at six different time points per day (zeitgeber time [ZT]: ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22). Mortality was monitored for 7-days after administration. The mice were tolerant to Zn administered at ZT2 and ZT6, and were highly sensitive at ZT14 and ZT18. Furthermore, when mice were administered a non-lethal dose of Zn, the levels of hepatic injury indicators (AST and ALT) were much higher at ZT14 than at ZT2. To explore the mechanism of Zn-induced diurnal hepatotoxicity, we performed an in vitro experiment, focusing on the clock genes. We found that Zn downregulated the expression levels of several clock genes, neuronal PAS domain protein 2 (Npas2) and Peroid2 (Per2), in Hepa1-6 cells. Interestingly, overexpression of both Npas2 and Per2 restored Zn-induced toxicity in Hepa1-6 cells. Since NPAS2 and PER2 are known to modulate the hepatic injury induced by carbon tetrachrolide or acetaminophen, our results suggest that Zn-induced diurnal toxicity may be associated with modulation of Npas2 and Per2 gene expression.

Identification of circadian clock genes as regulators of immune infiltration in Hepatocellular Carcinoma.

Background: Multiple studies have reported that the immune system is under the control of a circadian clock, especially in cancers, but how circadian clock genes shape tumor immune cell infiltration in hepatocellular carcinoma (HCC) remains unclear.Methods: The rhythmicity of circadian clock genes was investigated using the GETx database. The expression and methylation level of circadian clock genes in HCC and paracancerous was evaluated using the GETx and TCGA databases. The differential expression of circadian clock genes in HCC was analyzed using the “limma” package of the R 4.0.4 software. The prognosis of each circadian clock gene was accessed by Kaplan-Meier survival analysis and Cox proportional hazards regression analysis. Quantitative real-time PCR and immunohistochemistry (IHC) was carried out to confirm the results. The relationship between circadian rhythm and immune infiltration in HCC was evaluated using the TIMER database and the CIBERSORT algorithm.Results: In addition to RORA, RORB, and ARNTL2, there was a rhythmic expression of other circadian clock genes in liver tissue. The correlation between the expression of circadian clock genes differed when comparing HCC and liver tissue. HCC patients who express low levels of PER-1and CRY2 had a poor overall survival (OS). In contrast, patients with higher expression of NPAS2 had a poor prognosis. In HCC, the expression of the PER-1, CRY2, and NPAS2 genes was closely related to immune infiltration.Conclusion: Our study indicated the disruption of the expression of circadian clock-regulated genes in HCC and identified PER-1, CRY2, and NPAS2 as independent predictors of survival. These genes may be applied as candidate molecular targets for diagnosis and therapy of HCC.

Deletion of Npas2 in the liver alters the functional gut microbiome at light/dark timepoints

In utero exposure of a maternal high fat diet (mHFD) results in the targeted epigenetic reprograming and disruption of circadian clock genes in the fetal liver. However, it is unclear if this disruption of the circadian clock exacerbates the effect of the offspring’s post-weaning high fat diet (oHFD) on the function of its gut microbiome. To understand the interaction of the circadian clock and the offspring gut microbiome with HFD feeding, we disrupted liver Npas2 expression and analyzed the resultant microbiome in light and dark cycles of feeding.

Bioinformatics Analysis of Differentially Expressed Rhythm Genes in Liver Hepatocellular Carcinoma.

Liver Hepatocellular Carcinoma (LIHC), a malignant tumor with high incidence and mortality, is one of the most common cancers in the world. Multiple studies have found that the aberrant expression of rhythm genes is closely related to the occurrence of LIHC. This study aimed to use bioinformatics analysis to identify differentially expressed rhythm genes (DERGs) in LIHC. A total of 563 DERGs were found in LIHC, including 265 downregulated genes and 298 upregulated genes. KEGG pathway enrichment and GO analyses showed that DERGs were significantly enriched in rhythmic and metabolic processes. Survival analysis revealed that high expression levels of CNK1D, CSNK1E, and NPAS2 were significantly associated with the low survival rate in LIHC patients. Through cell experiment verification, the mRNA expression levels of CSNK1D, CSNK1E, and NPAS2 were found to be strongly upregulated, which was consistent with the bioinformatics analysis of LIHC patient samples. A total of 23 nodes and 135 edges were involved in the protein–protein interaction network of CSNK1D, CSNK1E, and NPAS2 genes. Clinical correlation analyses revealed that CSNK1D, CSNK1E, and NPAS2 expression levels were high-risk factors and independently connected with the overall survival rate in LIHC patients. In conclusion, the identification of these DERGs contributes to the exploration of the molecular mechanisms of LIHC occurrence and development and may be used as diagnostic and prognostic biomarkers and molecular targets for chronotherapy in LIHC patients in the future.

1128 Maternal high fat diet increases weight gain in female mice without Npas2 in the liver

Maternal high fat diet (MHFD) effects the metabolic health of offspring. We have previously demonstrated that in non-human primates, a MHFD is associated with the disruption of Npas2 in the fetal liver. We therefore developed a novel fetal hepatic Npas2 conditional knock out (cKO) mouse model to determine the role of hepatic Npas2 in adapting to the effects of a MHFD. In order to delete Npas2 during fetal life and specifically in the liver, a CreLox deletion was put under regulation of the AFP enhancer. Fetal cKO (n=70) and control mice (n=62) were thereby generated for this study from dams on either a MHFD or maternal control diet (MCD) during gestation until mice were weaned. At weaning, mice were placed onto HFD (42% fat) or a CD. To determine how a MHFD affects weight gain and adiposity mice were weighed weekly for 24 weeks and fat mass was determined by quantitative magnetic resonance (QMR). Since weight gain and adiposity can be linked to diabetes the mice were challenged with a glucose tolerance test (GTT) and an insulin tolerance test (ITT). Female fetal cKO mice exposed to a MHFD had significantly increased mass when weaned onto a CD or a HFD (Figure 1). However, female fetal cKO mice had significantly increased adiposity only when weaned onto a CD (p=0.002). Both female and male cKO mice exposed to a MHFD and weaned onto a CD had elevated blood glucose concentrations during a GTT (Figure 2). Despite these elevated glucose concentrations, no differences in glucose were measured during the ITTs. We have conditionally deleted a circadian gene (Npas2) during fetal development and have shown that a MHFD exposure alters weight gain, adiposity, and glucose response; this cannot be corrected with CD feeding in the postnatal period. These findings collectively suggest that Npas2 expression during fetal development has a role in adapting to metabolic stress during fetal and early development which has lifelong effects.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Polymethoxyflavones in Citrus Regulate Lipopolysaccharide-Induced Oscillating Decay of Circadian Rhythm Genes by Inhibiting Nlrp3 Expression.

The aim of this study is to compare the regulatory abilities of citrus flavonoids on the oscillating expression of circadian genes. Seven varieties of citrus fruits and twenty-five citrus flavonoids were selected and evaluated. Per2 luciferase bioluminescence report system and serum shock were used to induce circadian gene expression in mouse microglia BV-2 cells. In vivo experiments were carried out using C57BL6/J mice to evaluate the regulation of flavonoids on the oscillatory expression of liver biorhythm genes. Lipopolysaccharide was used to interfere the gene oscillating expression. QRT-PCR was performed to detect the expression of circadian rhythm-related genes, including Clock, Bmal1, Per1, Per2, Per3, Cry1, Cry2, Rev-erbα, Rev-erbβ, Rorα, Dbp, and Npas2. The results show that the polymethoxyflavones (PMFs) exerted stronger circadian gene regulatory capability, while the flavonoids containing glycosides showed no biological activity. Also, all tested flavonoids decreased LPS-induced nitric oxide release, but only polymethoxyflavones inhibited circadian rhythm disorder. PMFs inhibited Nlrp3 inflammasome-related genes and proteins, including Nlrp3, IL-1β, ASC, and Caspase1, while other flavonoids only affected IL-1β and Caspase1 expression. This mechanism was preliminarily verified using the Nlrp3 inhibitor INF39.

Abstract PR05: Epigenetic disruption of vitamin D receptor signaling in African American prostate cancer alters circadian signaling networks

Abstract The current study aimed to define the genomic functions of the vitamin D receptor (VDR) in African American (AA) prostate cancer (PCa) compared to European American (EA) counterparts. VDR-dependent ChIP-Seq and RNA-Seq gene was undertaken in EA (non-malignant HPr1AR and malignant LNCaP) and AA (non-malignant RC43N and malignant RC43T) prostate models, combined with analyses of three PCa cohorts. In AA prostate models the VDR is highly expressed, binds more frequently, and is enriched in active and poised enhancers. Motif analyses revealed selective enrichment, including ZBTB33/KAISO in AA cells and ERG family members in EA cells and, similarly, GIGGLE analyses revealed AA VDR cistromes were significantly overlapped with core circadian rhythm transcription factors (e.g. NONO). Combining VDR-dependent ChIP-Seq and RNA-Seq established that AA cells displayed a significantly stronger transcriptional response, compared to EA cells, and was most responsive in non-malignant RC43N. For example, RC43N transcriptional responses were enriched for circadian rhythm (NES 2.7) and inflammation, whereas in RC43T the same gene networks were repressed. To reveal how VDR/1,25(OH)2D3 signaling is corrupted in AA PCa, we mined TCGA data and revealed that the BAZ1A/SMARCA5 chromatin remodeling complex was uniquely altered in TMPRSS2:ERG fusion negative AA PCa. We are currently examining the impact of BAZ1A on the 1,25(OH)2D3 responsiveness. We also identified miRNA associated with progression from HGPIN to PCa in AA men, and that ~30% were bound by VDR and regulated by 1,25(OH)2D3, although ~5% of EA progression miRNA were VDR-responsive. For example, VDR binds to miR-199b, is uniquely 1,25(OH)2D3 up-regulated in RC43N but repressed in RC43T, and associates with AA progression from HGPIN to PCa. MiR-199b regulates expression of NPAS2, a core circadian transcription factor. Finally, leveraging a previously analyzed cohort of 1,25(OH)2D3-treated PCa patients revealed that AA tumors were intrinsically more 1,25(OH)2D3-responsive than EA counterparts, reflecting the cell line analyses. 1,25(OH)2D3 regulated circadian transcriptional regulators (e.g. NOCT and MYBBP1A) and inflammatory signals. Together, these data suggest VDR transcriptional control in AA men is more dynamic than in EA men, and is primed to govern inflammatory and circadian rhythm pathways. This is frequently disrupted, including by altered BAZ1A/SMARCA5 expression and/or reduced environmental-regulated serum vitamin D3 levels, and leads to altered regulation of circadian rhythm process, and inflammatory signals. Therefore, the VDR axis lies at the cross-roads of biopsychosocial processes that contributes to PCa health disparities. Citation Format: Manjunath Siddappa, Sajad D. Wani, Jaimie S. Gray, Mark D. Long, Hedieh Jafari, Hsuchang Wu, Honhe Wang, Rebecca Morgan, Gary Hardiman, James Marshall, Chanita Hughes-Halbert, Lara E. Sucheston-Campbell, Clayton L. Yates, Moray J. Campbell. Epigenetic disruption of vitamin D receptor signaling in African American prostate cancer alters circadian signaling networks [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PR05.

Whole transcriptome analysis of adrenal glands from prenatal glucocorticoid programmed hypertensive rodents

Prenatal glucocorticoid exposure is associated with the development of hypertension in adults. We have previously demonstrated that antenatal dexamethosone (DEX) administration in Wistar-Kyoto dams results in offspring with increased blood pressure coupled with elevated plasma epinephrine levels. In order to elucidate the molecular mechanisms responsible for prenatal DEX-mediated programming of hypertension, a whole-transcriptome analysis was performed on DEX programmed WKY male adrenal glands using the Rat Gene 2.0 microarray. Differential gene expression (DEG) analysis of DEX-exposed offspring compared with saline-treated controls revealed 142 significant DEGs (109 upregulated and 33 downregulated genes). DEG pathway enrichment analysis demonstrated that genes involved in circadian rhythm signaling were most robustly dysregulated. RT-qPCR analysis confirmed the increased expression of circadian genes Bmal1 and Npas2, while Per2, Per3, Cry2 and Bhlhe41 were significantly downregulated. In contrast, gene expression profiling of Spontaneously Hypertensive (SHR) rats, a genetic model of hypertension, demonstrated decreased expression of Bmal1 and Npas2, while Per1, Per2, Per3, Cry1, Cry2, Bhlhe41 and Csnk1D were all upregulated compared to naïve WKY controls. Taken together, this study establishes that glucocorticoid programmed adrenals have impaired circadian signaling and that changes in adrenal circadian rhythm may be an underlying molecular mechanism responsible for the development of hypertension.

Western diet-induced obesity disrupts the diurnal rhythmicity of hippocampal core clock gene expression in a mouse model

Western diet (WD) feeding disrupts core clock gene expression in peripheral tissues and contributes to WD-induced metabolic disease. The hippocampus, the mammalian center for memory, is also sensitive to WD feeding, but whether the WD disrupts its core clock is unknown. To this end, male mice were maintained on a WD for 16 weeks and diurnal metabolism, gene expression and memory were assessed. WD-induced obesity disrupted the diurnal rhythms of whole-body metabolism, markers of inflammation and hepatic gene expression, but did not disrupt diurnal expression of hypothalamic Bmal1, Npas2 and Per2. However, all measured core clock genes were disrupted in the hippocampus after WD feeding and the expression pattern of genes implicated in Alzheimer’s disease and synaptic function were altered. Finally, WD feeding disrupted hippocampal memory in a task- and time-dependent fashion. Our results implicate WD-induced alterations in the rhythmicity of hippocampal gene expression in the etiology of diet-induced memory deficits.

Low circadian clock genes expression in cancers: A meta-analysis of its association with clinicopathological features and prognosis.

BackgroundPer1, Per2, Per3, Cry1, Cry2, Bmal1, Npas2 and CLOCK genes are the eight core circadian clock genes. Low expression of these circadian clock genes plays an important role in the progression of cancers. However, its clinicopathological and prognostic value in patients with cancers remains controversial and inconclusive. We performed a meta-analysis of studies assessing the clinicopathological and prognostic significance of low expression of these genes in cancers.MethodsRelevant studies were searched from the Cochrane Central Register of Controlled Trials, Embase, EBSCO, Ovid, PubMed, Science Direct, Wiley Online Library database, CNKI and Wan Fang database. The meta-analysis was performed by using STATA version 12 software. A random-effect model was employed to evaluate all pooled hazard ratios (HRs) and odd ratios (ORs).ResultsA total of 36 studies comprising 7476 cases met the inclusion criteria. Meta-analysis suggested that low expression of Per1 was associated with poor differentiation (Per1: OR=2.30, 95%CI: 1.36∼3.87, P=0.002) and deeper invasion depth (Per1: OR=2.12, 95%CI: 1.62∼2.77, Ρ<0.001); low Per2 expression was correlated with poor differentiation (Per2: OR=2.41, 95%CI: 1.53∼3.79, Ρ<0.001), worse TNM stage (Per2:OR=3.47, 95%CI: 1.88∼6.42, P<0.001) and further metastasis (Per2:OR=2.35, 95%CI: 1.35∼4.11, Ρ=0.003). Furthermore, the results revealed that low expressions of Per1 and Per2 were also correlated with poor overall survival of cancers (Per1: HR=1.35, 95%CI: 1.06∼1.72, P=0.014; Per2: HR=1.43, 95%CI: 1.10∼1.85, P=0.007). Subgroup analysis indicated that low Per1 and Per2 expressions were especially associated with poor prognosis of gastrointestinal caners (Per1: HR=1.33, 95%CI: 1.14∼1.55, Ρ<0.001, Ι2=4.2%; Per2: HR=1.62, 95%CI: 1.25∼2.18, P<0.001, I2=0.0%).ConclusionsOur study suggested that low Per1, Per2 and Npas2 expression played a distinct and crucial role in progression of cancers. Low expressions of Per1 and Per2 could serve as unfavorable indicators for cancers prognosis, especially for gastrointestinal cancers.

Persistent myopathy despite release of partial obstruction: in vivo reversal of dysfunction and transcriptional responses using rapamycin.

Current and potential medical therapy for obstruction-induced myopathic bladder dysfunction (from benign prostatic hyperplasia or posterior urethral valves) focuses on symptoms. The persistent tissue pathology and dysfunction after release of obstruction is often deemed irreversible without any systematic therapeutic approaches. As rapamycin can attenuate bladder smooth muscle hypertrophy and dysfunction during the genesis of partial obstruction in vivo, we tested whether rapamycin could improve persistent function after release of obstruction (de-obstruction or REL). Female Sprague-Dawley rat bladderswere partiallyobstructed (PBO)by suturing around boththe urethra and apara-urethral steel rod, then removing the rod. One dayprior to release of obstruction (preREL), voiding parameters and residual urine volume of preREL+future rapa, preREL+future veh groups were recorded. Release of obstruction (REL) was performed by suture removal following 6weeks of PBO. For 4 more weeks afterthe de-obstruction, RELanimals were randomized to rapamycin (REL+rapa) or vehicle (REL+veh). PBO for 6weeks were used as positive controls. In shams, the urethra was exposed, but no suture tied. Voiding parameters and residual urine volume were measured prior to sacrifice of sham and REL+veh or REL+rapa, and PBO. Rapamycin efficacy was tested by pair-wise comparison of changes in individual voiding data from preREL+future veh or preREL+future rapa versus REL+veh or REL+rapa, respectively, as well as by comparisons of REL+veh to REL+rapa groups. Bladders were weighed and processed for a high-throughput QPCR array, and histopathology. Bladder/body mass ratios with PBO increased significantly and remained higher in the release phase in REL+veh animals. REL+rapa versus REL+veh improved residual volumes and micturition fractions toward sham levels. Three genes encoding extracellular proteins, BMP2, SOD3, and IGFBP7, correlated with functional improvement by Pearson's correlations. The promoters of these genes showed enrichment for several motifs including circadian E-boxes. While obstruction and REL augmented CLOCK and NPAS2 expression above sham levels, rapamycin treatment during release significantly blocked their expression. This experimental design of pharmaco-intervention during the de-obstruction phase revealed a novel pathway dysregulated during the clinically relevant treatment phase of obstructive bladder myopathy.

1213: Disruptions in Npas2 expression in the fetal liver alters the gut microbiome at light/dark timepoints

1213: Disruptions in Npas2 expression in the fetal liver alters the gut microbiome at light/dark timepoints