REGULATION OF CIRCADIAN CLOCKS AND METABOLISM BY ARYL HYDROCARBON RECEPTOR

The aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor, plays a crucial role in regulation of xenobiotic metabolism. AhR is also involved in dioxin-induced metabolic disorders and alteration of circadian rhythm. Furthermore, circadian clock disruption and metabolic dysfunction are integrally associated with each other. This study was designed to understand the mechanisms by which AhR contributes to regulation of circadian clocks, fat metabolism and glucose homeostasis. In the first aim, I have tested whether AhR interacts with the core clock gene, brain and muscle AhR nuclear translocator like-1(BMAL1), disrupting circadian locomotor output cycle kaput (CLOCK)/BMAL1 complex activity, and leading to the suppression of period1 gene (Per1) expression rhythm. My studies indicate that AhR activation by its agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and beta-naphoflavone (BNF) disrupts the rhythm and inhibits the expression of Per1 in mouse liver and hepatoma cell lines, respectively. Mechanistically, the disruption of the rhythm and the inhibition of Per1 expression occur secondary to the interaction between AhR and BMAL1, which attenuates transcriptional activity of the core clock complex CLOCK/BMAL1. These results suggest alteration of the circadian clock as a novel signaling event downstream of AhR activation. The integral relationship between the clock and metabolic function further suggest that AhR activation may cause metabolic dysfunction. In the second aim, I have tested whether AhR activation inhibits Per1 gene induction and influences circadian clock resetting through activation of JNK pathway. AhR activation by it agonists TCDD and BNF decreases light-induced phase shifts in the early night and inhibits light-induced Per1 expression in both suprachiasmatic nucleus (SCN) and liver. Inhibition of Per1 induction results from increased phospho-JNK induced by AhR activation. Taken together, activation of AhR disrupts circadian clock resetting which also could cause metabolic dysfunction. In the third aim, I have tested whether AhR deficiency regulates nuclear receptor peroxisome proliferator-activated receptor a; (PPARa) and alters glucose homeostasis. PPARa, a clock-controlled gene (CCG) that acts as a fat metabolism sensor, is important for lipid metabolism and glucose homeostasis. AhR knockout (AhRKO or AhR-/-) mice exhibit enhanced insulin sensitivity and glucose tolerance, accompanied by decreased expression of PPARa, key gluconeogenic genes, glucose-6 phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) and key fatty acid oxidation enzymes, carnitine palmitoyl transferase1b (CPT1b) and acyl-CoA oxidase (ACO). Conversely, AhR agonists increase PPARa; expression in a BMAL1-dependent manner. In addition, AhRKO mice display altered rhythm for clock genes, clock-controlled genes (CCGs) and physiological blood glucose. These data suggest that AhR may modulate the glucose homeostasis through regulation of CCGs such as PPARa and that PPARa may be an important link between the circadian clock and metabolism. In the fourth aim, I have tested the effects of AhR ablation or attenuation in high-fat diet (HFD)-induced obesity, insulin resistance and hepatic steatosis in mice. Recent studies suggest that PPARα expression is elevated with HFD feeding as an adaptive response to attenuate hepatic steatosis, and PPARa deficiency protects against HFD-induced insulin resistance. AhR-/- as well as AhR heterozygous (AhR+/-) mice are protected from the HFD-induced obesity, insulin resistance, hepatic steatosis and show reduced inflammatory cytokine expression. In addition, AhR-/- and AhR+/- mice display protected insulin signaling, a higher adiponectin and a lower leptin and insulin in serum. Food intake and physical activity are not significantly different among WT, AhR-/- and AhR+/- mice with HFD feeding. Indirect calorimetry has demonstrated that the AhR+/- mice have higher oxygen consumption, CO2 production and heat production. In addition, Real-time PCR data show that uncoupling protein 1(Ucp1) is higher in brown adipose tissue which supports the higher heat production; moreover, the muscle gene profile reveals that the fatty acid beta-oxidation genes and mitochondrial respiratory genes are higher in AHR+/- mice which further support higher energy expenditure in these mice. Collectively, these data suggest that AhR signaling could be a potential target for treatment of obesity and type 2 diabetes, and AhR antagonist may be developed into a drug for these metabolic diseases

FAAP100 is required for the resolution of transcription-replication conflicts in primordial germ cells

Abstract Background The maintenance of genome stability in primordial germ cells (PGCs) is crucial for the faithful transmission of genetic information and the establishment of reproductive reserve. Numerous studies in recent decades have linked the Fanconi anemia (FA) pathway with fertility, particularly PGC development. However, the role of FAAP100, an essential component of the FA core complex, in germ cell development is unexplored. Results We find that FAAP100 plays an essential role in R-loop resolution and replication fork protection to counteract transcription-replication conflicts (TRCs) during mouse PGC proliferation. FAAP100 deletion leads to FA pathway inactivation, increases TRCs as well as cotranscriptional R-loops, and contributes to the collapse of replication forks and the generation of DNA damage. Then, the activated p53 signaling pathway triggers PGC proliferation defects, ultimately resulting in insufficient establishment of reproductive reserve in both sexes of mice. Conclusions Our findings suggest that FAAP100 is required for the resolution of TRCs in PGCs to safeguard their genome stability

Gasdermin D mediates the pathogenesis of neonatal-onset multisystem inflammatory disease in mice.

Mutated NLRP3 assembles a hyperactive inflammasome, which causes excessive secretion of interleukin (IL)-1β and IL-18 and, ultimately, a spectrum of autoinflammatory disorders known as cryopyrinopathies of which neonatal-onset multisystem inflammatory disease (NOMID) is the most severe phenotype. NOMID mice phenocopy several features of the human disease as they develop severe systemic inflammation driven by IL-1β and IL-18 overproduction associated with damage to multiple organs, including spleen, skin, liver, and skeleton. Secretion of IL-1β and IL-18 requires gasdermin D (GSDMD), which-upon activation by the inflammasomes-translocates to the plasma membrane where it forms pores through which these cytokines are released. However, excessive pore formation resulting from sustained activation of GSDMD compromises membrane integrity and ultimately causes a pro-inflammatory form of cell death, termed pyroptosis. In this study, we first established a strong correlation between NLRP3 inflammasome activation and GSDMD processing and pyroptosis in vitro. Next, we used NOMID mice to determine the extent to which GSDMD-driven pyroptosis influences the pathogenesis of this disorder. Remarkably, all NOMID-associated inflammatory symptoms are prevented upon ablation of GSDMD. Thus, GSDMD-dependent actions are required for the pathogenesis of NOMID in mice

Gasdermin D mediates the pathogenesis of neonatal-onset multisystem inflammatory disease in mice

<div><p>Mutated NLRP3 assembles a hyperactive inflammasome, which causes excessive secretion of interleukin (IL)-1β and IL-18 and, ultimately, a spectrum of autoinflammatory disorders known as cryopyrinopathies of which neonatal-onset multisystem inflammatory disease (NOMID) is the most severe phenotype. NOMID mice phenocopy several features of the human disease as they develop severe systemic inflammation driven by IL-1β and IL-18 overproduction associated with damage to multiple organs, including spleen, skin, liver, and skeleton. Secretion of IL-1β and IL-18 requires gasdermin D (GSDMD), which—upon activation by the inflammasomes—translocates to the plasma membrane where it forms pores through which these cytokines are released. However, excessive pore formation resulting from sustained activation of GSDMD compromises membrane integrity and ultimately causes a pro-inflammatory form of cell death, termed pyroptosis. In this study, we first established a strong correlation between NLRP3 inflammasome activation and GSDMD processing and pyroptosis in vitro. Next, we used NOMID mice to determine the extent to which GSDMD-driven pyroptosis influences the pathogenesis of this disorder. Remarkably, all NOMID-associated inflammatory symptoms are prevented upon ablation of GSDMD. Thus, GSDMD-dependent actions are required for the pathogenesis of NOMID in mice.</p></div

MicroRNA-32 promotes calcification in vascular smooth muscle cells: Implications as a novel marker for coronary artery calcification

<div><p>Cardiovascular calcification is one of the most severe outcomes associated with cardiovascular disease and often results in significant morbidity and mortality. Previous reports indicated that epigenomic regulation of microRNAs (miRNAs) might play important roles in vascular smooth muscle cell (VSMC) calcification. Here, we identified potential key miRNAs involved in vascular calcification <i>in vivo</i> and investigated the role of miR-32-5p (miR-32). According to microarray analysis, we observed increased expression of miR-125b, miR-30a, and miR-32 and decreased expression of miR-29a, miR-210, and miR-320 during the progression of vascularcalcification. Additionally, gain- and loss-of-function studies of miR-32 confirmed promotion of VSMC calcification in mice through the enhanced expression of bonemorphogenetic protein-2, runt-related transcription factor-2(RUNX2), osteopontin, and the bone-specific phosphoprotein matrix GLA protein <i>in vitro</i>. Moreover, miR-32 modulated vascularcalcification progression by activating phosphoinositide 3-kinase (PI3K)signaling and increasing RUNX2 expression and phosphorylation by targeting the 3′-untranslated region of phosphatase and tensin homolog Mrna (PTEN) in mouse VSMCs. Furthermore, we detected higher miR-32 levels in plasmafrom patients with coronary artery disease with coronary artery calcification (CAC) as compared with levels observed in non-CAC patients (<i>P</i> = 0.016), further confirming miR-32 as a critical modulator and potential diagnostic marker for CAC.</p></div

MiR-32 induces the expression of vascular calcification markers.

<p><b>A&B</b>, The miR-32 mimics(pre-miR32),negative control of miR-32 mimics (pre-con), miR-32 inhibitors (anti-miR32), or negative control of miR-32 inhibitors (anti-con) were transfected into MVMSCs for 4 days and miR-32 expression level was determined by Q-PCR (<b>A</b>) and Northern Blot (<b>B</b>) (n = 3). <b>C</b>, The effect of miR-32 on the BMP2, Runx2, OPN and MGP mRNA level in MVSMCs were detected by Q-PCR at the time point of 4 days after the transfection of miR-32 mimics and miR-32 inhibitorsin MVMSCs (n = 3). <b>D&E</b>, The effect of miR-32 on ALP activity and calcium content of MVSMCs were also determined at the time point of 4 days after miR-32 mimics transfection(n = 3). Data are shown as mean±SEM of a representative experiment performed in triplicate, *<i>P</i>< 0.05, ** <i>P</i>< 0.01.</p

MiR-32 shows increased level in plasma of patients with coronary artery calcification.

<p><b>A</b>,The representative images of aortic stenosis of CADpatients confirmed by coronary arteriongraphy (n = 66). a&c (two pictures upper) represent the coronary artery before coronary arteriongraphy and b&d (two pictures lower) represent the coronary artery after coronary arteriongraphy; a&b, CAD patients without coronary calcification; c&d, CAD with calcification.<b>B</b>,Representatives of transverse (a&c, two left pictures) and maximum intensity projection (b&d, two right pictures) images of coronary arteries by CT scanning; a&b, examples of no coronary calcium; c&d, examples of coronary calcification; arrows indicate the calcification of coronary artery.<b>C</b>,Plasma from 66 patients with coronary heart disease of which 33 patients showed calcification in coronary artery confirmed by CT scanning were collected for miRNA-32 detection by Q-PCR. Data are shown as mean±SEM of experiments. <b>D</b>, The plasmaALP activity of patients was detected as described in methods, and data are shown as mean±SEM of experiments (n = 66). <b>E</b>,the scattering spots image showed the positive correlation between miRNA-32 and ALP activity in plasma of patients with coronary artery calcification (n = 33), which was performed by Spearman's Rank Correlation test.</p