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Oxidative Stress and Inflammation Regulate the Key Circadian Gene Rev-ERB? through Conserved NF?B and NrF2 Binding Sites
mitochondrial (TxnR2) thioredoxin reductase, and thioredoxininteracting protein (TXNIP) display diurnal rhythms, and the effect alcohol has on their expression. Single-housed male C57BL/6J mice were kept under a 12 hr light/dark cycle and pair-fed control or ethanol-containing diets for 5 wk. Liver was collected at 4 hr intervals for an entire 24 hr period and used for histology and gene expression by qRT-PCR. Liver histology showed that alcohol feeding for 5 wk established the first stage of ALD; steatosis. Our results showed that gene expression of TXNIP displayed diurnal rhythms, which peaked during the inactive (i.e., light) phase. Chronic alcohol resulted in a significant decrease in the expression of Txn1 and TxnR1, while inducing an increase in TXNIP expression. Txn2 and TxnR2 displayed no diurnal or alcohol treatment effects. These results indicate that chronic alcohol consumption alters the expression of Txn1, TxnR1, and TXNIP, and also disrupts the rhythmic gene expression of TXNIP. This rhythmicity is likely critical for maintaining proper antioxidant status and the redox state within the liver. Therefore, alcoholinduced disruption of this rhythm may contribute to the development and/or progression of ALD through enhanced oxidative stress.
doi:10.1016/j.freeradbiomed.2012.10.095
68 *OXFRVH3KRVSKDWH'HK\GURJHQDVH.QRFNGRZQ ,PSDLUV5HSURGXFWLRQDQG(PEU\RQLF'HYHORSPHQWLQ 1HPDWRGHCaenorhabditis Elegans 1
Hung-Chi Yang , Tzul-Ling Chen1, Nai-Jyun Ciou1, Mei-Ling Cheng1, Szecheng J. Lo1, and Tsun-Yee Daniel Chiu1,2 1 2 Chang Gung University, Taiwan Chang Gung Memorial Hospital, Taiwan The lack of an experimental animal model is a major hindrance for studying the physiologic role of Glucose 6-phosphate dehydrogenase (G6PD) in organisms due to the fact that G6PD deficiency is embryonic lethal. G6PD is the most highly expressed gene among other major NADPH producing systems, including isocitrate dehydrogenase and malic enzyme in Caenorhabditis elegans (C. elegans). We proposed that essential NADPH dependent systems, such as antioxidant defense and fatty acid biosynthesis are highly affected by G6PD deficiency. Using RNAi knockdown technique, G6PD-deficient C. elegans was established. Upon G6PD knockdown, such C. elegans displayed defective oogenesis and embryogenesis which were likely due to impaired redox homeostasis elicited by G6PD deficiency. However, oxidative stress was not the only determinant because selected antioxidants fail to rescue defective embryogenesis in G6PD-knockdown C. elegans. Signaling analysis showed that MAPK pathways were also involved in G6PD-mediated modulation of embryogenesis in C. elegans. In addition, embryos derived from G6PD-knockdown C. elegans exhibited permeability to small molecule dyes (Hoechst, FM4-64), polarity defect (symmetric division during cytokinesis) and egg shell deformation (disrupted lipid layer). Upon inhibition of fatty acid biosynthetic pathway, embryos derived from such mutants displayed similar phenotypes. The embryonic defects observed in the embryo of G6PD-deficient C. elegans is likely due to impairment of fatty acid synthesis because G6PD is crucial to provide NADPH for such reductive biosynthesis during embryonic development. In conclusion, we have demonstrated that C. elegans embryonic development is modulated by G6PD possibly through fatty acid biosynthesis.
69 2[LGDWLYH6WUHVVDQG,QIODPPDWLRQ5HJXODWHWKH.H\ &LUFDGLDQ*HQH5HY(5%ĮWKURXJK&RQVHUYHG1)ț% DQG1U)%LQGLQJ6LWHV 1
Guang Yang , Maurice D Hinson1, Manasa Muthu1, Clyde J Wright1, Ping La1, Amal P Fernando1, and Phyllis A Dennery1,2 1 2 Children's Hospital of Philadelphia, University of Pennsylvania The circadian rhythm is key in controlling cellular homeostasis because it alters cell metabolism and cell growth. Rev-ERBĮ, a nuclear receptor, is found in the lung and is a key gene for sustaining circadian rhythmicity. We have previously shown that the activity of a 0.9 kb promoter of Rev-ERBα was increased in hyperoxia. In fact, linear or targeted deletion mutants, aimed for the cis elements of NrF2 and NFκB transcription factor binding sites, showed decreased promoter activity at baseline and in hyperoxia. In this work we verified occupancy and DNA binding of NrF2 and NFκB on the Rev-ERBα promoter. Using serum shock as a model for circadian rhythmicity, we tested whether oxidative stress or inflammatory stimuli would alter Rev-ERBα circadian function. Mouse lung fibroblasts (Mlg) were exposed to hyperoxia (95%O2/5%CO2), normoxia (95%air/5%CO2) or incubated with 10 ng/ml TNFα for 24 hours. ChIP assay was performed for occupancy of NrF2, p65 and p50. In other experiments, nuclear fractions were evaluated for DNA binding to NrF2 and NFκB using EMSA. To test Rev-ERBα circadian function, Mlg cells were either exposed to hyperoxia for 4 h or stably disrupted with p50 shRNA prior to serum shock, where cells were starved with 0.5% FBS overnight and shocked with 50% horse serum for 2 h. Total RNA was collected 1 h after the shock and thereafter every 3 h for a total of 24 h. Levels of Rev-ERBα mRNA and protein were measured using RT-PCR. In hyperoxia, occupancy of NrF2 and p50 was increased on the proximal region of the Rev-ERBα promoter. DNA binding to a NrF2 sequence was also enhanced. However with TNFα incubation, p65 occupancy was diminished, suggesting that the NFκB binding site differentially regulates Rev-ERBα promoter activity under inflammatory conditions. In addition, hyperoxia increased the amplitude of the Rev-ERBα oscillations whereas p50 disruption increased the amplitude only on the peak of the rhythmicity of Rev-ERBα. We conclude that both NrF2 and NFκB binding sites mediate Rev-ERBα transcription. Furthermore, hyperoxia activates RevERBα transcription via the NrF2 binding site whereas inflammation suppresses Rev-ERBα transcription via the NFκB binding site on the promoter.
doi:10.1016/j.freeradbiomed.2012.10.097
doi:10.1016/j.freeradbiomed.2012.10.096
SFRBM 2012
S37
doi:10.1016/j.freeradbiomed.2012.10.095
68 *OXFRVH3KRVSKDWH'HK\GURJHQDVH.QRFNGRZQ ,PSDLUV5HSURGXFWLRQDQG(PEU\RQLF'HYHORSPHQWLQ 1HPDWRGHCaenorhabditis Elegans 1
Hung-Chi Yang , Tzul-Ling Chen1, Nai-Jyun Ciou1, Mei-Ling Cheng1, Szecheng J. Lo1, and Tsun-Yee Daniel Chiu1,2 1 2 Chang Gung University, Taiwan Chang Gung Memorial Hospital, Taiwan The lack of an experimental animal model is a major hindrance for studying the physiologic role of Glucose 6-phosphate dehydrogenase (G6PD) in organisms due to the fact that G6PD deficiency is embryonic lethal. G6PD is the most highly expressed gene among other major NADPH producing systems, including isocitrate dehydrogenase and malic enzyme in Caenorhabditis elegans (C. elegans). We proposed that essential NADPH dependent systems, such as antioxidant defense and fatty acid biosynthesis are highly affected by G6PD deficiency. Using RNAi knockdown technique, G6PD-deficient C. elegans was established. Upon G6PD knockdown, such C. elegans displayed defective oogenesis and embryogenesis which were likely due to impaired redox homeostasis elicited by G6PD deficiency. However, oxidative stress was not the only determinant because selected antioxidants fail to rescue defective embryogenesis in G6PD-knockdown C. elegans. Signaling analysis showed that MAPK pathways were also involved in G6PD-mediated modulation of embryogenesis in C. elegans. In addition, embryos derived from G6PD-knockdown C. elegans exhibited permeability to small molecule dyes (Hoechst, FM4-64), polarity defect (symmetric division during cytokinesis) and egg shell deformation (disrupted lipid layer). Upon inhibition of fatty acid biosynthetic pathway, embryos derived from such mutants displayed similar phenotypes. The embryonic defects observed in the embryo of G6PD-deficient C. elegans is likely due to impairment of fatty acid synthesis because G6PD is crucial to provide NADPH for such reductive biosynthesis during embryonic development. In conclusion, we have demonstrated that C. elegans embryonic development is modulated by G6PD possibly through fatty acid biosynthesis.
69 2[LGDWLYH6WUHVVDQG,QIODPPDWLRQ5HJXODWHWKH.H\ &LUFDGLDQ*HQH5HY(5%ĮWKURXJK&RQVHUYHG1)ț% DQG1U)%LQGLQJ6LWHV 1
Guang Yang , Maurice D Hinson1, Manasa Muthu1, Clyde J Wright1, Ping La1, Amal P Fernando1, and Phyllis A Dennery1,2 1 2 Children's Hospital of Philadelphia, University of Pennsylvania The circadian rhythm is key in controlling cellular homeostasis because it alters cell metabolism and cell growth. Rev-ERBĮ, a nuclear receptor, is found in the lung and is a key gene for sustaining circadian rhythmicity. We have previously shown that the activity of a 0.9 kb promoter of Rev-ERBα was increased in hyperoxia. In fact, linear or targeted deletion mutants, aimed for the cis elements of NrF2 and NFκB transcription factor binding sites, showed decreased promoter activity at baseline and in hyperoxia. In this work we verified occupancy and DNA binding of NrF2 and NFκB on the Rev-ERBα promoter. Using serum shock as a model for circadian rhythmicity, we tested whether oxidative stress or inflammatory stimuli would alter Rev-ERBα circadian function. Mouse lung fibroblasts (Mlg) were exposed to hyperoxia (95%O2/5%CO2), normoxia (95%air/5%CO2) or incubated with 10 ng/ml TNFα for 24 hours. ChIP assay was performed for occupancy of NrF2, p65 and p50. In other experiments, nuclear fractions were evaluated for DNA binding to NrF2 and NFκB using EMSA. To test Rev-ERBα circadian function, Mlg cells were either exposed to hyperoxia for 4 h or stably disrupted with p50 shRNA prior to serum shock, where cells were starved with 0.5% FBS overnight and shocked with 50% horse serum for 2 h. Total RNA was collected 1 h after the shock and thereafter every 3 h for a total of 24 h. Levels of Rev-ERBα mRNA and protein were measured using RT-PCR. In hyperoxia, occupancy of NrF2 and p50 was increased on the proximal region of the Rev-ERBα promoter. DNA binding to a NrF2 sequence was also enhanced. However with TNFα incubation, p65 occupancy was diminished, suggesting that the NFκB binding site differentially regulates Rev-ERBα promoter activity under inflammatory conditions. In addition, hyperoxia increased the amplitude of the Rev-ERBα oscillations whereas p50 disruption increased the amplitude only on the peak of the rhythmicity of Rev-ERBα. We conclude that both NrF2 and NFκB binding sites mediate Rev-ERBα transcription. Furthermore, hyperoxia activates RevERBα transcription via the NrF2 binding site whereas inflammation suppresses Rev-ERBα transcription via the NFκB binding site on the promoter.
doi:10.1016/j.freeradbiomed.2012.10.097
doi:10.1016/j.freeradbiomed.2012.10.096
SFRBM 2012
S37