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1.  Transcriptome Analysis of the Initial Stage of Acute WSSV Infection Caused by Temperature Change 
PLoS ONE  2014;9(3):e90732.
White spot syndrome virus (WSSV) is the most devastating virosis threatening the shrimp culture industry worldwide. Variations of environmental factors in shrimp culture ponds usually lead to the outbreak of white spot syndrome (WSS). In order to know the molecular mechanisms of WSS outbreak induced by temperature variation and the biological changes of the host at the initial stage of WSSV acute infection, RNA-Seq technology was used to analyze the differentially expressed genes (DEGs) in shrimp with a certain amount of WSSV cultured at 18°C and shrimp whose culture temperature were raised to 25°C. To analyze whether the expression changes of the DEGs were due to temperature rising or WSSV proliferation, the expression of selected DEGs was analyzed by real-time PCR with another shrimp group, namely Group T, as control. Group T didn’t suffer WSSV infection but was subjected to temperature rising in parallel. At the initial stage of WSSV acute infection, DEGs related to energy production were up-regulated, whereas most DEGs related to cell cycle and positive regulation of cell death and were down-regulated. Triose phosphate isomerase, enolase and alcohol dehydrogenase involved in glycosis were up-regulated, while pyruvate dehydrogenase, citrate synthase and isocitrate dehydrogenase with NAD as the coenzyme involved in TCA pathway were down-regulated. Also genes involved in host DNA replication, including DNA primase, DNA topoisomerase and DNA polymerase showed down-regulated expression. Several interesting genes including crustin genes, acting binding or inhibiting protein genes, a disintegrin and metalloproteinase domain-containing protein 9 (ADAM9) gene and a GRP 78 gene were also analyzed. Understanding the interactions between hosts and WSSV at the initial stage of acute infection will not only help to get a deep insight into the pathogenesis of WSSV but also provide clues for therapies.
doi:10.1371/journal.pone.0090732
PMCID: PMC3942461  PMID: 24595043
2.  Stochastic modeling of stem-cell dynamics with control 
Mathematical biosciences  2012;240(2):231-240.
Tissue development and homeostasis are thought to be regulated endogenously by control loops that ensure that the numbers of stem cells and daughter cells are maintained at desired levels, and that the cell dynamics are robust to perturbations. In this paper we consider several classes of stochastic models that describe stem/daughter cell dynamics in a population of constant size, which are generalizations of the Moran process that include negative control loops that affect differentiation probabilities for stem cells. We present analytical solutions for the steady-state expectations and variances of the numbers of stem and daughter cells; these results remain valid for non-constant cell populations. We show that in the absence of differentiation/proliferation control, the number of stem cells is subject to extinction or overflow. In the presence of linear control, a steady state may be maintained but no tunable parameters are available to control the mean and the spread of the cell population sizes. Two types of nonlinear control considered here incorporate tunable parameters that allow specification of the expected number of stem cells and also provide control over the size of the standard deviation. We show that under a hyperbolic control law, there is a trade-off between minimizing standard deviations and maintaining the system robustness against external perturbations. For the Hill-type control, the standard deviation is inversely proportional to the Hill coefficient of the control loop. Biologically this means that ultrasensitive response that is observed in a number of regulatory loops may have evolved in order to reduce fluctuations while maintaining the desired population levels.
doi:10.1016/j.mbs.2012.08.004
PMCID: PMC3921979  PMID: 22960597
3.  Dissociating Fatty Liver and Diabetes 
Fatty liver disease is epidemiologically associated with type 2 diabetes (T2D), leading to a speculation of a reciprocal cause-effect relationship and a vicious cycle of pathology. Here, we summarize recent literature reporting dissociation of hepatosteatosis from insulin resistance, in genetic mouse models and clinical studies. We highlight rhythmic flows of metabolic intermediates between hepatic lipid synthesis and glucose production in normal circadian physiology. Blocking triglyceride (TG) secretion, subcellular lipid sequestration, lipolysis deficiency, enhanced lipogenesis, gluconeogenesis defects, or inhibition of fatty acid oxidation, all result in hepatosteatosis without causing hyperglycemia or insulin resistance, suggesting that the cause-effect relationship between hepatosteatosis and diabetes does not exist in all situations.
doi:10.1016/j.tem.2012.09.005
PMCID: PMC3532558  PMID: 23043895
4.  Enzyme-processed Korean Red Ginseng extracts protects against skin damage induced by UVB irradiation in hairless mice 
Journal of Ginseng Research  2013;37(4):425-434.
UV irradiation is the main factor contributing to skin damages that are associated with an excessive production of matrix-degrading metalloproteinase (MMP)-1 and a deficient expression of collagens. To date, red ginseng has been revealed to possess many biomedical effects, such as anti-aging, anti-oxidation, and anti-inflammatory. In this study, we prepared the Korean Red Ginseng extracts treated with enzyme (KRGE) and investigated the effects of dietary KRGE on the formation of wrinkles generated by UVB irradiation in hairless mice. It was found that KRGE inhibited the UVB-induced formation of wrinkles, epidermal thickness, and skin dryness in hairless mice. Further results also showed that KRGE attenuated UVB-induced MMP-1 level, while accelerated procollagen type I, transforming growth factor-β1 secretion. Interestingly, the expression of profilaggrin and filaggrin in both the epidermis and dermis were decreased due to UVB exposure and reversed by KRGE. The KRGE 0.06% was prior to KRGE 0.24%. In view of these results, which indicated that KRGE protected skin from UVB-induced photodamages, which may not only mediated by regulating of MMP-1 and procollagen type I, but also by increasing the production of profilaggrin and filaggrin. In conclusion, our results suggest that KRGE may be a promising agent for the treatment of skin photodamages. The challenge of KRGE will be expected as cosmeceuticals and nutraceuticals in order to intervene in aging-related degenerative skin changes.
doi:10.5142/jgr.2013.37.425
PMCID: PMC3825857  PMID: 24233239
Panax ginseng; Red ginseng; Enzyme; Photoaging; Skin hydration
5.  Chemoprevention of 7,12-dimethylbenz[a]anthracene (DMBA)-induced Hamster Cheek Pouch Carcinogenesis by a 5-Lipoxygenase Inhibitor, Garcinol 
Nutrition and cancer  2012;64(8):1211-1218.
Our previous studies have shown that aberrant arachidonic acid metabolism, especially the 5-lipoxygenase (5-Lox) pathway, is involved in oral carcinogenesis, and can be targeted for cancer prevention. In order to develop potent topical agents for oral cancer chemoprevention, five known 5-Lox inhibitors from dietary and synthetic sources, Zileuton, ABT-761, Licofelone, Curcumin and Garcinol, were evaluated in silico for their potential efficacy. Garcinol, a polyisoprenylated benzophenone from the fruit rind of Garcinia spp., was found to be a promising agent based on the calculation of a theoretical activity index. Computer modeling showed that garcinol well fit the active site of 5-Lox, and potentially inhibited enzyme activity through interactions between the phenolic hydroxyl groups and the non-heme catalytic iron. In a short-term study on 7,12-dimethylbenz[a]anthracene (DMBA)-treated hamster cheek pouch, topical garcinol suppressed leukotriene B4 (LTB4) biosynthesis and inhibited inflammation and cell proliferation in the oral epithelium. In a long-term carcinogenesis study, topical garcinol significantly reduced the size of visible tumors, the number of cancer lesions, cell proliferation, and LTB4 biosynthesis. These results demonstrated that topical application of a 5-Lox inhibitor, garcinol, had chemopreventive effect on DMBA-induced hamster cheek pouch carcinogenesis.
doi:10.1080/01635581.2012.718032
PMCID: PMC3780792  PMID: 23137051
Oral cancer; Chemoprevention; Garcinol; 5-Lipoxygenase; Topical
6.  Circadian Epigenomic Remodeling and Hepatic Lipogenesis: Lessons from HDAC3 
Circadian rhythms have evolved to anticipate metabolic needs across the 24-hour light/dark cycle. This is accomplished by circadian expression of metabolic genes orchestrated by transcription factors through chromatin remodeling and histone modifications. Our recent genome-wide study on histone deacetylase 3 (HDAC3) in mouse liver provides novel insights into the molecular link between circadian rhythm and hepatic de novo lipogenesis. We found that liver-specific knockout of HDAC3 in adult mouse display severe hepatic steatosis associated with enhanced de novo lipogenesis and increased expression of lipogenic genes. Genome-wide analysis (ChIP-seq) revealed a pronounced circadian pattern of HDAC3 occupancy on genes involved in lipid metabolism, which is inversely related to histone acetylation and RNA polymerase II recruitment at these sites. The cistromes of HDAC3 and its binding partner, nuclear receptor co-repressor (NCoR), significantly overlap with that of Rev-erbα, a nuclear receptor directly involved in the core circadian machinery. Knockout of Rev-erbα in mouse also leads to hepatic steatosis and enhanced de novo lipogenesis. Collectively, these data suggest that the circadian epigenomic remodeling controlled by HDAC3, and largely directed by Rev-erbα, is essential for homeostasis of the lipogenic process in liver.
doi:10.1101/sqb.2011.76.011494
PMCID: PMC3755609  PMID: 21900149
7.  Nuclear Receptor Corepressors are Required for the Histone Deacetylase Activity of HDAC3 In Vivo 
Histone deacetylase 3 (HDAC3) is an epigenome-modifying enzyme that is required for normal mouse development and tissue-specific functions. In vitro, HDAC3 protein itself has minimal enzyme activity, but gains its histone deacetylation function from stable association with the conserved deacetylase activation domain (DAD) contained in nuclear receptor corepressors NCOR1 and SMRT. Here we show that HDAC3 enzyme activity is undetectable in mice bearing point mutations in the DAD of both NCOR1 and SMRT (NS-DADm), despite normal levels of HDAC3 protein. Local histone acetylation is increased, and genomic HDAC3 recruitment is reduced though not abrogated. Remarkably, the NS-DADm mice are born and live to adulthood, whereas genetic deletion of HDAC3 is embryonic lethal. These findings demonstrate that nuclear receptor corepressors are required for HDAC3 enzyme activity in vivo, and suggest that a deacetylase-independent function of HDAC3 may be required for life.
doi:10.1038/nsmb.2476
PMCID: PMC3565028  PMID: 23292142
8.  Cinnamoyl-based Nrf2-Activators Targeting Human Skin Cell Photo-oxidative Stress 
Free radical biology & medicine  2008;45(4):385-395.
Strong experimental evidence suggests the involvement of photo-oxidative stress mediated by reactive oxygen species as a crucial mechanism of solar damage relevant to human skin photoaging and photocarcinogenesis. Based on the established role of antioxidant response element (ARE)-mediated gene expression in cancer chemoprevention, we tested the hypothesis that small molecule Nrf2-activators may serve a photo-chemopreventive role by targeting skin cell photo-oxidative stress. A luciferase-based reporter gene assay was used as a primary screen for the identification of novel agents that modulate the Nrf2-Keap1 signaling pathway. A series of cinnamoyl-based electrophilic Michael acceptors including cinnamic aldehyde and methyl-1-cinnamoyl-5-oxo-2-pyrrolidine-carboxylate was identified as potent Nrf2-activators. Hit confirmation was performed in a secondary screen, based on immunodetection of Nrf2 protein upregulation in human Hs27 skin fibroblasts, HaCaT keratinocytes, and primary skin keratinocytes. Bioefficacy profiling of positive test compounds in skin cells demonstrated compound-induced upregulation of hemeoxygenase I and NAD(P)H-quinone oxidoreductase, two Nrf2 target genes involved in the cellular antioxidant response. Pretreatment with cinnamoyl-based Nrf2-activators suppressed intracellular oxidative stress and protected against photo-oxidative induction of apoptosis in skin cells exposed to high doses of singlet oxygen. Our pilot studies suggest feasibility of developing cinnamoyl-based Nrf2-activators as novel photo-chemopreventive agents targeting skin cell photo-oxidative stress.
doi:10.1016/j.freeradbiomed.2008.04.023
PMCID: PMC3710742  PMID: 18482591
Nrf2; skin cancer; photo-oxidative stress; photo-chemoprevention; Michael acceptor; cinnamic aldehyde; singlet oxygen
9.  Association among Polymorphisms in EGFR Gene Exons, Lifestyle and Risk of Gastric Cancer with Gender Differences in Chinese Han Subjects 
PLoS ONE  2013;8(3):e59254.
Background
The epidermal growth factor receptor (EGFR) gene plays a key role in tumor survival, invasion, angiogenesis, and metastatic spread. Recent studies showed that gastric cancer (GC) was associated with polymorphisms of the EGFR gene and environmental influences, such as lifestyle factors. In this study, seven known SNPs in EGFR exons were investigated in a high-risk Chinese population in Jiangsu province to test whether genetic variants of EGFR exons and lifestyle are associated with an increased risk of GC.
Methodology/Principal Findings
A hospital-based case-control study was performed in Jiangsu province. The results showed that smoking, drinking and preference for salty food were significantly associated with the risk of GC. The differences of lifestyle between males and females might be as the reason of higher incidence rates in males than those in females. Seven exon SNPs were genotyped rs2227983,rs2072454,rs17337023,rs1050171,rs1140475, rs2293347, and rs28384375. It was noted that the variant rs2072454 T allele and TT genotype were significantly associated with an increased risk of GC. Interestingly, our result suggested the ACAGCA haplotype might be associated with decreased risk of GC. However, no significant association was examined between the other six SNPs and the risk of GC both in the total population and the age-matching population even with gender differences.
Conclusions
Smoking, drinking and preference for salty food were significantly associated with the risk of GC in Jiangsu province with gender differences. Although only one SNP (rs2072454) was significantly associated with an increased risk of GC, combined the six EGFR exon SNPs together may be useful for predicting the risk of GC.
doi:10.1371/journal.pone.0059254
PMCID: PMC3612075  PMID: 23555641
10.  Transcriptome Analysis on Chinese Shrimp Fenneropenaeus chinensis during WSSV Acute Infection 
PLoS ONE  2013;8(3):e58627.
Previous studies have discovered a lot of immune-related genes responding to white spot syndrome virus (WSSV) infection in crustacean. However, little information is available in relation to underlying mechanisms of host responses during the WSSV acute infection stage in naturally infected shrimp. In this study, we employed next-generation sequencing and bioinformatic techniques to observe the transcriptome differences of the shrimp between latent infection stage and acute infection stage. A total of 64,188,426 Illumina reads, including 31,685,758 reads from the latent infection group and 32,502,668 reads from the acute infection group, were generated and assembled into 46,676 unigenes (mean length: 676 bp; range: 200–15,094 bp). Approximately 24,000 peptides were predicted and classified based on homology searches, gene ontology, clusters of orthologous groups of proteins, and biological pathway mapping. Among which, 805 differentially expressed genes were identified and categorized into 11 groups based on their possible function. Genes in the Toll and IMD pathways, the Ras-activated endocytosis process, the RNA interference pathway, anti-lipopolysaccharide factors and many other genes, were found to be activated in shrimp from latent infection stage to acute infection stage. The anti-bacterially proPO-activating cascade was firstly uncovered to be probably participated in antiviral process. These genes contain not only members playing function in host defense against WSSV, but also genes utilized by WSSV for its rapid proliferation. In addition, the transcriptome data provides detail information for identifying novel genes in absence of the genome database of shrimp.
doi:10.1371/journal.pone.0058627
PMCID: PMC3602427  PMID: 23527000
11.  Hepatic Hdac3 promotes gluconeogenesis by repressing lipid synthesis and sequestration 
Nature medicine  2012;18(6):934-942.
Fatty liver disease is associated with obesity and type 2 diabetes, and hepatic lipid accumulation may contribute to insulin resistance by a variety of mechanisms. Here we show that mice with liver-specific deletion of histone deacetylase 3 (Hdac3) display severe hepatosteatosis and, notably increased insulin sensitivity without changes in insulin signaling or body weight. Hdac3 deletion reroutes metabolic precursors towards lipid synthesis and storage within lipid droplets (LDs). Reduced hepatic glucose production in Hdac3-depleted liver is a result of the metabolic rerouting rather than due to inherently defective gluconeogenesis. The lipid-sequestering LDs-coating protein Perilipin 2 is markedly induced upon Hdac3 deletion and contributes to the development of both steatosis and improved tolerance to glucose. These findings suggest that the sequestration of hepatic lipids ameliorates insulin resistance, and establish Hdac3 as a pivotal epigenomic modifier that integrate signals from the circadian clock in regulation of hepatic intermediary metabolism.
doi:10.1038/nm.2744
PMCID: PMC3411870  PMID: 22561686
12.  Ethanol Promotes Chemically Induced Oral Cancer in Mice through Activation of the 5-Lipoxygenase Pathway of Arachidonic Acid Metabolism 
Alcohol drinking is a known risk factor for oral cancer in humans. However, previous animal studies on the promoting effect of ethanol on oral carcinogenesis were inconclusive. It is necessary to develop an animal model with which the molecular mechanism of ethanol-related oral carcinogenesis may be elucidated in order to develop effective prevention strategies. In this study, mice were first treated with 4-nitroquinoline-1-oxide (4NQO, 100μg/ml in drinking water) for 8 weeks, and then given water or ethanol (8%) as the sole drink for another 16 weeks. During the experiment, 8% ethanol was well tolerated by mice. The incidence of squamous cell carcinoma (SCC) increased from 20% (8/41) to 43% (17/40; p<0.05). Expression of 5-lipoxygenase (5-Lox) and cyclooxygenase 2 (Cox-2) was increased in dysplasia and SCC of 4NQO-treated tongues, and further enhanced by ethanol. Using this mouse model, we further demonstrated that fewer cancers were induced in Alox5−/− mice, as were cell proliferation, inflammation, and angiogenesis in the tongue, as compared with Alox5+/+ mice. Interestingly, Cox-2 expression was induced by ethanol in knockout mice, while 5-Lox and leukotriene A4 hydrolase (LTA4H) expression and leukotriene B4 (LTB4) biosynthesis were dramatically reduced. Moreover, ethanol enhanced expression and nuclear localization of 5-Lox and stimulated LTB4 biosynthesis in human tongue SCC cells (SCC-15 and SCC-4) in vitro. In conclusion, this study clearly demonstrated that ethanol promoted 4NQO-induced oral carcinogenesis, at least in part, through further activation of the 5-Lox pathway of arachidonic acid metabolism.
doi:10.1158/1940-6207.CAPR-11-0206
PMCID: PMC3208736  PMID: 21881027
Alcohol drinking; Ethanol; 4NQO; Oral cancer; 5-Lox
13.  SAP—A Sequence Mapping and Analyzing Program for Long Sequence Reads Alignment and Accurate Variants Discovery 
PLoS ONE  2012;7(8):e42887.
The third-generation of sequencing technologies produces sequence reads of 1000 bp or more that may contain high polymorphism information. However, most currently available sequence analysis tools are developed specifically for analyzing short sequence reads. While the traditional Smith-Waterman (SW) algorithm can be used to map long sequence reads, its naive implementation is computationally infeasible. We have developed a new Sequence mapping and Analyzing Program (SAP) that implements a modified version of SW to speed up the alignment process. In benchmarks with simulated and real exon sequencing data and a real E. coli genome sequence data generated by the third-generation sequencing technologies, SAP outperforms currently available tools for mapping short and long sequence reads in both speed and proportion of captured reads. In addition, it achieves high accuracy in detecting SNPs and InDels in the simulated data. SAP is available at https://github.com/davidsun/SAP.
doi:10.1371/journal.pone.0042887
PMCID: PMC3413671  PMID: 22880129
14.  A Circadian Rhythm Orchestrated By Histone Deacetylase 3 Controls Hepatic Lipid Metabolism 
Science (New York, N.y.)  2011;331(6022):1315-1319.
Disruption of the circadian clock exacerbates metabolic diseases including obesity and diabetes. Here we show that histone deacetylase 3 (HDAC3) recruitment to the genome displays a circadian rhythm in mouse liver. Histone acetylation is inversely related to HDAC3 binding, and this rhythm is lost when HDAC3 is absent. Although amounts of HDAC3 are constant, its genomic recruitment in liver corresponds to the expression pattern of the circadian nuclear receptor Rev-erbα. Rev-erbα colocalizes with HDAC3 near genes regulating lipid metabolism, and deletion of HDAC3 or Rev-erbα in mouse liver causes hepatic steatosis. Thus, genomic recruitment of HDAC3 by Rev-erbα directs a circadian rhythm of histone acetylation and gene expression required for normal hepatic lipid homeostasis.
doi:10.1126/science.1198125
PMCID: PMC3389392  PMID: 21393543
15.  Kava Components Down-Regulate Expression of AR and AR Splice Variants and Reduce Growth in Patient-Derived Prostate Cancer Xenografts in Mice 
PLoS ONE  2012;7(2):e31213.
Men living in Fiji and drinking kava have low incidence of prostate cancer (PCa). However, the PCa incidence among Fijian men who had migrated to Australia, increased by 5.1-fold. We therefore examined the potential effects of kava root extracts and its active components (kavalactones and flavokawains) on PCa growth and androgen receptor (AR) expression. PCa cell lines (LNCaP, LAPC-4, 22Rv1, C4-2B, DU145 and PC-3) with different AR expression, and a transformed prostate myofibroblast cell line (WPMY-1), were treated with a commercial kava extract, kavalactones (kawain, 5′6′-dehydrokawain, yangonin, methysticin) and flavokawain B. Expression of AR and its target genes (PSA and TMPRSS2) was examined. Two novel patient-derived PCa xenograft models from high grade PCa specimens were established by implanting the specimens into nude mice and passing tumor pieces through subcutaneous injection in nude mice, and then treated with kava extract and flavokawain B to examine their effects on tumor growth, AR expression and serum PSA levels. The kava extract and flavokawain B effectively down-regulated the expression of both the full-length AR and AR splice variants. The kava extract and kavalactones accelerated AR protein degradation, while flavokawain B inhibited AR mRNA transcription via decreasing Sp1 expression and the binding of Sp1 to the AR promoter. The kava root extract and flavokawain B reduce tumor growth, AR expression in tumor tissues and levels of serum PSA in the patient-derived PCa xenograft models. These results suggest a potential usefulness of a safe kava product or its active components for prevention and treatment of advanced PCa by targeting AR.
doi:10.1371/journal.pone.0031213
PMCID: PMC3276576  PMID: 22347450
16.  Metabolic Patterns and Biotransformation Activities of Resveratrol in Human Glioblastoma Cells: Relevance with Therapeutic Efficacies 
PLoS ONE  2011;6(11):e27484.
Background
Trans-resveratrol rather than its biotransformed monosulfate metabolite exerts anti-medulloblastoma effects by suppressing STAT3 activation. Nevertheless, its effects on human glioblastoma cells are variable due to certain unknown reason(s).
Methodology/Principal Findings
Citing resveratrol-sensitive UW228-3 medulloblastoma cell line and primarily cultured rat brain cells/PBCs as controls, the effect of resveratrol on LN-18 human glioblastoma cells and its relevance with metabolic pattern(s), brain-associated sulfotransferase/SULT expression and the statuses of STAT3 signaling and protein inhibitor of activated STAT3 (PIAS3) were elucidated by multiple experimental approaches. Meanwhile, the expression patterns of three SULTs (SULT1A1, 1C2 and 4A1) in human glioblastoma tumors were profiled immunohistochemically. The results revealed that 100 µM resveratrol-treated LN-18 generated the same metabolites as UW228-3 cells, while additional metabolite in molecular weight of 403.0992 in negative ion mode was found in PBCs. Neither growth arrest nor apoptosis was found in resveratrol-treated LN-18 and PBC cells. Upon resveratrol treatment, the levels of SULT1A1, 1C2 and 4A1 expression in LN-18 cells were more up-regulated than that expressed in UW228-3 cells and close to the levels in PBCs. Immunohistochemical staining showed that 42.0%, 27.1% and 19.6% of 149 glioblastoma cases produced similar SULT1A1, 1C2 and 4A1 levels as that of tumor-surrounding tissues. Unlike the situation in UW228-3 cells, STAT3 signaling remained activated and its protein inhibitor PIAS3 was restricted in the cytosol of resveratrol-treated LN-18 cells. No nuclear translocation of STAT3 and PIAS3 was observed in resveratrol-treated PBCs. Treatment with STAT3 chemical inhibitor, AG490, committed majority of LN-18 and UW228-3 cells but not PBCs to apoptosis within 48 hours.
Conclusions/Significance
LN-18 glioblastoma cells are insensitive to resveratrol due to the more inducible brain-associated SULT expression, insufficiency of resveratrol to suppress activated STAT3 signaling and the lack of PIAS3 nuclear translocation. The findings from PBCs suggest that an effective anticancer dose of resveratrol exerts little side effect on normal brain cells.
doi:10.1371/journal.pone.0027484
PMCID: PMC3214056  PMID: 22096581
17.  KPNA6 (Importin α7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿  
Molecular and Cellular Biology  2011;31(9):1800-1811.
The transcription factor Nrf2 has emerged as a master regulator of cellular redox homeostasis. As an adaptive response to oxidative stress, Nrf2 activates the transcription of a battery of genes encoding antioxidants, detoxification enzymes, and xenobiotic transporters by binding the cis-antioxidant response element in the promoter regions of genes. The magnitude and duration of inducible Nrf2 signaling is delicately controlled at multiple levels by Keap1, which targets Nrf2 for redox-sensitive ubiquitin-mediated degradation in the cytoplasm and exports Nrf2 from the nucleus. However, it is not clear how Keap1 gains access to the nucleus. In this study, we show that Keap1 is constantly shuttling between the nucleus and the cytoplasm under physiological conditions. The nuclear import of Keap1 requires its C-terminal Kelch domain and is independent of Nrf1 and Nrf2. We have determined that importin α7, also known as karyopherin α6 (KPNA6), directly interacts with the Kelch domain of Keap1. Overexpression of KPNA6 facilitates Keap1 nuclear import and attenuates Nrf2 signaling, whereas knockdown of KPNA6 slows down Keap1 nuclear import and enhances the Nrf2-mediated adaptive response induced by oxidative stress. Furthermore, KPNA6 accelerates the clearance of Nrf2 protein from the nucleus during the postinduction phase, therefore promoting restoration of the Nrf2 protein to basal levels. These findings demonstrate that KPNA6-mediated Keap1 nuclear import plays an essential role in modulating the Nrf2-dependent antioxidant response and maintaining cellular redox homeostasis.
doi:10.1128/MCB.05036-11
PMCID: PMC3133232  PMID: 21383067
18.  Artesunate inhibits growth and induces apoptosis in human osteosarcoma HOS cell line in vitro and in vivo 
This paper aims to investigate the effects of artesunate (ART) on growth and apoptosis in human osteosarcoma HOS cell line in vitro and in vivo and to explore the possible underlying mechanisms. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The induction of apoptosis was detected by light and transmission electron microscopy and flow cytometry. Western blot analysis was used to investigate the related mechanisms. Nude mice were further employed to investigate the antitumour activity of ART in vivo. MTT assay results demonstrated that ART selectively inhibits the growth of HOS cells in a dose- and time-dependent manner. Based on the findings of light and transmission electron microscopy, Hoechst 33258 staining, and fluorescein isothiocyanate (FITC)-annexin V staining, the cytotoxicity of ART in HOS cells occurs through apoptosis. With ART treatment, cytosolic cytochrome c was increased, Bax expression was gradually upregulated, Bcl-2 expression was downregulated, and caspase-9 and caspase-3 were activated. Thus, the intrinsic apoptotic pathway may be involved in ART-induced apoptosis. Cell cycle analysis by flow cytometry indicated that ART may induce cell cycle arrest at G2/M phase. In nude mice bearing HOS xenograft tumours, ART inhibited tumour growth and regulated the expressions of cleaved caspase-3 and survivin, in agreement with in vitro observations. ART has a selective antitumour activity against human osteosarcoma HOS cells, which may be related to its effects on induction of apoptosis via the intrinsic pathway. The results suggest that ART is a promising candidate for the treatment of osteosarcoma.
doi:10.1631/jzus.B1000373
PMCID: PMC3072588  PMID: 21462379
Artesunate; Human osteosarcoma HOS cells; Apoptosis; Cell cycle; Nude mice; Chemotherapy
19.  A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62▿  
Molecular and Cellular Biology  2010;30(13):3275-3285.
In response to stress, cells can utilize several cellular processes, such as autophagy, which is a bulk-lysosomal degradation pathway, to mitigate damages and increase the chances of cell survival. Deregulation of autophagy causes upregulation of p62 and the formation of p62-containing aggregates, which are associated with neurodegenerative diseases and cancer. The Nrf2-Keap1 pathway functions as a critical regulator of the cell's defense mechanism against oxidative stress by controlling the expression of many cellular protective proteins. Under basal conditions, Nrf2 is ubiquitinated by the Keap1-Cul3-E3 ubiquitin ligase complex and targeted to the 26S proteasome for degradation. Upon induction, the activity of the E3 ubiquitin ligase is inhibited through the modification of cysteine residues in Keap1, resulting in the stabilization and activation of Nrf2. In this current study, we identified the direct interaction between p62 and Keap1 and the residues required for the interaction have been mapped to 349-DPSTGE-354 in p62 and three arginines in the Kelch domain of Keap1. Accumulation of endogenous p62 or ectopic expression of p62 sequesters Keap1 into aggregates, resulting in the inhibition of Keap1-mediated Nrf2 ubiquitination and its subsequent degradation by the proteasome. In contrast, overexpression of mutated p62, which loses its ability to interact with Keap1, had no effect on Nrf2 stability, demonstrating that p62-mediated Nrf2 upregulation is Keap1 dependent. These findings demonstrate that autophagy deficiency activates the Nrf2 pathway in a noncanonical cysteine-independent mechanism.
doi:10.1128/MCB.00248-10
PMCID: PMC2897585  PMID: 20421418
20.  Nrf2 and p21 regulate the fine balance between life and death by controlling ROS levels 
Cell Cycle  2009;8(20):3255-3256.
doi:10.4161/cc.8.20.9565
PMCID: PMC3918967  PMID: 19806015
Nrf2; Keap1; p21; ROS; oxidative stress
21.  Marked Insulin Resistance in Obese Spontaneously Hypertensive Rat Adipocytes Is Ameliorated by in Vivo but Not in Vitro Treatment with Moxonidine 
The obese spontaneously hypertensive rat (SHROB) is a model of marked insulin resistance with normoglycemia. We sought to determine whether insulin resistance extends to adipocytes and the impact of an insulin-sensitizing imidazoline, moxonidine (4 mg/kg/days for 21 days). Gonadal adipocytes were isolated from SHROB and lean spontaneously hypertensive rat (SHR) littermates. In lean SHR adipocytes, Akt activation by 100 nM insulin peaked at 3 min at 25-fold, whereas SHROB adipocytes showed only 4-fold activation. In dose-response experiments, the maximal response (Emax) was markedly reduced 18.8 ± 2.3 versus 3.7 ± 0.8. Insulin sensitivity was also attenuated, with higher concentrations required for responses (EC50 = 3.5 ± 0.5 versus 29 ± 3.8 nM). Glucose uptake as determined with [3H]2-deoxyglucose was also less responsive to insulin in SHROB relative to lean SHR. Moxonidine had little or no effect when applied acutely in vitro, but adipocytes isolated from SHROB treated with moxonidine in vivo showed significantly improved responses to insulin, both in terms of Akt activation and facilitation of glucose uptake. Chronic but not acute moxonidine treatment partially restores insulin sensitivity in SHROB adipocytes, suggesting an indirect action of this agent.
doi:10.1124/jpet.106.111153
PMCID: PMC2930913  PMID: 17095615
22.  Direct interaction between Nrf2 and p21Cip1/WAF1 upregulates the Nrf2-mediated antioxidant response 
Molecular cell  2009;34(6):663-673.
Summary
In response to oxidative stress, Nrf2 and p21 Cip1/WAF1 are both upregulated to protect cells from oxidative damage. Nrf2 is constantly ubiquitinated by a Keap1 dimer that interacts with a weak-binding 29DLG motif and a strong-binding 79ETGE motif in Nrf2, resulting in degradation of Nrf2. Modification of the redox-sensitive cysteine residues on Keap1 disrupts the Keap1-29DLG binding, leading to diminished Nrf2 ubiquitination and activation of the antioxidant response. However, the underlying mechanism by which p21 protects cells from oxidative damage remains unclear. Here, we present molecular and genetic evidence suggesting that the antioxidant function of p21 is mediated through activation of Nrf2 by stabilizing the Nrf2 protein. The 154KRR motif in p21 directly interacts with the 29DLG and 79ETGE motifs in Nrf2, and thus, competes with Keap1 for Nrf2 binding, compromising ubiquitination of Nrf2. Furthermore, the physiological significance of our findings was demonstrated in vivo using p21-deficient mice.
doi:10.1016/j.molcel.2009.04.029
PMCID: PMC2714804  PMID: 19560419
23.  The Biological Effect of Contralateral Forepaw Stimulation in Rat Focal Cerebral Ischemia: A Multispectral Optical Imaging Study 
Our group has already published the possible neuroprotective effect of contralateral forepaw stimulation in temporary focal ischemia in a study. However, the background is still unclear. In the present study we investigated the possible mechanism by monitoring focal ischemia with multispectral [laser speckle, imaging of intrinsic signals (OIS)] imaging. Sprague–Dawley rats were prepared using 1.2% isoflurane anesthesia. The middle cerebral artery was occluded by photothrombosis (4 mW) and the common carotid artery was ligated permanently. Physiological variables were constantly monitored during the experiment. A 6 × 6 mm area centered 3 mm posterior and 4 mm lateral to Bregma was thinned for laser speckle and OIS imaging. Nine circular regions of interests (0.3 mm in diameter) were evenly spaced on the speckle contrast image for the analysis of peri-infarct flow transients, blood flow, and metabolic changes. Both the sham (n = 7) and forepaw-stimulated animals (n = 7) underwent neurological examinations 24 h after ischemia at which point all animals were sacrificed and the infarct size was determined by triphenyltetrazolium chloride. The physiological variables were in normal range and the experimental protocol did not cause significant differences between groups. Both the neurological scores (sham: 3.6 ± 1.7, stimulated: 4.3 ± 1.4) and the infarct volume (sham: 124 ± 39 mm3, stimulated: 147 ± 47 mm3) did not show significant differences between groups. The forepaw stimulation did not increase the intra-ischemic flow neither over the penumbral or the peri-ischemic area. However, the hemoglobin transients related metabolic load (CMRO2) was significantly lower (p < 0.001) while the averaged number of hyperemic flow transients were significantly (p = 0.013) higher in the forepaw (sham: 3.5 ± 2.2, stimulated: 7.0 ± 2.3) stimulated animals.
doi:10.3389/fnene.2010.00019
PMCID: PMC2922941  PMID: 20725601
optical imaging; focal cerebral ischemia; forepaw stimulation; middle cerebral artery occlusion; photothrombosis; speckle contrast; OIS; flow transients
24.  Acetylation of Nrf2 by p300/CBP Augments Promoter-Specific DNA Binding of Nrf2 during the Antioxidant Response▿ †  
Molecular and Cellular Biology  2009;29(10):2658-2672.
To maintain intracellular redox homeostasis, genes encoding many antioxidants and detoxification enzymes are transcriptionally upregulated upon deleterious oxidative stress through the cis antioxidant responsive elements (AREs) in their promoter regions. Nrf2 is the critical transcription factor responsible for ARE-dependent transcription. We and others have previously demonstrated that Nrf2 is targeted for ubiquitin-mediated degradation by Keap1 in a redox-sensitive manner through modifications of distinct cysteine residues of Keap1. Here, we report that p300/CBP directly acetylates Nrf2 in response to arsenite-induced stress. We have identified multiple acetylated lysine residues within the Nrf2 Neh1 DNA-binding domain. Combined lysine-to-arginine mutations on the acetylation sites, with no effects on Nrf2 protein stability, compromised the DNA-binding activity of Nrf2 in a promoter-specific manner. These findings demonstrated that acetylation of Nrf2 by p300/CBP augments promoter-specific DNA binding of Nrf2 and established acetylation as a novel regulatory mechanism that functions in concert with Keap1-mediated ubiquitination in modulating the Nrf2-dependent antioxidant response.
doi:10.1128/MCB.01639-08
PMCID: PMC2682049  PMID: 19273602
25.  Dual Roles of Nrf2 in Cancer 
In response to oxidative stress, the transcription factor NF-E2-related factor 2 (Nrf2) controls the fate of cells through transcriptional upregulation of antioxidant response element (ARE)-bearing genes, including those encoding endogenous antioxidants, phase II detoxifying enzymes, and transporters. Expression of the Nrf2-dependent proteins is critical for ameliorating or eliminating toxicants/carcinogens to maintain cellular redox homeostasis. As a result, activation of the Nrf2 pathway, by naturally-occurring compounds or synthetic chemicals at sub-toxic doses, confers protection against subsequent toxic/carcinogenic exposure. Thus, the use of dietary compounds or synthetic chemicals to boost the Nrf2-dependent adaptive response to counteract environmental insults has emerged to be a promising strategy for cancer prevention. Interestingly, recent emerging data has revealed the “dark” side of Nrf2. Nrf2 and its downstream genes are overexpressed in many cancer cell lines and human cancer tissues, giving cancer cells an advantage for survival and growth. Furthermore, Nrf2 is upregulated in resistant cancer cells and is thought to be responsible for acquired chemoresistance. Therefore, it may be necessary to inhibit the Nrf2 pathway during chemotherapy. This review is primarily focused on the role of Nrf2 in cancer, with emphasis on the recent findings indicating the cancer promoting function of Nrf2 and its role in acquired chemoresistance.
doi:10.1016/j.phrs.2008.09.003
PMCID: PMC2652397  PMID: 18838122

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