Publications by authors named "Pengfei Gong"

23 Publications

  • Page 1 of 1

Integrated transcriptome and proteome analysis reveals potential mechanisms for differential abdominal fat deposition between divergently selected chicken lines.

J Proteomics 2021 Jun 23;241:104242. Epub 2021 Apr 23.

Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture and Rural Affairs, Harbin 150030, PR China; Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, Harbin 150030, PR China; College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, PR China. Electronic address:

Genetic selection for meat production performance of broilers concomitantly causes excessive abdominal fat deposition, accompanied by several adverse effects, such as the reduction of feed conversion efficiency and reproduction performance. Our previous studies have identified important genes regulating chicken fat deposition, using the Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF) as an animal model. However, the molecular mechanism underlying fat deposition differences between fat and lean broilers remains largely unknown. Here, we integrated the transcriptome (RNA-Seq) and quantitative proteome (isobaric tags for relative and absolute quantitation, iTRAQ) profiling analyses on abdominal fat tissues from NEAUHLF chicken lines. Differentially expressed genes (2167 DEGs, corrected p-value < 0.01) and differentially abundant proteins (199 DAPs, corrected p-value < 0.05) were identified in lean line compared to fat line. Down-regulated DEGs and DAPs mainly enriched in pathways related to fatty acid metabolism, fatty acid biosynthesis, and PPAR signaling, and interestingly, up-regulated DEGs and DAPs enriched both in lysosome pathway. Moreover, numerous key DEGs and DAPs involved in long-chain fatty acid uptake, in situ lipogenesis (fatty acid and cholesterol synthesis), and lipid droplet accumulation were discovered after integrated transcriptome and proteome analysis. SIGNIFICANCE: Excessive abdominal fat deposition critically affects the health of broilers and causes economic loss to broiler producers, but the molecular mechanism of abdominal fat deposition is still unclear in chicken. We identified key DEGs/DAPs and potential pathways through an integration of chicken abdominal fat tissues transcriptome and proteome analyses. Our findings will facilitate a better revealing the mechanism and provide a novel insight into abdominal fat content discrepancy between the fat and lean chicken lines.
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http://dx.doi.org/10.1016/j.jprot.2021.104242DOI Listing
June 2021

Effect of Postnatal Nutritional Environment Due to Maternal Diabetes on Beta Cell Mass Programming and Glucose Intolerance Risk in Male and Female Offspring.

Biomolecules 2021 01 28;11(2). Epub 2021 Jan 28.

Laboratoire B2PE (Biologie et Pathologie du Pancréas Endocrine), Unité BFA (Biologie Fonctionnelle et Adaptive), Equipe 1, Université de Paris, UMR8251, CNRS, F-75013 Paris, France.

Besides the fetal period, the suckling period is a critical time window in determining long-term metabolic health. We undertook the present study to elucidate the impact of a diabetic suckling environment alone or associated with an in utero diabetic environment on beta cell mass development and the risk of diabetes in the offspring in the long term. To that end, we have compared two experimental settings. In setting 1, we used Wistar (W) rat newborns resulting from W ovocytes (oW) transferred into diabetic GK rat mothers (pGK). These oW/pGK neonates were then suckled by diabetic GK foster mothers (oW/pGK/sGK model) and compared to oW/pW neonates suckled by normal W foster mothers (oW/pW/sW model). In setting 2, normal W rat newborns were suckled by diabetic GK rat foster mothers (nW/sGK model) or normal W foster mothers (nW/sW model). Our data revealed that the extent of metabolic disorders in term of glucose intolerance and beta cell mass are similar between rats which have been exposed to maternal diabetes both pre- and postnatally (oW/pGK/sGK model) and those which have been exposed only during postnatal life (nW/sW model). In other words, being nurtured by diabetic GK mothers from birth to weaning was sufficient to significantly alter the beta cell mass, glucose-induced insulin secretion and glucose homeostasis of offspring. No synergistic deleterious effects of pre-and postnatal exposure was observed in our setting.
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http://dx.doi.org/10.3390/biom11020179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7911592PMC
January 2021

Whole-genome bisulfite sequencing of abdominal adipose reveals DNA methylation pattern variations in broiler lines divergently selected for fatness.

J Anim Sci 2021 Jan;99(1)

Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture and Rural Affairs, Harbin, P.R. China.

The methylation status of pivotal genes involved in fat deposition in chickens has been extensively studied. However, the whole-genome DNA methylation profiles of broiler abdominal adipose tissue remain poorly understood. Using whole-genome bisulfite sequencing, we generated DNA methylation profiles of chicken abdominal adipose tissue from Northeast Agricultural University broiler lines divergently selected for abdominal fat content. We aimed to explore whether DNA methylation was associated with abdominal fat deposition in broilers. The whole-genome DNA methylation profiles of fat- and lean-line broilers abdominal adipose tissue were constructed. The DNA methylation levels of functional genomic regions in the fat broiler were higher than those in the lean broiler, especially in the 3' untranslated regions (UTRs) and exons in the non-CG contexts. Additionally, we identified 29,631 differentially methylated regions and, subsequently, annotated 6,484 and 2,016 differentially methylated genes (DMGs) in the gene body and promoter regions between the two lines, respectively. Functional annotation showed that the DMGs in promoter regions were significantly enriched mainly in the triglyceride catabolic process, lipid metabolism-related pathways, and extracellular matrix signal pathways. When the DMG in promoter regions and differentially expressed genes were integrated, we identified 30 genes with DNA methylation levels that negatively correlated with their messenger RNA (mRNA) expression, of which CMSS1 reached significant levels (false discovery rate < 0.05). These 30 genes were mainly involved in fatty acid metabolism, peroxisome-proliferator-activated receptor signaling, Wnt signaling pathways, transmembrane transport, RNA degradation, and glycosaminoglycan degradation. Comparing the DNA methylation profiles between fat- and lean-line broilers demonstrated that DNA methylation is involved in regulating broiler abdominal fat deposition. Our study offers a basis for further exploring the underlying mechanisms of abdominal adipose deposition in broilers.
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http://dx.doi.org/10.1093/jas/skaa408DOI Listing
January 2021

Promoter Methylation Regulates ApoA-I Gene Transcription in Chicken Abdominal Adipose Tissue.

J Agric Food Chem 2019 Apr 10;67(16):4535-4544. Epub 2019 Apr 10.

Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture and Rural Affairs, Key Laboratory of Animal Genetics, Breeding and Reproduction of Education Department of Heilongjiang Province, College of Animal Science and Technology , Northeast Agricultural University , Harbin 150030 , Heilongjiang , China.

As a central constituent of HDL (high-density lipoprotein), apolipoprotein A-I (ApoA-I) has a vital function in lipid metabolism. Our previous studies confirmed that ApoA-I was differentially expressed in the adipose tissue of the abdomen of lean and fat broilers. The aim of the current work was to evaluate whether the transcription of ApoA-I in chicken abdominal adipose tissue was regulated by DNA methylation. The methylation status of ApoA-I promoter CpG island (PCGI) and promoter non-CpG island (PNCGI) as well as the ApoA-I expression level in adipose tissue of lean and fat broilers were determined using Sequenom MassARRAY and real-time PCR. The correlation analysis results showed that the methylation level of PCGI and the ApoA-I mRNA expression level were negatively correlated. Bisulfite sequencing PCR was used to assess the methylation level of ApoA-I promoter in the ICP1 cells treated with 5-aza-2'-deoxycytidine (5-Aza-CdR: an inhibitor of DNA methyltransferase). The result showed that 5-Aza-CdR caused a reduction in the methylation level of the ApoA-I promoter, thereby causing an increase in expression of the ApoA-I mRNA. Meanwhile, luciferase reporter assays indicated that in vitro methylation of the ApoA-I promoter containing CpG island with CpG methyltransferase led to transcriptional repression. Furthermore, the noticeable activation of NRF1 on ApoA-I transcription was largely enhanced  by the demethylation of the ApoA-I PCGI region. These observations indicated that the differential expression of ApoA-I gene in the adipose tissue of broilers could be mediated by transcription regulation, at least in part by DNA methylation in its PCGI region.
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http://dx.doi.org/10.1021/acs.jafc.9b00007DOI Listing
April 2019

Expression profile of long non-coding RNAs in cervical spondylotic myelopathy of rats by microarray and bioinformatics analysis.

Genomics 2019 12 5;111(6):1192-1200. Epub 2019 Jan 5.

Department of Orthopedics, The Second Hospital of Tianjin Medical University, Tianjin 300211, China. Electronic address:

Introduction: It has been reported that a wide range of long non-coding RNAs (lncRNAs) are implicated in numerous diseases such as tumor, cardiopathy and neurological disorders. Identifying the differentially expressed (DE) profile of lncRNAs in cervical spondylotic myelopathy (CSM) is essential to understand the mechanisms of CSM.

Methods: Microarray assay, quantitative real-time PCR (qRT-PCR) and bioinformatics analysis were employed to reveal the DE profile and potential functions of lncRNAs in CSM.

Results: Microarray analysis displayed the DE profiles of lncRNAs and mRNAs in rats between the CSM group and the control (CON) group. Thereinto, 1266 DE lncRNAs (738 up-regulation and 528 down-regulation) and 847 mRNAs (487 up-regulation and 360 down-regulation) with >1.1 fold change (FC) were finally identified. Moreover, 17 lncRNAs (13 up-regulation and 4 down-regulation) and 18 mRNAs (13 up-regulation and 5 down-regulation) were found deregulated by >2 FC. Further bioinformatics analysis showed the most remarkable biological processes among up-regulated RNAs contain cellular response to interferon-beta, inflammatory response and innate immune response, which may involve in CSM. Besides, related DE mRNAs of 17 DE lncRNAs in the genome were related to signaling pathway about NOD-like receptor, TNF, and apoptosis. In addition, a co-expression network of lncRNA-mRNA was established for analyzing the biological roles of lncRNAs. Among these, we found a ceRNA network related to CSM. Finally, the expressions of the DE lncRNAs and ceRNA network confirmed by qRT-PCR were in agreement with microarray data.

Conclusions: Our study revealed the DE profiles of lncRNAs and mRNAs for CSM. Those dysregulated RNAs may represent potential therapeutic targets of CSM for further study.
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http://dx.doi.org/10.1016/j.ygeno.2019.01.005DOI Listing
December 2019

A Potential Competitive Endogenous RNA Pathway Involved in Chronic Spinal Cord Injury.

Med Sci Monit 2018 Nov 9;24:8022-8032. Epub 2018 Nov 9.

Department of Orthopedics, The Second Hospital of Tianjin Medical University, Tianjin, China (mainland).

BACKGROUND Chronic spinal cord injury (CSCI) is a worldwide clinical problem. We aimed to reveal differentially expressed (DE) lncRNAs and to find associated pathways that may function as targets for CSCI therapy. MATERIAL AND METHODS After a CSCI rat model was confirmed by the Basso Beattie Bresnahan (BBB) scale and the Magnetic Resonance Imaging (MRI) test, microarray analysis was used to obtain the expression profile of DE lncRNAs between CSCI rats and corresponding control rats. Then, bioinformatics analyses, including GO and KEGG pathway analysis, DE lncRNAs-mRNAs co-expression analysis, and several databases, were used to examine the function of these DE lncRNAs. Finally, quantitative real-time PCR (qRT-PCR) was used to evaluate the expressions of the 5 most significantly changed lncRNAs, Col6a1, and miR-330-3p. RESULTS Our study identified 1266 DE lncRNAs and 847 DE mRNAs, among which lncRNA6032 was significant up-regulated. Furthermore, the expressions of miR-330-3p and Col6a1 associated with lncRNA6032 were down-regulated and up-regulated, respectively. CONCLUSIONS Our results showed that the abundance of DE lncRNAs may be associated with the risk of CSCI outcome and revealed a potential competitive endogenous RNA (ceRNA) pathway involved in CSCI. Further experiments in vivo and in vitro were essential to uncover the exact mechanism of this ceRNA pathway.
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http://dx.doi.org/10.12659/MSM.911536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6238548PMC
November 2018

Renal HIV expression is unaffected by serum LPS levels in an HIV transgenic mouse model of LPS induced kidney injury.

PLoS One 2011 16;6(6):e20688. Epub 2011 Jun 16.

Division of Nephrology, Department of Medicine, Mount Sinai School of Medicine, New York City, New York, United States of America.

Acute kidney injury (AKI) is associated with increased rates of mortality. For unknown reasons, HIV infected individuals have a higher risk of AKI than uninfected persons. We tested our hypothesis that increased circulating LPS increases renal expression of HIV and that HIV transgenic (Tg26) mice have increased susceptibility to AKI. Tg26 mice harbor an HIV transgene encoding all HIV genes except gag and pol, and develop a phenotype analogous to HIVAN. Mice were used at 4-6 weeks of age before the onset of gross renal disease. Mice were injected i.p. with LPS or sterile saline. Renal function, tubular injury, cytokine expression, and HIV transcription were evaluated in Tg26 and wild type (WT) mice. LPS injection induced a median 60.1-fold increase in HIV expression in spleen but no change in kidney. There was no significant difference in renal function, cytokine expression, or tubular injury scores at baseline or 24 hours after LPS injection. HIV transcription was also analyzed in vitro using a human renal tubular epithelial cell (RTEC) line. HIV transcription increased minimally in human RTEC, by 1.47 fold, 48 hours after LPS exposure. We conclude that Tg26 mice do not increase HIV expression or have increased susceptibility to LPS induced AKI. The increased risk of AKI in HIV infected patients is not mediated via increased renal expression of HIV in the setting of sepsis. Moreover, renal regulation of HIV transcription is different to that in the spleen.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0020688PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3116837PMC
November 2011

HIV-1 viral protein r induces ERK and caspase-8-dependent apoptosis in renal tubular epithelial cells.

AIDS 2010 May;24(8):1107-19

Department of Nephrology, Mount Sinai School of Medicine, New York, New York 10029, USA.

Objective: HIV-associated nephropathy (HIVAN) is the most common cause of end-stage renal disease in persons with HIV/AIDS and is characterized by focal glomerulosclerosis and dysregulated renal tubular epithelial cell (RTEC) proliferation and apoptosis. HIV-1 viral protein r (Vpr) has been implicated in HIV-induced RTEC apoptosis but the mechanisms of Vpr-induced RTEC apoptosis are unknown. The aim of this study was therefore to determine the mechanisms of Vpr-induced apoptosis in RTEC.

Methods: Apoptosis and caspase activation were analyzed in human RTEC (HK2) after transduction with Vpr-expressing and control lentiviral vectors. Bax and BID were inhibited with lentiviral shRNA, and ERK activation was blocked with the MEK1,2 inhibitor, U0126.

Results: Vpr induced apoptosis as indicated by caspase 3/7 activation, PARP-1 cleavage and mitochondrial injury. Vpr activated both caspases-8 and 9. Inhibition of Bax reduced Vpr-induced apoptosis, as reported in other cell types. Additionally, Vpr-induced cleavage of BID to tBID and suppression of BID expression prevented Vpr-induced apoptosis. Since sustained ERK activation can activate caspase-8 in some cell types, we studied the role of ERK in Vpr-induced caspase-8 activation. Vpr induced sustained ERK activation in HK2 cells and incubation with U0126 reduced Vpr-induced caspase-8 activation, BID cleavage and apoptosis. We detected phosphorylated ERK in RTEC in HIVAN biopsy specimens by immunohistochemistry.

Conclusions: These studies delineate a novel pathway of Vpr-induced apoptosis in RTEC, which is mediated by sustained ERK activation, resulting in caspase 8-mediated cleavage of BID to tBID, thereby facilitating Bax-mediated mitochondrial injury and apoptosis.
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http://dx.doi.org/10.1097/QAD.0b013e328337b0abDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2860650PMC
May 2010

The ubiquitin-like protein FAT10 mediates NF-kappaB activation.

J Am Soc Nephrol 2010 Feb 3;21(2):316-26. Epub 2009 Dec 3.

Division of Nephrology, Department of Medicine, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.

NF-kappaB is a central mediator of innate immunity and contributes to the pathogenesis of several renal diseases. FAT10 is a TNF-alpha-inducible ubiquitin-like protein with a putative role in immune response, but whether FAT10 participates in TNF-alpha-induced NF-kappaB activation is unknown. Here, using renal tubular epithelial cells (RTECs) derived from FAT10(-/-) and FAT10(+/+) mice, we observed that FAT10 deficiency abrogated TNF-alpha-induced NF-kappaB activation and reduced the induction of NF-kappaB-regulated genes. Despite normal IkBalpha degradation and polyubiquitination, FAT10 deficiency impaired TNF-alpha-induced IkBalpha degradation and nuclear translocation of p65 in RTECs, suggesting defective proteasomal degradation of polyubiquitinated IkBalpha. In addition, FAT10 deficiency reduced the expression of the proteasomal subunit low molecular mass polypeptide 2 (LMP2). Transduction of FAT10(-/-) RTECs with FAT10 restored LMP2 expression, TNF-alpha-induced IkBalpha degradation, p65 nuclear translocation, and NF-kappaB activation. Furthermore, LMP2 transfection restored IkBalpha degradation in FAT10(-/-) RTECs. In humans, common types of chronic kidney disease associated with tubulointerstitial upregulation of FAT10. These data suggest that FAT10 mediates NF-kappaB activation and may promote tubulointerstitial inflammation in chronic kidney diseases.
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http://dx.doi.org/10.1681/ASN.2009050479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2834541PMC
February 2010

FAT10: a novel mediator of Vpr-induced apoptosis in human immunodeficiency virus-associated nephropathy.

J Virol 2009 Nov 2;83(22):11983-8. Epub 2009 Sep 2.

Mount Sinai School of Medicine, Division of Nephrology, Box 1243, 1 Gustave L Levy Place, New York, New York 10029, USA.

Human immunodeficiency virus (HIV)-associated nephropathy is a significant cause of morbidity and mortality in HIV-infected persons. Vpr-induced cell cycle dysregulation and apoptosis of renal tubular epithelial cells are important components of the pathogenesis of HIV-associated nephropathy (HIVAN). FAT10 is a ubiquitin-like protein that is upregulated in renal tubular epithelial cells in HIVAN. In these studies, we report that Vpr induces increased expression of FAT10 in tubular cells and that inhibition of FAT10 expression prevents Vpr-induced apoptosis in human and murine tubular cells. Moreover, we found that Vpr interacts with FAT10 and that these proteins colocalize at mitochondria. These studies establish FAT10 as a novel mediator of Vpr-induced cell death.
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http://dx.doi.org/10.1128/JVI.00034-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2772664PMC
November 2009

Pyrazole induced oxidative liver injury independent of CYP2E1/2A5 induction due to Nrf2 deficiency.

Toxicology 2008 Oct 3;252(1-3):9-16. Epub 2008 Aug 3.

Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029, USA.

Pyrazole can induce CYP2E1 and 2A5, which produce reactive oxygen species (ROS). Nuclear factor erythroid 2-related factor 2 (Nrf2) regulates important antioxidant enzymes to remove ROS. In this study, we applied Nrf2 knockout mice to test the hypothesis that pyrazole will cause hepatotoxicity and elevate oxidative stress to a greater extent in Nrf2 knockout mice compared to wild type mice. Pyrazole induced severe oxidative liver damage in Nrf2 knockout mice but not in wild type mice. Activities and levels of CYP2E1 and 2A5 were elevated by pyrazole in the wild type mice but not in the Nrf2 knockout mice. However, expression or activity of Nrf2-regulated antioxidant enzymes, such as gamma-glutamylcysteine synthetase (GCS), heme oxygenase-1 (HO-1) and glutathione-S-transferase (GST), were upregulated in the pyrazole-treated wild type mice, but to a lesser extent or not at all in the pyrazole-treated Nrf2 knockout mice. Treatment with antioxidants such as vitamin C or S-adenosyl-l-methionine (SAM) or an inhibitor of iNOS prevented the pyrazole-induced oxidative liver damage, thus validating the role of oxidative/nitrosative stress in the pyrazole induced liver injury to the Nrf2 knockout mice. In summary, even though ROS-producing CYP2E1/2A5 were not elevated by pyrazole, impaired antioxidant capacity resulting from Nrf2 deficiency appear to be sufficient to promote pyrazole-induced oxidative liver injury.
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http://dx.doi.org/10.1016/j.tox.2008.07.058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2613847PMC
October 2008

Cadmium-responsive element of the human heme oxygenase-1 gene mediates heat shock factor 1-dependent transcriptional activation.

J Biol Chem 2007 Mar 23;282(12):8715-23. Epub 2007 Jan 23.

Mechanism of Health Effect Research Group, National Institute of Occupational Safety and Health, Kawasaki 214-8585, Japan.

Transcription of a number of mammalian genes is activated by heavy metals, but mechanisms of signaling and transcriptional regulation are not well understood. From a comparison of heavy metal responses of several human genes, it was noted that the heme oxygenase-1 (HO-1) gene is quite similar in the spectrum of metal response and induction kinetics to the heat shock protein 70 (HSP70) gene, suggesting a common regulatory mechanism shared by these genes. The cadmium-responsive element (CdRE) known to be responsible for the metal regulation of ho-1 formed complexes with proteins from heavy metal-treated HeLa cells in an electrophoretic mobility shift assay (EMSA). These complexes were indistinguishable in mobility from those formed by the heat shock factor 1 (HSF1) and the heat shock element involved in hsp70 regulation, suggesting the involvement of HSF1 also in the CdRE complexes. Competitive EMSA and supershift analysis with an anti-HSF1 antibody revealed that HSF1 was in fact a component of the CdRE complexes. A fine analysis on the affinity of HSF1 to a series of mutant CdRE sequences showed that HSF1 recognizes a sequence motif TnCTAGA. Transient transfection analysis with overexpressed recombinant HSF1 demonstrated that CdRE has HSF1-dependent enhancer-like activity that requires direct binding of HSF1. In the absence of overexpressed HSF1, however, CdRE by itself was insufficient to mediate heavy metal-induced transcription, suggesting requirement of additional regulatory sequences. The finding that HSF1 is directly involved in the regulation of ho-1 with an anti-oxidative role revealed a new aspect of the biological defense mechanism.
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http://dx.doi.org/10.1074/jbc.M609427200DOI Listing
March 2007

Transcription factor Nrf2 protects HepG2 cells against CYP2E1 plus arachidonic acid-dependent toxicity.

J Biol Chem 2006 May 21;281(21):14573-9. Epub 2006 Mar 21.

Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.

Nrf2 is a transcription factor that regulates important antioxidant and phase II detoxification genes. Arachidonic acid (AA) causes CYP2E1-dependent toxicity in HepG2 cells. The ability of Nrf2 to protect against CYP2E1-dependent AA toxicity and its possible mechanism were evaluated. AA activates Nrf2 in CYP2E1-expressing HepG2 cells (E47 cells), increasing Nrf2 protein and mRNA levels, Nrf2 nuclear translocation, and Nrf2-ARE binding activity. These increases in Nrf2 are associated with elevated expression of Nrf2-regulated antioxidant genes. Overexpression of Nrf2 by transient transfection of plasmid Nrf2 confers resistance of E47 cells against AA toxicity. Blocking Nrf2 with small interfering RNA (siRNA)-Nrf2 potentiates the CYP2E1-dependent AA toxicity. This enhanced toxicity is accompanied by decreases of cellular GSH levels and increases in production of reactive oxygen species and lipid peroxidation. There is also a potentiation of mitochondrial damage in the presence of siRNA-Nrf2. The protective effects of Nrf2 against CYP2E1-dependent toxicity can be blocked by l-buthionine-(S,R)-sulfoximine, a specific inhibitor of glutamate-cysteine ligase, which is a rate-limiting enzyme in the synthesis of GSH and is regulated by Nrf2. Elevation of GSH by supplementing with glutathione ethyl ester can partially reverse the enhanced AA toxicity by siRNA-Nrf2. Moreover, in contrast to AA, l-buthionine-(S,R)-sulfoximine toxicity is not prevented by plasmid Nrf2 probably because protective GSH cannot be synthesized. Together, these results suggest that Nrf2, through up-regulation of glutamate-cysteine ligase and increase of GSH levels, protects against CYP2E1-dependent AA toxicity.
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http://dx.doi.org/10.1074/jbc.M600613200DOI Listing
May 2006

Cross talk of heat shock and heavy metal regulatory pathways.

Biochem Biophys Res Commun 2006 Mar 25;341(4):1072-7. Epub 2006 Jan 25.

Department of Health Effects Research, National Institute of Industrial Health, 6-21-1 Nagao, Tama-ku, Kawasaki 214-8585, Japan.

Heavy metals induce transcription of human genes including those coding for metallothionein and heat shock protein 70 (HSP70). It has been suggested that these processes are mediated by metal-activated transcription factors, MTF-1 and HSF1, respectively, and are independent of each other. We raised an antibody against human MTF-1 which efficiently supershifts the protein-DNA complex formed by MTF-1 and its cognate binding sequence, MRE. We discovered that this antibody could also supershift complexes formed by HSF1 and its recognition sequence HSE, which suggested the involvement of MTF-1 in these complexes. This supershift was observed for HSF1/HSE complexes induced by Zn, Cd, Ag, and heat shock. Furthermore, overexpression of MTF-1 in HeLa cells markedly reduced metal-induced transcription from the hsp70-1 gene promoter which depends on HSF1. These data indicate that MTF-1 represses HSF1-mediated transcription probably through a direct protein-protein interaction, suggesting a cross talk of two lines of stress-responsive regulatory pathways.
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http://dx.doi.org/10.1016/j.bbrc.2006.01.066DOI Listing
March 2006

Nrf2 is increased by CYP2E1 in rodent liver and HepG2 cells and protects against oxidative stress caused by CYP2E1.

Hepatology 2006 Jan;43(1):144-53

Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, NY 10029, USA.

Induction of CYP2E1 by ethanol is one pathway through which ethanol generates oxidative stress. Nrf2 is a transcription factor that regulates important antioxidant and phase II detoxification genes. Nrf2 induction by CYP2E1 and its importance in the adaptive response to increased oxidative stress caused by CYP2E1 was studied. Increases in Nrf2 protein and mRNA were observed in livers or hepatocytes of chronic alcohol-fed mice or rats and of pyrazole-treated rats or mice, conditions known to elevate CYP2E1. HepG2 cells expressing CYP2E1 (E47 cells) showed increased Nrf2 mRNA and protein expression compared with control HepG2 C34 cells. Nrf2 is activated in E47 cells as shown by an increase in nuclear Nrf2 levels and Nrf2-antioxidant-responsive element binding activity, and upregulation of Nrf2-regultated genes, glutamate cysteine ligase catalytic subunit (GCLC), and heme oxygenase 1 (HO-1). Increases in Nrf2 protein and mRNA are blocked by inhibitors of CYP2E1 activity and a reactive oxygen species (ROS) scavenger, N-acetylcysteine, which decrease ROS levels as well as Nrf2 mRNA induction. Upregulation of GCLC and HO-1 in E47 cells is dependent on Nrf2 and is prevented by siRNA-Nrf2. Blocking Nrf2 by siRNA-Nrf2 decreases glutathione and increases ROS and lipid peroxidation, resulting in decreased mitochondrial membrane potential and loss of cell viability of E47 cells but not C34 cells. These results suggest that Nrf2 is activated and that levels of protein and mRNA are increased when CYP2E1 is elevated. In conclusion, Nrf2 plays a key role in the adaptive response against increased oxidative stress caused by CYP2E1.
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http://dx.doi.org/10.1002/hep.21004DOI Listing
January 2006

Diallyl sulfide induces heme oxygenase-1 through MAPK pathway.

Arch Biochem Biophys 2004 Dec;432(2):252-60

Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA.

Diallyl sulfide (DAS), is protective against chemically induced heptotoxicity, mutagenesis, and carcinogenesis. The mechanism of its protective effects is not fully understood. In this study, we found that DAS can induce the expression of heme oxygenase-1 (HO-1), which plays a critical role in the cell defense system against oxidative stress. DAS causes a dose- and time-dependent increase of HO-1 protein and mRNA level without toxicity in HepG2 cells. DAS-induced HO-1 protein expression is dependent on newly synthesized mRNA and newly synthesized protein. DAS increases Nrf2 protein expression, nuclear translocation, and DNA-binding activity. The MAP kinase ERK is activated by DAS. Both ERK and p38 pathways play an important role in DAS-induced Nrf2 nuclear translocation and ho-1 gene activation. DAS stimulates a transient increase of reactive oxygen species (ROS). N-Acetyl-cysteine blocked this increase of ROS production as well as DAS-induced ERK activation, Nrf2 protein expression and nuclear translocation, and ho-1 gene activation. The increase in HO-1 produced by DAS protected the HepG2 cells against toxicity by hydrogen peroxide or arachidonic acid. These results suggest that DAS induces ho-1 through production of ROS, and Nrf2 and MAPK (ERK and p38) mediate this induction. Induction of ho-1 may play a role in the protective effects of DAS.
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http://dx.doi.org/10.1016/j.abb.2004.09.024DOI Listing
December 2004

Heme oxygenase-1 protects HepG2 cells against cytochrome P450 2E1-dependent toxicity.

Free Radic Biol Med 2004 Feb;36(3):307-18

Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, NY 10029, USA.

The inducible form of heme oxygenase (HO-1) is increased during oxidative injury and HO-1 is believed to be an important defense mechanism against such injury. Arachidonic acid (AA) and l-buthionine-(S,R)-sulfoximine (BSO), which lowers GSH levels, cause cytochrome P450 2E1 (CYP2E1)-dependent oxidative injuries in HepG2 cells (E47 cells). Treatment of E47 cells with 50 microM AA or 100 microM BSO for 48 h was recently shown to increase HO-1 mRNA, protein, and activity. The possible functional significance of this increase in protecting against CYP2E1-dependent toxicity was evaluated in the current study. The treatment with AA and BSO caused loss of cell viability (40 and 50%, respectively) in E47 cells. Chromium mesoporphyrin (CrMP), an inhibitor of HO activity, significantly potentiated this cytotoxicity. ROS production, lipid peroxidation, and the decline in mitochondrial membrane potential produced by AA and BSO were also enhanced in the presence of CrMP in E47 cells. Infection with an adenovirus expressing rat HO-1 protected E47 cells from AA toxicity, increasing cell viability and reducing LDH release. HO catalyzes formation of CO, bilirubin, and iron from the oxidation of heme. Bilirubin was not protective whereas iron catalyzed the AA toxicity. The carbon monoxide (CO) scavenger hemoglobin enhanced AA toxicity in E47 cells analogous to CrMP, whereas exposure to exogenous CO partially reduced AA toxicity and the enhanced AA toxicity by CrMP. Addition of exogenous CO to the cells inhibited CYP2E1 catalytic activity, as did overexpression of the rat HO-1 adenovirus. These results suggest that induction of HO-1 protects against CYP2E1-dependent toxicity and this protection may be mediated in part via production of CO and CO inhibition of CYP2E1 activity.
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http://dx.doi.org/10.1016/j.freeradbiomed.2003.10.017DOI Listing
February 2004

The liver-selective nitric oxide donor O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) protects HepG2 cells against cytochrome P450 2E1-dependent toxicity.

Mol Pharmacol 2004 Jan;65(1):130-8

Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, NY 10029.

HepG2 cells expressing CYP2E1 (E47 cells) are more susceptible to toxicity by arachidonic acid (AA) or after glutathione depletion with an inhibitor of glutamate-cysteine ligase, l-buthionine-(S,R)-sulfoximine (BSO), compared with control HepG2 cells (C34 cells). The ability of nitric oxide (NO) to protect against CYP2E1-dependent toxicity has not been evaluated. We therefore studied the ability of O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), a liver-selective NO donor, to protect against CYP2E1-dependent toxicity and compared this with protection by chemical NO donors. E47 cells incubated with V-PYRRO/NO produced NO, whereas C34 cells did not. Incubation of E47 cells with 50 microM AA or 100 microM BSO for 2 days resulted in a 50% loss of cell viability. VPYRRO/NO (1 mM) blocked this toxicity of AA and BSO by a mechanism involving NO release via CYP2E1 metabolism of VPYRRO/NO. NO scavengers hemoglobin and 2-(4-carboxophenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide blocked the protective effects of V-PYRRO/NO. V-PYRRO/NO inhibited CYP2E1 activity and production of reactive oxygen species, whereas hemoglobin prevented these events. AA and BSO induced lipid peroxidation and decreased mitochondrial membrane potential; both of these effects were blocked by V-PYRRO/NO. Unlike V-PYRRO/NO, the chemical donors spermine/NO and (S)-nitroso-N-acetylpenicillamine release NO directly when added to the medium; however, they could partially protect against the CYP2E1-dependent toxicity. These results suggest that VPYRRO/NO protects HepG2 cells against CYP2E1-dependent toxicity through inhibition of CYP2E1-derived reactive oxygen species production and lipid peroxidation by the generated NO and that this compound may be valuable in protecting against CYP2E1-dependent toxicity via liver P450-specific generation of NO.
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http://dx.doi.org/10.1124/mol.65.1.130DOI Listing
January 2004

Increased expression of cytochrome P450 2E1 induces heme oxygenase-1 through ERK MAPK pathway.

J Biol Chem 2003 Aug 30;278(32):29693-700. Epub 2003 May 30.

Department of Pharmacology and Biological Chemistry, Mount Sinai School of Medicine, New York, New York 10029, USA.

The inducible form of heme oxygenase (HO-1) is increased during oxidative injury, and this may be an important defense mechanism against such injury. Cytochrome P450 2E1 (CYP2E1) generates reactive oxygen species and promotes lipid peroxidation. In this study induction of HO-1 by CYP2E1 and the possible role of mitogen-activated protein kinase (MAPK) in this process were evaluated. HO-1 induction was observed in the livers of chronic alcohol-fed mice or pyrazole-treated rats, conditions known to elevate CYP2E1 levels. Increased levels of HO-1 were observed in HepG2 cells overexpressing CYP2E1 (E47 cells) compared with control HepG2 cells or HepG2 cells expressing CYP3A4. Expression of CYP2E1 in HepG2 cells transcriptionally activated the HO-1 gene, increasing HO-1 mRNA and protein expression and activity of a HO-1 reporter construct. CYP2E1 inhibitors and catalase blocked the increased production of reactive oxygen species as well as HO-1 induction. Increasing oxidative stress by the addition of arachidonic acid or depletion of glutathione further increased HO-1 induction. The phosphorylated form of ERK MAPK but not that of p38 or JNK MAPK was increased in E47 cells compared with the control C34 HepG2 cells. PD98059, a specific inhibitor of ERK MAPK, blocked the activity of a HO-1 reporter in E47 cells but not in C34 cells. These results suggest that increased CYP2E1 activity leads to induction of the HO-1 gene, and the ERK MAPK pathway is important in mediating this process. This induction may serve as an adaptive mechanism to protect the E47 cells against the CYP2E1-dependent oxidative stress.
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http://dx.doi.org/10.1074/jbc.M304728200DOI Listing
August 2003

Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element.

Biochem J 2003 May;371(Pt 3):887-95

Vascular Biology Unit, Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow, Middlesex HA1 3UJ, UK.

The transcription factor Nrf2, which normally exists in an inactive state as a consequence of binding to a cytoskeleton-associated protein Keap1, can be activated by redox-dependent stimuli. Alteration of the Nrf2-Keap1 interaction enables Nrf2 to translocate to the nucleus, bind to the antioxidant-responsive element (ARE) and initiate the transcription of genes coding for detoxifying enzymes and cytoprotective proteins. This response is also triggered by a class of electrophilic compounds including polyphenols and plant-derived constituents. Recently, the natural antioxidants curcumin and caffeic acid phenethyl ester (CAPE) have been identified as potent inducers of haem oxygenase-1 (HO-1), a redox-sensitive inducible protein that provides protection against various forms of stress. Here, we show that in renal epithelial cells both curcumin and CAPE stimulate the expression of Nrf2 in a concentration- and time-dependent manner. This effect was associated with a significant increase in HO-1 protein expression and haem oxygenase activity. From several lines of investigation we also report that curcumin (and, by inference, CAPE) stimulates ho-1 gene activity by promoting inactivation of the Nrf2-Keap1 complex, leading to increased Nrf2 binding to the resident ho-1 AREs. Moreover, using antibodies and specific inhibitors of the mitogen-activated protein kinase (MAPK) pathways, we provide data implicating p38 MAPK in curcumin-mediated ho-1 induction. Taken together, these results demonstrate that induction of HO-1 by curcumin and CAPE requires the activation of the Nrf2/ARE pathway.
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http://dx.doi.org/10.1042/BJ20021619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1223348PMC
May 2003

Heme activates the heme oxygenase-1 gene in renal epithelial cells by stabilizing Nrf2.

Am J Physiol Renal Physiol 2003 Apr 26;284(4):F743-52. Epub 2002 Nov 26.

Department of Molecular Genetics, Ochsner Clinic Foundation, New Orleans 70121, USA.

The mechanism of heme oxygenase-1 gene (ho-1) activation by heme in immortalized rat proximal tubular epithelial cells was examined. Analysis of the ho-1 promoter identified the heme-responsive sequences as the stress-response element (StRE), multiple copies of which are present in two enhancer regions, E1 and E2. Electrophoretic mobility shift assays identified Nrf2, MafG, ATF3, and Jun and Fos family members as StRE-binding proteins; binding of Nrf2, MafG, and ATF3 was increased in response to heme. Dominant-negative mutants of Nrf2 and Maf, but not of c-Fos and c-Jun, inhibited basal and heme-induced expression of an E1-controlled luciferase gene. Heme did not affect the transcription activity of Nrf2, dimerization between Nrf2 and MafG, or the level of MafG, but did stimulate expression of Nrf2. Heme did not influence the level of Nrf2 mRNA but increased the half-life of Nrf2 protein from approximately 10 min to nearly 110 min. These results indicate that heme promotes stabilization of Nrf2, leading to accumulation of Nrf2. MafG dimers that bind to StREs to activate the ho-1 gene.
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http://dx.doi.org/10.1152/ajprenal.00376.2002DOI Listing
April 2003

Multiple basic-leucine zipper proteins regulate induction of the mouse heme oxygenase-1 gene by arsenite.

Arch Biochem Biophys 2002 Sep;405(2):265-74

Department of Molecular Genetics, Ochsner Clinic Foundation, 1516 Jefferson Highway, New Orleans, LA 70121, USA.

The mechanism of heme oxygenase-1 (ho-1) gene activation by arsenite was examined. Arsenite-stimulated expression of a ho-1 promoter/luciferase chimera in a dose-dependent manner in mouse hepatoma (Hepa) cells. Mutation analyses identified the arsenite-responsive sequence as the stress-response element (StRE), which resembles the binding sites for the AP-1 superfamily of basic-leucine zipper factors. In electrophoretic mobility shift assays, up to seven specific StRE-protein complexes were routinely detected using extracts from untreated Hepa cells whereas a single complex was typically observed after treatment with arsenite. Antibody "supershift" experiments identified Nrf2, JunD, and ATF3 in control complexes and the amount of these factors increased significantly in the arsenite-induced complex. MafG, ATF2, FosB, and JunB were also detected in the arsenite complex. Activation of a StRE-dependent luciferase gene by arsenite was inhibited to varying degrees by dominant-negative mutants of Nrf2, MafK, c-Fos, and CREB but most strongly with the latter. Together, these results implicate multiple basic-leucine zipper transcription factors in ho-1 gene activation by arsenite.
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http://dx.doi.org/10.1016/s0003-9861(02)00404-6DOI Listing
September 2002

Activation of the mouse heme oxygenase-1 gene by 15-deoxy-Delta(12,14)-prostaglandin J(2) is mediated by the stress response elements and transcription factor Nrf2.

Antioxid Redox Signal 2002 Apr;4(2):249-57

Department of Molecular Genetics, Alton Ochsner Medical Foundation, New Orleans, LA 70121, USA.

The mechanism of heme oxygenase-1 (ho-1) gene activation by 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) was examined. 15d-PGJ(2) stimulated expression of HO-1 mRNA and protein and of a mouse ho-1 gene promoter/luciferase fusion construct (HO15luc) in a dose-dependent manner in mouse hepatoma (Hepa) cells. HO15luc expression was not effected by troglitazone, a peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligand, but induction by 15d-PGJ(2) was abrogated by the antioxidant N-acetylcysteine. The primary 15d-PGJ(2) responsive sequences were localized to a 5' distal enhancer (E1) and identified as the stress-response element, previously shown to mediate ho-1 activation by several agents, including heme and heavy metals. Treatment of Hepa cells with 15d-PGJ(2) stimulated stress-response element-binding activity as judged by electrophoretic mobility shift assays. Antibody "supershift" experiments identified NF-E2 related factor 2 (Nrf2), but not Fos, Jun, or activating transcription factor/cyclic AMP response element binding protein transcription factors, within the 15d-PGJ(2)-induced complexes. Similarly, a dominant-negative mutant of Nrf2, but not of c-Jun or c-Fos, abrogated 15d-PGJ(2)-stimulated E1 transcription activity. Finally, prior induction of HO-1 in RAW264.7 mouse macrophages by 15d-PGJ(2) attenuated cell death caused by diesel exhaust particle extracts. These results demonstrate that induction of mouse HO-1 expression by 15d-PGJ(2) is independent of PPAR-gamma but dependent on oxidative stress, is regulated by the oxidative stress-activated transcription factor Nrf2, and provides cytoprotective activity.
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http://dx.doi.org/10.1089/152308602753666307DOI Listing
April 2002