Progressive Endoplasmic Reticulum Stress Contributes to Hepatocarcinogenesis in Fatty Acyl-CoA Oxidase 1–Deficient Mice
2011; Elsevier BV; Volume: 179; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2011.04.030
ISSN1525-2191
AutoresJiansheng Huang, Navin Viswakarma, Songtao Yu, Yuzhi Jia, Liang Bai, Aurore Vluggens, Mustapha Cherkaoui‐Malki, Mushfiquddin Khan, Inderjit Singh, Gongshe Yang, Mahendra S. Rao, Jayme Borensztajn, Janardan K. Reddy,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoFatty acyl-coenzyme A oxidase 1 (ACOX1) knockout (ACOX1−/−) mice manifest hepatic metabolic derangements that lead to the development of steatohepatitis, hepatocellular regeneration, spontaneous peroxisome proliferation, and hepatocellular carcinomas. Deficiency of ACOX1 results in unmetabolized substrates of this enzyme that function as biological ligands for peroxisome proliferator-activated receptor–α (PPARα) in liver. Here we demonstrate that sustained activation of PPARα in ACOX1−/− mouse liver by these ACOX1 substrates results in endoplasmic reticulum (ER) stress. Overexpression of transcriptional regulator p8 and its ER stress–related effectors such as the pseudokinase tribbles homolog 3, activating transcription factor 4, and transcription factor CCAAT/-enhancer-binding protein homologous protein as well as phosphorylation of eukaryotic translation initiation factor 2α, indicate the induction of unfolded protein response signaling in the ACOX1−/− mouse liver. We also show here that, in the liver, p8 is a target for all three PPAR isoforms (-α, -β, and -γ), which interact with peroxisome proliferator response elements in p8 promoter. Sustained activation of p8 and unfolded protein response–associated ER stress in ACOX1−/− mouse liver contributes to hepatocyte apoptosis and liver cell proliferation culminating in the development of hepatocarcinogenesis. We also demonstrate that human ACOX1 transgene is functional in ACOX1−/− mice and effectively prevents metabolic dysfunctions that lead to ER stress and carcinogenic effects. Taken together, our data indicate that progressive PPARα- and p8-mediated ER stress contribute to the hepatocarcinogenesis in ACOX1−/− mice. Fatty acyl-coenzyme A oxidase 1 (ACOX1) knockout (ACOX1−/−) mice manifest hepatic metabolic derangements that lead to the development of steatohepatitis, hepatocellular regeneration, spontaneous peroxisome proliferation, and hepatocellular carcinomas. Deficiency of ACOX1 results in unmetabolized substrates of this enzyme that function as biological ligands for peroxisome proliferator-activated receptor–α (PPARα) in liver. Here we demonstrate that sustained activation of PPARα in ACOX1−/− mouse liver by these ACOX1 substrates results in endoplasmic reticulum (ER) stress. Overexpression of transcriptional regulator p8 and its ER stress–related effectors such as the pseudokinase tribbles homolog 3, activating transcription factor 4, and transcription factor CCAAT/-enhancer-binding protein homologous protein as well as phosphorylation of eukaryotic translation initiation factor 2α, indicate the induction of unfolded protein response signaling in the ACOX1−/− mouse liver. We also show here that, in the liver, p8 is a target for all three PPAR isoforms (-α, -β, and -γ), which interact with peroxisome proliferator response elements in p8 promoter. Sustained activation of p8 and unfolded protein response–associated ER stress in ACOX1−/− mouse liver contributes to hepatocyte apoptosis and liver cell proliferation culminating in the development of hepatocarcinogenesis. We also demonstrate that human ACOX1 transgene is functional in ACOX1−/− mice and effectively prevents metabolic dysfunctions that lead to ER stress and carcinogenic effects. Taken together, our data indicate that progressive PPARα- and p8-mediated ER stress contribute to the hepatocarcinogenesis in ACOX1−/− mice. Fatty liver disease is a burgeoning chronic liver disorder, commencing with hepatic steatosis and steatohepatitis, that has a propensity to progress toward cirrhosis and liver cancer.1Grundy S.M. Obesity, metabolic syndrome, and cardiovascular disease.J Clin Endocrinol Metab. 2004; 89: 2595-2600Crossref PubMed Scopus (948) Google Scholar, 2Angulo P. Lindor K.D. 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Thus, sustained PPARα activation and its induction of p8 expression in the liver by the unmetabolized substrates of ACOX1 in mice nullizygous for ACOX1, contributes to ER stress and may contribute to the development of hepatocellular carcinoma. The data presented here also indicate that transgenic expression of human ACOX1 in ACOX1−/− mice prevents the development of metabolic dysfunctions including steatohepatitis spontaneous activation of PPARα, steatohepatitis, liver cancer, and unfolded protein response signaling. These observations indicate that human ACOX1 gene functionally replaces mouse ACOX1 gene and that this enzyme keeps the PPARα endogenous ligands in check and prevents sustained activation of this transcription factor. The human bacterial artificial chromosome (BAC) clone CTD2336I6, containing the human ACOX1 genomic DNA and its promoter, was purchased from Invitrogen Corporation (Carlsbad, CA). This BAC DNA was trimmed to ∼90 kb using homologous recombination in bacteria to excise flanking genes and then replaced with an ampicilin resistance gene using primers shown in Table 1. BAC transgenic mice were generated by zygotic pronuclear microinjection in the Transgenic and Targeted Mutagenesis Laboratory, Northwestern University (Chicago, IL). Human BAC transgenic founders were then backcrossed with ACOX1+/− mice8Fan C.Y. Pan J. Chu R. Lee D. Kluckman K.D. Usuda N. Singh I. Yeldandi A.V. Rao M.S. Maeda N. Reddy J.K. Hepatocellular and hepatic peroxisomal alterations in mice with a disrupted peroxisomal fatty acyl-coenzyme A oxidase gene.J Biol Chem. 1996; 271: 24698-24710Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 10Fan C.Y. Pan J. Usuda N. Yeldandi A.V. Rao M.S. Reddy J.K. Steatohepatitis, spontaneous peroxisome proliferation and liver tumors in mice lacking peroxisomal fatty acyl-CoA oxidase Implications for peroxisome proliferator-activated receptor alpha natural ligand metabolism.J Biol Chem. 1998; 273: 15639-15645Crossref PubMed Scopus (311) Google Scholar to generate ACOX1+/− mice containing human ACOX1 transgene (ACOX1+/−h+). The ACOX1+/−h+ mice were then mated with ACOX1+/− mice to generate ACOX1−/− humanized mice (ACOX1−/−h+), and ACOX1−/− and wild-type littermates. Mice used in this study were housed using 12-hour light, 12-hour dark cycle, in a pathogen-free animal facility and maintained on standard rodent chow (Harlan-Teklad, Indianapolis, IN) and water ad libitum. The PPARα synthetic ligand Wy-14,643 [4-chloro-6-(2,3-xylidino)−2-pyrimidinylthio]acetic acid] (0.125% w/w) was administered in chow.10Fan C.Y. Pan J. Usuda N. Yeldandi A.V. Rao M.S. Reddy J.K. 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Functional significance of the two ACOX1 isoforms and their crosstalks with PPARα and RXRα.Lab Invest. 2010; 90: 696-708Crossref PubMed Scopus (63) Google Scholar All animal procedures used in this study were reviewed and preapproved by the Northwestern University Institutional Animal Care and Use Committee.Table 1Oligonucleotide Sequences of Trimming BAC Clone, Genotyping, Real-Time PCR Primers, and Northern ProbesTrimming BAC clone5′-GGGTGCAAATTGCCCGGTGCCTTCTGTTTCCCAGGCAGCTCTGTG-3′5′-CAGCTTACCTCTCAGGAATGCTACGTTTTGAACATCAAGAATGGAAA-3′ACOX-amp Sense5′-GCCATGGATATGTTCCAGAAGGTAGCTTGGTCTGACAGTTACCAATGC-3′ Antisense5′-GCTGCCATTGAGGCTTTTAACAAAGGTGGCACTTTTC-3′Genotyping Neo Sense5′-TATTCGGCTATGACTGGGCACA-3′ Antisense5′-GATGGATACTTTCTCGGCAGGA-3′ mACOX1 exon Sense5′-CCGCAAGCCATCCGACATTC-3′ Antisense5′-ATTCAGTGGGTCAGGCGACTGC-3′ hACOX1BAC Sense5′-ATTGCCCGGTGCCTTCTGTTTC-3′ Antisense5′-AGCCGGTGAGCGTGGGTCTC-3′qPCR/Probe hACOX1 Forward5′-TCTGTCTGGGCCGCTGTCACTC-3′ Reverse5′-CCTAGGAGGCAGCCTCAGGACG-3′ mACOX1 Forward5′-GCCAAGGCGACCTGAGTGAGC-3′ Reverse5′-ACCGCAAGCCATCCGACATTC-3′ ATF6 Forward5′-CAGTTGCTCCATCTCCTCTCC-3′ Reverse5′-TGGGACACTGGCATTGGTTTG-3′ XBP1 Forward5′-CCTGAGCCCGGAGGAGAA-3′ Reverse5′-CTCGAGCAGTCTGCGCTG-3′ XBP1s Forward5′-ACACGCTTGGGAATGGACAC-3′ Reverse5′-CCATGGGAAGATGTTCTGGG-3′ ATF4 Forward5′-ACTATCTGGAGGTGGCCAAG-3′ Reverse5′-CATCCAACGTGGTCAAGAGC-3′ GRP78 Forward5′-CGTGGAGATCATAGCCAACG-3′ Reverse5′-ATACGCCTCAGCAGTCTCCT-3′ Trb3 Forward5′-CCCACAGGCACAGAGTACAC-3′ Reverse5′-CGTCCTCTCACAGTTGCTGA-3′ CHOP Forward5′-AGCCTGGTATGAGGATCTGC-3′ Reverse5′-CTCCTGCTCCTTCTCCTTCA-3′ Gadd45α Forward5′-CCAAGCTGCTCAACGTAGA-3′ Reverse5′-CCACTGATCCATGTAGCGAC-3′ P8 Forward5′-ACCAAGAGAGAAGCTGCTGC-3′ Reverse5′-CTCCCTCTCCAGAACCTCACT-3′ PPARγ Forward5′-CCACAGTTGATTTCTCCAGCATTTC-3′ Reverse5′-CAGGTTCTACTTTGATCGCACTTTG-3′ aP2 Forward5′-GAAGTGGGAGTGGGCTTTGC-3′ Reverse5′-TGTGGTCGACTTTCCATCCC-3′ Bax Forward5′-ACCAAGAAGCTGAGCGAGTG-3′ Reverse5′-CTCACGGAGGAAGTCCAGTG-3′ Bcl2 Forward5′-TCTTCTCCTTCCAGCCTGAG-3′ Reverse5′-CCCACCGAACTCAAAGAAGG-3′ 18S Forward5′-AAACGGCTACCACATCCAAG-3′ Reverse5′-CCTCCAATGGATCCTCGTTA-3′BAC = bacterial artificial chromosome. Open table in a new tab BAC = bacterial artificial chromosome. Livers were fixed in 4% paraformaldehyde and embedded in paraffin. Sections 5 μm in thickness were stained with hematoxylin and eosin (H&E) or Sirius Red, or processed for immunohistochemical localization of BrdUrd.31Jia Y. Qi C. Kashireddi P. Surapureddi S. Zhu Y.J. Rao M.S. Le Roith D. Chambon P. Gonzalez F.J. Reddy J.K. Transcription coactivator PBP, the peroxisome proliferator-activated receptor (PPAR)-binding protein, is required for PPARalpha-regulated gene expression in liver.J Biol Chem. 2004; 279: 24427-24434Crossref PubMed Scopus (99) Google Scholar, 32Qi C. Zhu Y. Pan J. Yeldandi A.V. Rao M.S. Maeda N. Subbarao V. Pulikuri S. Hashimoto T. Reddy J.K. 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Reddy J.K. Transcription coactivator PBP, the peroxisome proliferator-activated receptor (PPAR)-binding protein, is required for PPARalpha-regulated gene expression in liver.J Biol Chem. 2004; 279: 24427-24434Crossref PubMed Scopus (99) Google Scholar Sera were analyzed for very long chain fatty acids C26:0/C22:0 and C24:0/C22:0 as described previously.8Fan C.Y. Pan J. Chu R. Lee D. Kluckman K.D. Usuda N. Singh I. Yeldandi A.V. Rao M.S. Maeda N. Reddy J.K. Hepatocellular and hepatic peroxisomal alterations in mice with a disrupted peroxisomal fatty acyl-coenzyme A oxidase gene.J Biol Chem. 1996; 271: 24698-24710Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar For hepatic triglyceride and cholesterol levels, 100 mg of liver was homogenized for extraction of lipid by chloroform/methanol extraction assay. Total lipid was resuspended in 5% Triton X-100 in phosphate-buffered saline, and levels of triglyceride and total cholesterol were then quantified according to the manufacturer's procedures.34Huang J. Iqbal J. Saha P.K. Liu J. Chan L. Hussain M.M. Hussain M.M. Moore D.D. Wang L. Molecular characterization of the role of orphan receptor small heterodimer partner in development of fatty liver.Hepatology. 2007; 46: 147-157Crossref PubMed Scopus (120) Google Scholar Mouse primary hepatocytes from 1-month-old C57BL6/J mice were isolated by collagenase II perfusion and cultured as reported.33Vluggens A. Androletti P. Viswakarma N. Jia Y. Matsumoto K. Kulik W. Khan M. Huang J. Guo D. Yu S. Sarkar J. Singh I. Rao M.S. Wanders R.J. Reddy J.K. Che
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