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• W2052220384 abstract "Nur77, encoded by Nr4a1 (alias Nur77), plays roles in cell death, survival, and inflammation. To study the role of Nur77 in liver regeneration, wild-type (WT) and Nur77 knockout (KO) mice were subjected to standard two-thirds partial hepatectomy (PH). Nur77 mRNA and protein levels were markedly induced at 1 hour after PH in WT livers, coinciding with ERK1/2 activation. Surprisingly, Nur77 KO mice exhibited a higher liver-to-body weight ratio than WT mice at 24, 48, and 72 hours after PH. Nur77 KO livers exhibited increase in Ki-67–positive hepatocytes at 24 hours, with early induction of cell-cycle genes. Despite accelerated regeneration, Nur77 KO livers paradoxically incurred necrosis, hepatocyte apoptosis, elevated serum alanine aminotransferase activity, and Kupffer cell accumulation. Microarray analysis revealed up-regulation of genes modulating inflammation, cell proliferation, and apoptosis but down-regulation (due to Nur77 deficiency) of glucose and lipid homeostasis genes. Levels of proinflammatory cytokines IL-6, IL-12, IL-23, and CCL2 were increased and levels of anti-inflammatory IL-10 were decreased, compared with WT. Activated NF-κB and STAT3 and mRNA levels of target genes Myc and Bcl2l1 were elevated in Nur77 KO livers. Overall, Nur77 appears essential for regulating early signaling of liver regeneration by modulating cytokine-mediated inflammatory, apoptotic, and energy mobilization processes. The accelerated liver regeneration observed in Nur77 KO mice is likely due to a compensatory effect caused by injury. Nur77, encoded by Nr4a1 (alias Nur77), plays roles in cell death, survival, and inflammation. To study the role of Nur77 in liver regeneration, wild-type (WT) and Nur77 knockout (KO) mice were subjected to standard two-thirds partial hepatectomy (PH). Nur77 mRNA and protein levels were markedly induced at 1 hour after PH in WT livers, coinciding with ERK1/2 activation. Surprisingly, Nur77 KO mice exhibited a higher liver-to-body weight ratio than WT mice at 24, 48, and 72 hours after PH. Nur77 KO livers exhibited increase in Ki-67–positive hepatocytes at 24 hours, with early induction of cell-cycle genes. Despite accelerated regeneration, Nur77 KO livers paradoxically incurred necrosis, hepatocyte apoptosis, elevated serum alanine aminotransferase activity, and Kupffer cell accumulation. Microarray analysis revealed up-regulation of genes modulating inflammation, cell proliferation, and apoptosis but down-regulation (due to Nur77 deficiency) of glucose and lipid homeostasis genes. Levels of proinflammatory cytokines IL-6, IL-12, IL-23, and CCL2 were increased and levels of anti-inflammatory IL-10 were decreased, compared with WT. Activated NF-κB and STAT3 and mRNA levels of target genes Myc and Bcl2l1 were elevated in Nur77 KO livers. Overall, Nur77 appears essential for regulating early signaling of liver regeneration by modulating cytokine-mediated inflammatory, apoptotic, and energy mobilization processes. The accelerated liver regeneration observed in Nur77 KO mice is likely due to a compensatory effect caused by injury. Liver regeneration is a well-orchestrated and tightly regulated process that proceeds through distinct stages with priming of hepatocytes, cell-cycle progression, proliferation, and termination of regeneration.1Michalopoulos G.K. Liver regeneration.J Cell Physiol. 2007; 213: 286-300Crossref PubMed Scopus (1176) Google Scholar, 2Su A.I. Guidotti L.G. Pezacki J.P. Chisari F.V. Schultz P.G. Gene expression during the priming phase of liver regeneration after partial hepatectomy in mice.Proc Natl Acad Sci USA. 2002; 99: 11181-11186Crossref PubMed Scopus (170) Google Scholar Liver regeneration induced by partial hepatectomy (PH) involves multiple cell types interacting in coordination. Activated Kupffer cells (KCs) and hepatic stellate cells release a series of growth factors, including transforming growth factor β and hepatocyte growth factor, as well as proinflammatory cytokines such as IL-6 and tumor necrosis factor α (TNF-α) to drive the cell-cycle entry of quiescent hepatocytes.3Takeishi T. Hirano K. Kobayashi T. Hasegawa G. Hatakeyama K. Naito M. The role of Kupffer cells in liver regeneration.Arch Histol Cytol. 1999; 62: 413-422Crossref PubMed Scopus (76) Google Scholar, 4Xu C.S. Jiang Y. Zhang L.X. Chang C.F. Wang G.P. Shi R.J. Yang Y.J. The role of Kupffer cells in rat liver regeneration revealed by cell-specific microarray analysis.J Cell Biochem. 2012; 113: 229-237Crossref PubMed Scopus (23) Google Scholar In response to PH, KC-secreted TNF-α activates downstream target nuclear factor κB (NF-κB), which then up-regulates the transcription of Ccnd1.5Taub R. Greenbaum L.E. Peng Y. Transcriptional regulatory signals define cytokine-dependent and -independent pathways in liver regeneration.Semin Liver Dis. 1999; 19: 117-127Crossref PubMed Scopus (133) Google Scholar, 6Guttridge D.C. Albanese C. Reuther J.Y. Pestell R.G. Baldwin A.S. NF-kappa B controls cell growth and differentiation through transcriptional regulation of cyclin D1.Mol Cell Biol. 1999; 19: 5785-5799Crossref PubMed Google Scholar Meanwhile, IL-6–mediated activation of signal transducer and activator of transcription 3 (STAT3) also promotes hepatocyte proliferation after PH.1Michalopoulos G.K. Liver regeneration.J Cell Physiol. 2007; 213: 286-300Crossref PubMed Scopus (1176) Google Scholar, 7Li W. Liang X. Kellendonk C. Poli V. Taub R. STAT3 contributes to the mitogenic response of hepatocytes during liver regeneration.J Biol Chem. 2002; 277: 28411-28417Crossref PubMed Scopus (264) Google Scholar These findings suggest that the proinflammatory cytokines IL-6 and TNF-α and their downstream transcriptional regulators NF-κB and STAT3 are necessary for the priming and progression of liver regeneration.1Michalopoulos G.K. Liver regeneration.J Cell Physiol. 2007; 213: 286-300Crossref PubMed Scopus (1176) Google Scholar, 8Fausto N. Campbell J.S. Riehle K.J. Liver regeneration.Hepatology. 2006; 43: S45-S53Crossref PubMed Scopus (1283) Google Scholar, 9Scearce L.M. Laz T.M. Hazel T.G. Lau L.F. Taub R. RNR-1, a nuclear receptor in the NGFI-B/Nur77 family that is rapidly induced in regenerating liver.J Biol Chem. 1993; 268: 8855-8861Abstract Full Text PDF PubMed Google Scholar Furthermore, PH-induced liver regeneration is typically an injury-free process, with regeneration resulting from interactions between pro- and anti-inflammatory mediators to amplify the proliferative response elicited by growth factors.10DeAngelis R.A. Markiewski M.M. Lambris J.D. Liver regeneration: a link to inflammation through complement.Adv Exp Med Biol. 2006; 586: 17-34Crossref PubMed Scopus (23) Google Scholar, 11Markiewski M.M. DeAngelis R.A. Lambris J.D. Liver inflammation and regeneration: two distinct biological phenomena or parallel pathophysiologic processes?.Mol Immunol. 2006; 43: 45-56Crossref PubMed Scopus (88) Google Scholar, 12Taub R. Liver regeneration: from myth to mechanism.Nat Rev Mol Cell Biol. 2004; 5: 836-847Crossref PubMed Scopus (1282) Google Scholar Orphan nuclear receptor Nur77, a member of the nuclear receptor subfamily 4 (NR4A) encoded by Nr4a1 (alias Nur77), is an early immediate-response gene whose expression can be induced by diverse stimuli.13Martin L.J. Boucher N. El-Asmar B. Tremblay J.J. cAMP-induced expression of the orphan nuclear receptor Nur77 in MA-10 Leydig cells involves a CaMKI pathway.J Androl. 2009; 30: 134-145Crossref PubMed Scopus (43) Google Scholar, 14Moll U.M. Marchenko N. Zhang X.K. p53 and Nur77/TR3—transcription factors that directly target mitochondria for cell death induction.Oncogene. 2006; 25: 4725-4743Crossref PubMed Scopus (213) Google Scholar Together with Nurr1 (Nr4a2) and Nor1 (Nr4a3), Nur77 functions as a key transcriptional regulator of cell apoptosis, proliferation, inflammation, and energy metabolism.15Zhao Y. Bruemmer D. NR4A orphan nuclear receptors: transcriptional regulators of gene expression in metabolism and vascular biology.Arterioscler Thromb Vasc Biol. 2010; 30: 1535-1541Crossref PubMed Scopus (169) Google Scholar, 16Mohan H.M. Aherne C.M. Rogers A.C. Baird A.W. Winter D.C. Murphy E.P. Molecular pathways: the role of NR4A orphan nuclear receptors in cancer.Clin Cancer Res. 2012; 18: 3223-3228Crossref PubMed Scopus (133) Google Scholar, 17Pei L. Waki H. Vaitheesvaran B. Wilpitz D.C. Kurland I.J. Tontonoz P. NR4A orphan nuclear receptors are transcriptional regulators of hepatic glucose metabolism.Nat Med. 2006; 12: 1048-1055Crossref PubMed Scopus (260) Google Scholar NR4A receptors are highly conserved, with 97% homology in their DNA-binding domains, 60% to 65% homology in the C-terminal ligand-binding domains, and 20% to 30% homology in their N-terminal transactivation domains.18Maruyama K. Tsukada T. Ohkura N. Bandoh S. Hosono T. Yamaguchi K. The NGFI-B subfamily of the nuclear receptor superfamily (review).Int J Oncol. 1998; 12: 1237-1243PubMed Google Scholar Despite several common activities and high sequence similarity, each NR4A member exhibits divergent functions. Nurr1 is involved in the initiation and maintenance of midbrain dopamine neurons, and Nor1 regulates embryonic, inner ear, and hippocampus development.19Campos-Melo D. Galleguillos D. Sánchez N. Gysling K. Andrés M.E. Nur transcription factors in stress and addiction.Front Mol Neurosci. 2013; 6: 44Crossref PubMed Scopus (49) Google Scholar Distinct from its two subfamily members, Nur77 can modulate cell proliferation and apoptosis in lymphocytes, neurons, and tumor cells.20Li Y. Lin B. Agadir A. Liu R. Dawson M.I. Reed J.C. Fontana J.A. Bost F. Hobbs P.D. Zheng Y. Chen G.Q. Shroot B. Mercola D. Zhang X.K. Molecular determinants of AHPN (CD437)-induced growth arrest and apoptosis in human lung cancer cell lines.Mol Cell Biol. 1998; 18: 4719-4731Crossref PubMed Scopus (175) Google Scholar, 21Liu Z.G. Smith S.W. Mclaughlin K.A. Schwartz L.M. Osborne B.A. Apoptotic signals delivered through the T-cell receptor of a T-cell hybrid require the immediate–early gene Nur77.Nature. 1994; 367: 281-284Crossref PubMed Scopus (505) Google Scholar The opposing role of Nur77 in regulating cell survival and death is dependent on its intracellular location.14Moll U.M. Marchenko N. Zhang X.K. p53 and Nur77/TR3—transcription factors that directly target mitochondria for cell death induction.Oncogene. 2006; 25: 4725-4743Crossref PubMed Scopus (213) Google Scholar, 22Lin B. Kolluri S.K. Lin F. Liu W. Han Y.H. Cao X. Dawson M.I. Reed J.C. Zhang X.K. Conversion of Bcl-2 from protector to killer by interaction with nuclear orphan receptor Nur77/TR3.Cell. 2004; 116: 527-540Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar Growth factors such as epidermal growth factor rapidly induce the expression of Nur77 in the nucleus, where it serves as an oncogene to enhance cell survival and growth.23Kolluri S.K. Bruey-Sedano N. Cao X. Lin B. Lin F. Han Y.H. Dawson M.I. Zhang X.K. Mitogenic effect of orphan receptor TR3 and its regulation by MEKK1 in lung cancer cells.Mol Cell Biol. 2003; 23: 8651-8667Crossref PubMed Scopus (169) Google Scholar By contrast, apoptosis inducers increase cytosolic Nur77, which promotes cell death.24Yang H. Zhan Q. Wan Y.J. Enrichment of Nur77 mediated by retinoic acid receptor β leads to apoptosis of human hepatocellular carcinoma cells induced by fenretinide and histone deacetylase inhibitors.Hepatology. 2011; 53: 865-874Crossref PubMed Scopus (37) Google Scholar, 25Yang H. Nie Y. Li Y. Wan Y.J. ERK1/2 deactivation enhances cytoplasmic Nur77 expression level and improves the apoptotic effect of fenretinide in human liver cancer cells.Biochem Pharmacol. 2011; 81: 910-916Crossref PubMed Scopus (22) Google Scholar The role of Nor1 in the modulation of hepatocyte proliferation has been investigated recently. Nor1 promotes hepatocyte proliferation in mice through up-regulation of its target gene, Ccnd1, independently of TNF-α and IL-6–mediated inflammatory pathway.26Vacca M. Murzilli S. Salvatore L. Di Tullio G. D’Orazio A. Lo Sasso G. Graziano G. Pinzani M. Chieppa M. Mariani-Costantini R. Palasciano G. Moschetta A. Neuron-derived orphan receptor 1 promotes proliferation of quiescent hepatocytes.Gastroenterology. 2013; 144: 1518-1529 e1513Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar Despite the well-known role in regulating cell proliferation, the effect of Nur77 in liver regeneration has not been studied previously. As an immediate and transient growth factor–induced gene, Nur77 is also a key mediator of the inflammatory response in macrophages.27Bonta P.I. Matlung H.L. Vos M. Peters S.L.M. Pannekoek H. Bakker E.N.T.P. de Vries C.J.M. Nuclear receptor Nur77 inhibits vascular outward remodelling and reduces macrophage accumulation and matrix metalloproteinase levels.Cardiovasc Res. 2010; 87: 561-568Crossref PubMed Scopus (33) Google Scholar Moreover, Nur77 can also inhibit the expression of several proinflammatory genes by repressing the activity of NF-κB in vitro.28Harant H. Lindley I.J.D. Negative cross-talk between the human orphan nuclear receptor Nur77/NAK-1/TR3 and nuclear factor-kappaB.Nucleic Acids Res. 2004; 32: 5280-5290Crossref PubMed Scopus (53) Google Scholar In addition to the inflammatory response, Nur77 alters metabolism by modulating insulin sensitivity and glucose homeostasis via transcriptional regulation of genes involved in gluconeogenesis.17Pei L. Waki H. Vaitheesvaran B. Wilpitz D.C. Kurland I.J. Tontonoz P. NR4A orphan nuclear receptors are transcriptional regulators of hepatic glucose metabolism.Nat Med. 2006; 12: 1048-1055Crossref PubMed Scopus (260) Google Scholar, 29Chao C.Y. Cheing G.L. Microvascular dysfunction in diabetic foot disease and ulceration.Diabetes Metab Res Rev. 2009; 25: 604-614Crossref PubMed Scopus (138) Google Scholar Thus, Nur77 may be critical in modulating liver regeneration because of its regulatory effects on inflammatory signaling and energy metabolism. In the present study, the PH-induced mouse liver regeneration model was used to study the role of Nur77 in regulating liver regeneration. Our findings indicate that regenerating Nur77 knockout (KO) mouse livers exhibit transient inflammatory response and liver injury after PH that is accompanied by accelerated compensatory liver regeneration. Gene profiling revealed differentially expressed genes involved in cell proliferation, apoptosis, innate immune response, and energy metabolism between regenerating wild-type (WT) and Nur77 KO mouse livers. Our findings indicate that Nur77 is essential for controlling the early events of liver regeneration by modulating inflammatory, apoptotic, and metabolic processes. The compensatory early induction of cell-cycle genes may result from increased NF-κB and STAT3 signaling, contributing to complete restoration of liver mass in Nur77 KO mice. Taken together, these findings suggest that Nur77 is crucial for suppressing hepatic inflammation and preventing necrotic injury in PH-induced liver regeneration. C57BL/6 WT and Nur77 KO male mice30Lee S.L. Wesselschmidt R.L. Linette G.P. Kanagawa O. Russell J.H. Milbrandt J. Unimpaired thymic and peripheral T cell death in mice lacking the nuclear receptor NGFI-B (Nur77).Science. 1995; 269: 532-535Crossref PubMed Scopus (239) Google Scholar from the Jackson Laboratory (Bar Harbor, ME), aged 3 to 5 months, were housed in steel microisolator cages at 22°C with a 12 hours/12 hours light/dark cycle. Food and water were provided ad libitum. Standard two-thirds PH was performed as described previously,31Liu H.X. Fang Y.P. Hu Y. Gonzalez F.J. Fang J.W. Wan Y.J.Y. PPAR beta regulates liver regeneration by modulating Akt and E2f signaling.PLoS One. 2013; 8: e65644Crossref PubMed Scopus (30) Google Scholar and sham surgery was performed for control mice. Surgery was performed between 9:00 and 11:00 AM, and mice were sacrificed at specified time points (3 to 8 mice per time point). Liver and body weight were recorded at the time of death for calculating liver-to-body weight ratios. Serum and liver tissues were collected and kept at −20°C and −80°C, respectively, until assayed. A section of each liver was fixed in 10% formalin, embedded in paraffin, and stained for histological analysis. All animal experiments were conducted in accordance with the current edition of the Guide for the Care and Use of Laboratory Animals32National Research CouncilGuide for the Care and Use of Laboratory Animals.ed 8. National Academies Press, Washington, DC2011Google Scholar under protocols approved by the Animal Care and Use Committee of the University of California, Davis. Tissues were fixed in 10% formalin for 12 to 16 hours and washed in 70% ethanol for 24 hours. Tissues were then embedded in paraffin and cut into 5-μm sections. Standard hematoxylin and eosin staining was performed. To identify Nur77 intracellular localization, immunostaining was performed with anti-Nur77 antibody (Abcam, Cambridge, MA). To monitor hepatocyte proliferation, immunostaining was performed with anti–Ki-67 antibody (NeoMarkers, Fremont, CA). To assess KC activation, immunostaining with anti-F4/80 antibody (Abcam) was followed by labeling with biotinylated secondary antibodies (Jackson ImmunoResearch, West Grove, PA). The number of proliferating hepatocytes or activated KCs was determined by counting positive-staining cells in at least five random microscopic fields (×20) for each specimen. To monitor hepatocyte apoptosis, terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay was performed with an in situ cell death detection kit with tetramethylrhodamine (TMR red) (Roche Diagnostics, Mannheim, Germany; Indianapolis, IN), according to the manufacturer’s instructions. Nuclei were counterstained with DAPI (Life Technologies, Carlsbad, CA). The number of red fluorescent-labeled nuclei was counted under fluorescence microscopy in at least five random microscopic fields (×40) for each specimen. Serum was stored at −20°C and was used to assay alanine aminotransferase activities using a liquid alanine aminotransferase [ALT (SGPT)] reagent kit (Pointe Scientific, Brussels, Belgium). RNA was extracted using TRIzol reagent (Life Technologies). cDNA was synthesized using a high-capacity RNA-to-cDNA kit (Life Technologies). Real-time quantitative PCR with reverse transcription (RT-qPCR) was performed on an ABI 7900HT Fast real-time PCR system using Power SYBR Green PCR master mix (Life Technologies). Primers were designed using Primer3 Input software version 0.4.0; sequences are available in Table 1. For quantification, glyceraldehyde-3-phosphate dehydrogenase (Gapdh) mRNA level served as an internal control. Data generated from WT mice at time 0 were used to establish a baseline to calculate the relative expression levels between groups. The relative mRNA level at each time point was calculated by the 2−ΔΔCT method,33Schmittgen T.D. Livak K.J. Analyzing real-time PCR data by the comparative CT method.Nat Protoc. 2008; 3: 1101-1108Crossref PubMed Scopus (17798) Google Scholar where ΔΔCT = (CT,target − CT,Gapdh)Time t − (CT,target − CT,Gapdh)Time 0.Table 1Primers used for RT-qPCRTarget geneSequenceNur77F: 5′-TGCCTTCCTGGAACTCTTCATC-3′R: 5′-AGTACCAGGCCTGAGCAGAAGAT-3′Nurr1F: 5′-CCTCCAACTTGCAGAATATGAACA-3′R: 5′-CCGTGTCTCTCTGTGACCATAGC-3′Nor1F: 5′-AAGTGTCTCAGTGTCGGGATGGTT-3′R: 5′-TCCTGTTGTAGTGGGCTCTTTGGT-3′Ccnb1F: 5′-CCCCAAGTCTCACTATCA-3′R: 5′-CGAGGGAATGACTATGTT-3′Cdk1F: 5′-TCCGGTTGACATCTGGAGTA-3′R: 5′-TCCACTTGGGAAAGGTGTTC-3′Ccnd1F: 5′-CGTGGCCTCTAAGATGAAGGA-3′R: 5′-TCGGGCCGGATAGAGTTGT-3′Cdk4F: 5′-GCCAGAGATGGAGGAAGTCTG-3′R: 5′-TTGTGCAGGTAGGAGTGCTG-3′Ccne1F: 5′-GCAGCGAGCAGGAGACAGA-3′R: 5′-TGCTTCCACACCACTGTCTTTG-3′Cdk2F: 5′-TCCCCTCATCAAGAGCTATCTGTT-3′R: 5′-TCTGCATTGATAAGCAGGTTCTG-3′Casp8F: 5′-TGGAACCTGGTATATTCAGTCACTTT-3′R: 5′-CCAGTCAGGATGCTAAGAATGTCA-3′Cd14F: 5′-GGTCGAACAAGCCCGTGGAACC-3′R: 5′-AGCACACGCTCCATGGTCGG-3′Ccl2F: 5′-TGATCCCAATGAGTAGGCTGGAGA-3′R: 5′-ACCTCTCTCTTGAGCTTGGTGACA-3′Il6F: 5′-GTTGCCTTCTTGGGACTGATG-3′R: 5′-GGGAGTGGTATCCTCTGTGAAGTCT-3′Il12F: 5′-GGTGCAAAGAAACATGGACTTG-3′R: 5′-GGTGCAAAGAAACATGGACTTG-3′Il23F: 5′-AGTGTGAAGATGGTTGTGACCCAC-3′R: 5′-GAAGATGTCAGAGTCAAGCAGGTG-3′Il10F: 5′-GGAGCAGGTGAAGAGTGATTTTAATA-3′R: 5′-TGCAGTTGATGAAGATGTCAAATTC-3′Mmp9F: 5′-CGACGTGGGCTACGTGACCTAC-3′R: 5′-AGCACCTTTCCCTCGGATGGG-3′Mmp2F: 5′-ACACTGGGACCTGTCACTCC-3′R: 5′-TGTCACTGTCCGCCAAATAA-3′Map3k14F: 5′-AGAAGACCGAGCCCTTTACTACCT-3′R: 5′-ACAGGAGCACGTTGTCAGCTTTGA-3′IkkiF: 5′-CCCAGGCCGTTTTGCAT-3′R: 5′-GTCACGTTGGTCTGCTCATATACAG-3′MycF: 5′-AGTAATTCCAGCGAGAGGCA-3′R: 5′-AGCAGCTCGAATTTCTTCCA-3′Bcl2l1F: 5′-TCATCCTCTTATGCTTCCGGGCAT-3′R: 5′-ACTCCCTCTCCTAGAACCAGTCTT-3′F, forward; R, reverse. Open table in a new tab F, forward; R, reverse. Total RNA was isolated from WT and Nur77 KO mouse livers at 3 hours after PH. RNA quantity and quality were assessed with a Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA). Microarray and data analysis were performed as described previously.34Guo M. Gong L. He L. Lehman-McKeeman L. Wan Y.J. Hepatocyte RXRalpha deficiency in matured and aged mice: impact on the expression of cancer-related hepatic genes in a gender-specific manner.BMC Genomics. 2008; 9: 403Crossref PubMed Scopus (6) Google Scholar The GeneChip Mouse Genome 430 2.0 array (Affymetrix, Santa Clara, CA) was used. All biological function and pathway analyses were generated by the Functional Annotation tool in the Database for Annotation, Visualization and Integrated Discovery (DAVID version 6.7) (http://david.abcc.ncifcrf.gov). Functional pathways and processes with P < 0.05 and Bonferroni value <0.1 were accepted. Protein lysates obtained from liver homogenate (40 μg) were subjected to polyacrylamide gel electrophoresis under reducing conditions. Separated proteins from gels were transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk and incubated with specific primary antibody against Nur77, cyclin E, CDK2, β-actin (Santa Cruz Biotechnology, Santa Cruz, CA), ERK1/2, phosphorylated (p-) ERK1/2, STAT3, p-STAT3 (Tyr705), and p-NF-κB (Cell Signaling Technology, Danvers, MA). Membranes were then incubated with horseradish peroxidase–conjugated secondary antibodies. The signals were detected using an ECL enhanced chemiluminescence system with Pierce SuperSignal West Pico chemiluminescent substrates (Thermo Fisher Scientific, Waltham, MA). The differences between the two groups were analyzed with the Student’s t-test. P < 0.05 was considered statistically significant. Data are expressed as means ± SD. To study the role of Nur77 in hepatocyte proliferation, WT and Nur77 KO mice received two-thirds PH and were sacrificed at 1, 3, 8, 24, and 48 hours after surgery to determine Nur77 hepatic expression. In WT mice, Nur77 mRNA level was induced by 80-fold within 1 hour after PH and then dropped to basal level by 8 hours (Figure 1A). In accord, Nur77 protein level also showed a substantial induction at the same time point (Figure 1B). Additionally, Nur77 induction coincided with increased ERK1/2 phosphorylation, which peaked at 1 hour after PH. Because ERK1/2 activation is essential for the transcriptional effect of Nur77, PH-induced Nur77 could be a consequence of ERK1/2 activation.25Yang H. Nie Y. Li Y. Wan Y.J. ERK1/2 deactivation enhances cytoplasmic Nur77 expression level and improves the apoptotic effect of fenretinide in human liver cancer cells.Biochem Pharmacol. 2011; 81: 910-916Crossref PubMed Scopus (22) Google Scholar, 35Kovalovsky D. Refojo D. Liberman A.C. Hochbaum D. Pereda M.P. Coso O.A. Stalla G.K. Holsboer F. Arzt E. Activation and induction of NUR77/NURR1 in corticotrophs by CRH/cAMP: involvement of calcium, protein kinase A, and MAPK pathways.Mol Endocrinol. 2002; 16: 1638-1651Crossref PubMed Scopus (196) Google Scholar Immunohistochemistry staining revealed PH-induced Nur77 predominantly in the nuclei of hepatocytes, but not in KCs, stellate cells, or other nonparenchymal cells (Figure 1C). Localization of Nur77 in the hepatocyte nuclei suggests a role in gene regulation and cell proliferation. Nurr1 and Nor1 also showed marked induction at the mRNA level within 3 hours after PH in WT mouse livers. The induction of Nurr1 and Nor1 mRNA levels in Nur77 KO mouse livers was approximately twofold greater than that observed in WT controls at 3 hours after PH (Figure 1D). Overall, hepatic Nur77 exhibited a strong, transient induction during liver regeneration after PH, coupled with ERK1/2 activation to promote liver proliferation. These findings further support the notion that induced nuclear Nur77 in hepatocytes may be required to promote hepatocyte proliferation in response to PH. Liver-to-body weight ratios were significantly increased in regenerating Nur77 KO livers, compared with WT, at 24, 48, and 72 hours after PH (Figure 2A). Liver-to-body weight ratio did not statistically differ between WT and KO mice subjected to sham operation at the time points studied (data not shown). The increase in liver-to-body weight ratio in Nur77 KO livers correlated with increased cell proliferation, as demonstrated by significantly higher numbers of Ki-67–positive hepatocytes at 24 hours after PH (Figure 2, B and C). Although the number of Ki-67–positive hepatocytes did not statistically differ between WT and Nur77 KO livers at 48 and 72 hours, a higher trend toward increased Ki-67 expression was noted in KO mouse livers. Furthermore, examination under high magnification (×400) revealed that hepatocytes were the major population of proliferating cells within 48 hours after PH. Thus, loss of Nur77 accelerated PH-induced hepatocyte proliferation. To gain further insight into the effect of Nur77 on hepatocyte cell-cycle progression, we studied the expression of cyclins and cyclin-dependent kinases (CDKs) (Figure 3). These cell-cycle genes were markedly up-regulated in Nur77 KO mouse livers within 12 hours after PH, compared with WT counterparts (Figure 3A). However, Ccne1, Cdk1, Cdk2, and Cdk4 mRNA levels in Nur77 KO livers had returned to WT levels by 24 hours, and only Cdk4 levels differed again at 48 hours. The cyclin E protein level was slightly higher in Nur77 KO than in WT mouse livers at baseline (0 hour). Moreover, the PH-induced cyclin E and CDK2 levels were much higher in Nur77 KO than in WT mice overall (Figure 3B). Thus, the lack of Nur77 resulted in early up-regulation of a panel of cell-cycle genes in regenerating livers, which may account for enhanced hepatocyte proliferation. Despite accelerated liver regeneration, Nur77 KO mice paradoxically incurred liver damage after PH. An aberrant regenerative response accompanied by mononucleocyte infiltration and necrosis was observed in Nur77 KO mouse livers, compared with WT controls, as demonstrated by hematoxylin and eosin staining (Figure 4A). Strikingly, 9/49 Nur77 KO mice (18%) exhibited hepatic necrosis within 21 days after PH, but no necrotic injury occurred in WT mice (Table 2). Concurrently, Nur77 KO mice also had elevated serum alanine aminotransferase activity, compared with WT mice (Figure 4B). TUNEL staining revealed that Nur77 KO mouse livers exhibited more TUNEL-positive cells, compared with WT livers, at 3 and 48 hours after PH (Figure 4C). Consistent with TUNEL assay findings, both mRNA levels of Casp8 and protein levels of cleaved caspase 8 and cleaved caspase 3 were induced in Nur77 KO livers at 1 to 24 hours after PH, compared with WT counterparts (Figure 4, D and E). Overall, accelerated liver regeneration in Nur77 KO mouse livers after PH coincided with focal necrosis and hepatocyte apoptosis.Table 2The Incidence of Liver Necrosis in Regenerating WT and Nur77 KO Mouse LiversTime since PHNecrotic injury [n/N (%)]WTNur77 KO<24 hours0/255/2136 hours0/50/548 hours0/83/872 hours to 21 days0/131/15Total0/51 (0)9/49 (18) Open table in a new tab To test the hypothesis that Nur77 deficiency results in dysregulation of KCs and subsequent liver injury, F4/80 immunostaining was performed to assess macrophage activation in regenerating WT and Nur77 KO mouse livers. There were more F4/80-positive cells in Nur77 KO livers than in WT counterparts at 1, 3, and 48 hours after PH (Figure 5A). In accord, the mRNA levels of Cd14, chemokine (C-C Motif) ligand 2 (Ccl2), and proinflammatory interleukin genes Il6, Il12, and Il23 were elevated in Nur77 KO regenerating livers, compared with WT livers, at several time points (Figure 5B). Moreover, regenerating Nur77 KO livers also exhibited higher mRNA levels of matrix metallopeptidase genes Mmp2 and Mmp9 at 48 hours after PH, compared with WT livers. By degrading the extracellular matrix, MMPs facilitate KC infiltration and plasminogen activator activity, events necessary for initiating liver regeneration.36Mohammed F.F. Pennington C.J. Kassiri Z. Rubin J.S. Soloway P.D. Ruther U. Edwards D.R. Khokha R. Metalloproteinase inhibitor TIMP-1 affects hepatocyte cell cycle via HGF activation in murine liver regeneration.Hepatology. 2005; 41: 857-867Crossref PubMed Scopus (124) Google Scholar The elevated expression of these immune-response genes suggests greater KC activation in Nur77 KO regenerating livers, compared with WT. Moreover, the mRNA level of Il10 (encoding the anti-inflammatory cytokine IL-10) was suppressed in Nur77 KO regenerating livers, in contrast to WT. The reduced induction of IL-10 and increased expression of MMPs in Nur77 KO mice likely contributed to the liver injury and KC accumulation occurring in the early stages of PH-induced liver regeneration. To identify the potential Nur77-regulated pathways during liver regeneration, we performed a whole-genome microarray analysis to compare differential gene expression profiles between regenerating livers of WT and Nur77 KO mice at 3 hours" @default.
• W2052220384 created "2016-06-24" @default.
• W2052220384 creator A5020110618 @default.
• W2052220384 creator A5031817044 @default.
• W2052220384 creator A5032685603 @default.
• W2052220384 creator A5036116332 @default.
• W2052220384 creator A5061311927 @default.
• W2052220384 creator A5078004886 @default.
• W2052220384 date "2014-12-01" @default.
• W2052220384 modified "2023-10-06" @default.
• W2052220384 title "Accelerated Partial Hepatectomy–Induced Liver Cell Proliferation Is Associated with Liver Injury in Nur77 Knockout Mice" @default.
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