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• W2019910574 abstract "The AP-1 transcription factor modulates a wide range of cellular processes, including cellular proliferation, programmed cell death, and survival. JunD is a major component of the AP-1 complex following liver ischemia/reperfusion (I/R) injury; however, its precise function in this setting remains unclear. We investigated the functional significance of JunD in regulating AP-1 transcription following partial lobar I/R injury to the liver, as well as the downstream consequences for hepatocellular remodeling. Our findings demonstrate that JunD plays a protective role, reducing I/R injury to the liver by suppressing acute transcriptional activation of AP-1. In the absence of JunD, c-Jun phosphorylation and AP-1 activation in response to I/R injury were elevated, and this correlated with increased caspase activation, injury, and alterations in hepatocyte proliferation. The expression of dominant negative JNK1 inhibited c-Jun phosphorylation, AP-1 activation, and hepatic injury following I/R in JunD–/– mice but, paradoxically, led to an enhancement of AP-1 activation and liver injury in JunD+/– littermates. Enhanced JunD/JNK1-dependent liver injury correlated with the acute induction of diphenylene iodonium-sensitive NADPH-dependent superoxide production by the liver following I/R. In this context, dominant negative JNK1 expression elevated both Nox2 and Nox4 mRNA levels in the liver in a JunD-dependent manner. These findings suggest that JunD counterbalances JNK1 activation and the downstream redox-dependent hepatic injury that results from I/R, and may do so by regulating NADPH oxidases. The AP-1 transcription factor modulates a wide range of cellular processes, including cellular proliferation, programmed cell death, and survival. JunD is a major component of the AP-1 complex following liver ischemia/reperfusion (I/R) injury; however, its precise function in this setting remains unclear. We investigated the functional significance of JunD in regulating AP-1 transcription following partial lobar I/R injury to the liver, as well as the downstream consequences for hepatocellular remodeling. Our findings demonstrate that JunD plays a protective role, reducing I/R injury to the liver by suppressing acute transcriptional activation of AP-1. In the absence of JunD, c-Jun phosphorylation and AP-1 activation in response to I/R injury were elevated, and this correlated with increased caspase activation, injury, and alterations in hepatocyte proliferation. The expression of dominant negative JNK1 inhibited c-Jun phosphorylation, AP-1 activation, and hepatic injury following I/R in JunD–/– mice but, paradoxically, led to an enhancement of AP-1 activation and liver injury in JunD+/– littermates. Enhanced JunD/JNK1-dependent liver injury correlated with the acute induction of diphenylene iodonium-sensitive NADPH-dependent superoxide production by the liver following I/R. In this context, dominant negative JNK1 expression elevated both Nox2 and Nox4 mRNA levels in the liver in a JunD-dependent manner. These findings suggest that JunD counterbalances JNK1 activation and the downstream redox-dependent hepatic injury that results from I/R, and may do so by regulating NADPH oxidases. Transient tissue ischemia, caused by either surgical intervention or environmentally induced injury, can lead to a cascade of pathological events orchestrated by reoxygenation of the tissue. One example of this type of injury is liver ischemia/reperfusion (I/R) 3The abbreviations used are:I/Rischemia/reperfusionJNKc-Jun NH2-terminal kinasePCNAproliferating cell nuclear antigenROSreactive oxygen speciesEMSAelectrophoretic mobility shift assayGPTglutamic pyruvate transaminaseDPIdiphenylene iodoniumdndominant negativeNoxNADPH oxidases.3The abbreviations used are:I/Rischemia/reperfusionJNKc-Jun NH2-terminal kinasePCNAproliferating cell nuclear antigenROSreactive oxygen speciesEMSAelectrophoretic mobility shift assayGPTglutamic pyruvate transaminaseDPIdiphenylene iodoniumdndominant negativeNoxNADPH oxidases. following transplantation and/or surgical resection, and this can significantly influence patient survival (1Kretzschmar M. Kruger A. Schirrmeister W. Exp. Toxicol. Pathol. 2003; 54: 423-431Crossref PubMed Scopus (26) Google Scholar, 2Smyrniotis V. Kostopanagiotou G. Lolis E. Theodoraki K. Farantos C. Andreadou I. Polymeneas G. Genatas C. Contis J. J. Am. Coll. Surg. 2003; 197: 949-954Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 3Kim Y.I. J. Hepatobiliary Pancreat. Surg. 2003; 10: 195-199Crossref PubMed Scopus (72) Google Scholar, 4Kupiec-Weglinski J.W. Busuttil R.W. Transplant. Proc. 2005; 37: 1653-1656Crossref PubMed Scopus (207) Google Scholar). The pathology of liver I/R injury is biphasic, involving an acute stage of reperfusion-initiated cellular changes that are mediated by increased oxygen tension to the ischemic organ and an ensuing stage characterized by an inflammatory response (5Okaya T. Lentsch A.B. J. Investig. Surg. 2003; 16: 141-147Crossref PubMed Scopus (73) Google Scholar, 6Colletti L.M. Kunkel S.L. Walz A. Burdick M.D. Kunkel R.G. Wilke C.A. Strieter R.M. Hepatology. 1996; 23: 506-514Crossref PubMed Scopus (303) Google Scholar). Both of these phases result in organ damage, and both appear to involve the production of abnormal levels of reactive oxygen species (ROS). Indeed, numerous studies suggest that the formation of oxygen free radicals following reoxygenation may initiate the cascade of hepatocellular injury, necrosis/apoptosis, and inflammatory cell infiltration characteristic of this pathology (7Arthur M.J. Bentley I.S. Tanner A.R. Saunders P.K. Millward-Sadler G.H. Wright R. Gastroenterology. 1985; 89: 1114-1122Abstract Full Text PDF PubMed Scopus (180) Google Scholar, 8Atalla S.L. Toledo-Pereyra L.H. MacKenzie G.H. Cederna J.P. Transplantation. 1985; 40: 584-590Crossref PubMed Scopus (234) Google Scholar, 9Koo A. Komatsu H. Tao G. Inoue M. Guth P.H. Kaplowitz N. Hepatology. 1992; 15: 507-514Crossref PubMed Scopus (197) Google Scholar, 10Mathews W.R. Guido D.M. Fisher M.A. Jaeschke H. Free Radic. Biol. Med. 1994; 16: 763-770Crossref PubMed Scopus (162) Google Scholar). Therefore, modulating oxidative stress responses in transplanted organs has traditionally been considered as a rational approach to decreasing the complications associated with I/R damage.The AP-1 transcription complex is widely recognized as an important redox-activated factor involved in I/R liver injury. The family of AP-1 transcription factors consists of three main groups: the Jun proteins (c-Jun, JunB, and JunD), the Fos proteins (c-Fos, FosB, Fra1, and Fra2), and the activating transcription factors (ATF2, ATF3, and B-ATF) (11Chinenov Y. Kerppola T.K. Oncogene. 2001; 20: 2438-2452Crossref PubMed Scopus (570) Google Scholar). Members of these families can form homodimers or heterodimers that make up the active AP-1 complex. In addition to the regulation of AP-1 complexes by their dimer protein composition, post-translational modifications that alter the DNA binding and transactivation abilities of these complexes play important roles in controlling their activity (12Sheerin A. Thompson K.S. Goyns M.H. Mech. Ageing Dev. 2001; 122: 1813-1824Crossref PubMed Scopus (20) Google Scholar, 13Troen G. Nygaard V. Jenssen T.K. Ikonomou I.M. Tierens A. Matutes E. Gruszka-Westwood A. Catovsky D. Myklebost O. Lauritzsen G. Hovig E. Delabie J. J. Mol. Diagn. 2004; 6: 297-307Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). For example, c-Jun NH2-terminal kinase (JNK)-mediated phosphorylation of c-Jun on serines 63 and 73 of its NH2 terminus influences the transcriptional activity of the AP-1 complex (14Ronai Z. Mol. Cell. 2004; 15: 843-844Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). A second post-translational mechanism of AP-1 involves reduction-oxidation at a conserved cysteine residue within the DNA binding domains of Fos and Jun (15Abate C. Patel L. Rauscher III, F.J. Curran T. Science. 1990; 249: 1157-1161Crossref PubMed Scopus (1373) Google Scholar).Previous studies have shown that I/R injury promotes the activation of JNK1, as well as the induction of AP-1 DNA binding complexes composed of Jun heterodimers, during the reperfusion phases of injury (16Uehara T. Bennett B. Sakata S.T. Satoh Y. Bilter G.K. Westwick J.K. Brenner D.A. J. Hepatol. 2005; 42: 850-859Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 17Zhou W. Zhang Y. Hosch M.S. Lang A. Zwacka R.M. Engelhardt J.F. Hepatology. 2001; 33: 902-914Crossref PubMed Scopus (71) Google Scholar). In this context, we have previously shown that hepatic activation of AP-1 following I/R injury primarily induces heterodimers composed of JunD and c-Jun (17Zhou W. Zhang Y. Hosch M.S. Lang A. Zwacka R.M. Engelhardt J.F. Hepatology. 2001; 33: 902-914Crossref PubMed Scopus (71) Google Scholar). Although these two forms of Jun share a high degree of sequence homology, they have different transactivation properties and opposing effects on cellular proliferation, whereas c-Jun is a positive regulator of cell growth, JunD can function as a negative regulator of cell growth (18Pfarr C.M. Mechta F. Spyrou G. Lallemand D. Carillo S. Yaniv M. Cell. 1994; 76: 747-760Abstract Full Text PDF PubMed Scopus (285) Google Scholar, 19Weitzman J. Fiette L. Matsuo K. Yaniv M. Mol. Cell. 2000; 6: 1109-1119Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 20Bakiri L. Lallemand D. Bossy-Wetzel E. Yaniv M. EMBO J. 2000; 19: 2056-2068Crossref PubMed Scopus (330) Google Scholar, 21Ameyar-Zazoua M. Wisniewska M.B. Bakiri L. Wagner E.F. Yaniv M. Weitzman J.B. Oncogene. 2005; 24: 2298-2306Crossref PubMed Scopus (38) Google Scholar, 22Xiao L. Rao J.N. Zou T. Liu L. Marasa B.S. Chen J. Turner D.J. Passaniti A. Wang J.Y. Biochem. J. 2007; 403: 573-581Crossref PubMed Scopus (31) Google Scholar). In the context of liver I/R injury, hepatocytes undergo temporally regulated proliferation in a bimodal fashion, as evident from peaks in PCNA expression during the acute and subacute phases of reperfusion injury (23Schlossberg H. Zhang Y. Dudus L. Engelhardt J.F. Hepatology. 1996; 23: 1546-1555Crossref PubMed Google Scholar). However, it remains unclear whether these observed changes in cellular proliferation are coordinated by AP-1 transcriptional activity.In this study, we sought to address the role of AP-1 transcriptional activation following hepatic I/R injury by monitoring the expression of an AP-1-responsive luciferase reporter in vivo. Additionally, we wanted to better understand the functional significance of c-Jun/JunD heterodimers in the liver following I/R injury. Results from these studies demonstrate that JunD deletion significantly enhances hepatic transcriptional activation of AP-1 following I/R, and these changes correlated with enhanced cellular proliferation and increased liver injury. The expression of dominant negative JNK1 (dnJNK1) in the liver prior to I/R significantly altered the temporal pattern of AP-1 transcriptional activation, hepatic injury, and NADPH-dependent superoxide production by the liver in a JunD-dependent manner. These findings suggest that JNK1 and JunD collaborate to control NADPH-regulated redox stress in the liver following I/R and that these changes may influence AP-1 transcriptional programs that determine hepatocellular fates.EXPERIMENTAL PROCEDURESMouse Model of Partial Lobar I/R Injury—JunD knock-out mice used in this study were described previously (24Thepot D. Weitzman J.B. Barra J. Segretain D. Stinnakre M.G. Babinet C. Yaniv M. Development (Camb.). 2000; 127: 143-153Crossref PubMed Google Scholar). The JunD knock-out mice were bred onto a C57BL/6J background for 3–5 generations during the course of rederival into a specific pathogen-free barrier facility and maintained by breeding heterozygous JunD+/– males with knock-out JunD–/– females. Because these mice were not inbred, all experimental comparisons were performed with littermates from the colony (offspring from the mating scheme gave rise to litters composed of ∼50% heterozygous and ∼50% knock-out mice). Of note, male JunD–/– mice had reduced fertility and were not used for breeding. Male JunD+/– mice were compared with JunD–/– littermates in partial lobar liver I/R experiments as described previously (25Zwacka R.M. Zhou W. Zhang Y. Darby C.J. Dudus L. Halldorson J. Oberley L. Engelhardt J.F. Nat. Med. 1998; 4: 698-704Crossref PubMed Scopus (247) Google Scholar). In brief, mice were anesthetized and injected with heparin (100 μg/kg) to prevent clotting of blood during lobar ischemia. The largest medial lobe of the liver was clamped at its base with a micro-aneurysm clamp, followed by placement of the liver and clamp back into the peritoneal cavity for 45 or 60 min. Following surgically implemented ischemia, the clamp was removed and the abdominal wall sutured, and the animals were returned to their cages. Removal of the clamp signified the start of reperfusion. The ischemic lobe of livers were harvested at 0, 6, 9, 12, 18, and/or 24 h after the initiation of reperfusion for experimental analysis.Gene Delivery to the Liver Using Recombinant Adenovirus—Recombinant adenoviruse vectors Ad.LacZ (25Zwacka R.M. Zhou W. Zhang Y. Darby C.J. Dudus L. Halldorson J. Oberley L. Engelhardt J.F. Nat. Med. 1998; 4: 698-704Crossref PubMed Scopus (247) Google Scholar) and Ad.dnJNK1 (26Qiao L. Han S.I. Fang Y. Park J.S. Gupta S. Gilfor D. Amorino G. Valerie K. Sealy L. Engelhardt J.F. Grant S. Hylemon P.B. Dent P. Mol. Cell. Biol. 2003; 23: 3052-3066Crossref PubMed Scopus (137) Google Scholar) were described previously. Ad.AP-1Luc virus was generated using a fragment from the pAP1(PMA)-TA-Luc plasmid (Clontech) containing the firefly luciferase gene, driven by six tandem copies of the AP1 enhancer linked to the minimal TATA box promoter from the herpes simplex virus thymidine kinase gene. An XhoI/NotI fragment from pAP1(PMA)-TA-Luc was inserted into a promoterless adenoviral shuttle plasmid (pAd5mcspA), and Ad.AP-1Luc virus was generated by homologous recombination as described previously (25Zwacka R.M. Zhou W. Zhang Y. Darby C.J. Dudus L. Halldorson J. Oberley L. Engelhardt J.F. Nat. Med. 1998; 4: 698-704Crossref PubMed Scopus (247) Google Scholar, 27Anderson R.D. Haskell R.E. Xia H. Roessler B.J. Davidson B.L. Gene Ther. 2000; 7: 1034-1038Crossref PubMed Scopus (224) Google Scholar). Viral infections with Ad.LacZ and Ad.dnJNK1 were performed 3 days before liver I/R surgery, by tail vein injection with 1011 particles of purified virus per 25 g of body weight in 200 μl of phosphate-buffered saline. Ad.AP-1Luc infections were performed at a lower dose of 5 × 1010 particles per 25 g of body weight.Western Blot Analysis—Ischemic liver lobes were washed in phosphate-buffered saline and then homogenized in 2 ml of homogenization buffer (0.3 m sucrose, 10 mm HEPES (pH 7.6), 10 mm KCl, 0.74 mm spermidine, 0.15 mm spermine, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, and 1 Complete protease inhibitor mixture tablet (Roche Applied Science) for 50 ml). 100 μg of protein was separated on a denaturing 10% SDS-PAGE and transferred to a nitrocellulose membrane. PCNA and p53 protein levels were determined by Western blot analysis using anti-PCNA and anti-p53 antibodies (Santa Cruz Biotechnology). Analysis of c-Jun phosphorylation was performed by immunoprecipitation of c-Jun from 1 mg of ischemic liver lobe lysate followed by Western blotting with a phosphoserine 63 c-Jun-specific antibody. Both the pan c-Jun (catalog number sc-45) and p-c-Jun(Ser-63) (catalog number sc-822) antibodies were from Santa Cruz Biotechnology. Westerns blots were quantified on a LI-COR Biosciences Odyssey Infrared Imaging System using infrared-labeled secondary antibody.Luciferase Indicator Assays for AP-1 Transcriptional Activation—AP-1 transcriptional activity in the ischemic lobe of the liver was assessed using an Ad.AP-1Luc vector. 100 μg of total protein from each liver lysate was used to perform luciferase assays. Luciferase activity was measured using a kit from Promega (Madison, WI).Electrophoretic Mobility Shift Assays for AP-1 DNA Binding—Nuclear extracts were generated from the ischemic lobe of the liver as described previously (25Zwacka R.M. Zhou W. Zhang Y. Darby C.J. Dudus L. Halldorson J. Oberley L. Engelhardt J.F. Nat. Med. 1998; 4: 698-704Crossref PubMed Scopus (247) Google Scholar). Protein content was measured using a Bradford assay, and electrophoretic mobility shift assays (EMSAs) were performed as described previously with little modification (17Zhou W. Zhang Y. Hosch M.S. Lang A. Zwacka R.M. Engelhardt J.F. Hepatology. 2001; 33: 902-914Crossref PubMed Scopus (71) Google Scholar). In brief, 6 μl of nuclear extract was incubated in EMSA buffer (250 mm KCl, 100 mm HEPES (pH 7.9), 25% glycerol, 5 mm EDTA, 5 mmol/liter dithiothreitol) with bovine serum albumin (1 μg/μl), poly(dI-dC) (1 μg/μl) and double-stranded, 32P-end-labeled oligonucleotide (200,000 cpm) in a total volume of 20 μl. This mixture was incubated for 30 min at room temperature before being separated on a 4% native polyacrylamide gel. The oligonucleotide sequence used for the AP-1 double-stranded probe was 5′-CGCTTGATGACTCAGCCGGAA-3′.Serum GPT Assay for Liver Damage—Blood was collected from animals at the time of liver harvest at various times post-reperfusion. Blood samples were centrifuged for 2 min at 8,000 rpm to separate the serum from the cells. Serum transaminase (GPT) levels were measured using a microkinetic assay (2–6 μl of serum) as described previously (25Zwacka R.M. Zhou W. Zhang Y. Darby C.J. Dudus L. Halldorson J. Oberley L. Engelhardt J.F. Nat. Med. 1998; 4: 698-704Crossref PubMed Scopus (247) Google Scholar).Isolation of Liver Endomembranes and Chemiluminescence Assays for NADPH-dependent Superoxide (O2·¯) Production— Liver tissue (ischemic lobe of the liver) was lysed in the homogenization buffer described above by nitrogen cavitation. 600 μg of crude lysate was then centrifuged at 3,000 × g to remove the heavy mitochondria and nuclei, and the post-nuclear supernatant was subsequently centrifuged at 100,000 × g for 1 h to pellet total endomembranes. The membranes were washed three times in homogenization buffer before being resuspended in 100 μl of homogenization buffer. NADPH oxidase activities were analyzed by measuring the rate of O2·¯ generation using a chemiluminescent, lucigenin-based system as described previously (28Li Q. Harraz M.M. Zhou W. Zhang L.N. Ding W. Zhang Y. Eggleston T. Yeaman C. Banfi B. Engelhardt J.F. Mol. Cell. Biol. 2006; 26: 140-154Crossref PubMed Scopus (189) Google Scholar). In brief, 5 μm lucigenin in 50 μl of each endomembrane fraction was used to calculate the relative rate of O2·¯ production following the addition of β-NADPH at a final concentration of 100 μm, with or without 10 μm diphenylene iodonium (DPI).Quantification of Nox Transcripts—JunD+/– and JunD–/– mice were infected with 1.0 × 1011 particles (intravenously) of either Ad.LacZ or Ad.dnJNK 3 days prior to harvesting the livers. Total RNA was purified from the liver using a standard guanidine thiocyanate solution and ultracentrifugation CsCl2 banding procedure. Poly(A) mRNA was purified from the total mRNA using the Micro-FastTract 2.0 mRNA mini kit (Invitrogen). Reverse transcription was performed on the mRNA samples using the High Capacity cDNA reverse transcription kit (Applied Biosystems) with random priming and a reaction volume of 100 μl. Reverse transcriptase negative samples were also carried along as a control for mRNA specificity in the PCR. Each sample was evaluated by real time PCR for Nox2, Nox4, and β-actin mRNA copies. For the three respective amplification reactions a Nox2, Nox4, and β-actin master mix was generated (per reaction: 10 μl of iQ SYBR Green Supermix, 375 nm primer 1, 375 nm primer 2, in a total volume of 16 μl). The primers used were as follows: Nox2, 5′-ACGCCCTTTGCCTCCATTCTCAAGTC-3′ and 5′-ATGCGTGTCCCTGCACAGCCAGTAG-3′; Nox4, 5′-TTCGAGGATCACAGAAGGTCCCTAGCA-3′ and 5′-GGCGGCTACATGCACACCTGAGAAA-3′; and β-actin, 5′-CCTGAACCCTAAGGCCAACCGTGAAA-3′ and 5′-TACGACCAGAGGCATACAGGGACAGCA-3′.16 μl of a given master mix and 4 μl of a cDNA sample were combined for each reaction. For each cDNA sample, three replicate real time reactions were prepared using each of the three sets of primers, for a total of nine reactions per liver sample. Additionally, to accurately quantify copy number, a series of standards were created using known copies numbers of Nox2, Nox4, and β-actin plasmids. Samples were run on a Bio-Rad MyIQ real time machine (step 1, heat at 95 °C for 2 min; step 2, 45 cycles alternating between 95 °C for 30 s and 71 °C for 15 s). Ct values for each reaction were used to calculate copy number of each cDNA transcript against the standard curves. Finally, the copy number of each Nox transcript was normalized to the copy number of the β-actin internal control. This normalization controlled for slight variations in the quality/quantity of each cDNA preparation used in each PCR.RESULTSJunD Modulates AP-1 Transcriptional Activation following Liver I/R Injury—Following I/R injury to the liver, increased nuclear DNA binding activity by AP-1 heterodimers composed of c-Jun and JunD occurs within the first 3 h of reperfusion (17Zhou W. Zhang Y. Hosch M.S. Lang A. Zwacka R.M. Engelhardt J.F. Hepatology. 2001; 33: 902-914Crossref PubMed Scopus (71) Google Scholar, 25Zwacka R.M. Zhou W. Zhang Y. Darby C.J. Dudus L. Halldorson J. Oberley L. Engelhardt J.F. Nat. Med. 1998; 4: 698-704Crossref PubMed Scopus (247) Google Scholar). Using a recombinant adenoviral vector encoding an AP-1 responsive luciferase gene, we sought to determine the transcriptional status of AP-1 following liver I/R. In light of these previous findings, we were surprised to find that maximal AP-1 transcriptional activation in JunD+/– animals occurred at 12 h after the initiation of hepatic reperfusion (Fig. 1A). Interestingly, JunD-deficient littermates demonstrated significantly earlier onset (6–9 h) and enhanced (more than 3-fold) AP-1 transcriptional induction (Fig. 1A), suggesting that JunD suppresses the transcriptional activity of the AP-1 complex during the early stages of reperfusion injury. Comparative analysis in these animals demonstrated an inverse relationship between DNA binding by AP-1 (Fig. 1B) and in vivo transcriptional activation mediated by AP-1. In both JunD+/– and JunD–/– animals, DNA binding by AP-1 complexes exhibited a bimodal distribution, peaking at 3–6 h, diminishing at intermediate time points and rising again by 18–24 h. Thus, maximal transcriptional activation by AP-1, which occurred at 12 h post-reperfusion, correlated with a decline in AP-1 binding activity. We interpret these results to suggest that AP-1-mediated transcription following I/R undergoes both activating and inhibitory stages. During the activating stages (3–6 h), similar levels of DNA binding were seen in JunD-deficient and control animals, supporting the notion that the AP-1 complex was more active for transcription in the absence of JunD. Furthermore, JunD ablation led to an earlier and accentuated reduction in nuclear AP-1 DNA binding during the refractory stage when DNA binding activity is inhibited (9–12 h).JunD Protects the Liver from Injury following I/R—To determine the consequences of AP-1 activation in the liver following I/R injury and to investigate the role of JunD in this process, we evaluated the extent of liver injury by measuring serum transaminase (GPT) levels. These studies revealed significantly greater I/R liver injury in JunD-deficient mice as compared with heterozygous littermates, when the animals underwent 45 min of ischemia (Fig. 2A). Furthermore, when longer times of ischemia were used (1 h), lethality in JunD–/– mice within the first 90 min of reperfusion was significantly increased above that of JunD+/– littermates (Fig. 2B). This increased liver injury, as well as increased lethality, following liver I/R injury in the JunD–/– animals correlated with an elevation of caspase activity in liver homogenates during the early stages (6 h) of reperfusion (Fig. 2C). In JunD+/– littermates, by contrast, caspase activation in response to I/R treatment peaked much later (18 h post-reperfusion) and was attenuated in comparison with that seen in the JunD knock-out mice. In summary, these findings demonstrate that in the absence of JunD, enhanced AP-1-mediated transcriptional activation during the acute stage of reperfusion correlates with increased liver damage.FIGURE 2JunD protects against I/R-induced liver injury and alters hepatocellular remodeling. JunD–/– and JunD+/– mice were subjected to I/R injury, with partial lobar hepatic ischemia taking place for 45 min (A, C, and D) or 60 min(B), followed by the indicated times of reperfusion. A, serum samples were collected by cardiac puncture at the indicated time points following the start of reperfusion and assayed for GPT. Results depict the mean (±S.E.) units/liter of GPT for n = 4 animals in each group. B, survival of mice following 60 min of ischemia was plotted using a Kaplan-Meier survival curve, p < 0.0004. C, caspase 3/7 activity in whole cell lysates from liver was determined using a caspase-Glo detection kit, for which a substrate emits light when cleaved by either caspase 3 or 7. Results depict the mean (±S.E.) relative light units (RLU). D, for each experimental point, pooled liver lysates from three animals were evaluated for PCNA and p53 expression by Western blotting (WB). The 0-h time point in A, C, and D represents the respective values (serum glutathione S-transferase, caspase 3/7 activity, or PCNA/p53 levels) for non-I/R injured control animals (i.e. noninjured base-line values).View Large Image Figure ViewerDownload Hi-res image Download (PPT)JunD Alters Hepatocellular Responses following I/R Injury—JunD has been shown to slow cell cycle progression by inhibiting the G1-to-S phase transition (18Pfarr C.M. Mechta F. Spyrou G. Lallemand D. Carillo S. Yaniv M. Cell. 1994; 76: 747-760Abstract Full Text PDF PubMed Scopus (285) Google Scholar, 19Weitzman J. Fiette L. Matsuo K. Yaniv M. Mol. Cell. 2000; 6: 1109-1119Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 22Xiao L. Rao J.N. Zou T. Liu L. Marasa B.S. Chen J. Turner D.J. Passaniti A. Wang J.Y. Biochem. J. 2007; 403: 573-581Crossref PubMed Scopus (31) Google Scholar). Previous studies on I/R injury to the liver have also demonstrated that PCNA expression (an index of cellular proliferation) is tightly regulated during the early stages of reperfusion injury, as indicated by its bi-modal expression pattern (23Schlossberg H. Zhang Y. Dudus L. Engelhardt J.F. Hepatology. 1996; 23: 1546-1555Crossref PubMed Google Scholar). We hypothesized that the lack of JunD expression might alter cell cycle progression during the acute stages of I/R injury and thereby lead to impaired hepatic repair (remodeling) mechanisms. Analysis of JunD+/– animals reproduced the previously described bi-modal expression of PCNA (23Schlossberg H. Zhang Y. Dudus L. Engelhardt J.F. Hepatology. 1996; 23: 1546-1555Crossref PubMed Google Scholar), with peaks at 3 and 18 h post-reperfusion (Fig. 2D). In contrast, this I/R-induced bi-modal response was dysregulated in mice lacking JunD, remaining elevated up to 12 h following reperfusion. Furthermore, JunD knock-out mouse livers expressed PCNA at elevated base-line levels in the absence of I/R injury (Fig. 2D, 0 h time points), indicating a pre-existing dysregulation of the cell cycle. These findings suggest that JunD does indeed alter cell cycle regulation in the liver, both prior to and following I/R injury. However, despite previous studies in fibroblasts demonstrating that JunD can act through p53 to modulate growth arrest and DNA repair (19Weitzman J. Fiette L. Matsuo K. Yaniv M. Mol. Cell. 2000; 6: 1109-1119Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), we did not observe significant differences in p53 levels between the two JunD genotypes (Fig. 2D).The JNK Pathway Influences JunD-dependent AP-1 Transcriptional Activity following I/R Injury to the Liver—JNK plays a major role in regulating AP-1 activity by phosphorylating c-Jun on serine 63/73 (29Davis R.J. Biochem. Soc. Symp. 1999; 64: 1-12PubMed Google Scholar). Additionally, JunD has been shown to be a substrate of JNK phosphorylation (30Yazgan O. Pfarr C.M. J. Biol. Chem. 2002; 277: 29710-29718Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), but the functional consequences of this event remain largely unknown. Acute JNK1 activation occurs within the first 45–60 min following liver I/R and precedes the induction of nuclear AP-1 binding to DNA (17Zhou W. Zhang Y. Hosch M.S. Lang A. Zwacka R.M. Engelhardt J.F. Hepatology. 2001; 33: 902-914Crossref PubMed Scopus (71) Google Scholar). Chemical inhibitors of JNK appear to suppress DNA binding by AP-1 following I/R injury, and they have been shown to protect the liver from apoptotically mediated damage (16Uehara T. Bennett B. Sakata S.T. Satoh Y. Bilter G.K. Westwick J.K. Brenner D.A. J. Hepatol. 2005; 42: 850-859Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). To this end, we hypothesized that expressing dominant negative JNK1 using a recombinant adenoviral vector (26Qiao L. Han S.I. Fang Y. Park J.S. Gupta S. Gilfor D. Amorino G. Valerie K. Sealy L. Engelhardt J.F. Grant S. Hylemon P.B. Dent P. Mol. Cell. Biol. 2003; 23: 3052-3066Crossref PubMed Scopus (137) Google Scholar) might suppress the elevation of AP-1 transcriptio" @default.
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• W2019910574 date "2008-03-01" @default.
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• W2019910574 title "JunD Protects the Liver from Ischemia/Reperfusion Injury by Dampening AP-1 Transcriptional Activation" @default.
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