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1.
Hepatology ; 33(6): 1377-86, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11391526

ABSTRACT

Liver regeneration following 70% partial hepatectomy leads to rapid activation of genes in the remnant liver. Interleukin-6 deficient (IL-6 -/-) mice have impaired liver regeneration and abnormalities in immediate early gene expression. In this study, the gene expression program in the IL-6 +/+ and -/- livers at 2 hours posthepatectomy was examined with a cDNA array representing 588 highly regulated mouse genes. Thirty-six percent of the 103 immediate early genes were induced differently in IL-6 +/+ compared with IL-6 -/- livers, implying regulation by IL-6. IL-6 treatment of the IL-6 -/- mice in the absence of hepatectomy induced a much smaller set of genes in the liver, suggesting that IL-6 cooperates with other hepatectomy-induced factors to activate the large number of genes. Northern blot analyses were used to verify gene expression data obtained from the arrays. The expression of urokinase type plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1), critical components of the urokinase plasminogen activator (uPA) system, was lower and delayed in IL-6 -/- livers. Despite the fact that active uPAR/uPA complex is critical for hepatocyte growth factor (HGF) activation, no differences were detected between the IL-6 +/+ and -/- livers in HGF activation as measured by receptor phosphorylation. On the contrary, the mitogen-activated protein kinase (MAPK) pathway was activated in IL-6 +/+ livers early during regeneration but remarkably delayed in IL-6 -/- livers. Defective liver regeneration may be explained by the large number of gene activation pathways altered in IL-6 -/- livers and further supports the finding that IL-6 is necessary for normal liver regeneration.


Subject(s)
Gene Expression/physiology , Hepatectomy/methods , Interleukin-6/metabolism , Liver/physiology , Animals , Enzyme Activation , Genes, Immediate-Early/physiology , Hepatocyte Growth Factor/physiology , Interleukin-6/genetics , Liver Regeneration/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout/genetics , Mitogen-Activated Protein Kinases/metabolism , Oligonucleotide Array Sequence Analysis , Postoperative Period , Reference Values , Time Factors
2.
Hepatology ; 33(5): 1166-72, 2001 May.
Article in English | MEDLINE | ID: mdl-11343245

ABSTRACT

Apoptotic cell death in the liver in response to activation of the Fas pathway has been implicated in human disease states as well as liver remodeling and tissue repair. C/EBPbeta, a member of the CCAAT enhancer binding protein family of bZIP transcription factors has been linked to both growth response and apoptotic targets in the liver, and, therefore, is a likely candidate for the regulation of apoptotic liver injury. We investigated differences in apoptotic cell death in the livers of C/EBPbeta-null mice using the Jo-2 agonistic anti-Fas antibody. Apoptotic injury was dramatically reduced in C/EBPbeta -/- livers as shown by a nearly 20-fold reduction in apoptotic hepatocytes 6 hours post-Jo-2 treatment in C/EBPbeta -/- hepatocytes compared with controls (P < .04) and reduced activation of caspase 3. Bid cleavage occurred in Jo-2 treated C/EBPbeta -/- livers indicating a block of Fas-induced injury distal to the death-inducing signaling complex. The level of the antiapoptotic protein bcl-x(L) was increased greater than tenfold in the mutant animals (P < .04), which can, at least in part, account for the protection from Fas-mediated apoptosis. In contrast, bcl-x(L) mRNA levels were unchanged. These observations link C/EBPbeta to Fas-induced hepatocyte apoptosis through a mechanism that likely involves translational or posttranslational regulation of bcl-x(L).


Subject(s)
Apoptosis/physiology , CCAAT-Enhancer-Binding Protein-beta/physiology , Hepatocytes/physiology , fas Receptor/physiology , Animals , Antibodies/immunology , CCAAT-Enhancer-Binding Protein-beta/deficiency , CCAAT-Enhancer-Binding Protein-beta/genetics , Caspase 3 , Caspases/metabolism , Drug Resistance , Enzyme Activation/drug effects , Enzyme Activation/physiology , Hepatocytes/drug effects , In Vitro Techniques , Injections , Liver/drug effects , Liver/enzymology , Liver/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout/genetics , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger/metabolism , bcl-X Protein , fas Receptor/immunology , fas Receptor/pharmacology
3.
J Biol Chem ; 276(28): 26605-13, 2001 Jul 13.
Article in English | MEDLINE | ID: mdl-11349125

ABSTRACT

Previous studies showed that following acute carbon tetrachloride (CCl(4)) treatment, interleukin-6 null (IL-6-/-) mice develop increased hepatocellular injury, defective regeneration, delayed wound healing, and increased hepatocyte apoptosis. Pretreatment with IL-6 prior to CCl(4) reduces injury, hepatocyte apoptosis, and accelerates regeneration in both IL-6-/- and +/+ livers. To demonstrate whether IL-6 can prevent liver injury that involves direct stimulation of hepatocyte apoptosis, IL-6-/- and +/+ mice were treated with the Fas agonist, Jo-2 mAb. At low Fas agonist doses, IL-6+/+ mice developed mild hepatic injury and survived, whereas IL-6-/- mice developed severe apoptotic hepatitis within 12 h and died. Pretreatment with IL-6 improved survival in IL-6-/- mice and reduced injury in both IL-6-/- and +/+ livers. The direct anti-apoptotic effects of IL-6 were demonstrated in vitro as IL-6 decreased Fas-mediated apoptosis in both IL-6-/- and +/+ primary hepatocyte cultures, and suggested that IL-6-/- hepatocytes have a pre-existing defect in anti-apoptotic pathways. After Fas activation, IL-6-/- livers demonstrated evidence of both proximal and distal alterations in the apoptotic pathways including elevated caspase 8 and 3 activation-associated fragments, and loss of cytochrome c staining. IL-6-/- livers had reduced pre-existing protein expression of the anti-apoptotic factors Bcl-2 and Bcl-xL as well as more rapid degradation of FLIP following Fas treatment that appeared to be post-transcriptionally regulated. FLIP is a crucial proximal inhibitor of caspase 8 activation in Fas, tumor necrosis factor, and DR3/DR4-mediated apoptosis, and Bcl-2 and Bcl-xL more downstream anti-apoptotic regulators. IL-6 may function as a critical anti-apoptotic factor in the liver by its ability to establish and maintain an adequate level of FLIP and downstream anti-apoptotic factors.


Subject(s)
Carrier Proteins/metabolism , Interleukin-6/pharmacology , Intracellular Signaling Peptides and Proteins , Liver/pathology , Proto-Oncogene Proteins c-bcl-2/metabolism , Animals , CASP8 and FADD-Like Apoptosis Regulating Protein , Cell Death/drug effects , Interleukin-6/metabolism , Liver/metabolism , Mice , bcl-X Protein , fas Receptor/metabolism
4.
Hepatology ; 33(4): 915-24, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11283856

ABSTRACT

Nuclear factor kappaB (NF-kappaB) is rapidly activated during liver regeneration following partial hepatectomy or carbon tetrachloride (CCl(4))-mediated liver injury and is felt to be important in the antiapoptotic and regenerative responses. After partial hepatectomy, livers of mice deficient in the p50 subunit of NF-kappaB (p50(-/-)) showed a loss of NF-kappaB and decreased STAT3 transcription factor DNA binding activities. However, nuclear levels of the NF-kappaB p65 subunit were increased and peaked earlier in p50(-/-) livers. Both messenger RNA and cytoplasmic protein levels of the NF-kappaB inhibitor IkappaBalpha were lower in p50(-/-) livers, potentially accounting for the increase in p65 protein. Small effects on gene expression posthepatectomy were observed in p50(-/-) livers, but no effects were seen on hepatocyte DNA synthetic or mitotic responses, serum enzyme levels, or overall liver mass restoration. After CCl(4) treatment, hepatocyte DNA synthesis and mitosis and serum enzyme levels were similar in p50(-/-) and p50(+/+) mice, and histologic analysis indicated a slight decrease in overall damage in p50(-/-) livers. After injection of Fas antibody, p50(-/-) livers showed an earlier onset of nuclear changes consistent with apoptosis. These data indicate that absence of p50 affects certain protein and gene activation pathways following partial hepatectomy, CCl(4), and Fas treatment but does not impair overall liver regeneration. Interleukin 6 (IL-6) levels were reduced but still adequate to support regeneration. We hypothesize that increased levels of the NF-kappaB p65 subunit in p50(-/-) livers may provide compensation for the absence of p50, thereby allowing normal liver regeneration and repair following liver injury.


Subject(s)
Liver Regeneration/physiology , NF-kappa B/physiology , Alanine Transaminase/blood , Animals , Apoptosis , Carbon Tetrachloride/pharmacology , DNA/biosynthesis , DNA-Binding Proteins/metabolism , Gene Expression , Hepatectomy/methods , I-kappa B Proteins/metabolism , Injections, Intraperitoneal , Liver/drug effects , Liver/metabolism , Liver/pathology , Mice , Mice, Knockout/genetics , NF-kappa B/genetics , Protein Isoforms/genetics , Protein Isoforms/physiology , Reference Values , STAT3 Transcription Factor , Trans-Activators/metabolism , fas Receptor/pharmacology
5.
Hepatology ; 31(1): 149-59, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10613740

ABSTRACT

Interleukin-6 null (IL-6-/-) mice have impaired liver regeneration and increased liver necrosis following partial hepatectomy that is corrected with IL-6 treatment. Following acute carbon tetrachloride (CCl(4)) treatment, we found that IL-6-/- mice developed increased hepatocellular injury and defective regeneration with significant blunting of signal transducer-and-activator of transcription protein 3 (STAT3) and nuclear factor-kappaB (NF-kappaB) activation and reduced hepatocyte DNA synthetic and mitotic responses. After CCl(4) treatment, unlike partial hepatectomy, increased hepatocyte apoptosis was noted in IL-6-/- livers. Pretreatment with IL-6 before CCl(4) reduced acute CCl(4) injury and apoptosis and accelerated regeneration in both IL-6+/+ and -/- livers. Repetitive doses of CCl(4) in the presence or absence of phenobarbital resulted in increased injury and fibrosis in IL-6 -/- compared with +/+ livers. After acute and chronic injury, IL-6-/- livers showed the protracted presence of alpha-smooth muscle actin associated with activated stellate cells, indicating a disturbed response in wound healing that progressed to fibrosis. These data provide evidence for an important role for IL-6 in reducing CCl(4)-induced acute and chronic liver injury and fibrosis.


Subject(s)
Chemical and Drug Induced Liver Injury , Interleukin-6/deficiency , Liver Cirrhosis, Experimental/chemically induced , Actins/metabolism , Animals , Apoptosis , Carbon Tetrachloride , DNA/biosynthesis , DNA-Binding Proteins/metabolism , Interleukin-6/physiology , Kinetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitosis , NF-kappa B/metabolism , Phenobarbital/administration & dosage , STAT3 Transcription Factor , Trans-Activators/metabolism
6.
Hepatology ; 30(5): 1187-97, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10534340

ABSTRACT

The insulin-like growth factor binding protein-1 (IGFBP-1) gene is highly expressed in fetal, perinatal, and regenerating liver. Up-regulation is transcriptionally mediated in regenerating liver and occurs in the first few minutes to hours after partial hepatectomy. In transgenic mice a 970-bp region from -776 to +151 of the IGFBP-1 promoter was sufficient for tissue-specific and induced expression of the gene in fetal and hepatectomized livers. However weak and/or poorly regulated expression in some transgenic lines suggested the existence of other regulatory regions. Here, genomic clones containing large regions 5' of the mouse IGFBP-1 gene sequence were isolated, subcloned, and sequenced. Deoxyribonuclease I (DNaseI) hypersensitivity analyses identified clusters of tissue-specific nuclease-sensitive sites in the promoter region, -100 to -300, -2,300, -3,100, and -5,000 along with other weak sites. After partial hepatectomy, enhanced sensitivity and/or novel sites were detected in the -100/-300, -5,000, and -3,100 regions, the promoter region remaining the most hypersensitive. A subset of these sites was present in fetal and perinatal livers. Novel tissue-specific sites that interacted with C/EBP and hepatic nuclear factor 3 (HNF3) transcription factors were identified in the -3,100 region. A hepatectomy-induced DNA binding complex containing the transcription factor USF1 was identified within the -100 to -300 region of the promoter. These results suggested that a complex array of tissue-specific and hepatic proliferation-induced transcription factors combine to regulate both the proximal promoter and more distal regulatory elements of the IGFBP-1 gene.


Subject(s)
Gene Expression Regulation, Developmental , Insulin-Like Growth Factor Binding Protein 1/genetics , Liver Regeneration , Liver/cytology , Liver/metabolism , Promoter Regions, Genetic , Animals , Base Sequence , Carcinoma, Hepatocellular , Cloning, Molecular , Deoxyribonuclease I , Fetus , Hepatectomy , Humans , Kidney/metabolism , Liver Neoplasms , Mice , Mice, Transgenic , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Regulatory Sequences, Nucleic Acid , Restriction Mapping , Sequence Alignment , Sequence Homology, Nucleic Acid , Spleen/metabolism , Substrate Specificity , Transfection , Tumor Cells, Cultured
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