Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 38
Filter
Add more filters










Publication year range
1.
Cell Death Differ ; 20(7): 878-87, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23519075

ABSTRACT

Recent investigations have demonstrated a complex interrelationship between autophagy and cell death. A common mechanism of cell death in liver injury is tumor necrosis factor (TNF) cytotoxicity. To better delineate the in vivo function of autophagy in cell death, we examined the role of autophagy in TNF-induced hepatic injury. Atg7Δhep mice with a hepatocyte-specific knockout of the autophagy gene atg7 were generated and cotreated with D-galactosamine (GalN) and lipopolysaccharide (LPS). GalN/LPS-treated Atg7Δhep mice had increased serum alanine aminotransferase levels, histological injury, numbers of TUNEL (terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling)-positive cells and mortality as compared with littermate controls. Loss of hepatocyte autophagy similarly sensitized to GalN/TNF liver injury. GalN/LPS injury in knockout animals did not result from altered production of TNF or other cytokines. Atg7Δhep mice had accelerated activation of the mitochondrial death pathway and caspase-3 and -7 cleavage. Increased cell death did not occur from direct mitochondrial toxicity or a lack of mitophagy, but rather from increased activation of initiator caspase-8 causing Bid cleavage. GalN blocked LPS induction of hepatic autophagy, and increased autophagy from beclin 1 overexpression prevented GalN/LPS injury. Autophagy, therefore, mediates cellular resistance to TNF toxicity in vivo by blocking activation of caspase-8 and the mitochondrial death pathway, suggesting that autophagy is a therapeutic target in TNF-dependent tissue injury.


Subject(s)
Autophagy/physiology , Caspase 8/physiology , Chemical and Drug Induced Liver Injury/physiopathology , Hepatocytes/pathology , Hepatocytes/physiology , Tumor Necrosis Factor-alpha/adverse effects , Animals , Apoptosis Regulatory Proteins/metabolism , Autophagy-Related Protein 7 , Beclin-1 , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Cytokines/metabolism , Galactosamine/adverse effects , Galactosamine/pharmacology , In Vitro Techniques , Lipopolysaccharides/adverse effects , Lipopolysaccharides/pharmacology , MAP Kinase Signaling System/physiology , Macrophages/metabolism , Macrophages/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Microtubule-Associated Proteins/deficiency , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Models, Animal , Tumor Necrosis Factor-alpha/pharmacology
2.
Cell Death Differ ; 20(1): 3-11, 2013 Jan.
Article in English | MEDLINE | ID: mdl-22595754

ABSTRACT

Intracellular lipids are stored in lipid droplets (LDs) and metabolized by cytoplasmic neutral hydrolases to supply lipids for cell use. Recently, an alternative pathway of lipid metabolism through the lysosomal degradative pathway of autophagy has been described and termed lipophagy. In this form of lipid metabolism, LD triglycerides (TGs) and cholesterol are taken up by autophagosomes and delivered to lysosomes for degradation by acidic hydrolases. Free fatty acids generated by lipophagy from the breakdown of TGs fuel cellular rates of mitochondrial ß-oxidation. Lipophagy therefore functions to regulate intracellular lipid stores, cellular levels of free lipids such as fatty acids and energy homeostasis. The amount of lipid metabolized by lipophagy varies in response to the extracellular supply of nutrients. The ability of the cell to alter the amount of lipid targeted for autophagic degradation depending on nutritional status demonstrates that this process is selective. Intracellular lipids themselves regulate levels of autophagy by unclear mechanisms. Impaired lipophagy can lead to excessive tissue lipid accumulation such as hepatic steatosis, alter hypothalamic neuropeptide release to affect body mass, block cellular transdifferentiation and sensitize cells to death stimuli. Future studies will likely identify additional mechanisms by which lipophagy regulates cellular physiology, making this pathway a potential therapeutic target in a variety of diseases.


Subject(s)
Autophagy/physiology , Lipid Metabolism/physiology , Animals , Energy Metabolism/physiology , Humans , Lysosomes/metabolism
4.
J Biol Chem ; 275(51): 40155-62, 2000 Dec 22.
Article in English | MEDLINE | ID: mdl-11016920

ABSTRACT

The typical proliferative response of hepatocytes to tumor necrosis factor (TNF) can be converted to a cytotoxic one by transcriptional arrest. Although NF-kappaB activation is critical for hepatocyte resistance to TNF toxicity, the contribution of other TNF-inducible transcription factors remains unknown. To determine the function of c-Myc in hepatocyte sensitivity to TNF, stable transfectants of the rat hepatocyte cell line RALA255-10G containing sense and antisense c-myc expression vectors were isolated with increased (S-Myc cells) and decreased (AN-Myc cells) c-Myc transcriptional activity. While S-Myc cells proliferated in response to TNF treatment, AN-Myc cells underwent 32% cell death within 6 h. Fluorescent microscopic studies indicated that TNF induced apoptosis and necrosis in AN-Myc cells. Cell death was associated with DNA hypoploidy and poly(ADP-ribose) polymerase cleavage but occurred in the absence of detectable caspase-3, -7, or -8 activation. TNF-induced, AN-Myc cell death was dependent on Fas-associated protein with death domain and partially blocked by caspase inhibitors. AN-Myc cells had decreased levels of NF-kappaB transcriptional activity, but S-Myc cells maintained resistance to TNF despite NF-kappaB inactivation, suggesting that c-Myc and NF-kappaB independently mediate TNF resistance. Thus, in the absence of sufficient c-Myc expression, hepatocytes are sensitized to TNF-induced apoptosis and necrosis. These findings demonstrate that hepatocyte resistance to TNF is regulated by multiple transcriptional activators.


Subject(s)
Apoptosis/drug effects , Hepatocytes/drug effects , Proto-Oncogene Proteins c-myc/antagonists & inhibitors , Animals , Caspases/metabolism , Cell Line , Enzyme Activation , Hepatocytes/cytology , Hepatocytes/pathology , NF-kappa B/metabolism , Necrosis , Oligonucleotides, Antisense/genetics , Proto-Oncogene Proteins c-myc/genetics , Rats
5.
Am J Physiol Gastrointest Liver Physiol ; 278(5): G693-9, 2000 May.
Article in English | MEDLINE | ID: mdl-10801261

ABSTRACT

Reactive oxygen intermediates (ROI) have been implicated as mediators of hepatocyte death resulting from a variety of forms of liver injury. To delineate the mechanisms that underlie ROI-induced apoptosis, the roles of caspase activation and nuclear factor-kappaB (NF-kappaB) signaling were determined in the rat hepatocyte cell line RALA255-10G after treatment with H(2)O(2) or the superoxide generator menadione. By 8 h, H(2)O(2) and menadione caused 26% and 33% cell death, respectively. Death from both ROI occurred by apoptosis as indicated by morphology under fluorescence microscopy, the induction of caspase activation and DNA fragmentation, and the cleavage of poly(ADP-ribose) polymerase. Despite the presence of caspase activation in both forms of apoptosis, caspase inhibition blocked H(2)O(2)- but not menadione-induced apoptosis. In contrast, inhibition of NF-kappaB activation decreased cell death from both ROI. Different ROI, therefore, induce distinct apoptotic pathways in RALA hepatocytes that are both caspase dependent and independent. In contrast to the known protective effect of NF-kappaB activation in tumor necrosis factor-alpha-induced hepatocyte apoptosis, NF-kappaB promotes hepatocellular death from ROI in these cells.


Subject(s)
Apoptosis/physiology , Caspases/metabolism , Hydrogen Peroxide/pharmacology , Liver/cytology , Liver/physiology , NF-kappa B/metabolism , Superoxides/pharmacology , Vitamin K/pharmacology , Animals , Apoptosis/drug effects , Caspase 2 , Caspase 3 , Caspase 7 , Caspase 8 , Caspase 9 , Cell Death/drug effects , Cell Line , Liver/drug effects , Rats , Signal Transduction/drug effects , Tumor Necrosis Factor-alpha/pharmacology
6.
J Biol Chem ; 275(1): 705-12, 2000 Jan 07.
Article in English | MEDLINE | ID: mdl-10617670

ABSTRACT

Hepatocytes can be sensitized to tumor necrosis factor (TNF)-alpha toxicity by repression of NF-kappaB activation or inhibition of RNA synthesis. To determine whether both forms of sensitization lead to TNF-alpha cytotoxicity by similar mechanisms, TNF-alpha-induced cell death in RALA255-10G hepatocytes was examined following infection with an adenovirus, Ad5IkappaB, that blocks NF-kappaB activation or following cotreatment with actinomycin D (ActD). TNF-alpha treatment of Ad5IkappaB-infected cells resulted in 44% cell death within 6 h. ActD/TNF-alpha induced no death within 6 h but did lead to 37% cell death by 24 h. In both instances, cell death occurred by apoptosis and was associated with caspase activation, although caspase activation in ActD-sensitized cells was delayed. CrmA and chemical caspase inhibitors blocked Ad5IkappaB/TNF-alpha-induced cell death but did not inhibit ActD/TNF-alpha-induced apoptosis. A Fas-associated protein with death domain (FADD) dominant negative decreased Ad5IkappaB/TNF-alpha- and ActD/TNF-alpha-induced cell death by 81 and 47%, respectively. However, downstream events differed, since Ad5IkappaB/TNF-alpha but not ActD/TNF-alpha treatment caused mitochondrial cytochrome c release. These results suggest that NF-kappaB inactivation and inhibition of RNA synthesis sensitize RALA255-10G hepatocytes to TNF-alpha toxicity through distinct cell death pathways that diverge below the level of FADD. ActD-induced hepatocyte sensitization to TNF-alpha cytotoxicity occurs through a FADD-dependent, caspase-independent pathway of apoptosis.


Subject(s)
Adaptor Proteins, Signal Transducing , Apoptosis , Caspases/metabolism , Liver/physiology , Tumor Necrosis Factor-alpha/toxicity , Animals , Carrier Proteins/metabolism , Caspase Inhibitors , Cell Line , Cytochrome c Group/metabolism , Dactinomycin/pharmacology , Enzyme Activation , Fas-Associated Death Domain Protein , I-kappa B Proteins , Liver/cytology , NF-kappa B/metabolism , Ploidies , Poly(ADP-ribose) Polymerases/metabolism , Rats , Signal Transduction
7.
Hepatology ; 30(1): 215-22, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10385659

ABSTRACT

Ceramide has been implicated as a second messenger in intracellular signaling pathways leading to apoptosis in nonhepatic cells. To determine whether ceramide can mediate hepatocyte apoptosis, the cytotoxicity of ceramide was determined in rat hepatocytes. The rat hepatocyte cell line, RALA255-10G, and primary rat hepatocytes were completely resistant to toxicity from 10 to 100 micromol/L C2 ceramide. Resistance was not the result of a failure to take up ceramide, because ceramide treatment did cause nuclear factor-kappaB (NF-kappaB) activation. Because ceramide may mediate cell death from tumor necrosis factor alpha (TNF-alpha), the ability of RNA synthesis inhibition and NF-kappaB inactivation to sensitize hepatocytes to ceramide toxicity was examined. RALA hepatocytes were sensitized to ceramide toxicity by coadministration of actinomycin D (ActD). Cell death occurred by apoptosis as determined by the presence of morphological evidence of apoptosis, caspase activation, poly(ADP-ribose) polymerase (PARP) degradation, and DNA hypoploidy. Despite the induction of apoptosis associated with caspase activation, cell death from ActD/ceramide was not blocked by caspase inhibition. Inhibition of NF-kappaB activation also sensitized RALA hepatocytes to ceramide toxicity, but to a lesser extent than for TNF-alpha. Thus, unlike many nonhepatic cell types, rat hepatocytes are resistant to cell death from ceramide because of the transcriptionally dependent up-regulation of a protective gene(s). The ability of ActD and NF-kappaB inactivation to sensitize RALA hepatocytes to ceramide toxicity suggests that ceramide may act as a downstream mediator of TNF-alpha toxicity.


Subject(s)
Apoptosis/physiology , Caspases/metabolism , Dactinomycin/pharmacology , Liver/drug effects , Nucleic Acid Synthesis Inhibitors/pharmacology , Sphingosine/analogs & derivatives , Animals , Apoptosis/drug effects , Cell Line , Cysteine Proteinase Inhibitors/pharmacology , Enzyme Inhibitors/toxicity , Indoles/pharmacology , Liver/pathology , Liver/physiology , Male , NF-kappa B/metabolism , Oligopeptides/pharmacology , Ploidies , Poly(ADP-ribose) Polymerases/metabolism , Rats , Rats, Sprague-Dawley , Sphingosine/antagonists & inhibitors , Sphingosine/toxicity
8.
Am J Physiol ; 275(5): G874-8, 1998 11.
Article in English | MEDLINE | ID: mdl-9815013

ABSTRACT

Toxin-induced liver injury was formerly considered a passive biochemical event, but recent evidence has demonstrated that signal transduction pathways actively modulate the hepatocyte's response to this form of injury. Investigations have examined the effects of a variety of toxins on the activation of receptor-coupled signal transduction, mitogen-activated protein kinases, and Fas signaling, as well as the generation of second messengers such as ceramide and nitric oxide. Many of these pathways culminate in the activation of transcription factors such as activator protein-1, c-Myc, or nuclear factor-kappaB. This Themes article discusses the effects of toxic injury on these signaling pathways and their known functions in regulating hepatocyte death and proliferation following injury.


Subject(s)
Liver/pathology , Liver/physiopathology , Signal Transduction/physiology , Animals , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Fas Ligand Protein , Humans , Liver/injuries , Membrane Glycoproteins/physiology , Nitric Oxide/physiology , Sphingolipids/physiology , Transcription Factors/metabolism , fas Receptor/physiology
9.
Hepatology ; 28(5): 1347-56, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9794921

ABSTRACT

Mutant human hepatoblastoma cell lines resistant to copper toxicity were isolated from mutagenized HuH7. Two copper resistant cell lines (CuR), CuR 23 and CuR 27, had reduced basal expression of metallothionein (MT) messenger RNA (mRNA) and exhibited minimal or no increase in resistance to cadmium or zinc toxicity. Copper uptake, efflux of newly transported copper, glutathione content, and efflux rate were comparable with HuH7, whereas holoceruloplasmin synthesis and secretion were slightly decreased. Subcellular distribution of copper at steady-state showed an increase in organelle and membrane fractions with a reduction in cytosol. Expression of ATP7B mRNA was fivefold increased, and ATP7B protein approximately threefold increased in both CuR 23 and 27. Another cell line, CuR 41, showed increased basal expression of MT and ATP7B mRNA but not ATP7B protein, and resistance to cadmium and zinc toxicity. Copper uptake in CuR 41 was comparable with HuH7, but initial rates of efflux of copper and glutathione were reduced. The synthesis of holoceruloplasmin but not ceruloplasmin peptide was markedly diminished in CuR 41. Subcellular distribution of copper showed an increase in cytosolic and decreased organelle and membrane-associated copper. These data suggest that cellular resistance to copper toxicity was achieved in two independent cell lines without MT induction and that the induction of ATP7B may lead to the enhanced intracellular sequestration of copper by organelles.


Subject(s)
Adenosine Triphosphatases/genetics , Carrier Proteins/genetics , Cation Transport Proteins , Copper/pharmacology , Gene Expression , Hepatoblastoma/genetics , Liver Neoplasms/genetics , Metallothionein/genetics , Blotting, Northern , Blotting, Western , Cadmium/pharmacology , Ceruloplasmin/metabolism , Copper/metabolism , Copper-Transporting ATPases , Drug Resistance , Glutathione/metabolism , Humans , Kinetics , Mutagenesis , Protein Biosynthesis , RNA, Messenger/biosynthesis , Tumor Cells, Cultured , Zinc/pharmacology
10.
Gastroenterology ; 115(5): 1229-37, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9797379

ABSTRACT

BACKGROUND & AIMS: Tumor necrosis factor (TNF)-alpha causes much of the hepatocellular injury and cell death that follows toxin-induced liver damage. The mechanism by which toxic liver injury sensitizes hepatocytes to TNF-alpha cytotoxicity is unknown. The aim of this study was to determine the role of the antioxidant glutathione in this process. METHODS: A rat hepatocyte cell line and primary hepatocytes sensitized to TNF-alpha toxicity by the addition of actinomycin D were examined for changes in glutathione levels and for the effects of glutathione depletion or supplementation on cell death. The in vivo effects of glutathione depletion were determined in mice treated with galactosamine plus lipopolysaccharide. RESULTS: Treatment of hepatocytes with actinomycin D and TNF-alpha induced apoptotic cell death without affecting cellular glutathione levels or production of the reactive oxygen intermediate H2O2. Glutathione depletion induced by diethyl maleic acid significantly increased TNF-alpha-induced cell death even when this agent was administered 2 hours after TNF-alpha treatment. Hepatocyte cell death was not affected by glutathione supplementation. In mice treated with galactosamine plus lipopolysaccharide, glutathione depletion increased mortality from liver injury from 32% to 72%. CONCLUSIONS: TNF-alpha-induced cytotoxicity in hepatocytes occurs in the absence of glutathione depletion. However, a preexisting reduction in glutathione levels can significantly increase cell death from TNF-alpha.


Subject(s)
Glutathione/physiology , Liver/drug effects , Tumor Necrosis Factor-alpha/poisoning , Animals , Cells, Cultured , Dactinomycin/poisoning , Drug Combinations , Galactosamine/poisoning , Glutathione/deficiency , Glutathione/metabolism , Glutathione/pharmacology , Immunity, Innate/drug effects , Lipopolysaccharides/poisoning , Liver/cytology , Male , Mice , Mice, Inbred Strains , Rats , Rats, Sprague-Dawley
11.
Am J Physiol ; 275(4): C1058-66, 1998 10.
Article in English | MEDLINE | ID: mdl-9755059

ABSTRACT

Toxins convert the hepatocellular response to tumor necrosis factor-alpha (TNF-alpha) stimulation from proliferation to cell death, suggesting that hepatotoxins somehow sensitize hepatocytes to TNF-alpha toxicity. Because nuclear factor-kappaB (NF-kappaB) activation confers resistance to TNF-alpha cytotoxicity in nonhepatic cells, the possibility that toxin-induced sensitization to TNF-alpha killing results from inhibition of NF-kappaB-dependent gene expression was examined in the RALA rat hepatocyte cell line sensitized to TNF-alpha cytotoxicity by actinomycin D (ActD). ActD did not affect TNF-alpha-induced hepatocyte NF-kappaB activation but decreased NF-kappaB-dependent gene expression. Expression of an IkappaB superrepressor rendered RALA hepatocytes sensitive to TNF-alpha-induced apoptosis in the absence of ActD. Apoptosis was blocked by caspase inhibitors, and TNF-alpha treatment led to activation of caspase-2, caspase-3, and caspase-8 only when NF-kappaB activation was blocked. Although apoptosis was blocked by the NF-kappaB-dependent factor nitric oxide (NO), inhibition of endogenous NO production did not sensitize cells to TNF-alpha-induced cytotoxicity. Thus NF-kappaB activation is the critical intracellular signal that determines whether TNF-alpha stimulates hepatocyte proliferation or apoptosis. Although exogenous NO blocks RALA hepatocyte TNF-alpha cytotoxicity, endogenous production of NO is not the mechanism by which NF-kappaB activation inhibits this death pathway.


Subject(s)
Apoptosis/physiology , Cell Division/physiology , Gene Expression Regulation/drug effects , NF-kappa B/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Animals , Apoptosis/drug effects , Caspase Inhibitors , Cell Division/drug effects , Cell Line , Cell Survival/drug effects , Cysteine Proteinase Inhibitors/pharmacology , DNA/biosynthesis , DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/metabolism , Gene Expression Regulation/physiology , I-kappa B Proteins , NF-kappa B/antagonists & inhibitors , Nitric Oxide/physiology , Nitric Oxide Donors/pharmacology , Penicillamine/analogs & derivatives , Penicillamine/pharmacology , Rats , Recombinant Proteins/metabolism , S-Nitroso-N-Acetylpenicillamine , Thymidine/metabolism , Transfection
12.
Am J Physiol ; 273(4): G795-803, 1997 10.
Article in English | MEDLINE | ID: mdl-9357820

ABSTRACT

To determine whether intracellular signaling events involved in apoptosis may also mediate necrosis, the role of the transcription factor AP-1 was investigated in a hepatoma cell model of cellular necrosis induced by oxidant stress. Treatment of the human hepatoma cell line HuH-7 with H2O2 caused dose-dependent necrosis as determined by light microscopy, fluorescent staining, and an absence of DNA fragmentation. H2O2 treatment led to increases in c-fos and c-jun mRNA levels, Jun nuclear kinase activity, and AP-1 DNA binding. AP-1 transcriptional activity measured with an AP-1-driven luciferase reporter gene was also increased. To determine whether this AP-1 activation contributed to H2O2-induced cell necrosis, HuH-7 cells were stably transfected with an antisense c-jun expression vector. Cells expressing antisense c-jun had decreased levels of AP-1 activation and significantly increased survival after H2O2 exposure. These data indicate that AP-1 activation occurs during oxidant-induced cell necrosis and contributes to cell death. Necrosis is therefore not always a passive process but may involve the activation of intracellular signaling pathways similar to those that mediate apoptosis.


Subject(s)
Cell Survival/drug effects , Hydrogen Peroxide/toxicity , Proto-Oncogene Proteins c-jun/biosynthesis , Transcription Factor AP-1/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Carcinoma, Hepatocellular/pathology , Cycloheximide/pharmacology , DNA Fragmentation , Genes, Reporter , Genes, jun , Glutathione/metabolism , Humans , Liver/drug effects , Liver/metabolism , Liver/pathology , Liver Neoplasms/pathology , Luciferases/biosynthesis , Necrosis , Transfection , Tumor Cells, Cultured
13.
J Cell Physiol ; 170(2): 192-9, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9009148

ABSTRACT

Expression of c-myc regulates apoptotic cell death in the human hepatoma cell line HuH-7 during culture in serum-free medium (SFM) plus zinc. To understand the mechanism of this c-myc effect, the ability of various serum-contained factors to prevent apoptosis was determined. Apoptosis was not inhibited by growth factors and was even accelerated by supplementation with insulin-like growth factor I or insulin. Cell death was prevented by SFM supplementation with the amino acid glutamine but not serine or asparagine. Improved cell survival with glutamine was associated with increased levels of glutathione (GSH). In HuH-7 cells cultured in SFM plus zinc, c-myc expression led to decreased levels of GSH, and elevated intracellular levels of hydrogen peroxide (H2O2). Cell death induced by c-myc expression was inhibited by the addition of catalase or dimethyl sulfoxide, a hydroxyl radical scavenger, or by increased intracellular expression of catalase. In contrast to findings in fibroblasts, c-myc-dependent apoptosis during serum deprivation in HuH-7 hepatoma cells was unrelated to a loss of growth factors. Apoptosis resulted from H2O2-mediated oxidative stress with associated glutamine dependent intracellular GSH depletion.


Subject(s)
Apoptosis , Genes, myc , Growth Substances/pharmacology , Proto-Oncogene Proteins c-myc/biosynthesis , Apoptosis/drug effects , Carcinoma, Hepatocellular , Catalase/pharmacology , Cell Division , Culture Media, Serum-Free , Dimethyl Sulfoxide/pharmacology , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic/drug effects , Glutamine/metabolism , Glutamine/pharmacology , Glutathione/metabolism , Humans , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/toxicity , Liver Neoplasms , Oxidative Stress , Tumor Cells, Cultured , Zinc/pharmacology
14.
Gastroenterology ; 110(6): 1870-7, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8964413

ABSTRACT

BACKGROUND & AIMS: Monocyte chemoattractant protein 1 (MCP-1) is a potent monocyte/macrophage chemoattractant expressed by fat-storing cells (FSCs) in rat models of liver injury. This study investigated the mechanism of this activation of hepatic MCP-1 expression. METHODS: The regulation of MCP-1 messenger RNA (mRNA) expression and protein synthesis was examined in FSC lines derived from CCl4-induced cirrhotic rat liver (cirrhotic FSCs) and normal rat liver (normal FSCs). RESULTS: Northern blot hybridization analysis revealed low levels of MCP-1 mRNA in cultured cirrhotic FSCs that increased markedly after treatment with tumor necrosis factor alpha, interleukin 1 alpha, or transforming growth factor beta 1. All three cytokines increased the synthesis and secretion of MCP-1 protein. Oxygen free radical production also increased MCP-1 mRNA levels. These increases in MCP-1 mRNA were blocked by dexamethasone. In normal FSCs, levels of MCP-1 mRNA and secreted protein were increased in response to cytokines or oxygen free radical production, but the magnitude and duration of this increase was less than in cirrhotic FSCs. CONCLUSIONS: In liver injury, monocyte/macrophage recruitment and activation from FSC production of MCP-1 may be stimulated by cytokines and oxygen free radicals. During chronic liver injury leading to cirrhosis, FSCs may become hypersensitive to these stimuli, further fueling the inflammatory response.


Subject(s)
Chemokine CCL2/metabolism , Lipid Metabolism , Liver/metabolism , Reactive Oxygen Species/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Animals , Cell Line , Chemokine CCL2/genetics , Dexamethasone/pharmacology , Liver/cytology , Liver Cirrhosis, Experimental/metabolism , Liver Cirrhosis, Experimental/pathology , RNA, Messenger/metabolism , Rats , Rats, Wistar
15.
Am J Physiol ; 270(1 Pt 1): G60-70, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8772502

ABSTRACT

Since c-myc expression is increased during apoptosis in toxin-induced liver injury in vivo, the role of c-myc in liver cell apoptosis was investigated. The human hepatoma cell line HuH-7, which constitutively expresses c-myc, was stably transfected with sense and antisense c-myc expression vectors under the control of the zinc-inducible metallothionein promoter. None of the three cell types (wild-type, sense c-myc, or antisense c-myc) underwent apoptosis when cultured in serum-free medium (SFM). With the addition of SFM plus 37.5 microM zinc, wild-type and sense c-myc-expressing cells underwent rapid cell death, whereas antisense c-myc-expressing cells exhibited increased survival. This cell death had the light, fluorescent, and electron microscopic appearance of apoptosis, but did not result in DNA fragmentation. This apoptosis could be terminated by the addition of medium containing 2% fetal calf serum or the overexpression of bcl-2 but not by supplementation with specific growth factors. Altering c-myc expression did not affect cellular metallothionein mRNA levels or the rate of cell death from copper or cadmium. The requirement for zinc and absence of DNA fragmentation in c-myc-induced hepatoma cell apoptosis under serum-free conditions provides further evidence of the complex regulation of apoptosis in different cell types.


Subject(s)
Apoptosis , Carcinoma, Hepatocellular/pathology , DNA/physiology , Liver Neoplasms/pathology , Proto-Oncogene Proteins c-myc/physiology , Zinc/physiology , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Animals , Cadmium Poisoning/pathology , Cattle/blood , Cattle/embryology , Copper/poisoning , Culture Media, Serum-Free , Fetal Blood , Humans , Osmolar Concentration , Proto-Oncogene Proteins c-bcl-2/physiology , Transfection , Tumor Cells, Cultured/drug effects , Zinc/pharmacology
16.
Gastroenterology ; 108(6): 1849-54, 1995 Jun.
Article in English | MEDLINE | ID: mdl-7768392

ABSTRACT

BACKGROUND/AIMS: Considerable indirect evidence suggests that cytokine tumor necrosis factor alpha contributes to the hepatocellular damage caused by toxic liver injury. The effects of tumor necrosis factor alpha neutralization on liver cell injury were determined in an in vivo model of toxic liver injury. METHODS: The in vivo effects of tumor necrosis factor alpha were examined in carbon tetrachloride liver injury through the administration of a soluble tumor necrosis factor receptor to neutralize the effects of this cytokine. RESULTS: Soluble tumor necrosis factor receptor treatment decreased the degree of liver injury as measured by reduced levels of serum liver enzymes and improved histology. Soluble tumor necrosis factor receptor administration also lowered the mortality from a lethal dose of carbon tetrachloride from 60% to 16%. Tumor necrosis factor alpha neutralization had no detrimental effect on liver regeneration as determined by the timing of histone gene expression and postinjury liver weight. CONCLUSIONS: These data provide direct evidence for a role of tumor necrosis factor alpha in toxin-induced liver cell injury. In addition, these investigations suggest that soluble tumor necrosis factor receptor therapy may be of benefit in the treatment of human liver disease.


Subject(s)
Carbon Tetrachloride/toxicity , Liver/drug effects , Receptors, Tumor Necrosis Factor/physiology , Animals , Histones/genetics , Liver Regeneration , Male , Rats , Rats, Sprague-Dawley , Tumor Necrosis Factor-alpha/physiology
17.
Hepatology ; 19(5): 1282-9, 1994 May.
Article in English | MEDLINE | ID: mdl-8175153

ABSTRACT

Endogenous lipopolysaccharide has been implicated as a cofactor in the hepatocellular injury and death resulting from toxic liver injury. To prevent this lipopolysaccharide-induced injury and to further understand the mechanism of this effect, an anti-lipopolysaccharide antibody was administered to rats in which toxic hepatocellular injury was induced. Rats were given the hepatotoxin galactosamine together with an isotypic control antibody B55 or the anti-lipopolysaccharide antibody E5. E5 treatment resulted in reductions of serum AST levels of 43% at 36 hr (p < 0.02) and 60% at 48 hr (NS) after galactosamine administration. These decreases in AST values were accompanied by diminished histological evidence of injury and inflammation. In carbon tetrachloride-induced liver injury, E5 similarly reduced serum AST levels at 36 and 48 hr by 47% (p < 0.04) and 54% (p < 0.03), respectively. E5 treatment was equally effective in reducing AST levels 48 hr after administration of carbon tetrachloride, whether the initial dose of antibody was given 1 hr before or 3 or 6 hr after the administration of this toxin. To understand the mechanism of this E5 effect, the activation of the toxic cytokine tumor necrosis factor-alpha and the chemotactic cytokine monocyte chemoattractant protein 1 was examined by Northern-blot analysis of RNA from rat livers after galactosamine-induced injury and treatment with B55 or E5. Despite E5's efficacy in reducing hepatocellular damage, E5 treatment did not affect the timing or magnitude of tumor necrosis factor-alpha or monocyte chemoattractant protein 1 activation during galactosamine-induced injury.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Antibodies, Bacterial/pharmacology , Lipopolysaccharides/immunology , Liver/pathology , Animals , Aspartate Aminotransferases/blood , Blotting, Northern , Carbon Tetrachloride/administration & dosage , Carbon Tetrachloride/toxicity , Chemokine CCL2 , Chemotactic Factors/metabolism , Galactosamine/administration & dosage , Galactosamine/toxicity , Liver/drug effects , Liver/metabolism , Male , Rats , Rats, Sprague-Dawley , Tumor Necrosis Factor-alpha/metabolism
18.
Am J Physiol ; 266(4 Pt 1): G737-44, 1994 Apr.
Article in English | MEDLINE | ID: mdl-8179009

ABSTRACT

The model of toxic liver injury was used to examine the role of manganese superoxide dismutase (MnSOD) expression in cellular resistance to tumor necrosis factor (TNF)-alpha toxicity. The effects of the hepatotoxin D-galactosamine (GalN) and lipopolysaccharide (LPS) on hepatic and splenic TNF-alpha and MnSOD expression were studied. Treatment with GalN and LPS alone or in combination led to equivalent increases in hepatic and splenic TNF-alpha gene expression. Hepatic MnSOD mRNA levels were not affected by GalN or GalN with LPS but were increased 13-fold by LPS alone. Splenic MnSOD mRNA levels were increased twofold by GalN and 12-fold by either LPS alone or GalN plus LPS. The determination of MnSOD protein content, however, revealed no changes in hepatic or splenic steady-state levels of the protein with any of the treatments, despite the marked increases in MnSOD gene expression. Hepatic MnSOD enzyme activity was also unchanged by LPS or GalN plus LPS administration. Biosynthesis of MnSOD protein in rat hepatocytes isolated from an in vivo LPS-treated rat was unchanged compared with control. MnSOD mRNA levels were increased when GalN treatment was combined with uridine rescue, but again no change in protein was seen. The lack of any increase in MnSOD protein after GalN or LPS administration indicates that MnSOD upregulation is not involved in cellular resistance against TNF-alpha cytotoxicity in the liver in vivo.


Subject(s)
Gene Expression , Liver/drug effects , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Tumor Necrosis Factor-alpha/toxicity , Animals , Galactosamine/pharmacology , Lipopolysaccharides/pharmacology , Liver/cytology , Liver/metabolism , Male , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Tumor Necrosis Factor-alpha/genetics , Uridine/pharmacology
19.
J Leukoc Biol ; 55(1): 120-6, 1994 Jan.
Article in English | MEDLINE | ID: mdl-8283136

ABSTRACT

Considerable evidence suggests that monocytes/macrophages play a crucial role in the process of liver injury and repair. Recent investigations have focused on the function of various macrophage-produced cytokines in liver disease. Much is still unknown, however, about the mechanism of macrophage recruitment and activation during liver disease. To further define this process, the gene expression of the monocyte chemoattractant monocyte chemoattractant protein 1 (MCP-1) was examined in animal and human liver disease. MCP-1 mRNA was not found in normal rat liver by Northern blot analysis. After single-dose treatments with the hepatotoxins carbon tetrachloride and galactosamine, MCP-1 mRNA was detectable beginning at 2 and 4 h after treatment, respectively, and was expressed continuously until 60-72 h. During chronic carbon tetrachloride administration, MCP-1 mRNA levels were elevated for the entire 10 weeks of treatment with peak levels of expression occurring early (weeks 1-3) and late (weeks 8-10) in this model. Isolated liver cell fractions from rats treated for 3 weeks with carbon tetrachloride revealed the major cellular source of MCP-1 mRNA to be fat-storing or Ito cells, with some expression occurring in the endothelial cell fraction. Studies of potential inducers of hepatic MCP-1 expression showed that lipopolysaccharide, tumor necrosis factor-alpha, and interleukin-1 alpha and beta treatments all led to MCP-1 expression. Finally, studies of human liver samples revealed MCP-1 gene expression in nondiseased liver and greatly increased levels in livers from patients with fulminant hepatic failure. These data implicate MCP-1 from fat-storing cells as a modulator of the process of liver injury and further support a role for MCP-1 in the pathogenesis of human disease.


Subject(s)
Chemotactic Factors/genetics , Liver Diseases/metabolism , Liver/drug effects , Animals , Carbon Tetrachloride/toxicity , Chemokine CCL2 , Cytokines/pharmacology , Galactosamine/toxicity , Gene Expression , Humans , Lipopolysaccharides/pharmacology , Liver/metabolism , Male , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Rats, Wistar
20.
J Cell Physiol ; 154(2): 294-300, 1993 Feb.
Article in English | MEDLINE | ID: mdl-8425910

ABSTRACT

Hepatic expression of the protooncogenes c-fos and c-myc occurs within 2 h after partial hepatectomy, and these immediate early genes are thought to prime the hepatocytes for subsequent proliferation. To examine whether such gene activation occurred in the setting of hepatocyte proliferation after toxic liver injury, protooncogene expression was examined during the regenerative response following liver injury from carbon tetrachloride (CCl4) or galactosamine (GalN). The pattern of protooncogene expression after CCl4 mirrored that seen after partial hepatectomy, with rises in c-fos and c-myc mRNA content within 2 h, and then a rapid return to baseline levels. In contrast, early c-fos and c-myc expression did not occur after GalN injury. Instead GalN-induced regeneration led to a delayed, and prolonged c-fos and c-myc activation which peaked 24-48 h after injury. Increases in c-jun, jun-B, and jun-D mRNA levels also occurred in both models at times similar to the rises of c-fos and c-myc expression. Although the timing of DNA synthesis was identical after GalN or CCl4 treatment, the proliferative response after GalN injury was significantly less than that of CCl4, and marked by the histologic appearance of oval cells. The coadministration of 2-acetylaminofluorene, an inhibitor of differentiated hepatocyte proliferation, together with CCl4 altered the usual pattern of post-CCl4 protooncogene expression to one resembling that seen after GalN injury. Thus, the timing of protooncogene expression during liver regeneration may vary considerably. These variations may influence the nature of the proliferative response in terms of which cell type(s) proliferates, and the amount of regeneration that ensues.


Subject(s)
Carbon Tetrachloride/pharmacology , Gene Expression , Liver Regeneration , Liver/drug effects , Proto-Oncogenes , Animals , Galactosamine/pharmacology , Gene Expression/drug effects , Genes, fos , Genes, myc , Liver/metabolism , Male , Rats , Rats, Sprague-Dawley , Time Factors
SELECTION OF CITATIONS
SEARCH DETAIL
...