RESUMEN
We established a mouse model of developmental nonalcoholic steatohepatitis (NASH) by feeding a high polyunsaturated fat liquid diet to female glutathione-S-transferase 4-4 (Gsta4-/-)/peroxisome proliferator activated receptor α (Ppara-/-) double knockout 129/SvJ mice for 12 weeks from weaning. We used it to probe the importance of lipid peroxidation in progression of NASH beyond simple steatosis. Feeding Gsta4-/-/Ppara-/- double-knockout (dKO) mice liquid diets containing corn oil resulted in a percentage fat-dependent increase in steatosis and necroinflammatory injury (P < 0.05). Increasing fat to 70% from 35% resulted in increases in formation of 4-hydroxynonenal protein adducts accompanied by evidence of stellate cell activation, matrix remodeling, and fibrosis (P < 0.05). Comparison of dKO mice with wild-type (Wt) and single knockout mice revealed additive effects of Gsta4-/- and Ppara-/- silencing on steatosis, 4-hydroxynonenal adduct formation, oxidative stress, serum alanine amino transferase, expression of tumor necrosis factor alpha, Il6, interferon mRNA, and liver pathology (P < 0.05). Induction of Cyp2e1 protein by high-fat diet was suppressed in Gsta4-/- and dKO groups (P < 0.05). The dKO mice had similar levels of markers of stellate cell activation and matrix remodeling as Ppara-/- single KO mice. These data suggest that lipid peroxidation products play a role in progression of liver injury to steatohepatitis in NASH produced by high-fat feeding during development but appear less important in development of fibrosis.
Asunto(s)
Glutatión Transferasa/deficiencia , Peroxidación de Lípido/fisiología , Enfermedad del Hígado Graso no Alcohólico/enzimología , Enfermedad del Hígado Graso no Alcohólico/patología , Animales , Western Blotting , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Femenino , Hígado/patología , Ratones , Ratones Noqueados , PPAR alfa/deficiencia , Reacción en Cadena de la Polimerasa , TranscriptomaRESUMEN
Chronic alcohol consumption induces hepatic oxidative stress resulting in production of highly reactive electrophilic α/ß-unsaturated aldehydes that have the potential to modify proteins. A primary mechanism of reactive aldehyde detoxification by hepatocytes is through GSTA4-driven enzymatic conjugation with GSH. Given reports that oxidative stress initiates GSTA4 translocation to the mitochondria, we hypothesized that increased hepatocellular damage in ethanol (EtOH)-fed GSTA4(-/-) mice is due to enhanced mitochondrial protein modification by reactive aldehydes. Chronic ingestion of EtOH increased hepatic protein carbonylation in GSTA4(-/-) mice as evidenced by increased 4-HNE and MDA immunostaining in the hepatic periportal region. Using mass spectrometric analysis of biotin hydrazide conjugated carbonylated proteins, a total of 829 proteins were identified in microsomal, cytosolic and mitochondrial fractions. Of these, 417 were novel to EtOH models. Focusing on mitochondrial fractions, 1.61-fold more carbonylated proteins were identified in EtOH-fed GSTA4(-)(/-) mice compared to their respective WT mice ingesting EtOH. Bioinformatic KEGG pathway analysis of carbonylated proteins from the mitochondrial fractions revealed an increased propensity for modification of proteins regulating oxidative phosphorylation, glucose, fatty acid, glutathione and amino acid metabolic processes in GSTA4(-/-) mice. Additional analysis revealed sites of reactive aldehyde protein modification on 26 novel peptides/proteins isolated from either SV/GSTA4(-/-) PF or EtOH fed mice. Among the peptides/proteins identified, ACSL, ACOX2, MTP, and THIKB contribute to regulation of fatty acid metabolism and ARG1, ARLY, and OAT, which regulate nitrogen and ammonia metabolism having direct relevance to ethanol-induced liver injury. These data define a role for GSTA4-4 in buffering hepatic oxidative stress associated with chronic alcohol consumption and that this GST isoform plays an important role in protecting against carbonylation of mitochondrial proteins.
Asunto(s)
Aldehídos/metabolismo , Glutatión Transferasa/metabolismo , Hepatopatías Alcohólicas/metabolismo , Proteínas Mitocondriales/metabolismo , Animales , Modelos Animales de Enfermedad , Eliminación de Gen , Glutatión Transferasa/genética , Hepatopatías Alcohólicas/genética , Ratones , Carbonilación Proteica , Isoformas de Proteínas , Procesamiento Proteico-PostraduccionalRESUMEN
To test the significance of lipid peroxidation in the development of alcoholic liver injury, an ethanol (EtOH) liquid diet was fed to male 129/SvJ mice (wild-type, WT) and glutathione S-transferase A4-4-null (GSTA4-/-) mice for 40 days. GSTA4-/- mice were crossed with peroxisome proliferator-activated receptor-α-null mice (PPAR-α-/-), and the effects of EtOH in the resulting double knockout (dKO) mice were compared with the other strains. EtOH increased lipid peroxidation in all except WT mice (P < 0.05). Increased steatosis and mRNA expression of the inflammatory markers CXCL2, tumor necrosis factor-α (TNF-α), and α-smooth muscle actin (α-SMA) were observed in EtOH GSTA4-/- compared with EtOH WT mice (P < 0.05). EtOH PPAR-α-/- mice had increased steatosis, serum alanine aminotransferase (ALT), and hepatic CD3+ T cell populations and elevated mRNA encoding CD14, CXCL2, TNF-α, IL-6, CD138, transforming growth factor-ß, platelet-derived growth factor receptor-ß (PDGFR-ß), matrix metalloproteinase (MMP)-9, MMP-13, α-SMA, and collagen type 1 compared with EtOH WT mice. EtOH-fed dKO mice displayed elevation of periportal hepatic 4-hydroxynonenal adducts and serum antibodies against malondialdehyde adducts compared with EtOH feeding of GSTA4-/-, PPAR-α-/-, and WT mice (P < 0.05). ALT was higher in EtOH dKO mice compared with all other groups (P < 0.001). EtOH-fed dKO mice displayed elevated mRNAs for TNF-α and CD14, histological evidence of fibrosis, and increased PDGFR, MMP-9, and MMP-13 mRNAs compared with the EtOH GSTA4-/- or EtOH PPAR-α-/- genotype (P < 0.05). These findings demonstrate the central role lipid peroxidation plays in mediating progression of alcohol-induced necroinflammatory liver injury, stellate cell activation, matrix remodeling, and fibrosis.
Asunto(s)
Aldehídos/metabolismo , Glutatión Transferasa/metabolismo , Peroxidación de Lípido , Hepatopatías Alcohólicas/metabolismo , PPAR alfa/metabolismo , Actinas/genética , Actinas/metabolismo , Alanina Transaminasa/sangre , Aldehídos/inmunología , Animales , Anticuerpos/sangre , Quimiocina CXCL2/genética , Quimiocina CXCL2/metabolismo , Citocinas/genética , Citocinas/metabolismo , Fibrosis/metabolismo , Eliminación de Gen , Glutatión Transferasa/genética , Receptores de Lipopolisacáridos/genética , Receptores de Lipopolisacáridos/metabolismo , Hígado/metabolismo , Hígado/patología , Hepatopatías Alcohólicas/inmunología , Masculino , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Ratones , PPAR alfa/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Patients with severe burns are highly susceptible to bacterial infection. While immunosuppression facilitates infection, the contribution of soft tissues to infection beyond providing a portal for bacterial entry remains unclear. We showed previously that glutathione S-transferase S1 (gstS1), an enzyme with conjugating activity against the lipid peroxidation byproduct 4-hydroxynonenal (4HNE), is important for resistance against wound infection in Drosophila muscle. The importance of the mammalian functional counterpart of GstS1 in the context of wounds and infection has not been investigated. Here we demonstrate that the presence of a burn wound dramatically affects expression of both human (hGSTA4) and mouse (mGsta4) 4HNE scavengers. hGSTA4 is down-regulated significantly within 1 wk of thermal burn injury in the muscle and fat tissues of patients from the large-scale collaborative Inflammation and the Host Response to Injury multicentered study. Similarly, mGsta4, the murine GST with the highest catalytic efficiency for 4HNE, is down-regulated to approximately half of normal levels in mouse muscle immediately postburn. Consequently, 4HNE protein adducts are increased 4- to 5-fold in mouse muscle postburn. Using an open wound infection model, we show that deletion of mGsta4 renders mice more susceptible to infection with the prevalent wound pathogen Pseudomonas aeruginosa, while muscle hGSTA4 expression negatively correlates with burn wound infection episodes per patient. Our data suggest that hGSTA4 down-regulation and the concomitant increase in 4HNE adducts in human muscle are indicative of susceptibility to infection in individuals with severely thermal injuries.
Asunto(s)
Infecciones Bacterianas/enzimología , Infecciones Bacterianas/etiología , Quemaduras/complicaciones , Quemaduras/enzimología , Glutatión Transferasa/metabolismo , Músculo Esquelético/enzimología , Infección de Heridas/enzimología , Infección de Heridas/etiología , Aldehídos/metabolismo , Animales , Infecciones Bacterianas/genética , Secuencia de Bases , Quemaduras/genética , Estudios de Casos y Controles , Cartilla de ADN/genética , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Regulación hacia Abajo , Femenino , Glutatión Transferasa/deficiencia , Glutatión Transferasa/genética , Humanos , Estudios Longitudinales , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Estudios Prospectivos , Infecciones por Pseudomonas/enzimología , Infecciones por Pseudomonas/etiología , Infecciones por Pseudomonas/genética , Infección de Heridas/genéticaRESUMEN
This review begins with the premise that an organism's life span is determined by the balance between two countervailing forces: (i) the sum of destabilizing effects and (ii) the sum of protective longevity-assurance processes. Against this backdrop, the role of electrophiles is discussed, both as destabilizing factors and as signals that induce protective responses. Because most biological macromolecules contain nucleophilic centers, electrophiles are particularly reactive and toxic in a biological context. The majority of cellular electrophiles are generated from polyunsaturated fatty acids by a peroxidation chain reaction that is readily triggered by oxygen-centered radicals, but propagates without further input of reactive oxygen species (ROS). Thus, the formation of lipid-derived electrophiles such as 4-hydroxynon-2-enal (4-HNE) is proposed to be relatively insensitive to the level of initiating ROS, but to depend mainly on the availability of peroxidation-susceptible fatty acids. This is consistent with numerous observations that life span is inversely correlated to membrane peroxidizability, and with the hypothesis that 4-HNE may constitute the mechanistic link between high susceptibility of membrane lipids to peroxidation and shortened life span. Experimental interventions that directly alter membrane composition (and thus their peroxidizability) or modulate 4-HNE levels have the expected effects on life span, establishing that the connection is not only correlative but causal. Specific molecular mechanisms are considered, by which 4-HNE could (i) destabilize biological systems via nontargeted reactions with cellular macromolecules and (ii) modulate signaling pathways that control longevity-assurance mechanisms.
Asunto(s)
Envejecimiento/fisiología , Estrés Oxidativo , Aldehídos/metabolismo , Animales , Humanos , Peroxidación de Lípido , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
Many lifespan-modulating genes are involved in either generation of oxidative substrates and end-products, or their detoxification and removal. Among such metabolites, only lipoperoxides have the ability to produce free-radical chain reactions. For this study, fatty-acid profiles were compared across a panel of C. elegans mutants that span a tenfold range of longevities in a uniform genetic background. Two lipid structural properties correlated extremely well with lifespan in these worms: fatty-acid chain length and susceptibility to oxidation both decreased sharply in the longest-lived mutants (affecting the insulinlike-signaling pathway). This suggested a functional model in which longevity benefits from a reduction in lipid peroxidation substrates, offset by a coordinate decline in fatty-acid chain length to maintain membrane fluidity. This model was tested by disrupting the underlying steps in lipid biosynthesis, using RNAi knockdown to deplete transcripts of genes involved in fatty-acid metabolism. These interventions produced effects on longevity that were fully consistent with the functions and abundances of their products. Most knockdowns also produced concordant effects on survival of hydrogen peroxide stress, which can trigger lipoperoxide chain reactions.
Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Lípidos/biosíntesis , Longevidad/fisiología , Estrés Oxidativo , Animales , Caenorhabditis elegans/efectos de los fármacos , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Peróxido de Hidrógeno/farmacología , Oxidantes/farmacología , Interferencia de ARN , Tasa de Supervivencia , Transcripción GenéticaAsunto(s)
Aldehídos/farmacología , Inhibidores Enzimáticos/farmacología , Ácidos Grasos/metabolismo , Obesidad/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Aconitato Hidratasa/antagonistas & inhibidores , Aconitato Hidratasa/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Ácido Cítrico/metabolismo , Ácidos Grasos/biosíntesisRESUMEN
The lipid peroxidation product 4-hydroxynonenal (4-HNE) forms as a consequence of oxidative stress. By electrophilic attack on biological macromolecules, 4-HNE mediates signaling or may cause toxicity. A major route of 4-HNE disposal is via glutathione conjugation, in the mouse catalyzed primarily by glutathione transferase mGSTA4-4. Unexpectedly, mGsta4-null mice, in which 4-HNE detoxification is impaired, have an extended life span. This finding could be explained by the observed activation of the transcription factor Nrf2 in the knockout mice, which in turn leads to an induction of antioxidant and antielectrophilic defenses. Especially, the latter could provide a detoxification mechanism that contributes to enhanced longevity. We propose that disruption of 4-HNE conjugation elicits a hormetic response in which an initially increased supply of 4-HNE is translated into activation of Nrf2, leading to a new steady state in which the rise of 4-HNE concentrations is dampened, but life-extending detoxification mechanisms are concomitantly induced.
Asunto(s)
Aldehídos/farmacología , ADN/genética , Expresión Génica , Glutatión Transferasa/genética , Longevidad/genética , Animales , Inhibidores de Cisteína Proteinasa/farmacología , Genotipo , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Reacción en Cadena de la PolimerasaRESUMEN
BACKGROUND: Of the five human Alpha-class glutathione transferases, expression of hGSTA5 has not been experimentally documented, even though in silico the hGSTA5 sequence can be assembled into a mRNA and translated. The present work was undertaken to determine whether hGSTA5 is functional. METHODS: Human K562 cells were transfected with the hGSTA5 gene driven by the CMV promoter, and hGSTA5 cDNA was recovered from mature mRNA by reverse transcription. The cDNA was used in bacterial and eukaryotic protein expression systems. The resulting protein, after purification by glutathione affinity chromatography where appropriate, was tested for glutathione transferase activity. RESULTS: Human K562 cells transfected with the hGSTA5 gene under control of a CMV promoter produced a fully spliced mRNA which, after reverse transcription and expression in E. coli, yielded a protein that catalyzed the conjugation of the lipid peroxidation product 4-hydroxynonenal to glutathione. Similarly, transfection of human HEK-293 cells with the hGSTA5 gene driven by the CMV promoter led to an elevated 4-hydroxynonenal-conjugating activity in the cell lysate. In addition, translation of hGSTA5 cDNA in a cell-free eukaryotic system gave rise to a protein with 4-hydroxynonenal-conjugating activity. CONCLUSIONS: hGSTA5 can be processed to a mature mRNA which is translation-competent, producing a catalytically active enzyme. GENERAL SIGNIFICANCE: Because a functional gene would not be maintained in the absence of selective pressure, we conclude that the native hGSTA5 promoter is active but has a spatially or temporally restricted expression pattern, and/or is expressed only under specific (patho)physiological conditions.
Asunto(s)
Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Aldehídos/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Catálisis , Línea Celular , Cromatografía de Afinidad , Clonación Molecular , Electroforesis , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Glutatión/metabolismo , Humanos , Células K562 , Datos de Secuencia Molecular , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
BACKGROUND: The C-terminus of the serotonin transporter (SERT) contains binding domains for different proteins and is critical for its functional expression. In endogenous and heterologous expression systems, our proteomic and biochemical analysis demonstrated that an intermediate filament, vimentin, binds to the C-terminus of SERT. It has been reported that 5HT-stimulation of cells leads to disassembly and spatial reorientation of vimentin filaments. METHODOLOGY/PRINCIPAL FINDINGS: We tested the impact of 5HT-stimulation on vimentin-SERT association and found that 5HT-stimulation accelerates the translocation of SERT from the plasma membrane via enhancing the level of association between phosphovimentin and SERT. Furthermore a progressive truncation of the C-terminus of SERT was performed to map the vimentin-SERT association domain. Deletion of up to 20, but not 14 amino acids arrested the transporters at intracellular locations. Although, truncation of the last 14 amino acids, did not alter 5HT uptake rates of transporter but abolished its association with vimentin. To understand the involvement of 5HT in phosphovimentin-SERT association from the plasma membrane, we further investigated the six amino acids between Delta14 and Delta20, i.e., the SITPET sequence of SERT. While the triple mutation on the possible kinase action sites, S(611), T(613), and T(616) arrested the transporter at intracellular locations, replacing the residues with aspartic acid one at a time altered neither the 5HT uptake rates nor the vimentin association of these mutants. However, replacing the three target sites with alanine, either simultaneously or one at a time, had no significant effect on 5HT uptake rates or the vimentin association with transporter. CONCLUSIONS/SIGNIFICANCE: Based on our findings, we propose that phosphate modification of the SITPET sequence differentially, one at a time exposes the vimentin binding domain on the C-terminus of SERT. Conversely, following 5HT stimulation, the association between vimentin-SERT is enhanced which changes the cellular distribution of SERT on an altered vimentin network.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismo , Serotonina/farmacología , Vimentina/metabolismo , Biotinilación , Plaquetas , Western Blotting , Células Cultivadas , Cromatografía de Afinidad , Técnica del Anticuerpo Fluorescente , Humanos , Mutación/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Proteínas de Transporte de Serotonina en la Membrana Plasmática/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Vimentina/genéticaRESUMEN
4-Hydroxynonenal (4-HNE) has been suggested to be involved in stress-induced signaling for apoptosis. In present studies, we have examined the effects of 4-HNE on the intrinsic apoptotic pathway associated with p53 in human retinal pigment epithelial (RPE and ARPE-19) cells. Our results show that 4-HNE causes induction, phosphorylation, and nuclear accumulation of p53 which is accompanied with down regulation of MDM2, activation of the pro-apoptotic p53 target genes viz. p21 and Bax, JNK, caspase3, and onset of apoptosis in treated RPE cells. Reduced expression of p53 by an efficient silencing of the p53 gene resulted in a significant resistance of these cells to 4-HNE-induced cell death. The effects of 4-HNE on the expression and functions of p53 are blocked in GSTA4-4 over expressing cells indicating that 4-HNE-induced, p53-mediated signaling for apoptosis is regulated by GSTs. Our results also show that the induction of p53 in tissues of mGsta4 (-/-) mice correlate with elevated levels of 4-HNE due to its impaired metabolism. Together, these studies suggest that 4-HNE is involved in p53-mediated signaling in in vitro cell cultures as well as in vivo that can be regulated by GSTs.
Asunto(s)
Aldehídos/farmacología , Glutatión Transferasa/fisiología , Epitelio Pigmentado de la Retina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis , Glutatión Transferasa/metabolismo , Humanos , Peroxidación de Lípido , Ratones , Ratones Noqueados , Modelos Biológicos , Estrés Oxidativo , Fosforilación , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
It is widely (although not universally) accepted that organismal aging is the result of two opposing forces: (i) processes that destabilize the organism and increase the probability of death, and (ii) longevity assurance mechanisms that prevent, repair, or contain damage. Processes of the first group are often chemical and physico-chemical in nature, and are either inevitable or only under marginal biological control. In contrast, protective mechanisms are genetically determined and are subject to natural selection. Life span is therefore largely dependent on the investment into protective mechanisms which evolve to optimize reproductive fitness. Recent data indicate that toxicants, both environmental and generated endogenously by metabolism, are major contributors to macromolecular damage and physiological dysregulation that contribute to aging; electrophilic carbonyl compounds derived from lipid peroxidation appear to be particularly important. As a consequence, detoxification mechanisms, including the removal of electrophiles by glutathione transferase-catalyzed conjugation, are major longevity assurance mechanisms. The expression of multiple detoxification enzymes, each with a significant but relatively modest effect on longevity, is coordinately regulated by signaling pathways such as insulin/insulin-like signaling, explaining the large effect of such pathways on life span. The major aging-related toxicants and their cognate detoxification systems are discussed in this review.
Asunto(s)
Envejecimiento/metabolismo , Fase I de la Desintoxicación Metabólica/fisiología , Homeostasis/fisiología , Humanos , Longevidad/fisiología , Mutación/fisiologíaRESUMEN
The lipid peroxidation product 4-hydroxynonenal (4-HNE) is a signaling mediator with wide-ranging biological effects. In this paper, we report that disruption of mGsta4, a gene encoding the 4-HNE-conjugating enzyme mGSTA4-4, causes increased 4-HNE tissue levels and is accompanied by age-dependent development of obesity which precedes the onset of insulin resistance in 129/sv mice. In contrast, mGsta4 null animals in the C57BL/6 genetic background have normal 4-HNE levels and remain lean, indicating a role of 4-HNE in triggering or maintaining obesity. In mGsta4 null 129/sv mice, the expression of the acetyl-CoA carboxylase (ACC) transcript is enhanced several-fold with a concomitant increase in the tissue level of malonyl-CoA. Also, mitochondrial aconitase is partially inhibited, and tissue citrate levels are increased. Accumulation of citrate could lead to allosteric activation of ACC, further augmenting malonyl-CoA levels. Aconitase may be inhibited by 4-HNE or by peroxynitrite generated by macrophages which are enriched in white adipose tissue of middle-aged mGsta4 null 129/sv mice and, upon lipopolysaccharide stimulation, produce more reactive oxygen species and nitric oxide than macrophages from wild-type mice. Excessive malonyl-CoA synthesized by the more abundant and/or allosterically activated ACC in mGsta4 null mice leads to fat accumulation by the well-known mechanisms of promoting fatty acid synthesis and inhibiting fatty acid beta-oxidation. Our findings complement the recent report that obesity causes both a loss of mGSTA4-4 and an increase in the level of 4-HNE [Grimsrud, P. A., et al. (2007) Mol. Cell. Proteomics 6, 624-637]. The two reciprocal processes are likely to establish a positive feedback loop that would promote and perpetuate the obese state.
Asunto(s)
Aldehídos/metabolismo , Glutatión Transferasa/fisiología , Peroxidación de Lípido/fisiología , Obesidad/inducido químicamente , Acetil-CoA Carboxilasa/metabolismo , Aconitato Hidratasa/metabolismo , Envejecimiento/fisiología , Aldehídos/farmacología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Glucemia/metabolismo , Ácido Cítrico/metabolismo , Femenino , Prueba de Tolerancia a la Glucosa , Glutatión Transferasa/deficiencia , Resistencia a la Insulina/fisiología , Masculino , Malonil Coenzima A/metabolismo , Ratones , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Previously, we have shown that 4-hydroxynonenal (4-HNE) induces Fas-mediated apoptosis in HLE B-3 cells through a pathway which is independent of FasL, FADD, procaspase 8, and DISC (Li, J., et al. (2006) Biochemistry 45, 12253-12264). The involvement of Daxx has also been suggested in this pathway, but its role is not clear. Here, we report that Daxx plays an important regulatory role during 4-HNE-induced, Fas-mediated apoptosis in Jurkat cells. 4-HNE induces Fas-dependent apoptosis in procaspase 8-deficient Jurkat cells via the activation of ASK1, JNK, and caspase 3, and the apoptosis can be inhibited by masking Fas with the antagonistic anti-Fas antibodies. We demonstrate that 4-HNE exposure to Jurkat cells leads to the induction of both Fas and Daxx. 4-HNE binds to both Fas and Daxx and promotes the export of Daxx from the nucleus to the cytosol, where it binds to Fas and inhibits apoptosis. Depletion of Daxx results in an increase in the activation of ASK1, JNK, and caspase 3 along with exacerbation of 4-HNE-induced apoptosis, suggesting that Daxx inhibits apoptosis by binding to Fas. 4-HNE-induced translocation of Daxx is also accompanied by the activation of the transcription factor HSF1. The results of these studies are consistent with a model in which, by interacting with Fas, 4-HNE promotes proapoptotic signaling via ASK1, JNK, and caspase 3. In parallel, 4-HNE induces Daxx and promotes its export from the nucleus to the cytosol, where it interacts with Fas to self-limit the extent of apoptosis by inhibiting the downstream proapoptotic signaling. Cytoplasmic translocation of Daxx also results in up-regulation of HSF1-associated stress-responsive genes.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Aldehídos/farmacología , Apoptosis/efectos de los fármacos , Núcleo Celular/metabolismo , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte/metabolismo , Proteína Ligando Fas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Antracenos/farmacología , Western Blotting , Caspasa 3/metabolismo , Caspasa 8/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Proteínas Co-Represoras , Citosol/efectos de los fármacos , Citosol/metabolismo , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas HSP70 de Choque Térmico/metabolismo , Factores de Transcripción del Choque Térmico , Humanos , Inmunoprecipitación , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Células Jurkat , MAP Quinasa Quinasa Quinasa 5/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Chaperonas Moleculares , Proteínas Nucleares/genética , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/genética , Factores de Transcripción/metabolismoRESUMEN
Deposition and mobilization of fat in an organism are tightly controlled by multiple levels of endocrine and neuroendocrine regulation. Because these hormonal mechanisms ultimately act by affecting biochemical reactions of fat synthesis or utilization, obesity could be also modulated by altering directly the underlying lipid biochemistry. We have previously shown that genetically modified mice with an elevated level of the lipid peroxidation product 4-HNE become obese. We now demonstrate that the process is phylogenetically conserved and thus likely to be universal. In the nematode C. elegans, disruption of either conjugation or oxidation of 4-HNE leads to fat accumulation, whereas augmentation of 4-HNE conjugation results in a lean phenotype. Moreover, direct treatment of C. elegans with synthetic 4-HNE causes increased lipid storage, directly demonstrating a causative role of 4-HNE. The postulated mechanism, which involves modulation of acetyl-CoA carboxylase activity, could contribute to the triggering and maintenance of the obese phenotype on a purely metabolic level.
Asunto(s)
Tejido Adiposo/metabolismo , Aldehídos/metabolismo , Caenorhabditis elegans/metabolismo , Metabolismo de los Lípidos/fisiología , Tejido Adiposo/efectos de los fármacos , Aldehído Deshidrogenasa/genética , Aldehídos/farmacología , Animales , Caenorhabditis elegans/efectos de los fármacos , Ácido Cítrico/metabolismo , Ácidos Grasos/metabolismo , Glutatión/metabolismo , Glutatión Transferasa/genética , Metabolismo de los Lípidos/efectos de los fármacos , Peróxidos Lipídicos/metabolismo , Malonil Coenzima A/metabolismo , Oxidación-Reducción , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genética , Interferencia de ARNRESUMEN
The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) forms as a consequence of oxidative stress, and acts as a signaling molecule or, at superphysiological levels, as a toxicant. The steady-state concentration of the compound reflects the balance between its generation and its metabolism, primarily through glutathione conjugation. Using an RNAi-based screen, we identified in Caenorhabditis elegans five glutathione transferases (GSTs) capable of catalyzing 4-HNE conjugation. RNAi knock-down of these GSTs (products of the gst-5, gst-6, gst-8, gst-10, and gst-24 genes) sensitized the nematode to electrophilic stress elicited by exposure to 4-HNE. However, interference with the expression of only two of these genes (gst-5 and gst-10) significantly shortened the life span of the organism. RNAi knock-down of the other GSTs resulted in at least as much 4-HNE adducts, suggesting tissue specificity of effects on longevity. Our results are consistent with the oxidative stress theory of organismal aging, broadened by considering electrophilic stress as a contributing factor. According to this extended hypothesis, peroxidation of lipids leads to the formation of 4-HNE in a chain reaction which amplifies the original damage. 4-HNE then acts as an "aging effector" via the formation of 4-HNE-protein adducts, and a resulting change in protein function.
Asunto(s)
Aldehídos/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Glutatión Transferasa/metabolismo , Longevidad/fisiología , Aldehídos/química , Aldehídos/farmacología , Animales , Caenorhabditis elegans/química , Caenorhabditis elegans/efectos de los fármacos , Proteínas de Caenorhabditis elegans/genética , Glutatión Transferasa/genética , Interferencia de ARN , Estrés PsicológicoRESUMEN
The Fas (apo/CD95) receptor which belongs to the TNF-alpha family is a transmembrane protein involved in the signaling for apoptosis through the extrinsic pathway. During this study, we have examined a correlation between intracellular levels of 4-HNE and expression of Fas in human lens epithelial (HLE B-3) cells. Our results show that in HLE B-3 cells, Fas is induced by 4-HNE in a concentration- and time-dependent manner, and it is accompanied by the activation of JNK, caspase 3, and the onset of apoptosis. Fas induction and activation of JNK are also observed in various tissues of mGsta4 null mice which have elevated levels of 4-HNE. Conversely, when 4-HNE is depleted in HLE B-3 cells by a transient transfection with hGSTA4, Fas expression is suppressed. However, upon the cessation of hGSTA4 expression in these transiently transfected cells, Fas and 4-HNE return to their basal levels. Fas-deficient transformed HLE B-3 cells stably transfected with hGSTA4 show remarkable resistance to apoptosis. Also, the wild-type HLE B-3 cells in which Fas is partially depleted by siRNA acquire resistance to 4-HNE-induced apoptosis, suggesting an at least partial role of Fas in 4-HNE-induced apoptosis in HLE B-3 cells. We also demonstrate that during 4-HNE-induced apoptosis of HLE B-3 cells, Daxx is induced and it binds to Fas. Together, these results show an important role of 4-HNE in regulation of the expression and functions of Fas.
Asunto(s)
Aldehídos/metabolismo , Apoptosis/fisiología , Transducción de Señal/fisiología , Receptor fas/genética , Aldehídos/farmacología , Animales , Caspasa 3 , Caspasas/metabolismo , Transformación Celular Viral , Células Cultivadas , Regulación hacia Abajo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Cristalino , MAP Quinasa Quinasa 4/metabolismo , Ratones , Receptor fas/biosíntesisRESUMEN
Glutathione S-transferases (GSTs) play a key role in cellular detoxification of environmental toxicants through their conjugation to glutathione (GSH). Recent studies have shown that the alpha-class GSTs also provide protection against oxidative stress and lipid peroxidation (LPO). GSTA4-4 is a member of a sub group of the alpha-class GSTs. It has been shown to metabolize 4-hydroxynonenal (4-HNE) with high catalytic efficiency through its conjugation to glutathione (GSH) and has been suggested to be a major component of cellular defense against toxic electrophiles such as 4-HNE generated during LPO. Since the hepatotoxicity of carbon tetrachloride (CCl(4)) has been suggested to be due to the generation of free radicals leading to membrane LPO, the present studies were designed to compare hepatotoxicity of CCl(4) in GSTA4-4 null (-/-) and wild type (+/+) mice. The results show that administration of a single dose of CCl(4) (1 ml/kg i.p.) resulted in time dependent hepatotoxicity in both -/- and +/+ mice; the extent of cellular damage by serum enzymes suggests that progression was more rapid in -/- mice, although injury was similar by 24 h. Histopathologic examination showed similar degrees of centrilobular necrosis by 24 h but much greater surrounding degenerative change, including cellular swelling, disarray, and vacuolization, in the liver of -/- mice. As expected -/- mice did not show any expression of mGSTA4-4; after CCl(4) a compensatory increase in the activities of total GST activity was noted at 24 h. Major alterations in other antioxidant enzymes was not observed. 4-HNE levels in the liver of -/- mice were about four-fold higher than in +/+ mice, suggesting a positive correlation between 4-HNE levels and the altered course of CCl(4) hepatotoxicity. These studies suggest that GSTA4-4 is an important component during the early stages (1-6 h) of cellular defense against oxidative stress and LPO although, it is not effective in protecting against the ultimate degree of overall cell injury.