RESUMO
NF-E2-related factor 2 (Nrf2) is a transcriptional regulator of biologic defense proteins, such as antioxidant proteins and phase II detoxification enzymes. Cytochrome P450 (P450) enzymes have been shown to regulate phase I metabolism of various drugs and are partially regulated by Nrf2; however, the influence of Nrf2 on drug pharmacokinetics is not known. Here, we showed that Nrf2 depletion prolonged the effect of pentobarbital, a sleep-promoting drug. Pretreatment with phenobarbital, a P450 inducer, shortens the sleeping time associated with pentobarbital-induced sedation in wild-type (WT) mice; however, this effect was not observed in Nrf2-/- mice. Furthermore, the blood pentobarbital concentration was higher in Nrf2-/- mice than in WT mice at 30-60 minutes, and the phenobarbital-induced enhancement of its clearance was attenuated in Nrf2-/- mice compared with WT mice. Total P450 content was decreased in Nrf2-/- mouse livers, and the phenobarbital-induced increase in P450 content was lower in Nrf2-/- mice than WT mice. Cyp1a2, Cyp2a5, Cyp2c29, and Cyp2e1 gene expression levels under physiologic conditions and Cyp1a2, Cyp2a5, and Cyp2b10 gene expression levels under phenobarbital-treated conditions were lower in Nrf2-/- mice compared with WT mice. Additionally, pentobarbital metabolism in liver microsomes was attenuated by Nrf2 depletion. Taken together, these findings suggested that Nrf2 influenced pentobarbital pharmacokinetics through the regulation of drug metabolism and P450 gene expression. Thus, Nrf2-mediated regulation of P450 may contribute to the biologic defense against increased reactive oxygen species production. SIGNIFICANCE STATEMENT: NF-E2-related factor 2 (Nrf2) plays a critical role in the cellular defense against oxidative stress. Nrf2-/- mice with reduced ability to eliminate reactive oxygen species (ROS) showed a significant delay in emergence from pentobarbital-induced sleep, which was associated with decreased P450 activities and gene expression. Our findings provide that Nrf2 dysfunction or ROS that exceed a threshold level of the eliminating ability of the Nrf2 system may reduce P450 activity.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Hipnóticos e Sedativos/farmacocinética , Fator 2 Relacionado a NF-E2/metabolismo , Pentobarbital/farmacocinética , Espécies Reativas de Oxigênio/metabolismo , Animais , Hipnóticos e Sedativos/administração & dosagem , Masculino , Camundongos , Camundongos Knockout , Modelos Animais , Fator 2 Relacionado a NF-E2/genética , Oxirredução , Estresse Oxidativo , Pentobarbital/administração & dosagemRESUMO
Vascular smooth muscle cell (VSMC) migration contributes to neointimal formation after vascular injury. We previously demonstrated that copper (Cu) transporter ATP7A is involved in platelet-derived growth factor (PDGF)-induced VSMC migration in a Cu- and Rac1-dependent manner. The underlying mechanism is still unknown. Here we show that ATP7A interacts with IQGAP1, a Rac1 and receptor tyrosine kinase binding scaffolding proteins, which mediates PDGF-induced VSMC migration and vascular remodeling. In cultured rat aortic SMCs, PDGF stimulation rapidly promoted ATP7A association with IQGAP1 and Rac1 and their translocation to the lipid rafts and leading edge. Cotransfection assay revealed that ATP7A directly bound to NH2-terminal domain of IQGAP1. Functionally, either ATP7A or IQGAP1 depletion using siRNA significantly inhibited PDGF-induced VSMC migration without additive effects, suggesting that IQGAP1 and ATP7A are in the same axis to promote migration. Furthermore, IQGAP1 siRNA blocked PDGF-induced ATP7A association with Rac1 as well as its translocation to leading edge, while PDGF-induced IQGAP1 translocation was not affected by ATP7A siRNA or Cu chelator. Overexpression of mutant IQGAP1 lacking a Rac1 binding site prevented PDGF-induced translocation of Rac1, but not ATP7A, to the leading edge, thereby inhibiting lamellipodia formation and VSMC migration. In vivo, ATP7A colocalized with IQGAP1 at neointimal VSMCs in a mice wire injury model, while neointimal formation and extracellular matrix deposition induced by vascular injury were inhibited in ATP7A mutant mice with reduced Cu transporter function. In summary, IQGAP1 functions as ATP7A and Rac1 binding scaffolding protein to organize PDGF-dependent ATP7A translocation to the lamellipodial leading edge, thereby promoting VSMC migration and vascular remodeling.
Assuntos
ATPases Transportadoras de Cobre/genética , Fator de Crescimento Derivado de Plaquetas/genética , Remodelação Vascular/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas Ativadoras de ras GTPase/genética , Animais , Aorta/citologia , Aorta/metabolismo , Movimento Celular/genética , Cobre/química , Cobre/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Camundongos , Músculo Liso Vascular/crescimento & desenvolvimento , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima/genética , Fosforilação , Ligação Proteica , RatosRESUMO
OBJECTIVE: Reactive oxygen species are important mediators for platelet-derived growth factor (PDGF) signaling in vascular smooth muscle cells, whereas excess reactive oxygen species-induced oxidative stress contributes to the development and progression of vascular diseases, such as atherosclerosis. Activation of the redox-sensitive transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2), is pivotal in cellular defense against oxidative stress by transcriptional upregulation of antioxidant proteins. This study aimed to elucidate the role of Nrf2 in PDGF-mediated vascular smooth muscle cell migration and neointimal hyperplasia. APPROACH AND RESULTS: PDGF promoted nuclear translocation of Nrf2, followed by the induction of target genes, including NAD(P)H:quinone oxidoreductase-1, heme oxygenase-1, and thioredoxin-1. Nrf2 depletion by small interfering RNA enhanced PDGF-promoted Rac1 activation and reactive oxygen species production and persistently phosphorylated downstream extracellular signal-regulated kinase-1/2. Nrf2 depletion enhanced vascular smooth muscle cell migration in response to PDGF and wound scratch. In vivo, Nrf2-deficient mice showed enhanced neointimal hyperplasia in a wire injury model. CONCLUSIONS: These findings suggest that the Nrf2 system is important for PDGF-stimulated vascular smooth muscle cell migration by regulating reactive oxygen species elimination, which may contribute to neointimal hyperplasia after vascular injury. Our findings provide insight into the Nrf2 system as a novel therapeutic target for vascular remodeling and atherosclerosis.
Assuntos
Movimento Celular , Proliferação de Células , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neointima , Lesões do Sistema Vascular/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Antioxidantes/farmacologia , Becaplermina , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Artéria Femoral/lesões , Artéria Femoral/metabolismo , Artéria Femoral/patologia , Heme Oxigenase (Desciclizante)/metabolismo , Hiperplasia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , NAD(P)H Desidrogenase (Quinona)/metabolismo , Fator 2 Relacionado a NF-E2/deficiência , Fator 2 Relacionado a NF-E2/genética , Oxirredução , Fosforilação , Proteínas Proto-Oncogênicas c-sis/metabolismo , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Tiorredoxinas/metabolismo , Fatores de Tempo , Transfecção , Regulação para Cima , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/patologiaRESUMO
OBJECTIVE: Vascular smooth muscle cell (VSMC) migration is critically important for neointimal formation after vascular injury and atherosclerosis lesion formation. Copper (Cu) chelator inhibits neointimal formation, and we previously demonstrated that Cu transport protein antioxidant-1 (Atox1) is involved in Cu-induced cell growth. However, role of Atox1 in VSMC migration and neointimal formation after vascular injury is unknown. APPROACH AND RESULTS: Here, we show that Atox1 expression is upregulated in injured vessel, and it is colocalized with the Cu transporter ATP7A, one of the downstream targets of Atox1, mainly in neointimal VSMCs at day 14 after wire injury. Atox1(-/-) mice show inhibition of neointimal formation and extracellular matrix expansion, which is associated with a decreased VSMCs accumulation within neointima and lysyl oxidase activity. Mechanistically, in cultured VSMC, Atox1 depletion with siRNA inhibits platelet-derived growth factor-induced Cu-dependent VSMC migration by preventing translocation of ATP7A and small G protein Rac1 to the leading edge, as well as Cu- and Rac1-dependent lamellipodia formation. Furthermore, Atox1(-/-) mice show decreased perivascular macrophage infiltration in wire-injured vessels, as well as thioglycollate-induced peritoneal macrophage recruitment. CONCLUSIONS: Atox1 is involved in neointimal formation after vascular injury through promoting VSMC migration and inflammatory cell recruitment in injured vessels. Thus, Atox1 is a potential therapeutic target for VSMC migration and inflammation-related vascular diseases.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Chaperonas Moleculares/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima , Lesões do Sistema Vascular/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Transporte de Cátions/deficiência , Proteínas de Transporte de Cátions/genética , Movimento Celular , Células Cultivadas , Proteínas de Transporte de Cobre , ATPases Transportadoras de Cobre , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Artéria Femoral/lesões , Artéria Femoral/metabolismo , Artéria Femoral/patologia , Humanos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Knockout , Chaperonas Moleculares/genética , Músculo Liso Vascular/imunologia , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/patologia , Neuropeptídeos/metabolismo , Peritonite/induzido quimicamente , Peritonite/imunologia , Peritonite/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Transporte Proteico , Proteína-Lisina 6-Oxidase/metabolismo , Pseudópodes/metabolismo , Interferência de RNA , Ratos , Ratos Sprague-Dawley , Tioglicolatos , Fatores de Tempo , Transfecção , Regulação para Cima , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/imunologia , Lesões do Sistema Vascular/patologia , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTPRESUMO
Platelet-derived growth factor (PDGF) stimulates vascular smooth muscle cell (VSMC) migration and neointimal formation in response to injury. We previously identified IQ-domain GTPase-activating protein 1 (IQGAP1) as a novel VEGF receptor 2 binding scaffold protein involved in endothelial migration. However, its role in VSMC migration and neointimal formation in vivo is unknown. Here we show that PDGF stimulation rapidly promotes IQGAP1 association with PDGF receptor-ß (PDGFR) as well as IQGAP1 tyrosine phosphorylation in cultured VSMC. Overexpression or knockdown of IQGAP1 enhances or inhibits PDGFR autophosphorylation (p-PDGFR), respectively. Immunofluorescence and cell fractionation analysis reveals that PDGF-induced p-PDGFR localized in focal adhesions (FAs), but not caveolae/lipid rafts, is inhibited by IQGAP1 knockdown with siRNA. PDGF stimulation promotes IQGAP1 association with PDGFR/FA signaling protein complex. Functionally, IQGAP1 siRNA inhibits PDGF-induced FA formation as well as VSMC migration induced by PDGF. In vivo, IQGAP1 expression is markedly increased at neointimal VSMC in wire-injured femoral arteries. Mice lacking IQGAP1 exhibit impaired neointimal formation in response to vascular injury. In summary, IQGAP1, through interaction with PDGFR and FA signaling proteins, promotes activation of PDGFR in FAs as well as FA formation, which may contribute to VSMC migration and neointimal formation after injury. Our findings provide insight into IQGAP1 as a potential therapeutic target for vascular migration-related diseases.
Assuntos
Movimento Celular/fisiologia , Adesões Focais/fisiologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Neointima/patologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Animais , Células Cultivadas , Artéria Femoral/metabolismo , Artéria Femoral/fisiologia , Adesões Focais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Neointima/metabolismo , Fosforilação/fisiologia , Fator de Crescimento Derivado de Plaquetas/metabolismo , Ratos , Ratos Sprague-Dawley , Tirosina/metabolismo , Lesões do Sistema Vascular/metabolismo , Lesões do Sistema Vascular/patologiaRESUMO
Polyethylene glycol-23 glyceryl distearate (GDS-23), a diacylglycerol polyethylene glycol adduct, forms niosomes with a liposome-like structure and functions as an active ingredient in drug delivery systems. In addition, it upregulates antioxidant proteins such as heme oxygenase 1 and NAD(P)H-quinone dehydrogenase 1 in cells. However, the activation of nuclear factor E2-related factor-2 (Nrf2), which plays a role in inducing the expression of antioxidant proteins, and its protective effects induced by GDS-23 treatment against oxidative stress have not been elucidated. This study aimed at verifying the activation of Nrf2 by GDS-23 and clarifying its underlying mechanisms, and investigated whether GDS-23 protects against hydroquinone-induced cytotoxicity. Normal human epidermal keratinocytes were treated with GDS-23. Real-time reverse transcription-polymerase chain reaction, western blotting, and immunostaining were used to investigate the mechanism of Nrf2 activation, and neutral red assay was performed to evaluate cytotoxicity. GDS-23-treated cells showed an increase in antioxidant protein levels and stabilization of Nrf2 in the nucleus. During Nrf2 activation, p62, an autophagy-related adaptor protein, was phosphorylated at Ser349. Inhibition of the interaction between the phosphorylated p62 and Kelch-like ECH-associated protein 1 significantly suppressed the GDS-23-mediated induction of antioxidant protein expression. In addition, hydroquinone-induced cell toxicity was significantly attenuated by GDS-23. GDS-23 induced the intracellular antioxidant system by activating Nrf2 in a p62 phosphorylation-dependent manner without generating oxidative stress in the cells. GDS-23 may be applied as a multifunctional material for drug delivery system that enhances internal antioxidant systems.
Assuntos
Antioxidantes , Fator 2 Relacionado a NF-E2 , Humanos , Antioxidantes/metabolismo , Diglicerídeos/farmacologia , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Hidroquinonas/toxicidade , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Queratinócitos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Polietilenoglicóis/farmacologia , Polietilenoglicóis/metabolismoRESUMO
Hepatic multidrug transporters expressed on the canalicular membrane play a role in the hepatobiliary excretion of xenobiotics and endogenous substrates. The aim of this study was to elucidate the role of pro-inflammatory cytokines in the regulation of hepatic drug transporter expression after cecal ligation and puncture (CLP), a valuable tool for studying polymicrobial sepsis, and to compare CLP with lipopolysaccharide (LPS) treatment. CLP reduced the expression of Mdr2/Abcb4, Mrp2/Abcc2, Bsep/Abcb11, Bcrp/Abcg2, and Mate1/Slc47a1 mRNAs in wild-type (WT) mouse livers in a time-dependent manner up to 48 h postoperation. LPS also reduced the expression of all transporters in WT mouse livers 24 h posttreatment; thereafter, expression levels tended to return to normal by 48 h posttreatment. IL-6-/- mice exhibited inhibited downregulation of drug transporters following CLP, although IL-1-/- and TNFα-/- mice exhibited the reduced expression of all transporters in a manner similar to that found in WT mice. Compared with CLP, LPS treatment reduced the expression of all transporters in all cytokine-deficient mouse livers, except for the expression of Mrp2/Abcc2 in IL-6-/- mice. Overall, these findings suggest that IL-6 is major factor in the downregulation of hepatic multidrug transporters following the onset of polymicrobial sepsis but not after LPS treatment.
Assuntos
Interleucina-6 , Sepse , Animais , Camundongos , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Citocinas/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Ligadura , Lipopolissacarídeos/farmacologia , Fígado/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Neoplasias/metabolismo , Punções , Sepse/induzido quimicamente , Sepse/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Sepsis is a pathological condition that affects the metabolism of administered drugs, leading to changes in the duration and intensity of their intended efficacies. Proinflammatory cytokines downregulate the expression of cytochrome P450s (P450s). The effects of P450 expression under inflammatory conditions have been studied using prophlogistic substances such as lipopolysaccharide; however, few studies have focused on clinical models of sepsis. Here, we show that cecal ligation and puncture (CLP), an approach for the study of human polymicrobial sepsis, leads to the expression of interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor α (TNFα) at 24 h after the CLP operation. Following CLP, IL-6-/- mice exhibited markedly lower survival than WT mice. In addition, CLP led to the significant downregulation of Cyp2c29 and Cyp3a11 gene expression in IL-1α-/-/ß-/- (IL-1-/-) and TNFα-/- mice as well as in WT mice. In contrast, CLP elicited no significant effect on Cyp3a11 expression in IL-6-/- mice. Although CLP reduced the Cyp2c29 expression level in IL-6-/- mice, the expression of Cyp2c29 was lower in CLP-operated WT mice than in CLP-operated IL-6-/- mice. The reduction in the respective P450 protein levels and activities due to CLP-induced sepsis, reflected in the mRNA expression levels, was abolished by IL-6 depletion. Thus, CLP-induced sepsis downregulates P450 gene expression, particularly Cyp2c expression, and this effect is associated with IL-6 without affecting resistance to CLP-induced sepsis. These findings demonstrate the usefulness of CLP for studying the regulation of P450s and highlight IL-6 as a potential indicator of drug-metabolizing capacity under septic conditions.
Assuntos
Interleucina-6 , Sepse , Humanos , Camundongos , Animais , Interleucina-6/genética , Interleucina-6/metabolismo , Regulação para Baixo , Fator de Necrose Tumoral alfa/metabolismo , Punções , Ligadura , Expressão Gênica , Sepse/metabolismo , Ceco/metabolismo , Família 2 do Citocromo P450/genética , Família 2 do Citocromo P450/metabolismo , Proteínas de Membrana/metabolismo , Citocromo P-450 CYP3A/genéticaRESUMO
Olanzapine is widely used as a treatment for schizophrenia and other psychiatric disorders. Its metabolic side effects, including weight gain and hyperglycemia, are a clinical problem; however, their full mechanism is not yet clearly understood. Recently, it was reported that the accumulation of oxidative stress in the hypothalamus may cause obesity and diabetes mellitus. Epidemiologically, metabolic side effects are known to be more likely to occur in women. In the present study, we investigated and tested the hypothesis that olanzapine induces oxidative stress in the hypothalamus and induces metabolic side effects. We also examined its association with sex differences. Olanzapine was administered intraperitoneally to male and female C57BL/6 mice, and the expression levels of oxidative stress-responsible genes in the hypothalamus and cerebral cortex were measured by qRT-PCR. In addition, olanzapine was administered intraperitoneally to C57BL/6 and Nrf2 KO mice, and the expression level of total glutathione was measured. Gene expressions induced by the Keap1-Nrf2-regulated system showed different responses to olanzapine for each gene. Under the conditions of this experiment, cystine-glutamate transporter was decreased although heme oxygenase-1 and γ-glutamylcysteine synthetase were increased. It was also clear that these responses were not hypothalamus-specific. Long-term feeding with olanzapine suppressed weight gain in males but not females. No glucose intolerance was observed at 13 weeks of administration. Furthermore, deaths occurred only in females. In conclusion, this study failed to provide evidence that olanzapine induces oxidative stress in a hypothalamic-specific manner. Instead, sex differences were observed in response to long-term and high-dose olanzapine administration, suggesting that individual susceptibility to olanzapine toxicity occurred in female mice.
Assuntos
Antipsicóticos , Fator 2 Relacionado a NF-E2 , Feminino , Masculino , Camundongos , Animais , Olanzapina/toxicidade , Proteína 1 Associada a ECH Semelhante a Kelch , Caracteres Sexuais , Camundongos Endogâmicos C57BL , Aumento de Peso , Antipsicóticos/toxicidade , Antipsicóticos/uso terapêuticoRESUMO
RATIONALE: Copper, an essential nutrient, has been implicated in vascular remodeling and atherosclerosis with unknown mechanism. Bioavailability of intracellular copper is regulated not only by the copper importer CTR1 (copper transporter 1) but also by the copper exporter ATP7A (Menkes ATPase), whose function is achieved through copper-dependent translocation from trans-Golgi network (TGN). Platelet-derived growth factor (PDGF) promotes vascular smooth muscle cell (VSMC) migration, a key component of neointimal formation. OBJECTIVE: To determine the role of copper transporter ATP7A in PDGF-induced VSMC migration. METHODS AND RESULTS: Depletion of ATP7A inhibited VSMC migration in response to PDGF or wound scratch in a CTR1/copper-dependent manner. PDGF stimulation promoted ATP7A translocation from the TGN to lipid rafts, which localized at the leading edge, where it colocalized with PDGF receptor and Rac1, in migrating VSMCs. Mechanistically, ATP7A small interfering RNA or CTR small interfering RNA prevented PDGF-induced Rac1 translocation to the leading edge, thereby inhibiting lamellipodia formation. In addition, ATP7A depletion prevented a PDGF-induced decrease in copper level and secretory copper enzyme precursor prolysyl oxidase (Pro-LOX) in lipid raft fraction, as well as PDGF-induced increase in LOX activity. In vivo, ATP7A expression was markedly increased and copper accumulation was observed by synchrotron-based x-ray fluorescence microscopy at neointimal VSMCs in wire injury model. CONCLUSIONS: These findings suggest that ATP7A plays an important role in copper-dependent PDGF-stimulated VSMC migration via recruiting Rac1 to lipid rafts at the leading edge, as well as regulating LOX activity. This may contribute to neointimal formation after vascular injury. Our findings provide insight into ATP7A as a novel therapeutic target for vascular remodeling and atherosclerosis.
Assuntos
Adenosina Trifosfatases/fisiologia , Proteínas de Transporte de Cátions/fisiologia , Movimento Celular/fisiologia , Cobre/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Animais , Aterosclerose/enzimologia , Aterosclerose/patologia , Células Cultivadas , ATPases Transportadoras de Cobre , Humanos , Masculino , Microdomínios da Membrana/enzimologia , Microdomínios da Membrana/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and cholesterol 7α-hydroxylase (Cyp7a1) are rate-limiting enzymes for cholesterol biosynthesis and catabolism, respectively. Involvement of inflammatory cytokines, particularly interleukin-1 (IL-1), in alterations of HMGR and Cyp7a1 gene expression during development of lead nitrate (LN)-induced hypercholesterolemia was examined in IL-1α/ß-knockout (IL-1-KO) and wild-type (WT) mice. Lead nitrate treatment of WT mice led to not only a marked downregulation of the Cyp7a1 gene at 6-12 h, but also a significant upregulation of the HMGR gene at 12 h. However, such changes were not observed at significant levels in IL-1-KO mice, although a slight, transient downregulation of the Cyp7a1 gene and a minimal upregulation of the HMGR gene occurred at 6 h and 24 h, respectively. Consequently, LN treatment led to development of hypercholesterolemia at 24 h in WT mice, but not in IL-1-KO mice. Furthermore, in WT mice, significant LN-mediated increases were observed at 3-6 h in hepatic IL-1 levels, which can modulate gene expression of Cyp7a1 and HMGR. These findings indicate that, in mice, LN-mediated increases in hepatic IL-1 levels contribute, at least in part, to altered expressions of Cyp7a1 and HMGR genes, and eventually to hypercholesterolemia development.
Assuntos
Poluentes Ambientais/toxicidade , Hipercolesterolemia/induzido quimicamente , Interleucina-1/metabolismo , Chumbo/toxicidade , Nitratos/toxicidade , Animais , Colesterol/sangue , Colesterol 7-alfa-Hidroxilase/genética , Hidroximetilglutaril-CoA Redutases/genética , Hipercolesterolemia/metabolismo , Interleucina-1/deficiência , Interleucina-1/genética , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , RNA Mensageiro/metabolismo , Esterol 14-Desmetilase/genética , Fator de Necrose Tumoral alfa/genéticaRESUMO
Our previous study using interleukin-1α/ß-knockout (IL-1-KO) and wild-type (WT) mice demonstrated that IL-1 acts as a positive factor for constitutive gene expression of hepatic cytochrome P4507a1 (Cyp7a1). In this study, to clarify the role of IL-1 in the expression of the hepatic Cyp7a1 gene, we focused on Cyp7a1 transcriptional regulators such as α-fetoprotein transcription factor (FTF), liver X receptor α (LXRα), hepatocyte nuclear factor 4α (HNF4α) and small heterodimer partner (SHP) and examined the effects of IL-1 on their gene expression by real-time reverse-transcription polymerase chain reaction using IL-1-KO and WT mice. We observed no significant differences between sex-matched IL-1-KO and WT mice with regard to gene expression levels of FTF, LXRα, and HNF4α, all of which are positive transcriptional regulators for the Cyp7a1 gene. However, interindividual differences in hepatic FTF and LXRα expression were closely dependent on the gene expression level(s) of hepatic IL-1 and tumor necrosis factor-α (TNF-α), while interindividual differences in hepatic HNF4α were clearly correlated with the expression of IL-1, but not TNF-α. In contrast, the gene expression level of SHP, which is a negative transcriptional regulator of the Cyp7a1 gene through inhibition of FTF function, was higher in IL-1-KO mice than in sex-matched WT mice. These findings demonstrate that, like TNF-α, IL-1 positively controls the gene expression of Cyp7a1 transcriptional upregulators but, in contrast to the previously reported action of TNF-α, IL-1 also acts to downregulate SHP gene expression.
Assuntos
Colesterol 7-alfa-Hidroxilase/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Animais , Colesterol 7-alfa-Hidroxilase/biossíntese , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Expressão Gênica/fisiologia , Regulação da Expressão Gênica/fisiologia , Fator 4 Nuclear de Hepatócito/efeitos dos fármacos , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Fígado/metabolismo , Receptores X do Fígado , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores Nucleares Órfãos/efeitos dos fármacos , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , Receptores Citoplasmáticos e Nucleares/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Vascular smooth muscle cell (VSMC) migration contributes to vascular remodeling after injury, whereas oxidative stress generated through dysfunctional redox homeostasis induces hypermigration, leading to arteriosclerosis. Platelet-derived growth factor (PDGF)-induced reactive oxygen species (ROS) serve as intracellular signaling molecules in VSMCs. Reactive sulfur species (RSS) may serve as a biological defense system because of the antioxidative properties of highly nucleophilic sulfane sulfur. However, insufficient information is available on its function in PDGF-induced VSMC migration. Here we show that PDGF significantly increased the levels of intracellular sulfane sulfur and that intracellular sulfane sulfur donors, donor 5a and Na2S4, inhibited the increase in ROS levels in PDGF-treated VSMCs and inhibited their migration. Consistent with the migration results, sulfane sulfur donors inhibited Akt phosphorylation, a downstream signaling molecule in the PDGF cascade, without affecting the autophosphorylation of PDGF receptor-ß. Further, sulfane sulfur donors inhibited vinculin and paxillin recruitment to the leading edge of VSMCs in response to PDGF to decrease focal adhesion formation. These findings suggest that RSS are required for PDGF-stimulated VSMC migration through the regulation of the ROS-regulated Akt pathway, which may contribute to focal adhesion formation. Our findings provide insight into RSS as novel regulators of vascular redox homeostasis.
Assuntos
Músculo Liso Vascular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Enxofre/uso terapêutico , Movimento Celular , Humanos , Enxofre/farmacologiaRESUMO
We examined the role of hepatic interleukin (IL)-1alpha/beta in serum total cholesterol homeostasis using male and female IL-1-knockout (KO) mice and wild-type (WT) mice. Serum total cholesterol level was higher in males than in females in WT and KO mice. The difference between sexes was closely correlated with the difference in gene expression level of cholesterol 7alpha-hydroxylase (Cyp7a1), a rate-limiting enzyme for bile acid synthesis. No significant sex difference in gene expression level of 3-hydroxy-3-methylglutaryl-CoA reductase, a rate-limiting enzyme for cholesterol synthesis, was observed in WT mice. Interestingly, the gene expression level of hepatic Cyp7a1 was lower in KO mice than in sex-matched WT mice, while the serum total cholesterol level was the opposite. The present findings demonstrate that IL-1alpha and IL-1beta are positive regulators for the Cyp7a1 gene in steady-state mice and that Cyp7a1 is one of the factors that mediate the difference in serum total cholesterol level between sexes.
Assuntos
Colesterol 7-alfa-Hidroxilase/genética , Colesterol/sangue , Regulação da Expressão Gênica , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Fígado/enzimologia , Animais , Ácidos e Sais Biliares/biossíntese , Feminino , Homeostase/genética , Interleucina-1alfa/genética , Interleucina-1beta/genética , Masculino , Camundongos , Camundongos Knockout , Fatores SexuaisRESUMO
Heme oxygenase-1 (HO-1) is induced under infectious diseases in macrophages. We performed experiments using various gene deficient mouse-derived macrophages to determine a detailed induction mechanism of HO-1 by lipopolysaccharide (LPS) and the functional role of HO-1 induction in macrophages. LPS (1 microg/mL) maximally induced inducible nitric oxide synthase (iNOS) and HO-1 mRNAs in wild-type (WT) macrophages at 6h and 12h after treatment, respectively, and liberated tumor necrosis factor alpha (TNFalpha) from WT macrophages. LPS also induced iNOS and HO-1 in TNFalpha(-/-) macrophages, but not in iNOS(-/-) macrophages. Interestingly, although LPS strongly induced iNOS, it failed to induce HO-1 almost completely in nuclear-factor erythroid 2-related factor 2 (Nrf2)(-/-) macrophages. The LPS-induced iNOS gene expression was suppressed by pretreatment with HO-1 inducers, hemin and Co-protoporphyrin (CoPP), but not with HO-1 inhibitor, Sn-protoporphyrin in WT macrophages. In the Nrf2(-/-) macrophages, the ability of CoPP to induce HO-1 and its inhibitory effect on the LPS-induced iNOS gene expression were lower than seen in WT macrophages. The present findings suggest that HO-1 is induced via NO-induced nuclear translocation of Nrf2, and the enzymatic function of HO-1 inhibits the overproduction of NO in macrophages.
Assuntos
Retroalimentação Fisiológica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase-1/biossíntese , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Óxido Nítrico Sintase Tipo II/genética , Animais , Citocinas/metabolismo , Indução Enzimática/efeitos dos fármacos , Proteínas I-kappa B/metabolismo , Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Metaloporfirinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/metabolismo , Inibidor de NF-kappaB alfa , Óxido Nítrico Sintase Tipo II/deficiência , Fosforilação/efeitos dos fármacos , Protoporfirinas/farmacologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/deficiênciaRESUMO
Sepsis is an etiologically complex and often fatal inflammatory process involving a multitude of cytokine signaling pathways. Tumor necrosis factor α (TNFα) acts as a central regulator of the acute-phase inflammatory response by recruiting immune cells, including circulating monocyte/macrophages, to sites of infection or tissue damage. Inorganic polyphosphate (polyP), a linear polymer of orthophosphate residues, has been found in almost all cells and tissues, but its functions in immunity remain largely unknown. In this study, we show that pre- or post-treatment of mice with polyP150 (average chain length of 150 phosphate residues) markedly increases survival from lipopolysaccharide (LPS)-induced shock and inhibits macrophage recruitment to the liver and lungs, resulting in protection against tissue injury. In accord with these in vivo results, pretreatment of cultured peritoneal macrophages with polyP150 inhibited chemotaxis and actin polarization in response to TNFα. PolyP150 also inhibited phosphorylation of stress-activated protein kinases c-Jun N-terminal kinase (JNK) and p38, two downstream signaling molecules of the TNFα cascade, thereby preventing cyclooxygenase-2 gene expression by macrophages. These findings suggest that polyP150 inhibits recruitment of macrophages into organs by regulating the TNFα-JNK/p38 pathway, which may, in turn, protect against multi-organ dysfunction and lethality induced by LPS. Our findings identify polyP regulation as a novel therapeutic target for sepsis.
Assuntos
Lipopolissacarídeos/toxicidade , Macrófagos Peritoneais/efeitos dos fármacos , Polifosfatos/farmacologia , Choque Séptico/tratamento farmacológico , Animais , Quimiotaxia/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/patologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Masculino , Camundongos Endogâmicos , Óxido Nítrico Sintase Tipo II/genética , Fosfatos/química , Fosfatos/farmacologia , Polifosfatos/química , Choque Séptico/induzido quimicamente , Choque Séptico/mortalidade , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Rheumatoid arthritis is characterized by chronic inflammation of the synovial tissue. We examined the effect of interleukin (IL)-6 neutralization on the expression of cytochrome P450 or metallothionein-1/2 (metallothionein) during chronic phase inflammatory disease using rheumatoid arthritis model mice, human T-cell leukemia virus type I (HTLV-I) transgenic mice. Serum IL-6 concentrations of arthritis-developed HTLV-I transgenic mice were 129.9+/-26.1 pg/ml. Moreover, signal transducer and activator of transcription (STAT) 1/3 phosphorylations was observed in arthritic HTLV-I transgenic mouse livers. CYP3A11 mRNA was more strongly reduced by the development of arthritis in HTLV-I transgenic mouse livers as compared with CYP2C29 or CYP2E1 mRNAs. CYP3A protein and testosterone 6beta-hydroxylation activity also changed in a similar manner to the corresponding CYP3A11 mRNA level. On the other hand, metallothionein mRNA was significantly induced as compared with that of wild-type or non-arthritic mice. CYP3A suppression and metallothionein mRNA overexpression activity seen in the developed arthritic mice returned to the gene conditions of the non-arthritic HTLV-I transgenic mice by IL-6 antibody at 48 h after treatment. The present study has revealed that CYP3A11 and metallothionein expressions are affected by the release of IL-6 by arthritis and its systemic circulation, and neutralization of IL-6 recovered from the down-regulation of CYP3A11 mRNA and the induction of metallothionein mRNA in arthritic HTLV-I transgenic mice.
Assuntos
Artrite Reumatoide/metabolismo , Citocromo P-450 CYP3A/genética , Interleucina-6/fisiologia , Fígado/metabolismo , Proteínas de Membrana/genética , Metalotioneína/genética , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Fosforilação , RNA Mensageiro/análise , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Esteroide Hidroxilases/metabolismoRESUMO
A wide variety of drugs and chemicals have been shown to produce induction and inhibition of heme-metabolizing enzymes, and of drug-metabolizing enzymes, including cytochrome P450s (P450s, CYPs), which consist of many molecular species with lower substrate specificity. Such chemically induced enzyme alterations are coordinately or reciprocally regulated through the same and/or different signal transductions. From the toxicological point of view, these enzymatic changes sometimes exacerbate inherited diseases, such as precipitation of porphyrogenic attacks, although the induction of these enzymes is dependent on the animal species in response to the differences in the stimuli of the liver, where they are also metabolized by P450s. Since P450s are hemoproteins, their induction and/or inhibition by chemical compounds could be coordinately accompanied by heme synthesis and/or inhibition. This review will take a retrospective view of research works carried out in our department and current findings on chemical-induced changes in hepatic heme metabolism in many places, together with current knowledge. Specifically, current beneficial aspects of induction of heme oxygenase-1, a rate-limiting heme degradation enzyme, and its relation to reciprocal and coordinated changes in P450s, with special reference to CYP2A5, in the liver are discussed. Mechanistic studies are also summarized in relation to current understanding on these aspects. Emphasis is also paid to an example of a single chemical compound that could cause various changes by mediating multiple signal transduction systems. Current toxicological studies have been developing by utilizing a sophisticated "omics" technology and survey integrated changes in the tissues produced by the administration of a chemical, even in time- and dose-dependent manners. Toxicological studies are generally carried out step by step to determine and elucidate mechanisms produced by drugs and chemicals. Such approaches are correct; however, current "omics" technology can clarify overall changes occurring in the cells and tissues after treating animals with drugs and chemicals, integrate them and discuss the results. In the present review, we will discuss chemical-induced similar changes of heme synthesis and degradation, and of P450s and finally convergence to similar or different directions.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Heme/metabolismo , Fígado/enzimologia , Fígado/metabolismo , Compostos Orgânicos/farmacologia , Compostos Orgânicos/toxicidade , Ácido Aminolevulínico , Animais , Indução Enzimática , Heme/biossíntese , Heme Oxigenase (Desciclizante) , Heme Oxigenase-1/metabolismo , Humanos , Roedores , Toxicologia , Fatores de TranscriçãoRESUMO
Abnormal increases in vascular smooth muscle cells (VSMCs) in the intimal region after a vascular injury is a key event in developing neointimal hyperplasia. To maintain vascular function, proliferation and apoptosis of VSMCs is tightly controlled during vascular remodeling. NF-E2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) system, a key component of the oxidative stress response that acts in maintaining homeostasis, plays an important role in neointimal hyperplasia after a vascular injury; however, the role of Nrf2/Keap1 in VSMC apoptosis has not been clarified. Here we report that 14 days after arterial injury in mice, TUNEL-positive VSMCs are detected in both the neointimal and medial layers. These layers contain cells expressing high levels of Nrf2 but low Keap1 expression. In VSMCs, Keap1 depletion induces features of apoptosis, such as positive TUNEL staining and annexin V binding. These changes are associated with an increased expression of nuclear Nrf2. Simultaneous Nrf2 depletion inhibits Keap1 depletion-induced apoptosis. At 14 days after the vascular injury, Nrf2-deficient mice demonstrated fewer TUNEL-positive cells and increased neointimal formation in the neointimal and medial areas. The results suggest that the Nrf2/Keap1 system regulates VSMC apoptosis during neointimal formation, thereby inhibiting neointimal hyperplasia after a vascular injury.
Assuntos
Apoptose , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neointima , Lesões do Sistema Vascular/metabolismo , Animais , Células Cultivadas , Feminino , Homeostase , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fator 2 Relacionado a NF-E2/deficiência , Fator 2 Relacionado a NF-E2/genética , Interferência de RNA , Ratos Sprague-Dawley , Remodelação Vascular , Lesões do Sistema Vascular/genética , Lesões do Sistema Vascular/patologiaRESUMO
Phenobarbital (PB) induction of CYP2B, a representative target gene of constitutive androstane receptor (CAR), has been observed to be attenuated in preneoplastic lesions of rat liver; however, molecular basis for this attenuation is poorly understood. In this report, we provide evidence indicating that the CAR expressed in the hepatic preneoplastic lesions of rats and mice was resistant to nuclear translocation and transactivation of the PB-responsive enhancer module upon PB treatment. These observations suggest that the attenuation of the induction of CYP2B by PB in hepatic preneoplastic lesions is evidently a consequence of impaired nuclear translocation of CAR.