RESUMO
Cyclooxygenase (COX)-2 has been shown to be involved in regulating basal airway function, bacterial LPS-induced airway hyperresponsiveness (AHR) and lung inflammation, and bleomycin-induced lung fibrosis; however, the cellular source of COX-2 that underlies these effects is unknown. We generated mice with alveolar type II (ATII) cell-specific knockdown of COX-2 (AT2CC(-/-)), to examine the role of ATII cell-derived prostaglandins (PGs) in these processes. Specific knockdown of COX-2 was confirmed by real-time RT-PCR and Western blot analyses. LC/MS/MS analysis showed that ATII cells produced PGs. Basal airway responsiveness of AT2CC(-/-) mice was decreased compared to that of wild-type (WT) mice. LPS-induced hypothermic response, infiltration of inflammatory cells into the airway, and lung inflammation were enhanced in AT2CC(-/-) mice relative to WT controls; however, LPS-induced AHR and proinflammatory cytokine and chemokine expression were similar between the genotypes. After 21 d of bleomycin administration, AT2CC(-/-) mice behaved in a manner similar to WT mice. Thus, ATII cell-derived COX-2 plays an important role in regulating basal airway function and LPS-induced lung inflammation, but does not play a role in bleomycin-induced fibrosis. These findings provide insight into the cellular source of COX-2 related to these lung phenotypes.
Assuntos
Ciclo-Oxigenase 2/genética , Pneumonia/metabolismo , Fibrose Pulmonar/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Animais , Bleomicina/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Camundongos Transgênicos , Pneumonia/genética , Pneumonia/patologia , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologiaRESUMO
RATIONALE: Helper CD4(+) T cell subsets, including IL-9- and IL-10-producing T helper cell type 9 (Th9) cells, exist under certain inflammatory conditions. Cyclooxygenase (COX)-1 and COX-2 play important roles in allergic lung inflammation and asthma. It is unknown whether COX-derived eicosanoids regulate Th9 cells during allergic lung inflammation. OBJECTIVES: To determine the role of COX metabolites in regulating Th9 cell differentiation and function during allergic lung inflammation. METHODS: COX-1(-/-), COX-2(-/-), and wild-type (WT) mice were studied in an in vivo model of ovalbumin-induced allergic inflammation and an in vitro model of Th9 differentiation using flow cytometry, cytokine assays, confocal microscopy, real-time PCR, and immunoblotting. In addition, the role of specific eicosanoids and their receptors was examined using synthetic prostaglandins (PGs), selective inhibitors, and siRNA knockdown. MEASUREMENTS AND MAIN RESULTS: Experimental endpoints were not different between COX-1(-/-) and WT mice; however, the percentage of IL-9(+) CD4(+) T cells was increased in lung, bronchoalveolar lavage fluid, lymph nodes, and blood of allergic COX-2(-/-) mice relative to WT. Bronchoalveolar lavage fluid IL-9 and IL-10, serum IL-9, and lung IL-17RB levels were significantly increased in allergic COX-2(-/-) mice or in WT mice treated with COX-2 inhibitors. IL-9, IL-10, and IL-17RB expression in vivo was inhibited by PGD2 and PGE2, which also reduced Th9 cell differentiation of murine and human naive CD4(+) T cells in vitro. Inhibition of protein kinase A significantly increased Th9 cell differentiation of naive CD4(+) T cells isolated from WT mice in vitro. CONCLUSIONS: COX-2-derived PGD2 and PGE2 regulate Th9 cell differentiation by suppressing IL-17RB expression via a protein kinase A-dependent mechanism.
Assuntos
Asma/imunologia , Inibidores de Ciclo-Oxigenase 2/imunologia , Pulmão/imunologia , Receptores de Interleucina-17/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Citocinas/análise , Eicosanoides/imunologia , Eicosanoides/fisiologia , Citometria de Fluxo , Humanos , Immunoblotting , Inflamação/imunologia , Masculino , Camundongos , Microscopia Confocal , Modelos Animais , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The emergence of nanotechnology has produced a multitude of engineered nanomaterials such as carbon nanotubes (CNTs), and concerns have been raised about their effects on human health, especially for susceptible populations such as individuals with asthma. Multiwalled CNTs (MWCNTs) have been shown to exacerbate ovalbumin (OVA)-induced airway remodeling in mice. Moreover, cyclooxygenase-2 (COX-2) has been described as a protective factor in asthma. We postulated that COX-2-deficient (COX-2(-/-)) mice would be susceptible to MWCNT-induced exacerbations of allergen-induced airway remodeling, including airway inflammation, fibrosis, and mucus-cell metaplasia (i.e., the formation of goblet cells). Wild-type (WT) or COX-2(-/-) mice were sensitized to OVA to induce allergic airway inflammation before a single dose of MWCNTs (4 mg/kg) delivered to the lungs by oropharyngeal aspiration. MWCNTs significantly increased OVA-induced lung inflammation and mucus-cell metaplasia in COX-2(-/-) mice compared with WT mice. However, airway fibrosis after exposure to allergen and MWCNTs was no different between WT and COX-2(-/-) mice. Concentrations of certain prostanoids (prostaglandin D2 and thromboxane B2) were enhanced by OVA or MWCNTs in COX-2(-/-) mice. No differences in COX-1 mRNA concentrations were evident between WT and COX-2(-/-) mice treated with OVA and MWCNTs. Interestingly, MWCNTs significantly enhanced allergen-induced cytokines involved in Th2 (IL-13 and IL-5), Th1 (CXCL10), and Th17 (IL-17A) inflammatory responses in COX-2(-/-) mice, but not in WT mice. We conclude that exacerbations of allergen-induced airway inflammation and mucus-cell metaplasia by MWCNTs are enhanced by deficiencies in COX-2, and are associated with the activation of a mixed Th1/Th2/Th17 immune response.
Assuntos
Remodelação das Vias Aéreas/fisiologia , Alérgenos/imunologia , Ciclo-Oxigenase 2/imunologia , Nanotubos de Carbono , Remodelação das Vias Aéreas/genética , Remodelação das Vias Aéreas/imunologia , Animais , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/imunologia , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Citocinas/imunologia , Feminino , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Metaplasia/genética , Metaplasia/imunologia , Metaplasia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Muco/imunologia , Muco/metabolismo , Ovalbumina/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
Sex differences exist in the hypertrophic response, cardiac remodeling, and transition to heart failure of hypertensive patients, and while some of these differences are likely influenced by estrogen, the genetic pathways downstream of estrogen that impact on cardioprotection have yet to be fully elucidated. We have previously shown that the cardioprotective effects of adrenomedullin (AM), an emerging clinical biomarker for cardiovascular disease severity, vary with sex in mouse models. AM signaling during cardiovascular stress is strongly modulated by receptor activity-modifying protein 3 (RAMP3) via its interaction with the G protein-coupled receptor calcitonin receptor-like receptor (CLR). Like AM, RAMP3 expression is potently regulated by estrogen, and so we sought to determine the consequences of genetic Ramp3 loss on cardiac adaptation to chronic hypertension, with a particular focus on characterizing potential sex differences. We generated and bred RAMP3(-/-) mice to RenTgMK mice that consistently display severe angiotensin II-mediated CV disease and compared CV disease progression in RenTgMK to that of RenTgMK:RAMP3(-/-) offspring. As expected, RAMP3 gene expression was higher in cardiovascular tissues of RenTgMK mice and more strongly up-regulated in female RenTgMK mice relative to wildtype controls. RAMP3 loss did not affect the development of hypertension or the presence and severity of perivascular and interstitial fibrosis in the left ventricle (LV). However, echocardiography revealed that while RenTgMK mice developed concentric cardiac hypertrophy with sustained systolic function, male RenTgMK:RAMP3(-/-) mice showed evidence of LV chamber dilatation and depressed systolic function, suggestive of cardiac decompensation. Consistent with these measures of heart failure, male RenTgMK:RAMP3(-/-) mice had increased cardiac apoptosis and elevated activation of Akt. These phenotypes were not present in female RenTgMK:RAMP3(-/-) mice. Collectively, these data demonstrate a sex-dependant, cardioprotective role of RAMP3 in the setting of chronic hypertension.
Assuntos
Cardiomegalia/genética , Insuficiência Cardíaca/genética , Proteína 3 Modificadora da Atividade de Receptores/deficiência , Angiotensina II/efeitos adversos , Animais , Cardiomegalia/metabolismo , Doenças Cardiovasculares/induzido quimicamente , Doenças Cardiovasculares/genética , Modelos Animais de Doenças , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Estudos de Associação Genética , Insuficiência Cardíaca/metabolismo , Hipertensão/complicações , Hipertensão/genética , Hipertensão/metabolismo , Hipertrofia Ventricular Direita/genética , Nefropatias/etiologia , Nefropatias/genética , Nefropatias/patologia , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína 3 Modificadora da Atividade de Receptores/genética , Proteína 3 Modificadora da Atividade de Receptores/metabolismo , Renina/genética , Fatores SexuaisRESUMO
Epoxyeicosatrienoic acids (EET), the primary arachidonic acid metabolites of cytochrome P450 2J (CYP2J) epoxygenases, possess potent vasodilatory, anti-inflammatory, antiapoptotic, and mitogenic effects. To date, little is known about the role of CYP2J2 and EETs in tumor necrosis factor (TNF)-α-induced cardiac injury. We utilized cell culture and in vivo models to examine the effects of exogenously applied EETs or CYP2J2 overexpression on TNF-α-induced cardiac apoptosis and cardiac dysfunction. In neonatal rat cardiomyocytes, TNF-α-induced apoptosis was markedly attenuated by EETs or CYP2J2 overexpression, leading to significantly improved cell survival. Further studies showed that TNF-α decreased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL, decreased IκBα and PPARγ, and also inhibited PI3K-dependent Akt and EGFR signaling. Both EETs and CYP2J2 overexpression reversed the effects of TNF-α on these pathways. Furthermore, overexpression of CYP2J2 in rats prevented the decline in cardiac function that is normally observed in TNF-α-challenged animals. These results demonstrate that EETs or CYP2J2 overexpression can prevent TNF-α-induced cardiac cell injury and cardiac dysfunction by inhibiting apoptosis, reducing inflammation, and enhancing PPARγ expression. Targeting the CYP2J2 epoxygenase pathway may represent a novel approach to mitigate cardiac injury in diseases such as heart failure, where increased TNF-α levels are known to occur.
Assuntos
Apoptose/efeitos dos fármacos , Coração/efeitos dos fármacos , Fator de Necrose Tumoral alfa/toxicidade , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/urina , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Linhagem Celular , Células Cultivadas , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Eicosanoides , Citometria de Fluxo , Hemodinâmica/efeitos dos fármacos , Masculino , Receptores Ativados por Proliferador de Peroxissomo/genética , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
RATIONALE: Th17 cells comprise a distinct lineage of proinflammatory T helper cells that are major contributors to allergic responses. It is unknown whether cyclooxygenase (COX)-derived eicosanoids regulate Th17 cells during allergic lung inflammation. OBJECTIVES: To determine the role of COX metabolites in regulating Th17 cell differentiation and function during allergic lung inflammation. METHODS: COX-1(-/-), COX-2(-/-), and wild-type mice were studied in an in vivo model of ovalbumin-induced allergic inflammation and an in vitro model of Th17 differentiation using flow cytometry, cytokine assays, confocal microscopy, real-time polymerase chain reaction, and immunoblotting. In addition, the role of specific eicosanoids and their receptors was examined using synthetic prostaglandins (PGs), selective inhibitors, and siRNA knockdown. MEASUREMENTS AND MAIN RESULTS: Th17 cell differentiation in lung, lymph nodes, and bronchoalveolar lavage fluid was significantly lower in COX-2(-/-) mice after ovalbumin sensitization and exposure in vivo. In vitro studies revealed significantly impaired Th17 cell differentiation of COX-2(-/-) naive CD4(+) T cells with decreased Stat3 phosphorylation and RORγt expression. Synthetic PGF(2α) and PGI(2) enhanced Th17 cell differentiation of COX-2(-/-) CD4(+) T cells in vitro. The selective COX-2 inhibitor, NS-398, and PGF(2α) receptor and PGI(2) receptor siRNA knockdown significantly decreased Th17 cell differentiation in vitro. Administration of synthetic PGs restored accumulation of Th17 cells in lungs of allergic COX-2(-/-) mice in vivo. CONCLUSIONS: COX-2 is a critical regulator of Th17 cell differentiation during allergic lung inflammation via autocrine signaling of PGI(2) and PGF(2α) through their respective cell surface receptors.
Assuntos
Asma/imunologia , Ciclo-Oxigenase 2/fisiologia , Células Th17/imunologia , Animais , Asma/metabolismo , Asma/patologia , Diferenciação Celular , Ciclo-Oxigenase 1/fisiologia , Eicosanoides/metabolismo , Inflamação , Interleucina-17/metabolismo , Pulmão/patologia , Camundongos , Camundongos Knockout , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Ovalbumina/imunologia , Receptores de Prostaglandina/metabolismo , Fator de Transcrição STAT3/metabolismo , Células Th17/metabolismo , Células Th17/patologia , Células Th17/fisiologiaRESUMO
Prostaglandin E(2) (PGE(2)) is a lipid mediator that is produced via the metabolism of arachidonic acid by cyclooxygenase enzymes. In the lung, PGE(2) acts as an anti-inflammatory factor and plays an important role in tissue repair processes. Although several studies have examined the role of PGE(2) in the pathogenesis of pulmonary fibrosis in rodents, results have generally been conflicting, and few studies have examined the therapeutic effects of PGE(2) on the accompanying lung dysfunction. In this study, an established model of pulmonary fibrosis was used in which 10-12-wk-old male C57BL/6 mice were administered a single dose (1.0 mg/kg) of bleomycin via oropharyngeal aspiration. To test the role of prostaglandins in this model, mice were dosed, via surgically implanted minipumps, with either vehicle, PGE(2) (1.32 µg/h), or the prostacyclin analog iloprost (0.33 µg/h) beginning 7 days before or 14 days after bleomycin administration. Endpoints assessed at 7 days after bleomycin administration included proinflammatory cytokine levels and measurement of cellular infiltration into the lung. Endpoints assessed at 21 days after bleomycin administration included lung function assessment via invasive (FlexiVent) analysis, cellular infiltration, lung collagen content, and semiquantitative histological analysis of the degree of lung fibrosis (Ashcroft method). Seven days after bleomycin administration, lymphocyte numbers and chemokine C-C motif ligand 2 expression were significantly lower in PGE(2)- and iloprost-treated animals compared with vehicle-treated controls (P < 0.05). When administered 7 days before bleomycin challenge, PGE(2) also protected against the decline in lung static compliance, lung fibrosis, and collagen production that is associated with 3 wk of bleomycin exposure. However, PGE(2) had no therapeutic effect on these parameters when administered 14 days after bleomycin challenge. In summary, PGE(2) prevented the decline in lung static compliance and protected against lung fibrosis when it was administered before bleomycin challenge but had no therapeutic effect when administered after bleomycin challenge.
Assuntos
Bleomicina/efeitos adversos , Colágeno/biossíntese , Dinoprostona/farmacologia , Iloprosta/farmacologia , Pulmão/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Animais , Bleomicina/administração & dosagem , Líquido da Lavagem Broncoalveolar/citologia , Colágeno/análise , Citocinas/biossíntese , Dinoprostona/metabolismo , Modelos Animais de Doenças , Esquema de Medicação , Histocitoquímica , Humanos , Iloprosta/metabolismo , Bombas de Infusão Implantáveis , Contagem de Leucócitos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infiltração de Neutrófilos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Fibrose Pulmonar/patologia , Fibrose Pulmonar/fisiopatologia , Fibrose Pulmonar/prevenção & controle , Reação em Cadeia da Polimerase em Tempo Real , Índice de Gravidade de DoençaRESUMO
Cytochrome P450 2J2 (CYP2J2) epoxygenase converts arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EETs) that exert multiple biological effects in the cardiovascular system and in various human solid cancers. However, it is unknown whether this enzyme is expressed or plays any role in malignant hematological diseases. In this study, we found strong and highly selective CYP2J2 expression in five human-derived malignant hematological cell lines and in leukemia cells from peripheral blood and bone marrow in 36 of 42 patients (86%) with malignant hematologic diseases. Furthermore, increased levels of EETs were detected in urine and blood samples from these patients. Addition of exogenous EET or CYP2J2 overexpression in cultured human-derived malignant hematologic cell lines markedly accelerated proliferation and attenuated apoptosis. Addition of the selective CYP2J2 inhibitor compound 26 (C26; 1-[4-(vinyl) phenyl]-4-[4-(diphenyl-hydroxymethyl)-piperidinyl]-butanone hydrochloride) inhibited cell proliferation and increased apoptosis, an effect that was significantly reversed by EET. CYP2J2 overexpression and exogenous EET activated AMP-activated protein kinase, c-Jun NH(2)-terminal kinase, and phosphatidylinositol 3-kinase/Akt signaling pathways, and increased epidermal growth factor receptor phosphorylation levels. CYP2J2 overexpression also enhanced malignant xenograft growth, which was efficiently inhibited by oral administration of C26 in Tie2-CYP2J2 transgenic mice and in severe combined immunodeficiency (SCID) xenograft mice. Together, these results suggest that CYP2J2 plays a key role in the pathogenesis of human hematologic malignant diseases. Selective inhibition of CYP2J2 may be a promising therapeutic strategy for these conditions.
Assuntos
Sistema Enzimático do Citocromo P-450/fisiologia , Leucemia/enzimologia , Animais , Apoptose , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Citocromo P-450 CYP2J2 , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/genética , Humanos , Leucemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/análiseRESUMO
Pulmonary arterial hypertension (PAH) is a life-threatening disease that leads to progressive pulmonary hypertension, right heart failure, and death. Endothelial dysfunction and inflammation were implicated in the pathogenesis of PAH. Epoxyeicosatrienoic acids (EETs), products of the cytochrome P450 epoxygenase metabolism of arachidonic acid, are potent vasodilators that possess anti-inflammatory and other protective properties in endothelial cells. We investigated whether gene delivery with the human cytochrome P450 epoxygenase 2J2 (CYP2J2) ameliorates monocrotaline (MCT)-induced pulmonary hypertension in rats. Significant pulmonary hypertension developed 3 weeks after the administration of MCT, but gene therapy with CYP2J2 significantly attenuated the development of pulmonary hypertension and pulmonary vascular remodeling, without causing changes in systemic arterial pressure or heart rate. These effects were associated with increased pulmonary endothelial NO synthase (eNOS) expression and its activity, inhibition of inflammation in the lungs, and transforming growth factor (TGF)-ß/type II bone morphogenetic protein receptor (BMPRII)-drosophila mothers against decapentaplegic proteins (Smads) signaling. Collectively, these data suggest that gene therapy with CYP2J2 may have potential as a novel therapeutic approach to this progressive and oftentimes lethal disorder.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/uso terapêutico , Técnicas de Transferência de Genes , Terapia Genética , Hipertensão Pulmonar/terapia , Artéria Pulmonar/patologia , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido 8,11,14-Eicosatrienoico/urina , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Citocromo P-450 CYP2J2 , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Hemodinâmica/efeitos dos fármacos , Humanos , Hipertensão Pulmonar/sangue , Hipertensão Pulmonar/complicações , Hipertensão Pulmonar/fisiopatologia , Hipertrofia Ventricular Direita/sangue , Hipertrofia Ventricular Direita/complicações , Hipertrofia Ventricular Direita/fisiopatologia , Hipertrofia Ventricular Direita/urina , Interleucina-10/sangue , Interleucina-6/sangue , Monocrotalina , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise de Sobrevida , Extratos de Tecidos/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVE: 20-Hydroxyeicosatetraenoic acid has been shown to play an important role in cerebral vascular function. We hypothesized that polymorphisms in genes encoding 20-Hydroxyeicosatetraenoic acid synthesizing enzymes might confer susceptibility to stroke. METHODS AND RESULTS: To test the hypothesis, haplotype tagging single nucleotide polymorphisms and potential functional polymorphisms of CYP4A11 and CYP4F2 genes were genotyped in 558 ischemic stroke patients, 221 hemorrhagic stroke patients and 557 controls. The association analyses were performed at both single nucleotide polymorphism and haplotype levels. We further verified our findings in an independent cohort of 551 ischemic stroke cases and 48 hemorrhagic stroke cases and 694 unaffected controls. We identified CYP4A11 C-296T and CYP4F2 V433M were associated with significantly increased risk of ischemic stroke (CT+TT vs. CC, adjusted odds ratio: 1.50, 95% confidence interval: 1.17-1.93, Pcombined=0.001, Pcorr=0.008; V/M+M/M vs. V/V, odds ratio: 1.38, 95% confidence interval: 1.15-1.65, Pcombined=5.6x10, Pcorr=0.005, respectively). Interestingly, the effects of CYP4F2 V433M on ischemic stroke in our study was only evident in male individuals. CONCLUSION: Our results suggest that genetic variation in CYP4A11 and CYP4F2 alters susceptibility to stroke in the Han Chinese population.
Assuntos
Povo Asiático/genética , Sistema Enzimático do Citocromo P-450/genética , Acidente Vascular Cerebral/genética , Idoso , Estudos de Casos e Controles , China , Citocromo P-450 CYP4A , Família 4 do Citocromo P450 , Feminino , Variação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Fatores SexuaisRESUMO
AIM: To test the hypothesis that the epoxyeicosatrienoic acid (EET)-induced transactivation of EGF-R depends on the activation of metalloproteinases and the subsequent release of HB-EGF in cancer cells. METHODS: Exogenous 14,15-EET were added to four human-derived cancer cell lines Tca-8113, A549, HepG2, and MDA-MB-231, or these same cell lines were transfected with a mutant CYP epoxygenase (CYP102 F87V, an active 14,15-epoxygenase). The effects of elevated EET levels on the phosphorylation of tyrosine residues in the EGF receptor and on ERK1/2 activation were then assessed. RESULTS: Both the addition of 14,15-EET and the transfection of cells with CYP102 F87V markedly increased the phosphorylation of the tyrosine residues of EGF-R and ERK1/2, an effect that was blocked by a selective EGF-R tyrosine kinase inhibitor (tyrphostin AG1478), a broad-spectrum metalloproteinase inhibitor (1,10-phenanthroline), and an inhibitor of HB-EGF release (CRM197) in Tca-8113 cells. In addition, AG1478, 1,10-phenanthroline, and CRM197 also inhibited the tyrosine phosphorylation of EGF-R and ERK1/2 that was induced by 14,15-EET in the A549, HepG2, and MDA-MB-231 cell lines. CONCLUSION: These results suggest that the EET-induced transactivation of EGF-R depends on activation of metalloproteinases and the subsequent release of HB-EGF in cancer cell lines.
Assuntos
Ácidos Araquidônicos/farmacologia , Receptores ErbB/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Metaloproteases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Fenantrolinas/farmacologia , Fosforilação , Ativação TranscricionalRESUMO
Adrenomedullin (AM) is a multifunctional peptide vasodilator that is essential for life. To date, numerous in vitro studies have suggested that AM can mediate its biological effects through at least three different receptors. To determine the in vivo importance of the most likely candidate receptor, calcitonin receptor-like receptor, a gene-targeted knockout model of the gene was generated. Mice heterozygous for the targeted Calcrl allele appear normal, survive to adulthood, and reproduce. However, heterozygote matings fail to produce viable Calcrl-/- pups, demonstrating that Calcrl is essential for survival. Timed matings confirmed that Calcrl-/- embryos die between embryonic day 13.5 (E13.5) and E14.5 of gestation. The Calcrl-/- embryos exhibit extreme hydrops fetalis and cardiovascular defects, including thin vascular smooth muscle walls and small, disorganized hearts remarkably similar to the previously characterized AM-/- phenotype. In vivo assays of cellular proliferation and apoptosis in the hearts and vasculature of Calcrl-/- and AM-/- embryos support the concept that AM signaling is a crucial mediator of cardiovascular development. The Calcrl gene targeted mice provide the first in vivo genetic evidence that CLR functions as an AM receptor during embryonic development.
Assuntos
Anormalidades Cardiovasculares/metabolismo , Perda do Embrião/metabolismo , Hidropisia Fetal/metabolismo , Receptores da Calcitonina/deficiência , Adrenomedulina , Animais , Apoptose , Anormalidades Cardiovasculares/patologia , Proliferação de Células , Morte Fetal , Idade Gestacional , Camundongos , Camundongos Knockout , Músculo Liso Vascular/patologia , Miocárdio/patologia , Miócitos Cardíacos/patologia , Peptídeos/deficiência , Receptores da Calcitonina/genética , Recombinação GenéticaRESUMO
Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to biologically active cis-epoxyeicosatrienoic acids, which have potent vasodilatory, antiinflammatory, antiapoptotic, and antidiabetes properties. Here, we showed the effects of cardiac-specific overexpression of CYP epoxygenase 2J2 (CYP2J2) on diabetic cardiomyopathy and insulin resistance in high-fat (HF) diet fed, low-dose streptozotocin-treated mice. Diabetic cardiomyopathy was induced by HF and streptozotocin in cardiac-specific CYP2J2 transgenic mice. Physiological parameters and systemic metabolic parameters were monitored using ELISA kits. Intraperitoneal injection glucose tolerance test and hyperinsulinemic-euglycemic clamp study were implied to indicate insulin resistance. Cardiac function was assessed by echocardiography and Millar catheter system. Real-time PCR and Western blotting were used in signal pathway detection. αMHC-CYP2J2 transgenic mice showed significantly lower plasma glucose and insulin levels, improved glucose tolerance, and increased cardiac glucose uptake. Furthermore, αMHC-CYP2J2 transgenic mice were significantly protected from HF-streptozotocin-induced diabetic cardiomyopathy. Strikingly, CYP2J2 overexpression attenuated myocardial hypertrophy induced by diabetes. We conclude that cardiac-specific overexpression of CYP2J2 significantly protects against diabetic cardiomyopathy, which may be due to improved cardiac insulin resistance, glucose uptake, and reversal of cardiac hypertrophy. Relevant mechanisms may include up-regulation of peroxisome proliferator-activated receptor γ, activation of insulin receptor and AMP-activated protein kinase signaling pathways, and inhibition of nuclear factor of activated T cells c3 signal by enhanced atrial natriuretic peptide production. These results suggest that CYP2J2 epoxygenase metabolites likely play an important role in plasma glucose homeostasis, and enhancement of epoxyeicosatrienoic acids activation may serve as an effective therapeutic strategy to prevent diabetic cardiomyopathy.