RESUMO
Despite years of study and major research advances over the past 50 years, atherosclerotic diseases continue to rank as the leading global cause of death. Accumulation of cholesterol within the vascular wall remains the main problem and represents one of the early steps in the development of atherosclerotic lesions. There is a complex relationship between vesicular cholesterol transport and atherosclerosis, and abnormalities in cholesterol trafficking can contribute to the development and progression of the lesions. The dysregulation of vesicular cholesterol transport and lysosomal function fosters the buildup of cholesterol within various intracytoplasmic compartments, including lysosomes and lipid droplets. This, in turn, promotes the hallmark formation of foam cells, a defining feature of early atherosclerosis. Multiple cellular processes, encompassing endocytosis, exocytosis, intracellular trafficking, and autophagy, play crucial roles in influencing foam cell formation and atherosclerotic plaque stability. In this review, we highlight recent advances in the understanding of the intricate mechanisms of vesicular cholesterol transport and its relationship with atherosclerosis and discuss the importance of understanding these mechanisms in developing strategies to prevent or treat this prevalent cardiovascular disease.
Assuntos
Aterosclerose , Placa Aterosclerótica , Humanos , Aterosclerose/patologia , Colesterol , Placa Aterosclerótica/complicações , Placa Aterosclerótica/patologia , Células Espumosas/patologia , Lisossomos/patologiaRESUMO
BACKGROUND: Impairment of cellular cholesterol trafficking is at the heart of atherosclerotic lesions formation. This involves egress of cholesterol from the lysosomes and 2 lysosomal proteins, the NPC1 (Niemann-Pick C1) and NPC2 that promotes cholesterol trafficking. However, movement of cholesterol out the lysosome and how disrupted cholesterol trafficking leads to atherosclerosis is unclear. As the Wnt ligand, Wnt5a inhibits the intracellular accumulation of cholesterol in multiple cell types, we tested whether Wnt5a interacts with the lysosomal cholesterol export machinery and studied its role in atherosclerotic lesions formation. METHODS: We generated mice deleted for the Wnt5a gene in vascular smooth muscle cells. To establish whether Wnt5a also protects against cholesterol accumulation in human vascular smooth muscle cells, we used a CRISPR/Cas9 guided nuclease approach to generate human vascular smooth muscle cells knockout for Wnt5a. RESULTS: We show that Wnt5a is a crucial component of the lysosomal cholesterol export machinery. By increasing lysosomal acid lipase expression, decreasing metabolic signaling by the mTORC1 (mechanistic target of rapamycin complex 1) kinase, and through binding to NPC1 and NPC2, Wnt5a senses changes in dietary cholesterol supply and promotes lysosomal cholesterol egress to the endoplasmic reticulum. Consequently, loss of Wnt5a decoupled mTORC1 from variations in lysosomal sterol levels, disrupted lysosomal function, decreased cholesterol content in the endoplasmic reticulum, and promoted atherosclerosis. CONCLUSIONS: These results reveal an unexpected function of the Wnt5a pathway as essential for maintaining cholesterol homeostasis in vivo.
Assuntos
Aterosclerose/metabolismo , Colesterol/metabolismo , Lisossomos/metabolismo , Proteína Wnt-5a/metabolismo , Animais , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína C1 de Niemann-Pick/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteína Wnt-5a/genéticaRESUMO
The low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitously expressed cell surface receptor that protects from intracellular cholesterol accumulation. However, the underlying mechanisms are unknown. Here we show that the extracellular (α) chain of LRP1 mediates TGFß-induced enhancement of Wnt5a, which limits intracellular cholesterol accumulation by inhibiting cholesterol biosynthesis and by promoting cholesterol export. Moreover, we demonstrate that the cytoplasmic (ß) chain of LRP1 suffices to limit cholesterol accumulation in LRP1(-/-) cells. Through binding of Erk2 to the second of its carboxyl-terminal NPXY motifs, LRP1 ß-chain positively regulates the expression of ATP binding cassette transporter A1 (ABCA1) and of neutral cholesterol ester hydrolase (NCEH1). These results highlight the unexpected functions of LRP1 and the canonical Wnt5a pathway and new therapeutic potential in cholesterol-associated disorders including cardiovascular diseases.
Assuntos
Colesterol/metabolismo , Receptores de LDL/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Via de Sinalização Wnt , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Animais , Células HEK293 , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Receptores de LDL/química , Receptores de LDL/genética , Esterol Esterase/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/genética , Proteínas Wnt/metabolismo , Proteína Wnt-5aRESUMO
Src homology and collagen A (ShcA) is an adaptor protein that binds to tyrosine kinase receptors. Its germ line deletion is embryonic lethal with abnormal cardiovascular system formation, and its role in cardiovascular development is unknown. To investigate its functional role in cardiovascular development in mice, ShcA was deleted in cardiomyocytes and vascular smooth muscle cells by crossing ShcA flox mice with SM22a-Cre transgenic mice. Conditional mutant mice developed signs of severe dilated cardiomyopathy, myocardial infarctions, and premature death. No evidence of a vascular contribution to the phenotype was observed. Histological analysis of the heart revealed aberrant sarcomeric Z-disk and M-band structures, and misalignments of T-tubules with Z-disks. We find that not only the ErbB3/Neuregulin signaling pathway but also the baroreceptor reflex response, which have been functionally associated, are altered in the mutant mice. We further demonstrate that ShcA interacts with Caveolin-1 and the costameric protein plasma membrane Ca(2+)/calmodulin-dependent ATPase (PMCA), and that its deletion leads to abnormal dystrophin signaling. Collectively, these results demonstrate that ShcA interacts with crucial proteins and pathways that link Z-disk and costamere.
Assuntos
Costâmeros/metabolismo , Coração/embriologia , Miócitos Cardíacos/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Alelos , Animais , Aorta Torácica/metabolismo , Pressão Sanguínea , Sobrevivência Celular , Distrofina/metabolismo , Ecocardiografia , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Imageamento por Ressonância Magnética , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Receptor ErbB-3/metabolismo , Proteínas Adaptadoras da Sinalização Shc/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de SrcRESUMO
The low density lipoprotein receptor-related protein (LRP1) is a transmembrane receptor that integrates multiple signaling pathways. Its cytoplasmic domain serves as docking sites for several adaptor proteins such as the Src homology 2/α-collagen (ShcA), which also binds to several tyrosine kinase receptors such as the insulin-like growth factor 1 (IGF-1) receptor. However, the physiological significance of the physical interaction between LRP1 and ShcA, and whether this interaction modifies tyrosine kinase receptor signaling, are still unknown. Here we report that LRP1 forms a complex with the IGF-1 receptor, and that LRP1 is required for ShcA to become sensitive to IGF-1 stimulation. Upon IGF-1 treatment, ShcA is tyrosine phosphorylated and translocates to the plasma membrane only in the presence of LRP1. This leads to the recruitment of the growth factor receptor-bound protein 2 (Grb2) to ShcA, and activation of the Ras/MAP kinase pathway. Conversely, in the absence of ShcA, IGF-1 signaling bifurcates toward the Akt/mammalian target of rapamycin pathway and accelerates adipocyte differentiation when cells are stimulated for adipogenesis. These results establish the LRP1-ShcA complex as an essential component in the IGF-1-regulated pathway for MAP kinase and Akt/mammalian target of rapamycin activation, and may help to understand the IGF-1 signaling shift from clonal expansion to growth-arrested cells and differentiation during adipogenesis.
Assuntos
Regulação da Expressão Gênica , Receptor IGF Tipo 1/metabolismo , Receptores de LDL/metabolismo , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Transdução de Sinais , Proteínas Supressoras de Tumor/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Animais , Diferenciação Celular , Fibroblastos/metabolismo , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Camundongos Transgênicos , Fosforilação , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Serina-Treonina Quinases TOR/metabolismo , Tirosina/química , Proteínas ras/metabolismoRESUMO
Doxorubicin (Dox) is an antineoplastic agent that can cause cardiomyopathy in humans and experimental animals. As an inducer of reactive oxygen species and a DNA damaging agent, Dox causes elevated expression of p21(WAF1/Cip1/Sdi1) (p21) gene. Elevated levels of p21 mRNA and p21 protein have been detected in the myocardium of mice following Dox treatment. With chronic treatment of Dox, wild type (WT) animals develop cardiomyopathy evidenced by elongated nuclei, mitochondrial swelling, myofilamental disarray, reduced cardiac output, reduced ejection fraction, reduced left ventricular contractility, and elevated expression of ANF gene. In contrast, p21 knockout (p21KO) mice did not show significant changes in the same parameters in response to Dox treatment. In an effort to understand the mechanism of the resistance against Dox induced cardiomyopathy, we measured levels of antioxidant enzymes and found that p21KO mice did not contain elevated basal or inducible levels of glutathione peroxidase and catalase. Measurements of 6 circulating cytokines indicated elevation of IL-6, IL-12, IFNγ and TNFα in Dox treated WT mice but not p21KO mice. Dox induced elevation of IL-6 mRNA was detected in the myocardium of WT mice but not p21KO mice. While the mechanism of the resistance against Dox induced cardiomyopathy remains unclear, lack of inflammatory response may contribute to the observed cardiac protection in p21KO mice.
Assuntos
Antibióticos Antineoplásicos/toxicidade , Cardiomiopatias/induzido quimicamente , Inibidor de Quinase Dependente de Ciclina p21/genética , Doxorrubicina/toxicidade , Animais , Catalase/biossíntese , Catalase/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Indução Enzimática/efeitos dos fármacos , Citometria de Fluxo , Glutationa Peroxidase/biossíntese , Glutationa Peroxidase/metabolismo , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superóxido Dismutase/biossíntese , Superóxido Dismutase/metabolismoRESUMO
Caveolin-1 plays a checkpoint function in the regulation of processes often altered in cancer. Although increased expression of caveolin-1 seems to be the norm in the glioma family of malignancies, populations of caveolin-1 positive and negative cells coexist among glioblastoma specimens. As no data are available to date on the contribution of such cells to the phenotype of glioblastoma, we manipulated caveolin-1 in the glioblastoma cell line U87MG. We showed that caveolin-1 plays a critical role in the aggressiveness of glioblastoma. We identified integrins as the main set of genes affected by caveolin-1. We reported here that the phenotypic changes observed after caveolin-1 modulation were mediated by alpha(5)beta(1) integrins. As a consequence of the regulation of alpha(5)beta(1) levels by caveolin-1, the sensitivity of cells to the specific alpha(5)beta(1) integrin antagonist, SJ749, was affected. Mediator of caveolin-1 effects, alpha(5)beta(1) integrin, is also a marker for glioma aggressiveness and an efficient target for the treatment of glioma especially the ones exerting the highest aggressive phenotype.
Assuntos
Caveolina 1/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfa5beta1/metabolismo , Propionatos/farmacologia , Piridinas/farmacologia , Compostos de Espiro/farmacologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/enzimologia , Glioblastoma/genética , Humanos , Integrina alfa5beta1/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Invasividade Neoplásica/genética , Metástase Neoplásica/genética , Fenótipo , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ensaio Tumoral de Célula-TroncoRESUMO
Atherosclerosis, a pathology affecting large and medium-sized arteries, is the major cause of cardiovascular morbidity/mortality in industrialized countries. During atherosclerosis, cells accumulate large amounts of cholesterol through the uptake of modified low-density lipoprotein particles to form foam cells. This accumulation forms the basis for the development of the disease and for a large spectrum of other diseases in various organs. Massive research efforts have yielded valuable information about the underlying molecular mechanisms of atherosclerosis. In particular, newer discoveries on the early stage of lesion formation, cholesterol accumulation, reverse cholesterol transport, and local inflammation in the vascular wall have opened unanticipated horizons of understanding and raised novel questions and therapeutic opportunities. In this review, we focus on Wnt signaling, which has received little attention so far, yet affects lysosomal function and signalling pathways that limit cholesterol accumulation. This occurs in different tissues and cell types, including smooth muscle cells, endothelial cells and macrophages in the arterial wall, and thus profoundly impacts on atherosclerotic disease development and progression.
Assuntos
Aterosclerose , Células Endoteliais , Colesterol , Células Espumosas , Humanos , MacrófagosRESUMO
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMO
ShcA is an adaptor protein that binds to the cytoplasmic tail of receptor tyrosine kinases and of the Low Density Lipoprotein-related receptor 1 (LRP1), a trans-membrane receptor that protects against atherosclerosis. Here, we examined the role of endothelial ShcA in atherosclerotic lesion formation. We found that atherosclerosis progression was markedly attenuated in mice deleted for ShcA in endothelial cells, that macrophage content was reduced at the sites of lesions, and that adhesion molecules such as the intercellular adhesion molecule-1 (ICAM-1) were severely reduced. Our data indicate that transcriptional regulation of ShcA by the zinc-finger E-box-binding homeobox 1 (ZEB1) and the Hippo pathway effector YAP, promotes ICAM-1 expression independently of p-NF-κB, the primary driver of adhesion molecules expressions. In addition, ShcA suppresses endothelial Akt and nitric oxide synthase (eNOS) expressions. Thus, through down regulation of eNOS and ZEB1-mediated ICAM-1 up regulation, endothelial ShcA promotes monocyte-macrophage adhesion and atherosclerotic lesion formation. Reducing ShcA expression in endothelial cells may represent an obvious therapeutic approach to prevent atherosclerosis.
RESUMO
Vascular calcification is a hallmark of advanced atherosclerosis. Here we show that deletion of the nuclear receptor PPARγ in vascular smooth muscle cells of low density lipoprotein receptor (LDLr)-deficient mice fed an atherogenic diet high in cholesterol, accelerates vascular calcification with chondrogenic metaplasia within the lesions. Vascular calcification in the absence of PPARγ requires expression of the transmembrane receptor LDLr-related protein-1 in vascular smooth muscle cells. LDLr-related protein-1 promotes a previously unknown Wnt5a-dependent prochondrogenic pathway. We show that PPARγ protects against vascular calcification by inducing the expression of secreted frizzled-related protein-2, which functions as a Wnt5a antagonist. Targeting this signalling pathway may have clinical implications in the context of common complications of atherosclerosis, including coronary artery calcification and valvular sclerosis.
Assuntos
Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , PPAR gama/metabolismo , Calcificação Vascular/metabolismo , Animais , Humanos , Immunoblotting , Imunoprecipitação , Hibridização In Situ , Técnicas In Vitro , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Miócitos de Músculo Liso/efeitos dos fármacos , PPAR gama/agonistas , PPAR gama/genética , Rosiglitazona , Tiazolidinedionas/farmacologia , Calcificação Vascular/genética , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteína Wnt-5aRESUMO
AIMS: Numerous lines of evidence suggest a role of oxidative stress in initiation and progression of heart failure. We identify novel pathways of oxidative stress in cardiomyocytes using proteomic technology. METHODS AND RESULTS: Cardiomyocytes and cardiac fibroblasts isolated from rat hearts were treated with sublethal doses of H(2)O(2) for detection of secreted protein factors in the conditioned media by mass spectrometry-based proteomics. Comparison between the two cell types leads to the finding that H(2)O(2) caused an elevated cystatin C protein in the conditioned medium from cardiomyocytes. When cardiomyopathy was induced in mice by chronic administration of doxorubicin, elevated cystatin C protein was detected in the plasma. Myocardial ischaemia by left anterior descending coronary artery occlusion causes an increase in the level of cystatin C protein in the plasma. In myocardial tissue from the ischaemic area, an increase in cystatin C correlates with the inhibition of cathepsin B activity and accumulation of fibronectin and collagen I/III. Overexpressing cystatin C gene or exposing fibroblasts to cystatin C protein results in an inhibition of cathepsin B and accumulation of fibronectin and collagen I/III. CONCLUSION: Oxidants induce elevated cystatin C production from CMCs. Cystatin C plays a role in cardiac extracellular matrix remodelling.
Assuntos
Cardiomiopatias/metabolismo , Cistatina C/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Isquemia Miocárdica/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Estresse Oxidativo , Animais , Animais Recém-Nascidos , Cardiomiopatias/induzido quimicamente , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Catepsina B/metabolismo , Células Cultivadas , Cromatografia Líquida , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Meios de Cultivo Condicionados/metabolismo , Cistatina C/genética , Modelos Animais de Doenças , Doxorrubicina , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibronectinas/metabolismo , Peróxido de Hidrogênio/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Isquemia Miocárdica/patologia , Isquemia Miocárdica/fisiopatologia , Miocárdio/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteômica/métodos , Ratos , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Fatores de Tempo , Transfecção , Regulação para Cima , Remodelação VentricularRESUMO
Psychological or physical stress induces an elevation of corticosteroids in the circulating system. We report here that corticosterone (CT) protects cardiomyocytes from apoptotic cell death induced by doxorubicin (Dox), an antineoplastic drug known to induce cardiomyopathy possibly through reactive oxygen species production. The cytoprotection induced by CT is within the range of physiologically relevant doses. The lowest dose tested, 0.1 microM (or 3.5 microg/dl), inhibited apoptosis by approximately 25% as determined by caspase activity. With 1 microM CT, cardiomyocytes gain a cytoprotective effect after 8 h of incubation and remain protected for at least 72 h. Hydrocortisone, cortisone, dexamethasone, and aldosterone but not androstenedione or cholesterol also induced cytoprotection. Analyses of 20,000 gene expression sequences using Affymetrix high-density oligonucleotide array found that CT caused up-regulation of 140 genes and down-regulation of 108 genes over 1.5-fold. Among the up-regulated genes are bcl-xL, metallothioneins, glutathione peroxidase-3, and glutathione S-transferases. Western blot analyses revealed that CT induced an elevation of bcl-xL but not bcl-2 or proapoptotic factors bax, bak, and bad. Inhibiting the expression of bcl-xL reduced the cytoprotective effect of CT. Our data suggest that CT induces a cytoprotective effect on cardiomyocytes in association with reprogramming gene expression and induction of bcl-xL gene.
Assuntos
Corticosteroides/farmacologia , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Doxorrubicina/toxicidade , Inativação Metabólica/genética , Miócitos Cardíacos/efeitos dos fármacos , Animais , Apoptose/fisiologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Relação Dose-Resposta a Droga , Miócitos Cardíacos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND/AIM: Excitation-contraction coupling is modulated by nitric oxide (NO) which otherwise has either beneficial or detrimental effects on myocardial function during hypoxia-reoxygenation. This work aimed at characterizing the variations of electromechanical delay (EMD) induced by anoxia-reoxygenation within the developing heart and determining whether atrial and ventricular EMD are modulated by NO to the same extent. METHODS: Hearts of 4 or 4.5-day-old chick embryos were excised and submitted in vitro to normoxia (45 min), anoxia (30 min) and reoxygenation (60 min). Electrocardiogram and atrial and ventricular contractions were simultaneously recorded throughout experiment. Anoxia-reoxygenation-induced chrono-, dromo-and inotropic disturbances and changes in EMD in atrium (EMDa) and ventricle (EMDv) were investigated in control hearts and in hearts exposed to 0.1, 1, 10, 50 and 100 microM of DETA-NONOate (a NO donating agent) or to 50 microM of L-NAME (a NOS inhibitor). RESULTS: Under normoxia, heart rate, PR interval, ventricular shortening velocity, EMDa and EMDv were similar in control, L-NAME-treated and DETA-NONOate-treated hearts. Under anoxia, cardiac activity became markedly erratic within less than 10 min in all groups. At the onset of reoxygenation, EMDv was increased by about 300% with respect to the preanoxic value while EMDa did not vary significatively. Compared to control conditions, L-NAME or DETA-NONOate had no influence on the negative chrono-, dromo- and inotropic effects induced by anoxia-reoxygenation. However, L-NAME prolonged EMDv during anoxia and delayed EMDv recovery during reoxygenation while 100 microM DETA-NONOate had the opposite effects. EMDa was neither affected by NOS inhibitor nor NO donor. At the end of reoxygenation, all the investigated parameters returned to their basal values. CONCLUSION: This work provides evidence that a NO-dependent pathway is involved in regulation of the ventricular excitation-contraction coupling in the anoxic-reoxygenated developing heart.
Assuntos
Ventrículos do Coração/patologia , Coração/embriologia , Óxido Nítrico/química , Animais , Embrião de Galinha , Galinhas , Eletrocardiografia , Eletrofisiologia/métodos , Átrios do Coração/embriologia , Ventrículos do Coração/embriologia , Hipóxia/metabolismo , Técnicas In Vitro , Contração Miocárdica , Miocárdio/metabolismo , Miocárdio/patologia , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Compostos Nitrosos/farmacologia , Oxigênio/metabolismo , Traumatismo por Reperfusão , Transdução de Sinais , Fatores de TempoRESUMO
Oxidants cause activation of the AP-1 transcription factor in cardiomyocytes. c-Fos, a component of the AP-1 transcription factor, is transiently induced by H2O2 and the induction is sensitive to the protein synthesis inhibitor cycloheximide. With high percentage gel electrophoresis, multiple c-Fos bands were resolved by Western blot analyses, indicating post-translational modification of newly synthesized c-Fos protein after H2O2 exposure. Treatment of immunoprecipitated c-Fos protein with the type 2 serine/threonine phosphatase A (PP2A) and immunoblotting of c-Fos protein with antibodies against phosphorylated serine or threonine demonstrated that c-Fos was phosphorylated at serine residues. A pharmacological inhibitor of JNKs inhibited the formation of multiple c-Fos bands without affecting c-fos transcription. The proteasomal inhibitor MG132 and Proteasome Inhibitor I extended the time course of c-Fos protein elevation. An increase in ubiquitin was detectable in c-Fos protein from H2O2-treated cells. Interestingly, treating the whole cell lysates with PP2A, but not calcineurin (i.e. PP2B), resulted in disappearance of c-Fos protein and MG132 was able to prevent this loss. H2O2 caused an elevation of PP2B and total phosphatase activity. The phosphatase inhibitor okadaic acid, but not PP2B inhibiter cypermethrin, extended the time course of c-Fos protein elevation after H2O2 exposure. These data suggest that JNK-mediated phosphorylation of newly synthesized c-Fos protects the protein from being degraded by the proteasome. PP2B independent dephosphorylation contributes to degradation of c-Fos protein during oxidative stress response of cardiomyocytes.