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1.
Cardiovasc Pathol ; 27: 1-8, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27923151

RESUMO

Genome-wide association studies have shown that Krüppel-like factor 14 (KLF14) is associated with both Type 2 diabetes mellitus and lipid metabolism. However, its role in chronic inflammatory responses and the pathogenesis of atherosclerosis remains unknown. The present study was designed to investigate both in vivo and in vitro the impact of KLF14 on chronic inflammatory responses and atherosclerosis. ApoE KO mice, a well-established animal model of atherosclerosis, had higher expressions of KLF14 in aorta tissues than that in C57BL/6 J mice when fed the high-fat diet (HFD) or standard chow diet. Adenovirus-mediated KLF14 knockdown markedly reduced the circulating levels of proinflammatory cytokines and the formation of atherosclerotic lesions in HFD-fed ApoE KO mice. In the in vitro study, KLF14 overexpression in the RAW264.7 macrophages significantly increased the expressions of inflammatory cytokines, total cholesterol (TC), cholesteryl ester (CE), and the ratio of CE to TC in the cells treated with acetylated low-density lipoproteins (AcLDL). Conversely, KLF14 knockdown remarkably attenuated AcLDL-induced increase in TC, CE, and the ratio of CE to TC as well as the expressions of inflammatory cytokines. Furthermore, up-regulation or down-regulation of KLF14 markedly elevated or inhibited the phosphorylation levels of p38 MAPK and ERK1/2 in AcLDL-stimulated RAW264.7 macrophages, respectively. Importantly, treatment with p38 MAPK or ERK1/2 inhibitor nullified the effects of KLF14 on inflammatory cytokine expressions in the cells. These data demonstrate an important role for KLF14 expression in atherosclerotic lesion formation.


Assuntos
Aterosclerose/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Macrófagos/metabolismo , Animais , Aterosclerose/imunologia , Aterosclerose/patologia , Citocinas/biossíntese , Dieta Hiperlipídica , Modelos Animais de Doenças , Immunoblotting , Imuno-Histoquímica , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células RAW 264.7 , Reação em Cadeia da Polimerase em Tempo Real
2.
J Biol Chem ; 278(10): 8091-8, 2003 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-12506111

RESUMO

The apoptosome is a multiprotein complex comprising Apaf-1, cytochrome c, and caspase-9 that functions to activate caspase-3 downstream of mitochondria in response to apoptotic signals. Binding of cytochrome c and dATP to Apaf-1 in the cytosol leads to the assembly of a heptameric complex in which each Apaf-1 subunit is bound noncovalently to a procaspase-9 subunit via their respective CARD domains. Assembly of the apoptosome results in the proteolytic cleavage of procaspase-9 at the cleavage site PEPD(315) to yield the large (p35) and small (p12) caspase-9 subunits. In addition to the PEPD site, caspase-9 contains a caspase-3 cleavage site (DQLD(330)), which when cleaved, produces a smaller p10 subunit in which the NH(2)-terminal 15 amino acids of p12, including the XIAP BIR3 binding motif, are removed. Using purified proteins in a reconstituted reaction in vitro, we have assessed the relative impact of Asp(315) and Asp(330) cleavage on caspase-9 activity within the apoptosome. In addition, we characterized the effect of caspase-3 feedback cleavage of caspase-9 on the rate of caspase-3 activation, and the potential ramifications of Asp(330) cleavage on XIAP-mediated inhibition of the apoptosome. We have found that cleavage of procaspase-9 at Asp(330) to generate p35, p10 or p37, p10 forms resulted in a significant increase (up to 8-fold) in apoptosome activity compared with p35/p12. The significance of this increase was demonstrated by the near complete loss of apoptosome-mediated caspase-3 activity when a point mutant (D330A) of procaspase-9 was substituted for wild-type procaspase-9 in the apoptosome. In addition, cleavage at Asp(330) exposed a novel p10 NH(2)-terminal peptide motif (AISS) that retained the ability to mediate XIAP inhibition of caspase-9. Thus, whereas feedback cleavage of caspase-9 by caspase-3 significantly increases the activity of the apoptosome, it does little to attenuate its sensitivity to inhibition by XIAP.


Assuntos
Apoptose , Caspases/metabolismo , Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Fator Apoptótico 1 Ativador de Proteases , Caspase 3 , Caspase 9 , Inibidores de Caspase , Caspases/química , Grupo dos Citocromos c/metabolismo , Ativação Enzimática , Humanos , Hidrólise , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X
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