RESUMO
The peroxisome proliferator-activated receptor alpha (PPARalpha) belongs to the nuclear receptor family and plays a central role in the regulation of lipid metabolism, glucose homeostasis and inflammatory processes. In addition to its ligand-induced activation, PPARalpha is regulated by phosphorylation via ERK-MAPK, PKA and PKC. In this study we examined the effect of p38-MAPK on PPARalpha transcriptional activity. In COS-7 cells, anisomycin, a p38 activator, induced a dose-dependent phosphorylation of PPARalpha and a 50% inhibition of its transcriptional activity. In H4IIE hepatoma cells, anisomycin-induced p38 phosphorylation decreased both endogenous and PPARalpha ligand-enhanced L-CPTI and ACO gene expression. Interestingly, PPARalpha/p38 interaction required the molecular adapter ZIP/p62. Reducing ZIP/p62 expression by siRNA, partially reversed the inhibitory effect of anisomycin on L-CPTI gene expression. In conclusion, we showed that p38 activation induced PPARalpha phosphorylation and inhibition of its transcriptional activity through a trimeric interaction between p38-MAPK, ZIP/p62 and PPARalpha.
Assuntos
Proteínas de Choque Térmico/metabolismo , PPAR alfa/metabolismo , Transcrição Gênica , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Anisomicina/metabolismo , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas de Choque Térmico/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Inibidores da Síntese de Ácido Nucleico/metabolismo , PPAR alfa/genética , Proteína Quinase C/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Proteína Sequestossoma-1 , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Liver carnitine palmitoyl transferase (L-CPT) I is a key regulatory enzyme of long-chain fatty acid (LCFA) oxidation that ensures the first step of LCFA import into the mitochondrial matrix. In rat hepatocytes, we showed previously that L-CPT I gene expression was induced by LCFAs as well as by fibrates. The aim of this study was to determine whether LCFA-induced L-CPT I gene expression was mediated by PPARalpha. For this purpose, we constructed a PPARalpha-dominant negative receptor to inhibit endogenous PPARalpha signaling. Highly conserved hydrophobic and charged residues (Leu459 and Glu462) in helix 12 of the ligand-binding domain were mutated to alanine. These mutations led to a total loss of transcriptional activity due to impaired coactivator recruitment. Furthermore, competition studies confirmed that the mutated PPARalpha receptor abolished the wild-type PPARalpha receptor action and thus acted as a powerful dominant negative receptor. When overexpressed in rat hepatoma cells (H4IIE) using a recombinant adenovirus, the mutated PPARalpha receptor antagonized the clofibrate-induced L-CPT I gene expression, whereas it did not affect LCFA-induced L-CPT I. These results provide the first direct demonstration that LCFAs regulate L-CPT I transcription through a PPARalpha-independent pathway, at least in hepatoma cells.
Assuntos
Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Ácido Linoleico/farmacologia , PPAR alfa/metabolismo , Adenoviridae/genética , Animais , Células COS , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Chlorocebus aethiops , Clofibrato/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Hipolipemiantes/farmacologia , Neoplasias Hepáticas , PPAR alfa/genética , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ativação Transcricional/efeitos dos fármacosRESUMO
In addition to their ligand-mediated activation, nuclear receptor activity is finely tuned by their phosphorylation status. PPARs are phosphorylated by several kinases (PKA, PKC, MAPKs, and AMPK), which affect their activity in a ligand-dependent or -independent manner according to the isoform and cellular context. Molecular consequences are multiple, including changes in ligand affinity, DNA binding, recruitment of transcriptional cofactors, proteasome degradation... Finally, the physiological relevance of PPAR phosphorylation is discussed.
