RESUMO
Cytochrome P4502E1 (CYP2E1) plays a key role in the metabolism of numerous drug substrates, mostly in mammalian liver. Both the apoprotein and mRNA levels are increased in response to interleukin 4 (IL-4) in primary human hepatocyte cultures. We developed a human hepatoma cell model that faithfully reproduces the responsiveness of the CYP2E1 gene to IL-4 at least in part through transcriptional activation, upon treatment with 150 U/ml of IL-4. As expected, IL-4 induced tyrosine phosphorylation of the STAT6 transcription factor, an effect prevented by the tyrosine kinase inhibitor tyrphostin A25. However, this inhibitor as well as genistein (another inhibitor of tyrosine kinases) had no effect on the IL-4 induction of CYP2E1. Similarly, protein kinase A activators (forskolin and dibutyryl-cAMP) and inhibitor (H89) did not influence the response to IL-4. However, PKC inhibitors (H7 and calphostin C) strongly blocked any induction of the gene, as well as the IL-4-dependent translocation of PKCS. Taken together, our results show that IL-4 coordinately induces CYP2E1 transcription, mRNA and apoprotein levels in human hepatoma cells in a PKC-dependent manner, potentially through the activity of the PKCzeta isoform.
Assuntos
Citocromo P-450 CYP2E1/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Hepatócitos/enzimologia , Interleucina-4/farmacologia , Fígado/enzimologia , Proteína Quinase C/metabolismo , Transcrição Gênica/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , Bucladesina/farmacologia , Carcinoma Hepatocelular , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Neoplasias Hepáticas , Biossíntese de Proteínas , RNA Mensageiro/genética , Fator de Transcrição STAT6 , Acetato de Tetradecanoilforbol/farmacologia , Transativadores/metabolismo , Transcrição Gênica/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Phenobarbital (PB) has long been known as an inducer of drug-metabolizing enzymes in liver, but the molecular mechanism underlying this induction is still poorly understood. Using primary mouse hepatocyte culture, we have investigated the possible involvement of different regulatory pathways in PB action, by exposing PB-treated cells to various protein kinase/phosphatase modulators. Our results showed a negative role of the cAMP-dependent pathway, as treatment with cAMP-dependent protein kinase (PKA) activators (10 microM dibutyryl-cAMP and 50 microM forskolin) dramatically inhibited PB-induced Cyp2b9/10 mRNA accumulation, whereas PKA inhibitor potentiated the PB responsiveness of this gene. The cGMP-dependent protein kinase (PKG) seems to play a positive role as PKG inhibitor reduced the PB-induced level of Cyp2b9/10 mRNA. We also obtained two lines of evidence for the involvement of Ca2+ in modulating PB action. Firstly, measurements of intracellular Fura-2 fluorescence ratio in murine hepatocytes showed that long-term PB incubation (24 and 48 h) led to a significant increase of [Ca2+]i. Secondly, treatment with an intracellular Ca2+ chelator (BAPTA-AM) nearly completely abolished PB-induced Cyp2b9/10 expression. Ca2+ thus appeared to mediate PB action likely via Ca2+/calmodulin-dependent protein kinase II, as KN62, a specific inhibitor of this enzyme, also dramatically inhibited PB induction of the Cyp2b9/10 genes.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Sistema Enzimático do Citocromo P-450/genética , Fígado/enzimologia , Fenobarbital/farmacologia , Esteroide Hidroxilases , Ativação Transcricional/efeitos dos fármacos , Animais , Bucladesina/farmacologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Células Cultivadas , Quelantes/metabolismo , Quelantes/farmacologia , Colforsina/farmacologia , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico , Família 2 do Citocromo P450 , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Corantes Fluorescentes/metabolismo , Fura-2/metabolismo , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fenobarbital/antagonistas & inibidores , Inibidores de Proteínas Quinases , Proteínas Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
We investigated the involvement of diverse protein kinases and phosphatases in the transduction pathways elicited by phenobarbital (PB), a well-known inducer of some hepatic cytochromes P450 (CYP). Different inhibitors or activators of protein kinases or phosphatases were assessed for their ability to modulate PB-induction of CYP2B and CYP3A mRNA expression. Rat hepatocytes in primary culture were treated with the test compounds one hour prior to, and then continuously, in the absence or presence of 1 mmol/L PB for 24 h. By northern blot analysis of CYP2B1/2 and 3A1/2 gene expression, we first confirmed the negative role of the adenosine 3':5' cyclic monophosphate (cAMP)/protein kinase A pathway and the positive role of some serine/threonine protein phosphatases in the mechanism of PB-induction. The present data further suggested that Ca2+/calmodulin-dependent protein kinases II (independently of Ca2+) and extracellular signal-regulated kinases 1/2 (ERK1/2) might function respectively as positive and negative regulator in the PB-induction of CYP2B and CYP3A. In contrast, protein kinases C and phosphatidylinositol-3-kinase did not appear to be involved, while the role of tyrosine kinases remained unclear. We conclude that a complex network of phosphorylation/dephosphorylation events might be crucial for PB-induction of rat CYP2B and CYP3A.
