RESUMO
Acidic sphingomyelinase (ASMase) catalyses the generation of ceramide from sphingomyelin. Ceramide is a lipid mediator and is implicated in mediating and regulating various cellular processes including cell proliferation, differentiation, stress response and inflammation. We have previously reported that electrophiles including diethyl maleate (DEM), heavy metals and cigarette smoke extracts induced ASMase expression in human bladder carcinoma ECV-304 cells, but the mechanism of ASMase mRNA induction by electrophiles remains unknown. In this study, we clarified the involvement of NF-E2-related factor 2 (Nrf2) in the induction of ASMase mRNA by DEM. Promoter analysis using a series of deletion mutants of the human ASMase gene showed that ARE-like element1 located in a region between -200 and -160 bp upstream of the transcription start point is mainly a DEM-responsive element. Moreover, an electrophoretic mobility shift assay using ARE-like element1 revealed that Nrf2 is a candidate transcription factor that binds to ARE-like element1 in response to DEM. Finally, alteration of Nrf2 expression by overexpression and knockdown could regulate the induction of ASMase mRNA by DEM. This is the first evidence that supports the possibility that sphingolipid metabolism is affected via the induction of ASMase by the Nrf2 pathway.
Assuntos
Maleatos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Esfingomielina Fosfodiesterase/genética , Regulação para Cima , Sequência de Bases , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Esfingomielina Fosfodiesterase/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Electrophiles in environmental pollutants or cigarette smoke are high risk factors for various diseases caused by cell injuries such as apoptosis and inflammation. Here we show that electrophilic compounds such as diethyl malate (DEM), methyl mercury and cigarette smoke extracts significantly enhanced the expression of acidic sphingomyelinase (ASMase). ASMase activity and the amount of ceramide of DEM-treated cells were approximately 6 times and 4 times higher than these of non-treated cells, respectively. Moreover, we found that DEM pretreatment enhanced the production of IL-6 induced by TNF-α. Knockdown of ASMase attenuated the enhancement of TNF-α-dependent IL-6 production. On the other hand, enhancement of TNF-α-induced IL-6 production was observed in ASMase-overexpressing cells without DEM. Fractionation of the lipid raft revealed that the TNF receptor 1 (TNFR1) was migrated into the lipid raft in DEM-treated cells or ASMase-overexpressing cells. The TNF-α-induced IL-6 expression required the clustering of TNFR1 since IL-6 expression were decreased by the destruction of the lipid raft with filipin. These results demonstrated a new role for ASMase in the acceleration of the production of TNF-induced IL-6 as a pro-inflammatory cytokine and indicated that electrophiles could potentiate inflammation response by up-regulating of ASMase expression following formation of lipid rafts.