IL-4-mediated transcriptional regulation of human CYP2E1 by two independent signaling pathways.
Cytochrome P450 2E1 (CYP2E1), the alcohol-inducible member of the cytochrome P450 super family, plays an important role in both physiological and pathophysiological processes. The present study focused on the induction of human CYP2E1 transcription by the anti-inflammatory cytokine interleukin-4 (IL-4) in human hepatoma B16A2 cells and revealed that this regulation is mediated by two independent pathways. RNA interference and overexpression of STAT6, indicated that the JAK-STAT signaling pathway is involved in IL-4-dependent induction and mutagenesis revealed the presence of a STAT6 binding site in CYP2E1 proximal promoter region (-583/-574-bp). However, inhibition of the JAK-STAT6 pathway using JAK1 siRNA constructs could only partially inhibit the induction of CYP2E1 promoter constructs indicating the presence of a second IL-4 responsive element. Indeed by using a series of truncated CYP2E1 promoter constructs a second more distal IL-4 responsive element (-1604/-1428-bp) was identified, which was further shown to involve the activation of IRS1/2. This induction was dependent on the transcription factor NFATc1 as IL-4-induced CYP2E1 expression was altered by silencing or overexpressing NFATc1. A NFATc1 binding site was identified in the second distal IL-4 responsive element (-1551/-1545-bp) by chromatin immunoprecipitation (ChIP) analysis. Finally simultaneous siRNA-mediated down-regulation of both STAT6 and NFATc1 or mutation of both STAT6 and NFATc1 binding sites abolished the IL-4-dependent transcriptional induction of CYP2E1, demonstrating that both pathways are required for maximal activation. In conclusion, the present study indicates that the induction of CYP2E1 transcription by IL-4 is mediated through two independent parallel pathways, involving JAK-STAT6 and IRS1/2 and NFATc1.
Wang
Jue
J
Section of Pharmacogenetics, Department of Physiology and Pharmacology, Karolinska Institute, Nanna Svartz väg 2, 171 77 Stockholm, Sweden.
Hu
Yin
Y
Nekvindova
Jana
J
Ingelman-Sundberg
Magnus
M
Neve
Etienne P A
EP
D015152:Blotting, Northern; D045744:Cell Line, Tumor; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D000072467:Cytochrome P450 Family 2; D017930:Genes, Reporter; D006801:Humans; D055504:Insulin Receptor Substrate Proteins / Q000235:genetics / Q000378:metabolism; D015847:Interleukin-4 / Q000494:pharmacology / Q000502:physiology*; D053613:Janus Kinase 1 / Q000235:genetics / Q000378:metabolism; D049409:Luciferases, Firefly / Q000235:genetics; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D020218:Response Elements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050800:STAT6 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014158:Transcription, Genetic; D014162:Transfection
D055504:Insulin Receptor Substrate Proteins; D050778:NFATC Transcription Factors; C494003:NFATC1 protein, human; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human; D015847:Interleukin-4; D003577:Cytochrome P-450 Enzyme System; D049409:Luciferases, Firefly; C110332:CYP2F1 protein, human; D000072467:Cytochrome P450 Family 2; C507904:JAK1 protein, human; D053613:Janus Kinase 1