Impaired transactivation of the human CYP2J2 arachidonic acid epoxygenase gene in HepG2 cells subjected to nitrative stress.
OBJECTIVE
Human cytochrome P450 2J2 (CYP2J2) generates epoxyfatty acids that modulate cellular apoptosis and proliferation. CYP2J2 regulation has not been intensively studied but induction of the activator protein-1 (AP-1) subunit c-fos mediates CYP2J2 down-regulation in hypoxia, a component of ischaemic injury. Decreased CYP2J2 expression may contribute to tissue injury.


METHODS
HepG2 cells were treated with sodium nitroprusside (SNP) to induce nitrative stress, which has been associated with inflammation and infection in liver and other tissues. CYP2J2 protein and mRNA expression were evaluated by immunoblotting and real-time PCR respectively. The role of mitogen-activated protein (MAP) kinases in CYP2J2 dysregulation was assessed using specific inhibitors and dominant negative MAP kinase expression plasmids. CYP2J2-luciferase reporter constructs and electromobility shift assays (EMSAs) were used to identify SNP-regulated regions in the CYP2J2 gene.


RESULTS
Cytochrome P450 2J2 was down-regulated by SNP while the AP-1 proteins c-jun and c-fos were up-regulated; inhibition of p38 and ERK MAP kinases normalized their expression. The gene elements at -105/-95 and -56/-63 were required for the down-regulation of CYP2J2 induced by nitrative stress.


CONCLUSIONS
p38 and ERK MAP kinases transduce stress stimuli that down-regulate CYP2J2. Targeting these kinases may prevent the loss of CYP2J2 and epoxy-fatty acids that protect cells against deleterious stresses.
Cui
Pei H
PH
Pharmacogenomics and Drug Development Group, Faculty of Pharmacy, University of Sydney, NSW, Australia.
Lee
Andy C
AC
Zhou
Fanfan
F
Murray
Michael
M
D001483:Base Sequence; D045744:Cell Line, Tumor; D000090762:Cytochrome P-450 CYP2J2; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D017931:DNA Primers; D015536:Down-Regulation; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism*; D009599:Nitroprusside / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015533:Transcriptional Activation
C000717350:CYP2J2 protein, human; D017931:DNA Primers; D009566:Nitrates; D009599:Nitroprusside; D003577:Cytochrome P-450 Enzyme System; D000090762:Cytochrome P-450 CYP2J2; D020928:Mitogen-Activated Protein Kinases