Impaired transactivation of the human CYP2J2 arachidonic acid epoxygenase gene in HepG2 cells subjected to nitrative stress

Abstract

Background and purpose: 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.

Experimental approach: 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.

Key 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 and implications: 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.

Figure 1

(A) Western immunoblot analysis of CYP2J2 expression in microsomal fractions from 12 individual human livers. (B) Cell viability (MTT assay) and apoptosis (caspase-3 activity) in HepG2 cells after treatment with SNP (1 mM, 24 h) and/or 11,12-EET (10 µM, 2 h prior to SNP). (C) Expression of CYP2J2, c-fos and c-jun mRNAs in HepG2 cells 1, 6 or 24 h after application of SNP (1 mM) (open columns, control; solid columns, SNP). (D) Immunoblot and densitometric analysis showing the down-regulation of CYP2J2 and up-regulation of c-fos and c-jun immunoreactive protein expression in SNP-treated HepG2 cells. A representative of at least two independent experiments is shown. Different from time-matched control: *P < 0.05, †P < 0.01, ‡P < 0.001. CYP, cytochrome P450; EET, epoxyeicosatrienoic acid; MTT, thiazolyl blue tetrazolium bromide; SNP, sodium nitroprusside.

Figure 2

Down-regulation of (A) CYP2J2 mRNA and up-regulation of (B) c-fos mRNA and (C) c-jun mRNA in HepG2 cells that were subjected to nitrative stress induced by SNP (1 mM, at 24 h intervals up to 72 h). MAP kinase inhibitors: PD98059 (10 µM, ERK), SB203580 (20 µM, p38) or SP600125 (50 µM, JNK) were included 2 h prior to SNP treatment and replenished at 24 h intervals. Different from time-matched control: *P < 0.05, †P < 0.01, ‡P < 0.001. Different from inhibitor alone: ^aP < 0.05, ^bP < 0.01, ^cP < 0.001. CYP, cytochrome P450; ERK, extracellular signal-regulated kinase; JNK, c-jun N-terminal kinase; MAP kinase, mitogen-activated protein kinase; SNP, sodium nitroprusside.

Figure 3

(A) Immunoblots showing phospho-MAP kinase and total MAP kinase expression in HepG2 lysates harvested at intervals (0–120 min) after SNP administration; the lower panel shows findings from densitometric analysis of a representative of three independent experiments. (B) Immunoblots showing phospho-MAP kinase and total MAP kinase expression in lysates from HepG2 cells that had been treated with SNP (1 mM) at 24 h intervals prior to harvest at 72 h. MAP kinase inhibitors: PD98059 (10 µM, ERK), SB203580 (20 µM, p38) or SP600125 (50 µM, JNK) were included 2 h prior to SNP treatment and replenished at 24 h intervals along with SNP. The lower panel shows findings from densitometric analysis of the immunoblots and aligns with the key above; a representative experiment from two independent experiments is shown. (C) Effect of SNP treatment (1 mM) on CYP2J2 mRNA expression in HepG2 cells that had been transiently transfected with dominant negative mutant p38 and ERK MAP kinase expression plasmids (1 µg DNA) 24 h before the first application of SNP; SNP (1 mM) was replenished at 24 h intervals. Different from control: *P < 0.05. CYP, cytochrome P450; ERK, extracellular signal-regulated kinase; JNK, c-jun N-terminal kinase; MAP kinase, mitogen-activated protein kinase; SNP, sodium nitroprusside.

Figure 4

(A) CYP2J2-luciferase reporter constructs showing the intact AP-1-like sites (solid boxes) in the 5′-proximal region [CYP2J2(−152/+98)] that are absent in the CYP2J2(−49/+98) construct and mutagenized (hatched boxes) in the CYP2J2(−152/+98; mt −105/−95, mt −56/−63) construct. (B) Responsiveness of the CYP2J2-luciferase constructs in transfected HepG2 cells to SNP (1 mM, 24 h; open boxes) relative to control (solid boxes). Different from corresponding untreated control: ‡P < 0.001. AP-1, activator protein-1; CYP, cytochrome P450.

Figure 5

Electromobility shift assay analysis of the binding of double stranded oligonucleotides corresponding to the AP-1-like elements in the CYP2J2 upstream region to nuclear proteins from untreated (CTL) or SNP-treated (SNP) HepG2 cells: (A) CYP2J2-105/-96 element (B) CYP2J2-63/-56 element and (C) the AP-1 consensus element. Gel lanes containing 200-fold excess of the indicated oligonucleotide probe as a cold competitor (self) or the STAT5 element from the β-casein promoter (STAT5) are indicated. Shifted complexes in lanes containing the anti-c-fos or anti-c-jun IgG are also indicated. AP-1, activator protein-1; CYP, cytochrome P450; SNP, sodium nitroprusside.