Peroxisome proliferator-activated receptor (PPAR)-a is a fatty acid-activated nuclear receptor with roles regulating lipid metabolism, proliferation and inflammation (Bishop-Bailey, 2000). We have previously shown that CYP2J2, an abundant epoxygenase in human cardiovascular and pulmonary systems, activates PPARa (Wray et al., 2003). We have therefore, investigated products of CYP2J2 on PPAR mediated transcriptional activation.
Human embryonic kidney cells (HEK)293 were maintained in DMEM supplemented with antibiotics/ anti-mycotics and 10% FCS (37°C; 5% CO2; 95% air). Cells were transfected with combinations (0.5µg of each) of pACO.luc PPAR luciferase reporter gene, mPPAR or dominant negative PPAR (h6/29; a gift from Dr. Ruth Roberts; AstraZeneca) using NovaFector, as described previously (Wray et al., 2003). Cells were then incubated with 1µM of 8,9-, 11,12-, 14,15 epoxyeicosatrienoic acid (EET), or 5,6-dihydroxyeicosatrienoic acid (DiHETE) for 16h. Cells were then lysed, and luciferase activity measured and normalised to protein content (Wray et al., 2003).
Transfection of PPAR alone induced PPAR transcriptional activation. 8,9-, and 11,12-EET did not effect transcriptional activation alone, but significantly induced PPAR activation in the presence of PPAR. These effects were abolished when dominant negative h6/29PPAR was present (figure 1). 14,15-EET or 5,6-DiHETE did not effect PPAR activation under any condition tested (figure 1).
Figure 1. 8,9-EET and 11,12 EET-activate PPARa mediated reporter gene (fold luciferase/µg protein). HEK293 cells were transfected with pACO.luc, pCMX-mPPAR and/or h6/29PPAR. Activation was measured after 16h of incubation with 8,9-, 11,12-, 14,15-EET or 5,6-DiHETE (all 1µM). Data represents mean ± SEM of n=9 from 3 experiments. * indicates p<0.05 by Wilcoxon matched pairs test between PPARa in the absence and presence of drug treatment and † indicates p<0.05 by Wilcoxon matched pairs test between treatment in the presence or absence of h6/29PPAR.
The CYP2J2 products 8,9- and 11,12-EET activate PPAR. CYP2J2 products may represent novel endogenous mediators for PPAR activation and its subsequent vascular/pulmonary anti-inflammatory and anti-proliferative effects.
(2000). Br. J. Pharmacol. 129, 823-34.
Funded by the William Harvey Medical Research Foundation and the BHF (BS/02/002).