PPARδ activation inhibits homocysteine-induced p22(phox) expression in EA.hy926 cells through reactive oxygen...

Increased expression of the p22(phox) subunit of the NADPH oxidase complex may possibly contribute to both the enzyme׳s increased activation and the occurrence of oxidative stress during hyperhomocysteinaemia. However, the activation of peroxisome proliferator-activated receptor (PPAR) δ has been shown to inhibit p22(phox) expression. The purpose of this study was to elucidate the signaling pathway by which PPARδ activation regulated homocysteine-induced expression of p22(phox). EA.hy926 cells were stimulated with homocysteine (Hcy) in the presence or absence of the PPARδ-specific agonist, GW0742, or of various signaling inhibitors, including the antioxidants N-acetylcysteine (NAC), NADPH oxidase inhibitor, diphenyleneiodonium (DPI), and the p38MAPK inhibitor, SB203580. Expression of p22(phox) mRNA and phospho-p38MAPK protein were measured by real-time PCR and western blot analysis, respectively, and reactive oxygen species were measured by fluorescence microscopy. Our data indicate that Hcy increased both the expression of p22(phox) in a concentration-dependent manner and also increased phosphoryation of p38 MAPK and reactive oxygen species production in a time-dependent manner. However, activation of the PPARδ signaling pathway by the agonist GW0742 reversed all these changes induced by Hcy. Furthermore, SB203580 prevented the increase in p22(phox) expression, and NAC and DPI not only inhibited Hcy-induced phosphorylation of p38MAPK, but also prevented expression of p22(phox). These findings indicate that Hcy-induced expression of p22(phox) is regulated by the reactive oxygen species/p38MAPK pathway and that PPARδ activation is capable of attenuating this pathway by eliminating Hcy-induced reactive oxygen species production.