Abstract
It has been reported that oxidized low density lipoprotein (Ox-LDL) can activate both peroxisome proliferator-activated receptor-alpha (PPARalpha) and PPARgamma. However, the detailed mechanisms of Ox-LDL-induced PPARalpha and PPARgamma activation are not fully understood. In the present study, we investigated the effect of Ox-LDL on PPARalpha and PPARgamma activation in macrophages. Ox-LDL, but not LDL, induced PPARalpha and PPARgamma activation in a dose-dependent manner. Ox-LDL transiently induced cyclooxygenase-2 (COX-2) mRNA and protein expression, and COX-2 specific inhibition by NS-398 or meloxicam or small interference RNA of COX-2 suppressed Ox-LDL-induced PPARalpha and PPARgamma activation. Ox-LDL induced phosphorylation of ERK1/2 and p38 MAPK, and ERK1/2 specific inhibition abrogated Ox-LDL-induced COX-2 expression and PPARalpha and PPARgamma activation, whereas p38 MAPK-specific inhibition had no effect. Ox-LDL decreased the amounts of intracellular long chain fatty acids, such as arachidonic, linoleic, oleic, and docosahexaenoic acids. On the other hand, Ox-LDL increased intracellular 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) level through ERK1/2-dependent overexpression of COX-2. Moreover, 15d-PGJ(2) induced both PPARalpha and PPARgamma activation. Furthermore, COX-2 and 15d-PGJ(2) expression and PPAR activity were increased in atherosclerotic lesions of apoE-deficient mice. Finally, we investigated the involvement of PPARalpha and PPARgamma on Ox-LDL-induced mRNA expression of ATP-binding cassette transporter A1 and monocyte chemoattractant protein-1. Interestingly, specific inhibition of PPARalpha and PPARgamma suppressed Ox-LDL-induced ATP-binding cassette transporter A1 mRNA expression and enhanced Ox-LDL-induced monocyte chemoattractant protein-1 mRNA expression. In conclusion, Ox-LDL-induced increase in 15d-PGJ(2) level through ERK1/2-dependent COX-2 expression is one of the mechanisms of PPARalpha and PPARgamma activation in macrophages. These effects of Ox-LDL may control excess atherosclerotic progression.
Highlights
Accumulation of modified low density lipoprotein (LDL),2 such as oxidized LDL (Ox-LDL), and recruitment of monocytes in the arterial subendothelial spaces are early events in atherogenesis [1]
Ox-LDL Induces Peroxisome proliferator-activated receptors (PPARs)␣ and PPAR␥ Activation in Macrophages—We first examined the effect of Ox-LDL, which was prepared with 5 M CuSO4 for 20 h, on PPAR␣ and PPAR␥ activation in RAW264.7 cells by full-length PPAR␣ and PPAR␥ systems
Ox-LDL-induced COX-2 Expression Was Mediated by extracellular signal-regulated kinase 1/2 (ERK1/2) Signals—We have previously reported that Ox-LDL can activate ERK1/2 and p38 mitogen-activated protein kinase (MAPK) [32], which are known to be involved in COX-2 expression induced by several stimuli in macrophages
Summary
Materials—PD98059, SB203580, meloxicam, NS-398, T0070907, and 15d-PGJ2 were purchased from Calbiochem. To measure the PPAR␣ or PPAR␥ ligand binding activity, RAW264.7 cells (2 ϫ 106 cells/well) were transfected with p4xUASg-tk-luc and pM-PPAR␣ or pM-PPAR␥, and subjected to luciferase assays as described below. RAW264.7 cells (2 ϫ 106 cells/well) or mouse peritoneal macrophages (2 ϫ 106 cells/well) were transfected with the siRNA of COX-2, PPAR␣, PPAR␥, or control, in the absence or presence of appropriate plasmids using Lipofectamine 2000 (Invitrogen). Adenoviral Vectors—Cells were infected with a recombinant replication-deficient adenovirus containing each of the following genes: dominant negative ERK (DN-ERK) and p38 MAPK (DN-p38 MAPK) for 48 h at the multiplicity of infection of ϳ50, as described previously [26, 30], and were allowed to recover in medium A for 3 h This condition conferred expression of LacZ as a marker gene in nearly 100% of transfected cells [26, 30]. Differences between groups were examined for statistical significance by one-factor analysis of variance. p Ͻ 0.05 was considered to indicate a statistically significant difference
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