Abstract
The peroxisomal proliferator-activated nuclear receptor-alpha (PPARalpha), the target for most hypolipidemic drugs in current clinical use, regulates the transcription of genes involved in lipid metabolism and transport, and energy homeostasis. More recently, PPARalpha and its ligands have been implicated in inflammatory responses and the regulation of cell proliferation. PPARalpha also regulates the expression of Cyp4a fatty acid omega-hydroxylases and Cyp2c arachidonic acid epoxygenase genes. To study the role of the PPARalpha receptor and of its Cyp2c epoxygenase gene target in tumorigenesis, we treated mice injected with tumor cells with Wy-14,643, a PPARalpha-selective ligand. Compared with untreated controls, Wy-14643-treated animals showed marked reductions in tumor growth and vascularization, which were accompanied by decreases in the plasma levels of pro-angiogenic epoxygenase metabolites (EETs), hepatic EET biosynthesis, and Cyp2c epoxygenase expression. All these Wy-14643-induced responses were absent in PPARalpha(-/-) mice and are thus PPARalpha-mediated. Primary cultures of mouse lung endothelial cells treated with Wy-14643 showed reductions in cell proliferation and in the formation of capillary-like structures. These effects were absent in cells obtained from PPRAalpha(-/-) mice and reversed by the addition of EETs. These results identify important anti-angiogenic and anti-tumorigenic roles for PPARalpha, characterize the contribution of its Cyp2c epoxygenases gene target to these responses, and suggest potential anti-cancer roles for this nuclear receptor and its ligands.
Highlights
The peroxisomal proliferator activated nuclear receptors (PPARs),2 namely PPAR␣, PPAR/␦, and PPAR␥, regulate the transcription of several genes involved in lipid metabolism, as
The regulation of CYP2C epoxygenase expression and epoxyeicosatrienoic acids (EETs) biosynthesis by the PPAR␣ nuclear receptor and its ligands [5,6,7], offered an opportunity to study the role of PPAR␣ and the epoxygenase pathway in tumorigenesis
Wy-14643 Inhibits the Proliferation of Endothelial Cells Isolated from Wild-type but Not from peroxisomal proliferator-activated nuclear receptor-␣ (PPAR␣)KO Mice—To determine whether Wy-14643 and the PPAR␣ receptor had an effect on endothelial cell proliferation, primary cultures of lung endothelial cells isolated from wild-type and PPAR␣KO mice were incubated with different concentrations of Wy-14643, and their proliferation was evaluated by measuring [3H]thymidine incorporation
Summary
Cell Culture—Primary murine pulmonary microvascular endothelial cells were isolated from sex- and age-matched adult wild-type and PPAR␣Ϫ/Ϫ (PPAR␣KO) mice (a gift from Dr Frank Gonzalez, NCI, National Institutes of Health, Bethesda, MD) [29] and cultured in EGM-2-MV (Clonetics) containing 5% fetal calf serum as described [30]. Due to limitations in the number of primary endothelial cells that can be isolated and cultured from mouse lungs, conditionally immortalized wildtype cells were used as surrogates for the analysis of epoxygenase protein expression and EET/DHET biosynthesis. After 4 days, the cells were incubated in serum-free medium containing AA (10 M, final concentration) and, 4 h after, the cells and the media were removed from the plates, and the EETs and DHETs present in the cells and the media were extracted, purified, and quantified as described below. The EETs and DHETs present in cultured cells, mouse liver, or plasma were extracted, purified, analyzed, and quantified by the isotope ratio method using gas chromatography/mass spectrometry as described [41]. Statistical Analysis—One-tailed Student’s t test was used for group comparisons and for the analysis of variance using Sigma-Stat software for statistical differences between multiple groups. p Ն 0.05 was considered statistically significant
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