13-hydroxy linoleic acid increases expression of the cholesterol transporters ABCA1, ABCG1 and SR-BI and stimulates apoA-I-dependent cholesterol efflux in RAW264.7 macrophages

Abstract

Background: Synthetic activators of peroxisome proliferator-activated receptors (PPARs) stimulate cholesterol removal from macrophages through PPAR-dependent up-regulation of liver × receptor α (LXRα) and subsequent induction of cholesterol exporters such as ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type 1 (SR-BI). The present study aimed to test the hypothesis that the hydroxylated derivative of linoleic acid (LA), 13-HODE, which is a natural PPAR agonist, has similar effects in RAW264.7 macrophages.

Methods: RAW264.7 macrophages were treated without (control) or with LA or 13-HODE in the presence and absence of PPARα or PPARγ antagonists and determined protein levels of LXRα, ABCA1, ABCG1, SR-BI, PPARα and PPARγ and apolipoprotein A-I mediated lipid efflux.

Results: Treatment of RAW264.7 cells with 13-HODE increased PPAR-transactivation activity and protein concentrations of LXRα, ABCA1, ABCG1 and SR-BI when compared to control treatment (P < 0.05). In addition, 13-HODE enhanced cholesterol concentration in the medium but decreased cellular cholesterol concentration during incubation of cells with the extracellular lipid acceptor apolipoprotein A-I (P < 0.05). Pre-treatment of cells with a selective PPARα or PPARγ antagonist completely abolished the effects of 13-HODE on cholesterol efflux and protein levels of genes investigated. In contrast to 13-HODE, LA had no effect on either of these parameters compared to control cells.

Conclusion: 13-HODE induces cholesterol efflux from macrophages via the PPAR-LXRα-ABCA1/SR-BI-pathway.

Figure 1

Effects of 13-HODE, LA and WY-14,643 on PPAR/PPRE transactivation activity and PPAR protein levels in RAW264.6 macrophages. A, RAW264.7 cells were transiently transfected with 3 × ACO-PPRE reporter vector. After transfection, cells were treated or not with 0.1-2.5 μmol/L 13-HODE, 1-100 μmol/L LA and 50 μmol/L WY-14,643 for 24 h. Afterwards, cells were lysed, and luciferase activities of the ACO-PPRE firefly luciferase vector and a co-transfected renilla luciferase vector determined by a dual luciferase assay. Bars represent means ± SD from four independent experiments (n = 4). Data are expressed as percentage of relative luciferase activity of vehicle control cells. Results from statistical analysis are indicated: Significant effects are denoted with an asterisk (P < 0.05). B, RAW264.7 cells were treated with 2.5 μmol/L 13-HODE, 100 μmol/L LA or vehicle (ethanol) for 24 h. Afterwards, cells were lysed and subsequently processed for western blotting as described in the materials and methods section. Representative immunoblots specific for PPARα, PPARγ, and β-actin which was used for normalization are shown. Bars represent data from densitometric analysis and are means ± SD from three independent experiments (n = 3). Data are expressed as percentage of protein concentration of vehicle control cells.

Figure 2

Effects of 13-HODE and LA in the presence and absence of PPARα and PPARγ selective antagonists on molecular markers of cholesterol homeostasis in RAW264.7 macrophages. RAW264.7 cells were pre-treated without or with the PPARα selective antagonist GW6471 or the PPARγ selective antagonist GW9662 and subsequently treated without (vehicle control) or with 2.5 μmol/L 13-HODE or 100 μmol/L LA for 24 h. Afterwards, cells were lysed and subsequently processed for western blotting as described in the materials and methods section. A, Representative immunoblots specific for ABCA1, ABCG1, SR-BI, LXRα, and β-actin which was used for normalization are shown. B, Bars represent data from densitometric analysis and are means ± SD from three independent experiments (n = 3). Data are expressed as percentage of protein concentration of vehicle control cells. Results from statistical analysis are indicated: Significant effects are denoted with superscript letters. Bars marked without a common superscript letter significantly differ (P < 0.05).

Figure 3

Effects of 13-HODE and LA on cholesterol concentrations in cells and medium of macrophages in macrophages in the presence and absence of apoA-I and PPARα and PPARγ antagonists. After pre-treatment without or with PPAR antagonists for 4 h and treatment of RAW264.7 macrophage cells without (vehicle control) or with 2.5 μmol/L 13-HODE or 100 μmol/L LA for 20 h, cells were incubated again without or with the antagonists for 4 h and afterwards without or with the corresponding fatty acids in the presence or absence of apolipoprotein A-I (apoA-I) (30 μg/mL) for 24 h. Afterwards, medium was collected, and cells were washed with PBS. Total lipids were extracted from medium and cells and concentrations of cholesterol determined as described in the materials and methods section. A, Cellular and B, medium cholesterol concentrations were related to cellular protein content. Bars represent means ± SD from four independent experiments (n = 4). Data are expressed as percentage of cholesterol concentration in cells and medium of control cells. Results from statistical analysis are indicated: Significant effects are denoted with an asterisk (P < 0.05).