RU486 antagonizes the inhibitory effect of peroxisome proliferator-activated receptor α on interleukin-6 production in vascular endothelial cells

https://doi.org/10.1016/S0960-0760(02)00055-9Get rights and content

Abstract

Peroxisome proliferator-acitivated receptor α (PPARα) is a member of nuclear receptor superfamily. Recent studies have shown that the activators for PPARα inhibit the expression of some inflammatory molecules in vascular endothelial cells (ECs) and vascular smooth muscle cells, indicating the anti-inflammatory roles of PPARα on vascular walls. In this investigation, we showed that RU486, already proved to be an active anti-glucocorticoid and anti-progesterone agent, blocked the inhibition of tumor necrosis factor (TNF)-α-stimulated interleukin-6 (IL-6) production by the PPARα activator fenofibrate in human umbilical vein ECs. Transient transfection of bovine aortic ECs with an IL-6 promoter construct demonstrated that RU486 blocked the inhibitory effect of fenofibrate on TNF-α-induced IL-6 promoter activity. By fluorescence microscopy, RU486 was found to prevent fenofibrate-induced nuclear translocation of PPARα. Thus, RU486 has an antagonizing effect on PPARα-mediated down-regulation of IL-6 in vascular ECs. This effect may be exerted by its interference with the nuclear translocation of PPARα.

Introduction

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily, which consist of at least three isoforms (α, γ, and δ) with different tissue distribution and ligand specificity [1], [2]. Out of these three isoforms, PPARα is widely expressed in various tissues such as liver, heart, muscle, kidney and intestine [3]. This receptor is activated by fatty acids, eicosanoids and hypolipidemic fibric acid derivatives such as fenofibrate, bezafibrate, clofibrate and gemfibrozil [4], [5], [6]. Recently, it has been shown that PPARα is also expressed in vascular endothelial cells (ECs) [7], [8], [9], [10], [11] as well as vascular smooth muscle cells [12]. In these vascular cells, the expression of various inflammatory molecules including cytokines and adhesion molecules is up-regulated in response to proinflammatory cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-1, which represents part of the inflammatory process affecting vascular walls [13], [14], [15]. The activators for PPARα are shown to repress the expression of IL-6, vascular cell adhesion molecule-1, prostaglandin and cyclooxygenase-2 in these cells [9], [11], [12]. These results suggest that PPARα can work as an anti-inflammatory modulator in vascular walls.

RU486 (MIFEPRISTONE) is an active anti-glucocorticoid and anti-progesterone agent [16], [17]. This agent is known to bind to glucocorticoid receptor (GR) and progesterone receptor (PR) [16], [17]. Several biochemical studies revealed that RU486 stabilizes association of GR with heat shock protein 90 K (hsp90) and masks the functional DNA binding domain of the receptor in vivo [18], [19], [20]. Other studies also indicated that RU486 acts at steps subsequent to PR-DNA binding [21], [22], [23]. Thus, RU486 exerts its antagonizing effect via affecting different steps of the hormone action, probably dependent upon the receptor types.

To date, it has not been reported whether or not RU486 antagonizes PPARα. In order to reveal this issue, we examined the effects of RU486 on the repression of TNF-α-stimulated IL-6 production by the PPARα activator fenofibrate in vascular ECs.

Section snippets

Materials

Human TNF-α was obtained from Dainippon Pharmaceutical (Osaka, Japan). Basic fibroblast growth factor (bFGF) and fenofibrate were from Kaken Pharmaceutical (Osaka, Japan). RU486 was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). Rabbit antibody against PPARα was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cy3-conjugated anti-rabbit IgG antibody was from Jackson Immunoresearch Laboratories (West Grove, PA). Seapansy luciferase control plasmid was obtained from

RU486 blocks the inhibition of TNF-α-stimulated IL-6 production by fenofibrate in human vascular ECs

We previously reported that fenofibrate inhibited TNF-α-induced IL-6 production in HUVECs [11]. RU486 (10−5 M) alone had no effect on IL-6 concentrations in the culture supernatant of HUVECs. RU486 also showed no significant effect on TNF-α-induced IL-6 production (Fig. 1A). However, RU486 significantly blocked the inhibition of TNF-α-induced IL-6 production by 10−4 or 2.5×10−4 M of fenofibrate (Fig. 1A). RU486 showed significant blockade against the fenofibrate-mediated inhibition of the IL-6

Discussion

RU486 is known to elicit anti-glucocorticoid and anti-progesterone effects [16], [17]. In the present study, we for the first time demonstrated that RU486 antagonized the inhibitory effect of the PPARα activator fenofibrate on the TNF-α-stimulated IL-6 production in human vascular ECs. As we reported recently [11], the inhibitory effect of the PPARα activator is exerted at least partly as a result of transcriptional inhibition of the IL-6 gene. In the experiments involving transient

Acknowledgements

We thank Dr. Shizuo Akira for providing us the IL-6 reporter construct. We also thank Ms. Miho Sasaki for her secretarial assistance in preparing the manuscript. This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports Science and Technology of Japan (S.K.) and the Osaka Foundation for Promotion of Clinical Immunology (S.K.).

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