So Youn Park; Jin Ung Bae; Ki Whan Hong; Chi Dae Kim

doi:10.4196/kjpp.2011.15.2.83

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Park, Bae, Hong, and Kim: HO-1 Induced by Cilostazol Protects Against TNF-α-associated Cytotoxicity via a PPAR-γ-dependent Pathway in Human Endothelial Cells

Original Article

Korean J Physiol Pharmacol 2011;15(2):83-88.

Published online: 18 February 2011

DOI: https://doi.org/10.4196/kjpp.2011.15.2.83

HO-1 Induced by Cilostazol Protects Against TNF-α-associated Cytotoxicity via a PPAR-γ-dependent Pathway in Human Endothelial Cells

So Youn Park, Jin Ung Bae, Ki Whan Hong, Chi Dae Kim

Department of Pharmacology, School of Medicine, and MRC for Ischemic Tissue Regeneration and Medical Research Institute, Pusan National University, Yangsan 626-770, Korea

Corresponding to: Chi Dae Kim, Department of Pharmacology, School of Medicine, Pusan National University, Beomeo-ri, Mulgeum-eup, Yangsan 626-770, Korea. (Tel) 82-51-510-8063, (Fax) 82-51-510-8068, (E-mail) chidkim@pusan.ac.kr

Received 23 January 2011 Revised 10 March 2011 Accepted 14 March 2011

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

A large body of evidence has indicated that induction of endogenous antioxidative proteins seems to be a reasonable strategy for delaying the progression of cell injury. In our previous study, cilostazol was found to increase the expression of the antioxidant enzyme heme oxygenase-1 (HO-1) in synovial cells. Thus, the present study was undertaken to examine whether cilostazol is able to counteract tumor necrosis factor-α (TNF-α)-induced cell death in endothelial cells via the induction of HO-1 expression. We exposed human umbilical vein endothelial cells (HUVECs) to TNF-α (50 ng/ml), with or without cilostazol (10 μM). Pretreatment with cilostazol markedly reduced TNF-α-induced viability loss in the HUVECs, which was reversed by zinc protoporphyrine IX (ZnPP), an inhibitor of HO-1. Moreover, cilostazol increased HO-1 protein and mRNA expression. Cilostazol-induced HO-1 induction was markedly attenuated not only by ZnPP but also by copper-protoporphyrin IX (CuPP). In an assay measuring peroxisome proliferator-activated receptor-γ (PPAR-γ) transcription activity, cilostazol directly increased PPAR-γ transcriptional activity which was completely abolished by HO-1 inhibitor. Furthermore, increased PPAR-γ activity by cilostazol and rosiglitazone was completely abolished in cells transfected with HO-1 siRNA. Taken together, these results indicate that cilostazol up-regulates HO-1 and protects cells against TNF-α-induced endothelial cytotoxicity via a PPAR-γ-dependent pathway.

Keywords: Cilosatzol, PPAR-γ, HO-1, Endothelial cells

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Fig. 1.

Dose-dependent cytotoxic effects of TNF-α (1∼100 ng/ml) on HUVEC_s (A) and suppression of these effects by cilostazol (B). Cells were pretreated with cilostazol (1∼100 μM) for 4 h, and then stimulated with TNF-α (50 ng/mL) for 18 h. Results are expressed as the mean±S.E.M. of four experiments. ^∗p<0.05, ^∗∗p<0.01, ^∗∗∗p<0.001 vs. none, ^##p<0.01 vs. TNF-α alone.

Fig. 2.

Effect of an HO-1 inhibitor on the protective effects of cilostazol against TNF-α-mediated cytotoxicity in HUVECs. Cells were pretreated with cilostazol in the presence or absence of an HO-1 inhibitor (ZnPP, 1 μM) and then stimulated with TNF-α (50 ng/ml) for 18 h. Results are expressed as the mean±S.E.M. of four experiments. ^∗∗p<0.01 vs. none, ^#p<0.05 vs. TNF-α alone, ^††p<0.01 vs. TNF-α + cilostazol.

Fig. 3.

Cilostazol induces HO-1 expression. HUVECs were stimulated with different concentrations of cilostazol for 12 h. The expression of HO-1 mRNA and protein was determined using reverse transcription PCR and Western blotting, respectively. GAPDH and β-actin were used as loading controls. Results are expressed as the mean±S.E.M. of four experiments. ^∗p<0.05, ^∗∗p<0.01 vs. vehicle.

Fig. 4.

Effect of an HO-1 inhibitor on cilostazol-induced HO-1 expression. HUVECs were pretreated with an HO-1 inhibitor (ZnPP), and then stimulated with cilostazol (10 μM) for 12 h. The expression of HO-1 mRNA and protein was determined using reverse transcription PCR and Western blotting, respectively. GAPDH and μ-actin were used as loading controls. Results are expressed as the mean±S.E.M. of three or four experiments. ^∗p<0.05; ^∗∗p<0.01, ^∗∗∗p <0.001 vs. vehicle, ^#p<0.05; ^###p <0.001 vs. cilostazol alone.

Fig. 5.

HO-1 mediates the effect of cliostazol and rosiglitazone on PPAR-γ transcriptional activity. (A) HUVECs were transiently transfected with PPRE-pGL3, a renilla luciferase control reporter vector, and then treated either with cilostazol (10 μM) or rosiglitazone (10 μM) for 24 h. PPAR-γ transcriptional activity was determined using a luciferase activity assay. (B.) HUVECs were transfected with siRNA specific for HO-1 then treated with cilostazol (10 μM) or rosiglitazone (10 μM) for 24 h. ^∗∗p<0.01, ^∗∗∗p<0.001 vs. no treatment, ^###p<0.001 vs. cilostazol, ^†††p<0.001 vs. rosiglitazone.

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