Toxicology Letters 190 (2009) 172–178 Contents lists available at ScienceDirect Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet Cholesterol-3-beta, 5-alpha, 6-beta-triol induced PI 3 K-Akt-eNOS-dependent cyclooxygenase-2 expression in endothelial cells Po-Lin Liao a,1 , Yu-Wen Cheng b,1 , Ching-Hao Li a , Yi-Ling Lo b , Jaw-Jou Kang a,∗ a Institute of Toxicology, College of Medicine, National Taiwan University, Jen-Ai Road, Section 1, Taipei 100, Taiwan, ROC b Department of Pharmaceutical Analysis, School of Pharmacy, Taipei Medical University, Taipei, Taiwan, ROC article info Article history: Received 5 June 2009 Received in revised form 6 July 2009 Accepted 8 July 2009 Available online 16 July 2009 Keywords: Atherosclerosis Cholesterol-3-beta, 5-alpha, 6-beta-triol Cyclooxygenase-2 Nitric oxide abstract Oxidized cholesterols belong to a subgroup of oxLDLs which play major roles in atherosclerosis. In order to investigate the contribution of oxysterols from oxLDLs in atherosclerosis, cholesterol-3-beta, 5-alpha, 6-beta-triol (-Triol) was studied in human umbilical vein endothelial cells. We found that - Triol concentration- and time-dependently enhanced COX-2 protein expression and mRNA production followed by PGE 2 generation in human umbilical vein endothelial cells. In addition, -Triol upregulated peNOS 1177 protein phosphorylation and concentration-dependently increased nitric oxide production. eNOS 1177 phosphorylation was abrogated by the PI3K inhibitor, LY294002. In studying the mechanisms involved in -Triol-induced COX-2/PGE 2 production, inhibitors of NOS, PI3K, p38, and NF-B, effectively attenuated COX-2 protein induction and mRNA expression, suggesting that the PI 3 K-Akt-eNOS pathway, p38MAPK, and NF-B are involved in -Triol-induced COX-2 expression, and following increases in p38 and Akt phosphorylation, the concentration-dependent inhibition of COX-2 protein expression by L-NAME further suggested their involvement at the translation level. We concluded that -Triol increases COX-2 mRNA and protein expression via coordination with the PI 3 K-Akt-eNOS pathway and NF-B. Moreover, COX-2 gene expression might be regulated by activated p38 MAPK in another unknown regulation path- way. Our findings also suggested that -Triol might contribute to the effect of induced atherosclerosis in humans through COX-2 production in endothelial cells. © 2009 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Oxysterols are oxygenated cholesterol derivatives and constitute a family of compounds with various biological activities (Guardiola et al., 1996). They have been ascribed a number of important roles in connection with atherosclerosis, apoptosis, necrosis, inflamma- tion, immunosuppression, cholesterol turnover, and carcinogenesis (Brown and Jessup, 1999; Schroepfer, 2000; Wang and Afdhal, 2001; Yoon et al., 2004). They are generated by enzymatic mechanisms in cells as well as by nonezymatic mechanisms in various kinds of food during processing or storage for long periods (van de Bovenkamp et al., 1988). Oxysterols accumulate in the subendothelial level of the arterial wall during atherogenic processes (Berliner and Heinecke, 1996) and are believed to play important roles in the development of atherosclerosis (Witztum and Steinberg, 2001). They are also a major component of oxLDLs, which are some of the most notorious atherogenic factors, suggesting that oxysterols might be responsi- ble for the toxicity of oxLDLs (Hubbard et al., 1989; Imai et al., 1980). ∗ Corresponding author. Tel.: +886 2 23123456x88603; fax: +886 2 23410217. E-mail addresses: jjkang@ntu.edu.tw, ywcheng@tmu.edu.tw (J.-J. Kang). 1 These authors have equal contribution in this work. COX-2, an essential enzyme involved in inflammatory and other pathogenetic processes (Kuwano et al., 2004), is detectable only in certain types of tissues, and is the inducible form of a biphasic enzyme responsible for catalyzing the conversion of arachidonic acid to PGH 2 . PGH 2 is then subsequently catalyzed to other prostanoids, including PGE 2 . Prostanoids are potent mediators of inflammatory responses and increase vascular permeability. COX- 2 is found in macrophages, vascular endothelial cells, and vascular smooth muscle cells (LaPointe et al., 2004). Since atherosclerosis is a chronic inflammatory condition (Li, 2001), it is possible that COX-2 is involved in the formation of atherosclerotic plaques. Endothelial cells are known to possess both COX isoforms, and their induction has been demonstrated to occur in response to different proin- flammatory cytokines, such as interleukin IL-1 and , and TNF- (Caughey et al., 2001; Eligini et al., 2001). Therefore, the induction of COX-2 in endothelial cells might result from an inflammatory response. Nitric oxide (NO) produced in the endothelium was consid- ered as an endothelium-derived relaxing factor (Furchgott and Zawadzki, 1980). Three isoforms of NOS have been identified: two constitutive NOSs, endothelial (e)NOS and neuronal (n)NOS, which are regulated by Ca 2+ , and one inducible (i)NOS, which is inde- pendent of Ca 2+ regulation (Marletta, 1993). NO is now recognized 0378-4274/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2009.07.012