Roles of Cyclooxygenase (COX)-1 and COX-2 in Prostanoid Production by Human Endothelial Cells: Selective Up-Regulation of Prostacyclin Synthesis by COX-2 This information is current as of October 2, 2021. Gillian E. Caughey, Leslie G. Cleland, Peter S. Penglis, Jennifer R. Gamble and Michael J. James J Immunol 2001; 167:2831-2838; ; doi: 10.4049/jimmunol.167.5.2831 http://www.jimmunol.org/content/167/5/2831 Downloaded from References This article cites 36 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/167/5/2831.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 2, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Roles of Cyclooxygenase (COX)-1 and COX-2 in Prostanoid Production by Human Endothelial Cells: Selective Up-Regulation of Prostacyclin Synthesis by COX-21 Gillian E. Caughey,2* Leslie G. Cleland,* Peter S. Penglis,* Jennifer R. Gamble,† and Michael J. James* The two cyclooxygenase (COX) isoforms, COX-1 and COX-2, both metabolize arachidonic acid to PGH2, the common substrate for thromboxane A2 (TXA2), prostacyclin (PGI2), and PGE2 synthesis. We characterized the synthesis of these prostanoids in HUVECs in relation to COX-1 and COX-2 activity. Untreated HUVEC expressed only COX-1, whereas addition of IL-1␤ caused induction of COX-2. TXA2 was the predominant COX-1-derived product, and TXA2 synthesis changed little with up-regulation ␤ of COX-2 by IL-1 (2-fold increase). By contrast, COX-2 up-regulation was associated with large increases in the synthesis of PGI2 Downloaded from and PGE2 (54- and 84-fold increases, respectively). Addition of the selective COX-2 inhibitor, NS-398, almost completely abolished ␤ PGI2 and PGE2 synthesis, but had little effect on TXA2 synthesis. The up-regulation of COX-2 by IL-1 was accompanied by specific up-regulation of PGI synthase and PGE synthase, but not TX synthase. An examination of the substrate concentration dependencies showed that the pathway of TXA2 synthesis was saturated at a 20-fold lower arachidonic acid concentration than that for PGI2 and PGE2 synthesis. In conclusion, endothelial prostanoid synthesis appears to be differentially regulated by the induction of COX-2. The apparent PGI2 and PGE2 linkage with COX-2 activity may be explained by a temporal increase in total http://www.jimmunol.org/ COX activity, together with selective up-regulation of PGI synthase and PGE synthase, and different kinetic characteristics of the terminal synthases. These findings have particular importance with regard to the potential for cardiovascular consequences of COX-2 inhibition. The Journal of Immunology, 2001, 167: 2831–2838. 3 he prostanoids prostacyclin (PGI2) and thromboxane A2 bacterial endotoxin and cytokines. Also, several different prosta- (TXA2) play an essential role in the maintenance of vas- noid terminal synthases can be present within the one cell, and it cular homeostasis. PGI is a vasodilator and an inhibitor T 2 is not known what determines the relative rate of production of of platelet aggregation, whereas TXA2 is a vasoconstrictor and a each individual prostanoid within the same cell. In monocytic promoter of platelet aggregation (1). As a consequence of their cells, it has been observed that the ratio of PGE2/TXA2 produced by guest on October 2, 2021 opposing roles, an imbalance in PGI2 or TXA2 production has is not fixed, but varies according to which COX isoform is present. been implicated in the pathophysiology of many thrombotic and For example, in rat peritoneal macrophages, under conditions in cardiovascular disorders (1–3). Therefore, it is important to under- which only COX-1 was expressed, TXA2 was synthesized in ex- stand factors and conditions that might affect the balance of PGI / 2 cess of PGE2. However, under conditions of stimulation in which TXA2 synthesis. COX-2 was induced, the profile of prostanoid production shifted to PGI2 and TXA2 are products of arachidonic acid (AA) metab- favor PGE2 over TXA2 production (4, 5). olism by cyclooxygenase (COX), followed by metabolism of the It is considered that PGI2 is the main prostanoid synthesized by COX product, PGH2, by the terminal synthase enzymes, prosta- vascular endothelium and TXA2 is the main prostanoid produced cyclin or TX synthase, respectively. Two isoforms of COX have by platelets. However, the endothelium has been reported to syn- been identified: COX-1 is expressed constitutively in most cell thesize TXA2 in addition to PGI2 (6), and both COX isoforms have types, whereas COX-2 is induced by inflammatory stimuli such as been observed, with only COX-1 being detectable in unstimulated cells (6, 7). Endothelial COX-2 can be up-regulated in vitro by inflammatory stimuli (6, 8) and shear stress (7, 9). Because the *Rheumatology Unit, Royal Adelaide Hospital, and †Division of Human Immunol- balance between PGI2 and TXA2 production is central in the main- ogy, Hanson Centre for Cancer Research, Adelaide, South Australia, Australia tenance of vascular tone and platelet aggregation, determination of Received for publication November 27, 2000. Accepted for publication June the roles of endothelial COX isozymes, particularly with regard to 20, 2001. the contribution of COX-2 in the regulation of prostanoid biosyn- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance thesis by the endothelium, is important. with 18 U.S.C. Section 1734 solely to indicate this fact. In the current study, we examined the synthesis of prostanoids 1 This work was funded by the Alan Watkinson Memorial Postdoctoral Fellowship derived from either COX-1 or COX-2 by HUVECs. We observed from the Arthritis Foundation of Australia (to G.E.C.), and grants from the National Health and Medical Research Council of Australia and the National Heart Foundation that TXA2 is the predominant COX-1 product, whereas up-regu- of Australia. lation of COX-2 by IL-1␤ is associated with a greater increase in 2 Address correspondence and reprint requests to Dr. Gillian E. Caughey, Rheuma- the synthesis of PGI2 and PGE2 than TXA2. Both PGI synthase tology Unit, Royal Adelaide Hospital, Adelaide, South Australia 5000, Australia. and PGE synthase, but not TX synthase, were up-regulated by E-mail address: [email protected] IL-1␤. Additionally, an examination of the substrate concentration 3 Abbreviations used in this paper: PGI2, prostacyclin; TX, thromboxane; AA, ara- chidonic acid; ASA, acetylsalicylic acid; COX, cyclooxygenase; MAPK, mitogen- dependencies of PGI2, PGE2, and TXA2 synthesis suggests that activated protein kinase. different kinetic parameters of the terminal synthases are a major Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 2832 COX-1 AND COX-2 IN ENDOTHELIAL CELLS determinant of the dominance of PGI2 and PGE2 production when COX-2 was induced by IL-1␤. Materials and Methods Materials Human rIL-1␤ was from Genzyme (Cambridge, MA). AA, NS-398 (N-(2- cyclohexyloxy-4-nitrophenyl) methanesulfonamide, rabbit polyclonal Ab against human COX-2, and murine mAb against COX-1 were all purchased from Cayman Chemicals (Ann Arbor, MI). Peroxidase-labeled donkey anti- 3 rabbit and goat anti-mouse Abs, ECL Western blotting system, [ H]PGE2, 3 3 [ H]6-keto PGF1␣, and [ H]TXB2 were purchased from Amersham Phar- macia Biotech (Piscataway, NJ). PGH2 and (4-(4-fluorophenyl)-2-(4-meth- ylsulfinyl phenyl)-5-(4-pyridyl)1H-imidazole) (SB 203580) were obtained FIGURE 1. Time course of COX-2 protein induction by IL-1␤. from Calbiochem (San Diego, CA). Abs to the phosphorylated forms of HUVECs (4.5 ϫ 105) were stimulated with IL-1␤ (1 ng/ml) at time 0. At p38 and p44/42 mitogen-activated protein kinases (MAPKs) and the the indicated times, cells were processed for Western blot analysis, as mitogen-activated protein/extracellular signal-related kinase 1 inhibitor described in Materials and Methods. Results are representative of at least PD98059 were purchased from New England Biolabs (Beverly, MA). Rab- five separate experiments. bit anti-serum against PGE2 and 6-keto PGF1␣ and mouse mAb against ␤-actin were obtained from Sigma (St. Louis, MO). Cell culture Downloaded from HUVECs were isolated as described (10). The cells were cultured on gel- period examined (data not shown). Treatment of HUVECs with atin-coated culture flasks in medium M199 with Earle’s salts supplemented IL-1␤ (1 ng/ml) for increasing times resulted in expression of ␮ with 20% FCS, 25 g/ml endothelial growth supplement (Genome Ther- COX-2, which was maximal by 16 h (Fig. 1). By comparison, apeutics, Waltham, MA), and 25 ␮g/ml heparin. Cells between passages 2 and 4 were plated in 24-well dishes (1.5 ϫ 105/ml) and allowed to reach COX-1 was expressed in unstimulated cells, and its expression did confluence (24 h). not change with IL-1␤ treatment (Fig. 1). A time course of pro- stanoid production by HUVECs in response to IL-1␤ treatment Cell stimulation http://www.jimmunol.org/ indicated very different synthesis profiles between TXA2 and PGI2 HUVECs were incubated with RPMI 1640 medium (containing 10 mM or PGE2.