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Prostaglandins & other Lipid Mediators 68–69 (2002) 165–175 Distinct functions of COX-1 and COX-2 Ikuo Morita Department of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8549, Japan Abstract The enzymes that convert arachidonic acid to prostaglandin H2 are named cyclooxygenase-1 (COX-1) and COX-2. The properties of COX-1 are different from those of COX-2. It was originally thought that the function of COX-1 was involved in physiological phenomena, whereas that of COX-2 was involved in various pathologies. However, studies with COX-2 knockout mouse suggest that COX-2 also plays important roles in development and homeostasis. This chapter focuses on the distinct functions of COX-1 and COX-2. © 2002 Elsevier Science Inc. All rights reserved. Keywords: COX-1; COX-2; Knockout mouse 1. Introduction Prostaglandins are known to be involved in many physiological and pathological pro- cesses including inflammation [1], bone resorption [2], ovulation [3], and angiogenesis [4]. Since the discovery of prostaglandin H synthase-2, which is referred to as cyclooxygenase-2 (COX-2) in this review, numerous studies have focused on delineating the distinct roles of COX-1 and COX-2. These studies have been of four general types: (a) expression of either COX-1 or COX-2 mRNA and protein in tissues and organs; (b) pharmacological inhibition of COX-1 and/or COX-2; (c) COX-1 and COX-2 gene disruptions in mice; and (d) overexpression of COX-1 and COX-2 in various cells. These studies led to the con- clusion that these two closely related enzymes have distinct functions in the tissues and organs and have raised the possibility that selective inhibition of either COX isozyme may have useful therapeutic outcomes. In this chapter, I focus on the distinct functions of COX-1 and COX-2 and discuss the reason why two COX isozymes are necessary in the mammals. Tel.: +81-3-5803-5575; fax: +81-3-5803-0212. E-mail address: [email protected] (I. Morita). 0090-6980/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII:S0090-6980(02)00029-1

Distinct functions of COX-1 and COX-2

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Page 1: Distinct functions of COX-1 and COX-2

Prostaglandins & other Lipid Mediators 68–69 (2002) 165–175

Distinct functions of COX-1 and COX-2

Ikuo Morita∗Department of Cellular Physiological Chemistry, Graduate School, Tokyo Medical and Dental University,

Bunkyo-ku, Tokyo 113-8549, Japan

Abstract

The enzymes that convert arachidonic acid to prostaglandin H2 are named cyclooxygenase-1(COX-1) and COX-2. The properties of COX-1 are different from those of COX-2. It was originallythought that the function of COX-1 was involved in physiological phenomena, whereas that ofCOX-2 was involved in various pathologies. However, studies with COX-2 knockout mouse suggestthat COX-2 also plays important roles in development and homeostasis. This chapter focuses onthe distinct functions of COX-1 and COX-2.© 2002 Elsevier Science Inc. All rights reserved.

Keywords:COX-1; COX-2; Knockout mouse

1. Introduction

Prostaglandins are known to be involved in many physiological and pathological pro-cesses including inflammation[1], bone resorption[2], ovulation[3], and angiogenesis[4].Since the discovery of prostaglandin H synthase-2, which is referred to as cyclooxygenase-2(COX-2) in this review, numerous studies have focused on delineating the distinct rolesof COX-1 and COX-2. These studies have been of four general types: (a) expression ofeither COX-1 or COX-2 mRNA and protein in tissues and organs; (b) pharmacologicalinhibition of COX-1 and/or COX-2; (c) COX-1 and COX-2 gene disruptions in mice; and(d) overexpression of COX-1 and COX-2 in various cells. These studies led to the con-clusion that these two closely related enzymes have distinct functions in the tissues andorgans and have raised the possibility that selective inhibition of either COX isozymemay have useful therapeutic outcomes. In this chapter, I focus on the distinct functions ofCOX-1 and COX-2 and discuss the reason why two COX isozymes are necessary in themammals.

∗ Tel.: +81-3-5803-5575; fax:+81-3-5803-0212.E-mail address:[email protected] (I. Morita).

0090-6980/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.PII: S0090-6980(02)00029-1

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2. Expression of COX-1 and COX-2

2.1. Expression of COX-1 in various tissues and cells

It is recognized that COX-1 mRNA and protein are present at relatively stable levels inmany tissues and cells. Because fewcis-acting response elements and no TATA box havebeen identified in the 5′-flanking region of the COX-1 gene[5], COX-1 gene has beenconsidered to be a “housekeeping” gene. Many but not all tissues and cells express COX-1[6]. Moreover, COX-1 is inducible in some systems[7–18]. As shown inTable 1, in somecell lines COX-1 expression is increased during differentiation, while in endothelial cellsCOX-1 is increased in response to shear stress, VEGF and thrombin. Gel shift and promoterdeletion assays have demonstrated that Sp1cis-regulatory element at−610/−604 in thehuman COX-1 promoter is involved in transcription in endothelial cells[19]. It is widelyrecognized that Sp1 protein levels in nuclei are constitutive, but the ratio of Sp1/Sp3, thephosphorylated/glycosylated state of Sp1 or the coordinate binding of other transcriptionalfactors and Sp1 regulates the binding of Sp1 to its cognate sites in the promoters, andthis, in turn, alters transcription rates[20–22]. Unfortunately, the mechanism underlyingtranscriptional regulation of COX-1 by Sp1 has not been determined.

2.2. Expression of COX-2 in various tissues and cells

In contrast to COX-1, numerous regulatory elements have been identified in the 5′-flankingregion of COX-2 genes. Among them, there are two NF�B, one Sp1, one NF-IL-6 and oneCRE binding sites[23]. Several growth factors, cytokines and mechanical stress activatethese transcriptional factors, and as a consequence, upregulate the COX-2 gene expres-sion. As an example, the regulation of COX-2 expression in endothelial cells is summa-rized inTable 2. Expression of the COX-2 gene can be suppressed by glucocorticoids andanti-inflammatory cytokines such as IL-4 and IL-10[24]. The transcriptional regulationof COX-2 by glucocorticoids has been investigated and in some cases COX-2 expression

Table 1Induction of COX-1

Species Cell Stimulus Reference

Human THP-1 Phorbol ester [7]Rat EGV-6 Phorbol ester [8]Mouse Immature mast cells Ligands (c-kit) [9]Human Megakaryocytes Phorbol ester [10]Human Endothelial cells Shear stress [11]Bovine andhuman Endothelial cells VEGF [12]Human Lung fibroblasts TGF� [13]Rat Osteocytes Mechanical stress [14]Ovine Endothelial cells 17�-Estradiol [15]Guinea pig Gallbladder Bradykinin [16]Human Synovial cells (primary culture) IL-1� [17]Bovine Endothelial cells Thrombin [18]

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Table 2Inducers of COX-2 expression in endothelial cells

Cytokines Growth factors or tumor promoters Others

IL-1� Serum factor PGE2IL-1� aFGF NOIL-6 bFGF Anti-phospholipid antibodyIL-8 Insulin HypoxiaIL-11 IGF Mechanical stressTNF� EGF 25-HydroxycholesterolLPS PDGFIFN� TGF�

VEGFPhorbol ester

is inhibited. Glucocorticoids increase I�B proteins and suppress NF�B-mediated COX-2mRNA expression[25]. What is more, the glucocorticoid receptor–glucocorticoid complexcan bind to c-jun proteins and suppresses IL-1-mediated COX-2 mRNA expression[26].Glucocorticoids also regulate COX-2 protein expression by modifying COX-2 mRNA sta-bility. The 3′-untranslated region of COX-2 is extremely AT rich; for instance, there are 17copies of the ATTTA (Shaw–Kamens) sequence in the human COX-2 gene[27]. This motifis common in many inducible genes, such as those for interleukins and for inducible NOsynthase, and contributes to the mRNA instability.

3. Properties of COX-1 and COX-2 enzymes

Small differences in the structure of COX-1 and COX-2 lead to their important pharma-cological and biological differences (Table 3). The active site of COX-1 is smaller than thatof COX-2. Several substitutions including replacement of Ile434 in COX-1 with Val434 inCOX-2 increase the relative volume of the active site of COX-1[28]. In part, the discoverythat the active sites of COX-1 and COX-2 are of different sizes led to the development ofthe COX-2 specific inhibitors[29]. Moreover, the size difference between the active sitesis consistent with the finding that COX-1 is completely inhibited by aspirin acetylation,whereas COX-2 is still able to convert arachidonic acid to 15-R-HETE after aspirin treat-ment[30], and that dihomo-�-linolenic acid and eicosapentaenoic acid are somewhat bettersubstrates for human COX-2 than COX-1[31]. However, the difference in the size of theactive sites does not affect the gross kinetic properties of these two isozymes; COX-1 andCOX-2 have similarKm values with arachidonic acid[32].

Despite the fact that there are no gross differences in kinetics between COX-1 and COX-2,prostaglandins can be produced via either COX-1 or COX-2 in cells and tissues dependingas the conditions. Under serum-free conditions, cultured bovine endothelial cells expressedonly COX-1. Treatment of these cells with calcium ionophore (phospholipase activation)does not cause prostaglandin formation indicating that COX-1 is not able to synthesizeprostaglandins from endogenously released arachidonic acid. When the cells are treatedwith phorbol ester, an induction of COX-2 protein occurs that parallels an increase in

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Table 3Properties of COX-1 and COX-2

COX-1 COX-2

mRNA size 3 kb 4–4.5 kbmRNA stability Stable UnstableAmino acid 576 581Properties of enzyme Constitutive InducibleCells Almost all Induced-stimulated cells

Many tumor cellsMain biological functions Platelet aggregation Platelet disaggregation

Renal water balance InflammationGastric cytoprotection Vasodilation

Bone resorption and manypathological events

Subcellular localization Endoplasmic reticulum andnuclear membrane

Mainly nuclear membrane

Arachidonic acid utilized Mainly exogenous Endogenous and exogenousSubstrates Mainly arachidonic acid Arachidonic acidAspirin treatment No metabolite 15(R) hydroperoxy-

eicosatetra-enoic acidGlucocorticoid treatment No effect Inhibition (induction)

prostaglandin production[33]. There are three major possible explanations for these results:(a) differences in subcellular localizations; (b) differential coupling of phospholipases toCOX-1 and COX-2 and (c) different utilization of arachidonic acids by COX-1 and COX-2within cells.

The subcellular localization of COX-1 and COX-2 is an important consideration postulat-ing in distinct functions for COX-1 and COX-2. Confocal fluorescence imaging microscopyand histofluorescence staining techniques reveal that COX-1 and COX-2 are located in theendoplasmic reticulum and nuclear envelope but that COX-2 is more highly concentrated inthe nuclear envelope[34]. It is commonly recognized that activated cytosolic phospholipaseA2 (cPLA2) translocates to the perinuclear envelope and arachidonic acid is released fromphospholipids in the nuclear membrane[35].

Arachidonic acid can be mobilized by several different phospholipase A2s. Among themcPLA2 and type IIA and type V secreted PLA2 (sPLA2) predominantly contribute toprostaglandin production. Early work focused on the coupling of cPLA2 and COX-2 andthe coupling of sPLA2 and COX-1. However, coexpression of either COX-1 or COX-2 withvarious PLA2 has clearly demonstrated that the prostaglandin production induced by IL-1and dependent on COX-2 can involve any one of several PLA2s (cPLA2 and IIA, V and XsPLA2)[36]. These latter studies indicated that prostaglandin production via COX-2 in acti-vated cells is unlikely to be controlled by the specific coupling of phospholipases and COXs.

Our laboratory and others have reported that low concentrations of arachidonic acid(<2.5�M) are not oxygenated by COX-1 but rather are oxygenated exclusively by COX-2in intact cells. In contrast, higher concentrations of arachidonic acid (>10�M) are predom-inantly acted on by COX-1 rather than COX-2. The concentration of released arachidonicacid (i.e. via phospholipase catalyses) is usually less than 1�M, thus the endogenously

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released arachidonic acid at this level is mostly utilized by COX-2[33,37]. The mechanismfor selective metabolism of arachidonic acid by COX-2 probably dose not depend solelyon kinetic properties because theKm value for the two isozymes are nearly the same. Onepossible explanation is the different dependency of COX-1 and COX-2 on radical tone[38]. Prostaglandin production via COX-1, but not via COX-2, is inhibited by the combinedpresence of glutathione and glutathione peroxidase[39]. This is because COX-1 requiresabout 10-fold higher hydroperoxide levels to be activated than required by COX-2[40].

4. Distinct roles of COX-1 and COX-2

4.1. Phenotypic changes in COX-1 and COX-2 deficient mice

Using gene disruption experiments, the biological roles of proteins can be tested. In thecase of COX-1 and COX-2 the phenotypes of deficient mice have supported the data obtainedfrom pharmacological and epidemiological experiments. Overall, for the maintenance ofnormal physiology, it appears that a deficiency of COX-2 has more profound effects than adeficiency of COX-1.

COX-1(−/−) mice have reduced platelet aggregation and decreased arachidonic acid-induced inflammation but phorbol-induced inflammation is unaffected[41]. These data areconsistent with the fact that platelets have only COX-1, which contributes to platelet aggre-gation and high concentrations of arachidonic acid are able to be converted to prostaglandinsby COX-1. COX-1(−/−) mice are also sensitive to the radiation injury. Crypt stem cell sur-vival after gamma-irradiation decreased, and crypt epithelial cell apoptosis increased inCOX-1(−/−) mice[42].

However, COX-1(−/−) mice have no gastric pathology and are resistant to indomethacin-induced gastric ulceration[41]. This was a surprising result because in animal model andclinical studies classical NSAIDs induce gastric ulcers, but COX-2 specific inhibitors donot. The discrepancy between gene disruption and pharmacological results leads to thetesting of novel NSAIDs which inhibit specifically COX-1 and were found not to causegastric damage. COX-1(−/−) and COX-1(−/−) pairings lead to few live offspring, andthus COX-1 is important in reproduction[43].

In contrast to COX-1 deficient mice, COX-2 deficient mice have more dramatic pheno-typic changes. In COX-2 deficient mice female reproductive functions including ovulation,fertilization, implantation and decidualization are defective[44,45]. In the nervous systemCOX-2 deficient mice have a significant reduction in brain injury induced by ischemia[46].Among the phenotype changes in COX-2 deficient mice, the suppression of tumorigenesis isparticularly exciting, because the data confirm the epidemiological studies in which NSAIDhas been shown to suppress the incidence of colon cancer. Introduction of a COX-2 genemutation to the Apo�715 knockout mice reduced the number and size of intestinal polypsdramatically[47]. Other phenotype changes observed in the COX-2 deficient mice are (a)renal nephropathy, (b) cardiac fibrosis, (c) peritonitis, and (d) failure of ductus arteriosusclosure[48,49].

One problem with gene disruption approaches is that they are often complicated bycompensation by other enzymes. In COX-2 deficient mice, COX-1 is the alternative source

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for prostaglandin synthesis. In fact, the effects of COX-2 specific inhibitors on femalereproduction in the control mice are less than those observed with COX-1(−/−) mice[43]. Moreover, gene disruption experiments have led to the recognition that there arenovel mechanisms for NSAID activities in tumorigenesis. Primary fibroblasts derived fromCOX-1 and COX-2 double knockout mice are readily transformed by Ha-ras and SV-40,but even in these double knockout cells, NSAIDs inhibit colony formation in soft agar andinduce apoptosis[50]. These results indicate that transformation is independent of the statusof COX expression and that COX is involved in tumorigenesis at later stage. One third ofCOX-2(−/−) mice died with a patent ductus arteriosus within 48 h after birth, however,this ductus arteriosus significantly increased in COX-2(−/−) mice with inactivation of onecopy of the gene encoding COX-1 (79%). Of course, the ductus arteriosus is not observedin COX-1(−/−) mice[51].

4.2. Inflammation

Prostaglandins are produced in the inflamed tissues, and treatment with NSAIDs in-hibits the production of prostaglandins and down-regulates inflammation-related patholog-ical symptoms such as pain and swelling. During inflammation, COX-1 mRNA, proteinand activity levels do not change, but COX-2 levels increase dramatically, and, as a result,prostaglandin production increases. Moreover, when COX-2 specific inhibitors are admin-istered, prostaglandin production and subsequent inflammation are significantly reduced.These data have led to the conclusion that COX-2 is involved in inflammation, whereasCOX-1 is not[52].

During the inflammation process, COX-1 is thought to contribute to “resolution”. Inexperimental mesangioproliferative glomerulonephritis COX-1 is expressed in glomeruliduring the repair period[53]. In the process of ulcer healing, the COX-1 specific inhibitorsas well as the COX-2 specific ones delay healing. These results implicate the role of COX-1in the resolution, but not the progression, of inflammation.

The COX-2 gene is particularly responsive to mediators of inflammation. For example,IL-1�, IL-1�, TNF�, and LPS induce COX-2 gene expression and subsequent prostaglandinsproduction[54–56]. Therefore, COX-2 specific inhibitors have been used to attenuate thesymptoms of inflammation such as osteoarthritis, rheumatoid arthritis and musculoskeletalpain in patients[57,58]. In inflammation-related cells, the membrane bound type of PGEsynthase (mPGE2 synthase) is also induced by these cytokines[59]. The large amount ofPGE2 produced at the inflammation site by the coupling of COX-2 and mPGE2 synthasemay be involved in the progression of inflammation.

4.3. Cardiovascular system

It is well-known that the platelet has COX-1 protein alone, and thromboxane A2 producedvia COX-1 has an important role in thrombosis. In certain stages of megakaryogenesis,COX-2 as well as COX-1 are detectable[60], but the reason why demarcated plateletshave COX-1, but not COX-2 is unclear. It may be attributable to the different subcellularlocalizations of COX-1 and COX-2 in magakaryocytes, because COX-2 is more highlylocalized in the nuclear membrane than COX-1[34]. Alternatively, there may be differences

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in the stabilities of COX-1 and COX-2 or attenuation of COX-2 gene expression at the laterstage of platelet-formation.

It has been thought that vascular endothelial cells and smooth muscle cells have COX-1,and that prostacyclin formed via COX-1 has an important role in blood flow, blood pressureand anti-aggregation of platelets. However, a recent work has shown that prostacyclin invascular cells is produced by COX-2 as well as COX-1 under both physiological and patho-logical conditions. Treatment of volunteers with a COX-2 specific inhibitor decreased thelevels of urinary prostacyclin metabolites without affecting thromboxane A2 metabolites.In contrast, indomethacin decreased metabolites of both prostacyclin and thromboxane A2[61]. These results raised the possibility of an increased risk of cardiovascular events asso-ciated with COX-2 specific inhibitors. However, two major randomized trials have shownthe opposite results. VIGOR (8076 patients) showed that the relative risk of an adjudicatedthrombotic cardiovascular event with COX-2 specific inhibitor treatment compared withnaproxen was 2.38, whereas CLASS (8059 patients) showed the numbers of events associ-ated with COX-2 specific inhibitors and classical NSAIDs were not significantly different[62,63]. Further trial evaluation will be needed to determine the magnitude of the risk, if any.The reports that NO synthase gene therapy ameliorated several markers of arteriosclerosis[64] and NO-releasing NSAIDs were more effective than the traditional NSAIDs againstcancer cell proliferation[65] may lead to the development of NO-releasing COX-2 specificinhibitors.

In pathological conditions, COX-2 expression is enhanced, prostacyclin and PGE2 areproduced, and glucocorticoids prevent the hypotension caused by endotoxin. These obser-vations suggest that COX-2 contributes to hypotension in pathological conditions[66].

4.4. Tumorigenesis

COX has been implicated in the development of malignant tumors by epidemiologicalstudies, work with gene-disrupted mice and COX overexpression and pharmacological stud-ies. In tumorigenesis the role of COX-1 is distinct from that of COX-2. COX-1 is expressedin vascular endothelial cells and contributes to angiogenesis, which is an endothelial cellfunction and is involved in growth of tumors, endometrial growth, wound healing and in-flammation. The origin of the neovasculum is thought to be microvessel endothelial cellsand circulating endothelial cell precursors[67]. Human umbilical endothelial cells or aorticendothelial cells are commonly used in vitro as an angiogenesis model. In this system, ananti-sense oligonucleotide to COX-1 suppresses tube formation induced by colon cancercells overexpressing COX-2[68]. However, in our laboratory NSAIDs treatment of endothe-lial cells cultured between collagen gels did not cause inhibition of tube formation inducedby high concentration of vascular endothelial growth factor (VEGF) (unpublished results).One possibility to explain this apparent discrepancy is that the contribution of COX-1 toangiogenesis may be dependent on the stimulator of angiogenesis.

In contrast to the relatively small contribution of COX-1 in tumorigenesis, COX-2 isfunctional in tumorigenesis and tumor growth. Overexpression of COX-2 in tumor cellscauses cells to escape from apoptosis and to invade the matrix[69,70]. The contribution ofCOX-2 to tumorigenesis is mainly through three processes: (a) induction of angiogenic fac-tors such as VEGF, (b) anti-apoptosis, and (c) development of malignancy. These processes

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are closely linked to each other. In vivo tumor-associated macrophages as well as tumorcells produce PGE2 via COX-2, and the PGE2 produced induces VEGF, and finally VEGFstimulates angiogenesis[71]. The phenomenon is also observed in carrageenen-inducedinflammation[72].

4.5. Renal function

Although the prevalence of nephrotoxicity in patients treated with NSAIDs is relativelylow, the extensive use profile of these agents implies that many persons are at risk. At basalstates of normal renal function, the role of renal prostaglandin production in maintenanceof stable renal hemodynamic functions is relatively limited. Using immunohistochemistryin adult human kidney, COX-1 was detected in the collecting ducts, the loops of Henle,interstitial cells, endothelial cells, smooth muscle cells and pre- or post-glomerular vessels.In fetal kidney, COX-1 was primarily expressed in podocytes and collecting duct cells[73,74]. These data suggest that COX-1 is involved in glomerulogenesis. And some datashow that COX-1 regulates renal blood flow.

COX-2 expression in the human kidney was detected in the renal vascularture, medullaryinterstitial cells, and the macula densa. Glomerular staining showed that COX-2 was de-tectable in podocytes only in the final stage of renal development[73,74]. These data sug-gest that COX-2 modulated by podocytes will be involved in renal perfusion and glomerularhemodynamics.

5. Conclusion

In intact cells, COX-2 utilizes a low concentration of arachidonic acid about equivalentto the concentration of arachidonic acid released endogenously. This means the productionof prostaglandins via COX-2 is regulated by the activation of phospholipases and the ex-pression of the COX-2 gene. Many cytokines and growth factors affect the phospholipasesand COX-2, and glucocorticoids inhibit both phospholipase activity and the induction ofCOX-2. In contrast, COX-1 functions only at relatively high concentrations of arachidonicacid, corresponding to those observed when arachidonic acid is added exogenously or duringplatelet aggregation or cell injury or acute inflammation.

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