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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 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 processes 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 conclusion 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. ∗
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0090-6980/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 0 - 6 9 8 0 ( 0 2 ) 0 0 0 2 9 - 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 in many tissues and cells. Because few cis-acting response elements and no TATA box have been identified in the 5 -flanking region of the COX-1 gene [5], COX-1 gene has been considered 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 in Table 1, in some cell lines COX-1 expression is increased during differentiation, while in endothelial cells COX-1 is increased in response to shear stress, VEGF and thrombin. Gel shift and promoter deletion assays have demonstrated that Sp1 cis-regulatory element at −610/−604 in the human COX-1 promoter is involved in transcription in endothelial cells [19]. It is widely recognized that Sp1 protein levels in nuclei are constitutive, but the ratio of Sp1/Sp3, the phosphorylated/glycosylated state of Sp1 or the coordinate binding of other transcriptional factors and Sp1 regulates the binding of Sp1 to its cognate sites in the promoters, and this, in turn, alters transcription rates [20–22]. Unfortunately, the mechanism underlying transcriptional 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 -flanking region of COX-2 genes. Among them, there are two NFB, one Sp1, one NF-IL-6 and one CRE binding sites [23]. Several growth factors, cytokines and mechanical stress activate these transcriptional factors, and as a consequence, upregulate the COX-2 gene expression. As an example, the regulation of COX-2 expression in endothelial cells is summarized in Table 2. Expression of the COX-2 gene can be suppressed by glucocorticoids and anti-inflammatory cytokines such as IL-4 and IL-10 [24]. The transcriptional regulation of COX-2 by glucocorticoids has been investigated and in some cases COX-2 expression Table 1 Induction of COX-1 Species
Cell
Stimulus
Reference
Human Rat Mouse Human Human Bovine and human Human Rat Ovine Guinea pig Human Bovine
THP-1 EGV-6 Immature mast cells Megakaryocytes Endothelial cells
Phorbol ester Phorbol ester Ligands (c-kit) Phorbol ester Shear stress
[7] [8] [9] [10] [11]
Endothelial cells Lung fibroblasts Osteocytes Endothelial cells Gallbladder Synovial cells (primary culture) Endothelial cells
VEGF TGF Mechanical stress 17-Estradiol Bradykinin IL-1␣ Thrombin
[12] [13] [14] [15] [16] [17] [18]
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Table 2 Inducers of COX-2 expression in endothelial cells Cytokines
Growth factors or tumor promoters
Others
IL-1␣ IL-1 IL-6 IL-8 IL-11 TNF␣ LPS IFN␥
Serum factor aFGF bFGF Insulin IGF EGF PDGF TGF VEGF Phorbol ester
PGE2 NO Anti-phospholipid antibody Hypoxia Mechanical stress 25-Hydroxycholesterol
is inhibited. Glucocorticoids increase IB proteins and suppress NFB-mediated COX-2 mRNA expression [25]. What is more, the glucocorticoid receptor–glucocorticoid complex can 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 stability. The 3 -untranslated region of COX-2 is extremely AT rich; for instance, there are 17 copies of the ATTTA (Shaw–Kamens) sequence in the human COX-2 gene [27]. This motif is common in many inducible genes, such as those for interleukins and for inducible NO synthase, 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 pharmacological and biological differences (Table 3). The active site of COX-1 is smaller than that of COX-2. Several substitutions including replacement of Ile434 in COX-1 with Val434 in COX-2 increase the relative volume of the active site of COX-1 [28]. In part, the discovery that the active sites of COX-1 and COX-2 are of different sizes led to the development of the COX-2 specific inhibitors [29]. Moreover, the size difference between the active sites is 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 treatment [30], and that dihomo-␥-linolenic acid and eicosapentaenoic acid are somewhat better substrates for human COX-2 than COX-1 [31]. However, the difference in the size of the active sites does not affect the gross kinetic properties of these two isozymes; COX-1 and COX-2 have similar Km 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 depending as the conditions. Under serum-free conditions, cultured bovine endothelial cells expressed only COX-1. Treatment of these cells with calcium ionophore (phospholipase activation) does not cause prostaglandin formation indicating that COX-1 is not able to synthesize prostaglandins from endogenously released arachidonic acid. When the cells are treated with phorbol ester, an induction of COX-2 protein occurs that parallels an increase in
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Table 3 Properties of COX-1 and COX-2 COX-1
COX-2
mRNA size mRNA stability Amino acid Properties of enzyme Cells
3 kb Stable 576 Constitutive Almost all
Main biological functions
Platelet aggregation Renal water balance Gastric cytoprotection
Subcellular localization Arachidonic acid utilized Substrates Aspirin treatment
Endoplasmic reticulum and nuclear membrane Mainly exogenous Mainly arachidonic acid No metabolite
4–4.5 kb Unstable 581 Inducible Induced-stimulated cells Many tumor cells Platelet disaggregation Inflammation Vasodilation Bone resorption and many pathological events Mainly nuclear membrane
Glucocorticoid treatment
No effect
Endogenous and exogenous Arachidonic acid 15(R) hydroperoxyeicosatetra-enoic acid Inhibition (induction)
prostaglandin production [33]. There are three major possible explanations for these results: (a) differences in subcellular localizations; (b) differential coupling of phospholipases to COX-1 and COX-2 and (c) different utilization of arachidonic acids by COX-1 and COX-2 within cells. The subcellular localization of COX-1 and COX-2 is an important consideration postulating in distinct functions for COX-1 and COX-2. Confocal fluorescence imaging microscopy and histofluorescence staining techniques reveal that COX-1 and COX-2 are located in the endoplasmic reticulum and nuclear envelope but that COX-2 is more highly concentrated in the nuclear envelope [34]. It is commonly recognized that activated cytosolic phospholipase A2 (cPLA2) translocates to the perinuclear envelope and arachidonic acid is released from phospholipids in the nuclear membrane [35]. Arachidonic acid can be mobilized by several different phospholipase A2s. Among them cPLA2 and type IIA and type V secreted PLA2 (sPLA2) predominantly contribute to prostaglandin production. Early work focused on the coupling of cPLA2 and COX-2 and the coupling of sPLA2 and COX-1. However, coexpression of either COX-1 or COX-2 with various PLA2 has clearly demonstrated that the prostaglandin production induced by IL-1 and dependent on COX-2 can involve any one of several PLA2s (cPLA2 and IIA, V and X sPLA2) [36]. These latter studies indicated that prostaglandin production via COX-2 in activated 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-2 in intact cells. In contrast, higher concentrations of arachidonic acid (>10 M) are predominantly acted on by COX-1 rather than COX-2. The concentration of released arachidonic acid (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 mechanism for selective metabolism of arachidonic acid by COX-2 probably dose not depend solely on kinetic properties because the Km value for the two isozymes are nearly the same. One possible 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 combined presence of glutathione and glutathione peroxidase [39]. This is because COX-1 requires about 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 the case of COX-1 and COX-2 the phenotypes of deficient mice have supported the data obtained from pharmacological and epidemiological experiments. Overall, for the maintenance of normal physiology, it appears that a deficiency of COX-2 has more profound effects than a deficiency of COX-1. COX-1(−/−) mice have reduced platelet aggregation and decreased arachidonic acidinduced inflammation but phorbol-induced inflammation is unaffected [41]. These data are consistent with the fact that platelets have only COX-1, which contributes to platelet aggregation and high concentrations of arachidonic acid are able to be converted to prostaglandins by COX-1. COX-1(−/−) mice are also sensitive to the radiation injury. Crypt stem cell survival after gamma-irradiation decreased, and crypt epithelial cell apoptosis increased in COX-1(−/−) mice [42]. However, COX-1(−/−) mice have no gastric pathology and are resistant to indomethacininduced gastric ulceration [41]. This was a surprising result because in animal model and clinical studies classical NSAIDs induce gastric ulcers, but COX-2 specific inhibitors do not. The discrepancy between gene disruption and pharmacological results leads to the testing of novel NSAIDs which inhibit specifically COX-1 and were found not to cause gastric damage. COX-1(−/−) and COX-1(−/−) pairings lead to few live offspring, and thus COX-1 is important in reproduction [43]. In contrast to COX-1 deficient mice, COX-2 deficient mice have more dramatic phenotypic changes. In COX-2 deficient mice female reproductive functions including ovulation, fertilization, implantation and decidualization are defective [44,45]. In the nervous system COX-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 is particularly exciting, because the data confirm the epidemiological studies in which NSAID has been shown to suppress the incidence of colon cancer. Introduction of a COX-2 gene mutation to the Apo715 knockout mice reduced the number and size of intestinal polyps dramatically [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 arteriosus closure [48,49]. One problem with gene disruption approaches is that they are often complicated by compensation 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 female reproduction 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 are novel mechanisms for NSAID activities in tumorigenesis. Primary fibroblasts derived from COX-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 and induce apoptosis [50]. These results indicate that transformation is independent of the status of COX expression and that COX is involved in tumorigenesis at later stage. One third of COX-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 one copy of the gene encoding COX-1 (79%). Of course, the ductus arteriosus is not observed in COX-1(−/−) mice [51]. 4.2. Inflammation Prostaglandins are produced in the inflamed tissues, and treatment with NSAIDs inhibits the production of prostaglandins and down-regulates inflammation-related pathological symptoms such as pain and swelling. During inflammation, COX-1 mRNA, protein and 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 administered, prostaglandin production and subsequent inflammation are significantly reduced. These data have led to the conclusion that COX-2 is involved in inflammation, whereas COX-1 is not [52]. During the inflammation process, COX-1 is thought to contribute to “resolution”. In experimental mesangioproliferative glomerulonephritis COX-1 is expressed in glomeruli during the repair period [53]. In the process of ulcer healing, the COX-1 specific inhibitors as well as the COX-2 specific ones delay healing. These results implicate the role of COX-1 in 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 prostaglandins production [54–56]. Therefore, COX-2 specific inhibitors have been used to attenuate the symptoms of inflammation such as osteoarthritis, rheumatoid arthritis and musculoskeletal pain in patients [57,58]. In inflammation-related cells, the membrane bound type of PGE synthase (mPGE2 synthase) is also induced by these cytokines [59]. The large amount of PGE2 produced at the inflammation site by the coupling of COX-2 and mPGE2 synthase may 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 produced via 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 platelets have COX-1, but not COX-2 is unclear. It may be attributable to the different subcellular localizations of COX-1 and COX-2 in magakaryocytes, because COX-2 is more highly localized 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 later stage 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 pressure and anti-aggregation of platelets. However, a recent work has shown that prostacyclin in vascular cells is produced by COX-2 as well as COX-1 under both physiological and pathological conditions. Treatment of volunteers with a COX-2 specific inhibitor decreased the levels 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 associated with COX-2 specific inhibitors. However, two major randomized trials have shown the opposite results. VIGOR (8076 patients) showed that the relative risk of an adjudicated thrombotic cardiovascular event with COX-2 specific inhibitor treatment compared with naproxen was 2.38, whereas CLASS (8059 patients) showed the numbers of events associated 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 against cancer cell proliferation [65] may lead to the development of NO-releasing COX-2 specific inhibitors. In pathological conditions, COX-2 expression is enhanced, prostacyclin and PGE2 are produced, and glucocorticoids prevent the hypotension caused by endotoxin. These observations 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 epidemiological studies, work with gene-disrupted mice and COX overexpression and pharmacological studies. In tumorigenesis the role of COX-1 is distinct from that of COX-2. COX-1 is expressed in vascular endothelial cells and contributes to angiogenesis, which is an endothelial cell function and is involved in growth of tumors, endometrial growth, wound healing and inflammation. The origin of the neovasculum is thought to be microvessel endothelial cells and circulating endothelial cell precursors [67]. Human umbilical endothelial cells or aortic endothelial cells are commonly used in vitro as an angiogenesis model. In this system, an anti-sense oligonucleotide to COX-1 suppresses tube formation induced by colon cancer cells overexpressing COX-2 [68]. However, in our laboratory NSAIDs treatment of endothelial cells cultured between collagen gels did not cause inhibition of tube formation induced by high concentration of vascular endothelial growth factor (VEGF) (unpublished results). One possibility to explain this apparent discrepancy is that the contribution of COX-1 to angiogenesis may be dependent on the stimulator of angiogenesis. In contrast to the relatively small contribution of COX-1 in tumorigenesis, COX-2 is functional in tumorigenesis and tumor growth. Overexpression of COX-2 in tumor cells causes cells to escape from apoptosis and to invade the matrix [69,70]. The contribution of COX-2 to tumorigenesis is mainly through three processes: (a) induction of angiogenic factors 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 tumor cells produce PGE2 via COX-2, and the PGE2 produced induces VEGF, and finally VEGF stimulates angiogenesis [71]. The phenomenon is also observed in carrageenen-induced inflammation [72]. 4.5. Renal function Although the prevalence of nephrotoxicity in patients treated with NSAIDs is relatively low, the extensive use profile of these agents implies that many persons are at risk. At basal states of normal renal function, the role of renal prostaglandin production in maintenance of stable renal hemodynamic functions is relatively limited. Using immunohistochemistry in 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 data show that COX-1 regulates renal blood flow. COX-2 expression in the human kidney was detected in the renal vascularture, medullary interstitial cells, and the macula densa. Glomerular staining showed that COX-2 was detectable in podocytes only in the final stage of renal development [73,74]. These data suggest that COX-2 modulated by podocytes will be involved in renal perfusion and glomerular hemodynamics.
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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 processes 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 conclusion 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: S 0 0 9 0 - 6 9 8 0 ( 0 2 ) 0 0 0 2 9 - 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 in many tissues and cells. Because few cis-acting response elements and no TATA box have been identified in the 5 -flanking region of the COX-1 gene [5], COX-1 gene has been considered 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 in Table 1, in some cell lines COX-1 expression is increased during differentiation, while in endothelial cells COX-1 is increased in response to shear stress, VEGF and thrombin. Gel shift and promoter deletion assays have demonstrated that Sp1 cis-regulatory element at −610/−604 in the human COX-1 promoter is involved in transcription in endothelial cells [19]. It is widely recognized that Sp1 protein levels in nuclei are constitutive, but the ratio of Sp1/Sp3, the phosphorylated/glycosylated state of Sp1 or the coordinate binding of other transcriptional factors and Sp1 regulates the binding of Sp1 to its cognate sites in the promoters, and this, in turn, alters transcription rates [20–22]. Unfortunately, the mechanism underlying transcriptional 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 -flanking region of COX-2 genes. Among them, there are two NFB, one Sp1, one NF-IL-6 and one CRE binding sites [23]. Several growth factors, cytokines and mechanical stress activate these transcriptional factors, and as a consequence, upregulate the COX-2 gene expression. As an example, the regulation of COX-2 expression in endothelial cells is summarized in Table 2. Expression of the COX-2 gene can be suppressed by glucocorticoids and anti-inflammatory cytokines such as IL-4 and IL-10 [24]. The transcriptional regulation of COX-2 by glucocorticoids has been investigated and in some cases COX-2 expression Table 1 Induction of COX-1 Species
Cell
Stimulus
Reference
Human Rat Mouse Human Human Bovine and human Human Rat Ovine Guinea pig Human Bovine
THP-1 EGV-6 Immature mast cells Megakaryocytes Endothelial cells
Phorbol ester Phorbol ester Ligands (c-kit) Phorbol ester Shear stress
[7] [8] [9] [10] [11]
Endothelial cells Lung fibroblasts Osteocytes Endothelial cells Gallbladder Synovial cells (primary culture) Endothelial cells
VEGF TGF Mechanical stress 17-Estradiol Bradykinin IL-1␣ Thrombin
[12] [13] [14] [15] [16] [17] [18]
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Table 2 Inducers of COX-2 expression in endothelial cells Cytokines
Growth factors or tumor promoters
Others
IL-1␣ IL-1 IL-6 IL-8 IL-11 TNF␣ LPS IFN␥
Serum factor aFGF bFGF Insulin IGF EGF PDGF TGF VEGF Phorbol ester
PGE2 NO Anti-phospholipid antibody Hypoxia Mechanical stress 25-Hydroxycholesterol
is inhibited. Glucocorticoids increase IB proteins and suppress NFB-mediated COX-2 mRNA expression [25]. What is more, the glucocorticoid receptor–glucocorticoid complex can 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 stability. The 3 -untranslated region of COX-2 is extremely AT rich; for instance, there are 17 copies of the ATTTA (Shaw–Kamens) sequence in the human COX-2 gene [27]. This motif is common in many inducible genes, such as those for interleukins and for inducible NO synthase, 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 pharmacological and biological differences (Table 3). The active site of COX-1 is smaller than that of COX-2. Several substitutions including replacement of Ile434 in COX-1 with Val434 in COX-2 increase the relative volume of the active site of COX-1 [28]. In part, the discovery that the active sites of COX-1 and COX-2 are of different sizes led to the development of the COX-2 specific inhibitors [29]. Moreover, the size difference between the active sites is 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 treatment [30], and that dihomo-␥-linolenic acid and eicosapentaenoic acid are somewhat better substrates for human COX-2 than COX-1 [31]. However, the difference in the size of the active sites does not affect the gross kinetic properties of these two isozymes; COX-1 and COX-2 have similar Km 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 depending as the conditions. Under serum-free conditions, cultured bovine endothelial cells expressed only COX-1. Treatment of these cells with calcium ionophore (phospholipase activation) does not cause prostaglandin formation indicating that COX-1 is not able to synthesize prostaglandins from endogenously released arachidonic acid. When the cells are treated with phorbol ester, an induction of COX-2 protein occurs that parallels an increase in
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Table 3 Properties of COX-1 and COX-2 COX-1
COX-2
mRNA size mRNA stability Amino acid Properties of enzyme Cells
3 kb Stable 576 Constitutive Almost all
Main biological functions
Platelet aggregation Renal water balance Gastric cytoprotection
Subcellular localization Arachidonic acid utilized Substrates Aspirin treatment
Endoplasmic reticulum and nuclear membrane Mainly exogenous Mainly arachidonic acid No metabolite
4–4.5 kb Unstable 581 Inducible Induced-stimulated cells Many tumor cells Platelet disaggregation Inflammation Vasodilation Bone resorption and many pathological events Mainly nuclear membrane
Glucocorticoid treatment
No effect
Endogenous and exogenous Arachidonic acid 15(R) hydroperoxyeicosatetra-enoic acid Inhibition (induction)
prostaglandin production [33]. There are three major possible explanations for these results: (a) differences in subcellular localizations; (b) differential coupling of phospholipases to COX-1 and COX-2 and (c) different utilization of arachidonic acids by COX-1 and COX-2 within cells. The subcellular localization of COX-1 and COX-2 is an important consideration postulating in distinct functions for COX-1 and COX-2. Confocal fluorescence imaging microscopy and histofluorescence staining techniques reveal that COX-1 and COX-2 are located in the endoplasmic reticulum and nuclear envelope but that COX-2 is more highly concentrated in the nuclear envelope [34]. It is commonly recognized that activated cytosolic phospholipase A2 (cPLA2) translocates to the perinuclear envelope and arachidonic acid is released from phospholipids in the nuclear membrane [35]. Arachidonic acid can be mobilized by several different phospholipase A2s. Among them cPLA2 and type IIA and type V secreted PLA2 (sPLA2) predominantly contribute to prostaglandin production. Early work focused on the coupling of cPLA2 and COX-2 and the coupling of sPLA2 and COX-1. However, coexpression of either COX-1 or COX-2 with various PLA2 has clearly demonstrated that the prostaglandin production induced by IL-1 and dependent on COX-2 can involve any one of several PLA2s (cPLA2 and IIA, V and X sPLA2) [36]. These latter studies indicated that prostaglandin production via COX-2 in activated 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-2 in intact cells. In contrast, higher concentrations of arachidonic acid (>10 M) are predominantly acted on by COX-1 rather than COX-2. The concentration of released arachidonic acid (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 mechanism for selective metabolism of arachidonic acid by COX-2 probably dose not depend solely on kinetic properties because the Km value for the two isozymes are nearly the same. One possible 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 combined presence of glutathione and glutathione peroxidase [39]. This is because COX-1 requires about 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 the case of COX-1 and COX-2 the phenotypes of deficient mice have supported the data obtained from pharmacological and epidemiological experiments. Overall, for the maintenance of normal physiology, it appears that a deficiency of COX-2 has more profound effects than a deficiency of COX-1. COX-1(−/−) mice have reduced platelet aggregation and decreased arachidonic acidinduced inflammation but phorbol-induced inflammation is unaffected [41]. These data are consistent with the fact that platelets have only COX-1, which contributes to platelet aggregation and high concentrations of arachidonic acid are able to be converted to prostaglandins by COX-1. COX-1(−/−) mice are also sensitive to the radiation injury. Crypt stem cell survival after gamma-irradiation decreased, and crypt epithelial cell apoptosis increased in COX-1(−/−) mice [42]. However, COX-1(−/−) mice have no gastric pathology and are resistant to indomethacininduced gastric ulceration [41]. This was a surprising result because in animal model and clinical studies classical NSAIDs induce gastric ulcers, but COX-2 specific inhibitors do not. The discrepancy between gene disruption and pharmacological results leads to the testing of novel NSAIDs which inhibit specifically COX-1 and were found not to cause gastric damage. COX-1(−/−) and COX-1(−/−) pairings lead to few live offspring, and thus COX-1 is important in reproduction [43]. In contrast to COX-1 deficient mice, COX-2 deficient mice have more dramatic phenotypic changes. In COX-2 deficient mice female reproductive functions including ovulation, fertilization, implantation and decidualization are defective [44,45]. In the nervous system COX-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 is particularly exciting, because the data confirm the epidemiological studies in which NSAID has been shown to suppress the incidence of colon cancer. Introduction of a COX-2 gene mutation to the Apo715 knockout mice reduced the number and size of intestinal polyps dramatically [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 arteriosus closure [48,49]. One problem with gene disruption approaches is that they are often complicated by compensation 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 female reproduction 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 are novel mechanisms for NSAID activities in tumorigenesis. Primary fibroblasts derived from COX-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 and induce apoptosis [50]. These results indicate that transformation is independent of the status of COX expression and that COX is involved in tumorigenesis at later stage. One third of COX-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 one copy of the gene encoding COX-1 (79%). Of course, the ductus arteriosus is not observed in COX-1(−/−) mice [51]. 4.2. Inflammation Prostaglandins are produced in the inflamed tissues, and treatment with NSAIDs inhibits the production of prostaglandins and down-regulates inflammation-related pathological symptoms such as pain and swelling. During inflammation, COX-1 mRNA, protein and 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 administered, prostaglandin production and subsequent inflammation are significantly reduced. These data have led to the conclusion that COX-2 is involved in inflammation, whereas COX-1 is not [52]. During the inflammation process, COX-1 is thought to contribute to “resolution”. In experimental mesangioproliferative glomerulonephritis COX-1 is expressed in glomeruli during the repair period [53]. In the process of ulcer healing, the COX-1 specific inhibitors as well as the COX-2 specific ones delay healing. These results implicate the role of COX-1 in 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 prostaglandins production [54–56]. Therefore, COX-2 specific inhibitors have been used to attenuate the symptoms of inflammation such as osteoarthritis, rheumatoid arthritis and musculoskeletal pain in patients [57,58]. In inflammation-related cells, the membrane bound type of PGE synthase (mPGE2 synthase) is also induced by these cytokines [59]. The large amount of PGE2 produced at the inflammation site by the coupling of COX-2 and mPGE2 synthase may 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 produced via 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 platelets have COX-1, but not COX-2 is unclear. It may be attributable to the different subcellular localizations of COX-1 and COX-2 in magakaryocytes, because COX-2 is more highly localized 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 later stage 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 pressure and anti-aggregation of platelets. However, a recent work has shown that prostacyclin in vascular cells is produced by COX-2 as well as COX-1 under both physiological and pathological conditions. Treatment of volunteers with a COX-2 specific inhibitor decreased the levels 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 associated with COX-2 specific inhibitors. However, two major randomized trials have shown the opposite results. VIGOR (8076 patients) showed that the relative risk of an adjudicated thrombotic cardiovascular event with COX-2 specific inhibitor treatment compared with naproxen was 2.38, whereas CLASS (8059 patients) showed the numbers of events associated 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 against cancer cell proliferation [65] may lead to the development of NO-releasing COX-2 specific inhibitors. In pathological conditions, COX-2 expression is enhanced, prostacyclin and PGE2 are produced, and glucocorticoids prevent the hypotension caused by endotoxin. These observations 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 epidemiological studies, work with gene-disrupted mice and COX overexpression and pharmacological studies. In tumorigenesis the role of COX-1 is distinct from that of COX-2. COX-1 is expressed in vascular endothelial cells and contributes to angiogenesis, which is an endothelial cell function and is involved in growth of tumors, endometrial growth, wound healing and inflammation. The origin of the neovasculum is thought to be microvessel endothelial cells and circulating endothelial cell precursors [67]. Human umbilical endothelial cells or aortic endothelial cells are commonly used in vitro as an angiogenesis model. In this system, an anti-sense oligonucleotide to COX-1 suppresses tube formation induced by colon cancer cells overexpressing COX-2 [68]. However, in our laboratory NSAIDs treatment of endothelial cells cultured between collagen gels did not cause inhibition of tube formation induced by high concentration of vascular endothelial growth factor (VEGF) (unpublished results). One possibility to explain this apparent discrepancy is that the contribution of COX-1 to angiogenesis may be dependent on the stimulator of angiogenesis. In contrast to the relatively small contribution of COX-1 in tumorigenesis, COX-2 is functional in tumorigenesis and tumor growth. Overexpression of COX-2 in tumor cells causes cells to escape from apoptosis and to invade the matrix [69,70]. The contribution of COX-2 to tumorigenesis is mainly through three processes: (a) induction of angiogenic factors 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 tumor cells produce PGE2 via COX-2, and the PGE2 produced induces VEGF, and finally VEGF stimulates angiogenesis [71]. The phenomenon is also observed in carrageenen-induced inflammation [72]. 4.5. Renal function Although the prevalence of nephrotoxicity in patients treated with NSAIDs is relatively low, the extensive use profile of these agents implies that many persons are at risk. At basal states of normal renal function, the role of renal prostaglandin production in maintenance of stable renal hemodynamic functions is relatively limited. Using immunohistochemistry in 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 data show that COX-1 regulates renal blood flow. COX-2 expression in the human kidney was detected in the renal vascularture, medullary interstitial cells, and the macula densa. Glomerular staining showed that COX-2 was detectable in podocytes only in the final stage of renal development [73,74]. These data suggest that COX-2 modulated by podocytes will be involved in renal perfusion and glomerular hemodynamics.
5. Conclusion In intact cells, COX-2 utilizes a low concentration of arachidonic acid about equivalent to the concentration of arachidonic acid released endogenously. This means the production of prostaglandins via COX-2 is regulated by the activation of phospholipases and the expression of the COX-2 gene. Many cytokines and growth factors affect the phospholipases and COX-2, and glucocorticoids inhibit both phospholipase activity and the induction of COX-2. In contrast, COX-1 functions only at relatively high concentrations of arachidonic acid, corresponding to those observed when arachidonic acid is added exogenously or during platelet aggregation or cell injury or acute inflammation. References [1] Weissmann G. Prostaglandins as modulators rather than mediators of inflammation. J Lipid Med 1993;6: 275–86. [2] Raisz LG. Physiologic and pathologic roles of prostaglandins and other eicosanoids in bone metabolism. J Nutr 1995;125:2024S–7S. [3] Richards JS, Russell DL, Robker RL, Dajee M, Alliston TN. Molecular mechanisms of ovulation and luteinization. Mol Cell Endocrinol 1998;145:47–54. [4] Williams CS, Mann M, DuBois RN. The role of cyclooxygenases in inflammation, cancer, and development. Oncogene 1999;18:7908–16. [5] Kraemer SA, Meade EA, DeWitt DL. Prostaglandin endoperoxide synthase gene structure: identification of the transcriptional start site and 5 -flanking regulatory sequences. Arch Biochem Biophys 1992;293:391–400.
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