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Phenotypes of the COX-deficient mice indicate physiological and pathophysiological roles for COX-1 and COX-2
Prostaglandins & other Lipid Mediators 68–69 (2002) 177–185
Phenotypes of the COX-deficient mice indicate physiological and pathophysiological roles for COX-1 and COX-2 Charles D. Loftin, Howard F. Tiano, Robert Langenbach∗ Laboratory of Environmental Carcinogenesis and Mutagenesis, National Institutes of Health, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709, USA
Abstract The development of mice deficient in either cyclooxygenase-1 (COX-1) or COX-2, as well as mice deficient in both COX isoforms, has provided models to elucidate the physiological and pathophysiological roles of these enzymes. The findings obtained with the COX-deficient mice suggest that COX-2 may be more important than COX-1 for supplying prostaglandins (PGs) to maintain tissue homeostasis. Furthermore, both isoforms may be involved in the development of diseases, such as inflammation and cancer. Therefore, the contribution of each isoform to the prevention or development of disease is more complex than originally described. Studies with the COX-deficient mice suggest that in addition to COX-2-selective inhibition, therapeutic advances may also be achieved with COX-1-selective inhibitors which lack gastrointestinal side effects. © 2002 Published by Elsevier Science Inc. Keywords: Cyclooxygenase; Mice; Isoform; Inflammation; Reproduction; Cancer
1. Introduction The two isoforms of prostaglandin G/H synthase, also known as cyclooxygenase (COX)-1 and -2, catalyze the first committed step in the pathway leading to the production of prostaglandins (PGs) [1]. Generally, it has been believed that COX-1 is responsible for homeostatic functions, whereas the inducible isoform, COX-2, is involved in the development and progression of various pathological conditions. Because the inhibition of PG synthesis is considered the primary mechanism responsible for the therapeutic and toxic effects of nonsteroidal anti-inflammatory drugs (NSAIDs), many of the functions associated ∗
Corresponding author. Tel.: +1-919-541-7558; fax: +1-919-541-1460. E-mail address: [email protected] (R. Langenbach).
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Table 1 Characteristics of mice deficient in either COX-1 or COX-2 Physiological/pathogical process
COX-1(−/−) mice
COX-2(−/−) mice
Neonatal mortality Adult mortality Spontaneous gastric ulceration Patent ductus arteriosus Postnatal kidney development Ovulation Implantation Parturition Wound healing Platelet aggregation Tumor development Inflammation Peritonitis incidence Constitutive PG synthesis Inducible PG synthesis Autoimmune arthritis Bone resorption Induced hematopoiesis Intestinal stem cell survival Febrile response Colonic inflammation Induced cerebral blood flow Resting cerebral blood flow Ischemic brain injury Ischemia/reperfusion injury
Normal [2] Normal [2] Normal [2] Normal [4] Normal [2] Normal [12] Normal [12] Delayed [11] Unknown Impaired [2] Decreased [33] Altered [2,8,38] Normal [2] Decreased [2] Normal [2] Normal [39] Normal [40,41] Unknown Decreased [29] Normal [43,44] Increased [45] Normal [46] Decreased [46] Unknown Increased [49]
Increased [3,4,37] Increased [3] Normal [3] Increased [4] Impaired [3,14,37] Impaired [12,13] Impaired [12] Unknown Impaired [8] Normal [3] Decreased [32,33] Altered [8,38] Increased [3] Normal [3] Decreased [3] Decreased [39] Decreased [40,41] Decreased [42] Normal [29] Decreased [43,44] Increased [45] Decreased [47] Unknown Decreased [48] Increased [49]
with each COX isoform have been defined by the therapeutic or adverse effects resulting from NSAIDs. To better characterize the physiological and pathological functions of each COX isoform in vivo, we utilized gene targeting to generate mice deficient in either COX-1 or COX-2 [2,3], or deficient in both COX isoforms [4]. Characteristics of mice deficient in either COX-1 or COX-2 are shown in Table 1. The findings with the COX-deficient mice indicate that COX-1, as well as COX-2, is involved in the development of various pathologies; and that COX-2 has significant roles in perinatal development and in the maintenance of homeostasis. The study of COX-deficient mice has provided valuable insight into the in vivo functions of the COX isoforms.
2. Gastric ulceration Gastric ulceration is a serious complication which has long been associated with the use of NSAIDs. PG synthesis dependent on COX-1 is involved in regulating physiological processes important for maintenance of mucosal integrity [5,6], and thus the inhibition of COX-1 was thought responsible for ulcerative effects of NSAIDs. The reduced gastric toxicity of COX-2-selective inhibition [7] further supported the hypothesis that inhibition
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of COX-1 was responsible for the gastric side effects of NSAIDs. However, our studies have demonstrated that the genetic deficiency of COX-1 does not increase spontaneous gastric ulceration in mice [2]. The gastric mucosa of COX-1-deficient mice appears normal despite a > 99% reduction in gastric PGE2 , which is a level similar to that observed following administration of an ulcerative dose of indomethacin. The gastric pH in COX-1-deficient mice is, however, significantly reduced compared to wild-type and COX-2-deficient mice [8], which is in agreement with a role for COX-1 in the secretion of acid and/or bicarbonate in the stomach. The findings from studies with the COX-1-deficient mice indicate that although the elimination of COX-1 derived PGs increases stomach acidity, this alone is not sufficient to cause gastric lesions. Recent studies using isoform-selective COX inhibitors further support our observation in COX-1-deficient mice that a reduction in the level of PGs derived from COX-1 is not sufficient to induce gastric ulcers. Selective inhibition of COX-1 in healthy rats with SC-560 does not induce gastric ulcers even though gastric prostaglandin synthesis is significantly reduced [9,10]. However, the combined treatment of the COX-1-selective inhibitor with a selective inhibitor of COX-2 does result in gastric damage of comparable severity to a dual COX isoform inhibitor [9,10]. Therefore, genetic and pharmacological studies in rodents suggest that the combined inhibition of both COX-1 and COX-2 may be responsible for the development of gastric ulcers following the administration of NSAIDs.
3. Female reproduction The deficiency of COX-1 in female mice does not impede conception or fetal development, but does significantly prolong gestation and thereby reduce offspring survival [2]. As compared to wild-type mice, the gestation length of COX-1-deficient mice is prolonged 2–3 days due to a delay in the initiation of parturition [11]. Although delayed parturition significantly reduces litter survival following natural delivery, offspring from COX-1-deficient female mice show normal survival when delivered by Cesarean section after a normal gestation length [4]. Therefore, in mice, the maternal expression of COX-1 is not required for fertility or fetal development, but is important for the initiation labor and the generation of viable offspring. In contrast to the COX-1-deficient mice, female mice deficient in COX-2 show numerous reproductive abnormalities with defects in ovulation, implantation and decidualization [12]. It was observed that few eggs were recovered following superovulation of COX-2-deficient mice, and only 2% of the released eggs were fertilized [12]. In preovulatory follicles, COX-2 expression is normally induced by pituitary gonadotropins and this results in the increased synthesis of PGE2 [13]. Reduced PGE2 synthesis in the ovaries of COX-2-deficient mice disrupts follicle expansion and ovulation, which can be restored by treatment with PGE2 [13]. In contrast to the homozygous mutants, female mice heterozygous for the COX-2 gene disruption show normal fertility and offspring survival. The findings with the COX-deficient mice indicate that although both isoforms are essential for normal female reproduction, each isoform generates PGs which function in different reproductive processes.
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4. Renal development and function The kidney has long been known as a tissue rich in PG synthesis and the development of COX-1 or COX-2-deficient mice has contributed to a better understanding of the roles of the individual COX isoforms in renal function. Because of high levels of COX-1 expression in the renal vasculature, PG synthesis dependent on this isoform was thought to be important for regulating fluid and electrolyte balance. However, recent studies with the COX-deficient mice have identified a prominent role for COX-2 in both the development and function of the kidney. Although COX-1 has been thought to be the isoform primarily responsible for the maintenance of physiological processes in the kidney, the genetic deficiency of COX-1 fails to produce an identifiable renal pathology [2]. In contrast to the COX-1-deficient mice, mice deficient in COX-2 show severe developmental defects in the kidney. Kidney development in mice continues after birth and it is during postnatal renal development that COX-2 function appears most critical. In the neonatal mouse kidney, the expression of COX-2 increases during the first 4 days after birth and thereafter declines to undetectable levels [14]. Although the kidneys of COX-2-deficient mice are indistinguishable from wild-type neonatal kidneys at birth, as the kidneys develop postnatally, significant differences in renal morphology are observed [3,14,15]. The initial defects in kidney development in COX-2-deficient mice are observed at 10 days after birth, with the cortex being abnormally thin with regions of small immature glomeruli and tubular atrophy [3,14,15]. Progression of the nephropathy in adult COX-2-deficient mice leads to tubular dilation, interstitial fibrosis and glomerular sclerosis [15]. The kidney pathology observed in adult COX-2-deficient mice is distinct from renal toxicity previously reported following NSAID treatment of adult mice. However, when a COX-2-selective inhibitor is administered to neonatal wild-type mice, the resulting renal pathology is identical to that observed in COX-2-deficient mice [14]. In addition to their role in postnatal renal development, PGs generated via COX-2 also contribute to the modulation of specific functions in the adult kidney. The deficiency of COX-2 disrupts the signaling pathway in the salt sensing region of the kidney, which is important for activation of the renin–angiotensin system. In the renal cortex, COX-2 expression is localized to the macula densa and the adjacent thick ascending limb, a region important for monitoring NaCl levels [16]. COX-2 expression in these regions of the kidney is induced by reductions in the NaCl concentrations. Recent studies have examined the role of COX-2 in the stimulation of renin production and secretion following reduced dietary sodium [17] or the inhibition of angiotensin-converting enzyme [18]. Under conditions where renin expression and activity are induced in wild-type mice, renin production is not stimulated in the kidneys of mice deficient in COX-2 [17,18]. Therefore, COX-2 appears to be the isoform primarily responsible for maintaining renal homeostatic functions involved in the regulation of salt resorption, fluid volume and blood pressure.
5. Ductus arteriosus closure To identify physiological processes dependent on PG synthesis from either COX-1 or COX-2, we developed mice with deficiencies in both COX isoforms and characterized
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their phenotypes. All COX-deficient genotypes, including mice deficient in both COX isoforms (COX-1−/− /COX-2−/− ) are produced in the expected Mendelian ratio, indicating that in mice, embryo implantation and survival in utero does not require fetal PG synthesis [4]. Although COX-1−/− /COX-2−/− mice are born alive, respiratory distress develops after birth and all of these mice die during the first day of life. Pathological analysis of the COX-1−/− /COX-2−/− mice indicates a patent ductus arteriosus, a condition in which the ductus arteriosus fails to close after birth. Patent ductus arteriosus and resulting neonatal mortality is increased in all COX-2-deficient genotypes, however, the incidence varies with the COX-1 genotype. For COX-2-deficient mice that are either wild-type or heterozygous for the COX-1 gene, neonatal mortality is 35 or 79%, respectively. Survival is normal in all mice that express COX-2, including mice deficient in COX-1. Therefore, the expression of COX-2 has a profound effect on neonatal survival. In the ductus arteriosus, COX-2 expression is localized in smooth muscle cells and is likely responsible for contraction and closure of the vessel [4]. These findings indicate that PG synthesis essential for normal closure of the ductus arteriosus depends primarily on COX-2, although COX-1 provides partial compensation in mice deficient in COX-2. The human implications of these studies are that the administration of COX-2-selective inhibitors during pregnancy, which has been suggested for the treatment of premature labor [19–24], may adversely affect ductus arteriosus closure after birth and thereby compromise neonatal health.
6. Cellular proliferation and tissue repair PGs modulate the mitogenic responses of specific growth factors during the proliferation of cells in vitro [25,26] and recent studies utilizing COX-deficient mice have indicated that PGs may also function in the proliferative response during tissue regeneration following injury. During liver regeneration following partial hepatectomy, cellular proliferation is not altered by the deficiency of COX-1, but is significantly reduced when COX-1-deficient mice are treated with a COX-2 inhibitor [27]. Liver regeneration is more significantly attenuated in COX-2 inhibitor treated COX-1deficient mice than COX-2 inhibitor treated wild-type mice, suggesting that during hepatocyte proliferation COX-2 may compensate for the genetic deficiency of COX-1 [27]. In the intestine, cytotoxic damage normally induces proliferation of the stem cell population with the subsequent differentiation of daughter cells into mature epithelial cell types for repopulation of the intestinal lining [28]. However, following ␥-irradiation of COX-1-deficient mice, this process of intestinal epithelial cell repopulation is attenuated, whereas the response to ␥-irradiation is not altered in mice deficient in COX-2 [29]. An explanation for these observations is that the deficiency of COX-1 increases the level of programmed cell death of the intestinal crypt cell population following irradiation and also reduces the clonogenic proliferative response of stem cells which survive irradiation [29]. Therefore, in the intestinal epithelium, COX-1 is the isoform responsible for protecting the stem cell population, which allows for regeneration following radiation-induced injury.
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7. Inflammation To better characterize the contributions of each COX isoform to the development of inflammation, inflammatory responses in either COX-1 or COX-2-deficient mice were compared to wild-type mice. The initial studies with the COX-deficient mice utilized arachidonic acid and the tumor promotor, 12-O-tetradecanoylphorbol-13-acetate (TPA), applied topically to the ear to induce edema [2,3]. Arachidonic acid induces an equivalent inflammatory response in wild-type and COX-2-deficient mice, but edema is reduced by 70% in mice deficient in COX-1. These findings indicate that acute inflammation in response to topically applied arachidonic acid results primarily from COX-1-dependent PG synthesis. The level of edema induced by TPA was not significantly different between wild-type, COX-1-deficient and COX-2-deficient mice. These findings were surprising, particularly for COX-2-deficient mice, because TPA is a potent inducer of COX-2 expression and was expected to mediate, at least in part, the inflammatory response to TPA [30]. The responses observed following TPA treatment suggest that PG synthesis from either COX isoform is sufficient to generate the PGs responsible for TPA-induced edema, or alternatively, TPA-induced edema results from mechanisms independent of PG synthesis. The ear inflammation studies in the COX-deficient mice indicate that COX-1, as well as COX-2, may contribute to inflammatory responses and the isoform responsible for the inflammation may depend on the type of inflammatory stimuli and/or the relative levels of each isoform in the target tissue. The development of inflammation in the COX-deficient mice has also been studied using the subdermal air pouch model [8]. Six hours following carrageenan-induced inflammation, an increase in PGE2 production is observed in the air pouch exudate of all COX genotypes. When compared to wild-type mice, the deficiency of COX-2 reduces the level of PGE2 production by approximately 75%, while the deficiency of COX-1 reduces the PGE2 level by 25%. Studies with the COX-deficient mice also helped to identify isoform-specific functions during the resolution of carrageenan-induced inflammation. Significantly higher numbers of inflammatory cells, including apoptotic neutrophils, are observed in air pouch exudate from COX-2-deficient mice, compared to COX-1-deficient or wild-type mice [8]. Therefore, studies with the COX-deficient mice indicate that both COX isoforms may contribute to PG production during acute inflammation and that COX-2 derived PGs may also be important in the resolution of inflammation.
8. Intestinal tumorigenesis Because the expression of COX-2 is induced in many types of cancers, the majority of research on the role of PG synthesis in the development of cancer has emphasized COX-2, rather than COX-1. The overexpression of COX-2 in cultured intestinal epithelial cells has also been shown to increase cellular adhesion and produce a resistance to apoptosis, both of which are cellular characteristics that may contribute to tumor development [31]. Furthermore, in a mouse model of human familial adenomatous polyposis, intestinal tumor development is reduced by the genetic deficiency of COX-2 [32]. However, by utilizing COX-deficient mice in the study of intestinal tumorigenesis, we have demonstrated
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that the deficiency of COX-1, as well as COX-2, significantly reduces tumor development [33]. For determining the roles of the COX isoforms in the development of intestinal tumorigenesis, the COX-deficient mouse lines were bred into the Min mouse strain. Min mice have a chemically induced mutation in the Apc gene, which results in a 100% incidence of intestinal neoplasia [34]. However, intestinal tumorigenesis is reduced by approximately 80% in Min mice deficient in either COX-1 or COX-2 [33]. Furthermore, the heterozygous deficiency of COX-1 is more effective than the heterozygous deficiency of COX-2 in reducing the number of intestinal polyps in Min mice [33]. These findings of the important role of COX-1 in the development of intestinal tumorigenesis may, in part, explain the mechanism of reduced colon cancer risk for humans using NSAIDs such as aspirin [35,36], which are more effective for the inhibition of COX-1 than of COX-2. References [1] Smith WL, DeWitt DL. Prostaglandin endoperoxide H synthases-1 and-2. Adv Immunol 1996;62:167–215. [2] Langenbach R, Morham SG, Tiano HF, Loftin CD, Ghanayem BI, Chulada PC, et al. Prostaglandin synthase-1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin-induced gastric ulceration. Cell 1995;83:483–92. [3] Morham SG, Langenbach R, Loftin CD, Tiano HF, Vouloumanos N, Jennette JC, et al. Prostaglandin synthase-2 gene disruption causes severe renal pathology in the mouse. Cell 1995;83:473–82. [4] Loftin CD, Trivedi DB, Tiano HF, Clark JA, Lee CA, Epstein JA, et al. Failure of ductus arteriosus closure and remodeling in neonatal mice deficient in cyclooxygenase-1 and cyclooxygenase-2. Proc Natl Acad Sci USA 2001;98:1059–64. [5] Wallace JL, Tigley AW. New insights into prostaglandins and mucosal defence. Aliment Pharmacol Ther 1995;9:227–35. [6] Kargman S, Charleson S, Cartwright M, Frank J, Riendeau D, Mancini J, et al. Characterization of prostaglandin G/H synthase 1 and 2 in rat, dog, monkey, and human gastrointestinal tracts. Gastroenterology 1996;111:445–54. [7] Masferrer JL, Zweifel BS, Manning PT, Hauser SD, Leahy KM, Smith WG, et al. Selective inhibition of inducible cyclooxygenase-2 in vivo is anti-inflammatory and nonulcerogenic. Proc Natl Acad Sci USA 1994;91:3228–32. [8] Langenbach R, Loftin C, Lee C, Tiano H. Cyclooxygenase knockout mice-Models for elucidating isoform-specific functions. Biochem Pharmacol 1999;58:1237–46. [9] Gretzer B, Maricic N, Respondek M, Schuligoi R, Peskar BM. Effects of specific inhibition of cyclooxygenase-1 and cyclooxygenase-2 in the rat stomach with normal mucosa and after acid challenge. Br J Pharmacol 2001;132:1565–73. [10] Wallace JL, Mcknight W, Reuter BK, Vergnolle N. NSAID-induced gastric damage in rats: requirement for inhibition of both cyclooxygenase 1 and 2. Gastroenterology 2000;119:706–14. [11] Gross GA, Imamura T, Luedke C, Vogt SK, Olson LM, Nelson DM, Sadovsky Y, et al. Opposing actions of prostaglandins and oxytocin determine the onset of murine labor. Proc Natl Acad Sci USA 1998;95:11875–9. [12] Lim H, Paria BC, Das SK, Dinchuk JE, Langenbach R, Trzaskos JM, et al. Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 1997;91:197–208. [13] Davis BJ, Lennard DE, Lee CA, Tiano HF, Morham SG, Wetsel WC, et al. Anovulation in cyclooxygenase-2-deficient mice is restored by prostaglandin E-2 and interleukin-1 beta. Endocrinology 1999;140:2685–95. [14] Komhoff M, Wang JL, Cheng HF, Langenbach R, Mckanna JA, Harris RC, et al. Cyclooxygenase-2-selective inhibitors impair glomerulogenesis and renal cortical development. Kidney Int 2000;57:414–22. [15] Norwood VF, Morham SG, Smithies O. Postnatal development and progression of renal dysplasia in cyclooxygenase-2 null mice. Kidney Int 2000;58:2291–300.
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Phenotypes of the COX-deficient mice indicate physiological and pathophysiological roles for COX-1 and COX-2 Charles D. Loftin, Howard F. Tiano, Robert Langenbach∗ Laboratory of Environmental Carcinogenesis and Mutagenesis, National Institutes of Health, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709, USA
Abstract The development of mice deficient in either cyclooxygenase-1 (COX-1) or COX-2, as well as mice deficient in both COX isoforms, has provided models to elucidate the physiological and pathophysiological roles of these enzymes. The findings obtained with the COX-deficient mice suggest that COX-2 may be more important than COX-1 for supplying prostaglandins (PGs) to maintain tissue homeostasis. Furthermore, both isoforms may be involved in the development of diseases, such as inflammation and cancer. Therefore, the contribution of each isoform to the prevention or development of disease is more complex than originally described. Studies with the COX-deficient mice suggest that in addition to COX-2-selective inhibition, therapeutic advances may also be achieved with COX-1-selective inhibitors which lack gastrointestinal side effects. © 2002 Published by Elsevier Science Inc. Keywords: Cyclooxygenase; Mice; Isoform; Inflammation; Reproduction; Cancer
1. Introduction The two isoforms of prostaglandin G/H synthase, also known as cyclooxygenase (COX)-1 and -2, catalyze the first committed step in the pathway leading to the production of prostaglandins (PGs) [1]. Generally, it has been believed that COX-1 is responsible for homeostatic functions, whereas the inducible isoform, COX-2, is involved in the development and progression of various pathological conditions. Because the inhibition of PG synthesis is considered the primary mechanism responsible for the therapeutic and toxic effects of nonsteroidal anti-inflammatory drugs (NSAIDs), many of the functions associated ∗
Corresponding author. Tel.: +1-919-541-7558; fax: +1-919-541-1460. E-mail address: [email protected] (R. Langenbach).
0090-6980/02/$ – see front matter © 2002 Published by Elsevier Science Inc. PII: S 0 0 9 0 - 6 9 8 0 ( 0 2 ) 0 0 0 2 8 - X
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Table 1 Characteristics of mice deficient in either COX-1 or COX-2 Physiological/pathogical process
COX-1(−/−) mice
COX-2(−/−) mice
Neonatal mortality Adult mortality Spontaneous gastric ulceration Patent ductus arteriosus Postnatal kidney development Ovulation Implantation Parturition Wound healing Platelet aggregation Tumor development Inflammation Peritonitis incidence Constitutive PG synthesis Inducible PG synthesis Autoimmune arthritis Bone resorption Induced hematopoiesis Intestinal stem cell survival Febrile response Colonic inflammation Induced cerebral blood flow Resting cerebral blood flow Ischemic brain injury Ischemia/reperfusion injury
Normal [2] Normal [2] Normal [2] Normal [4] Normal [2] Normal [12] Normal [12] Delayed [11] Unknown Impaired [2] Decreased [33] Altered [2,8,38] Normal [2] Decreased [2] Normal [2] Normal [39] Normal [40,41] Unknown Decreased [29] Normal [43,44] Increased [45] Normal [46] Decreased [46] Unknown Increased [49]
Increased [3,4,37] Increased [3] Normal [3] Increased [4] Impaired [3,14,37] Impaired [12,13] Impaired [12] Unknown Impaired [8] Normal [3] Decreased [32,33] Altered [8,38] Increased [3] Normal [3] Decreased [3] Decreased [39] Decreased [40,41] Decreased [42] Normal [29] Decreased [43,44] Increased [45] Decreased [47] Unknown Decreased [48] Increased [49]
with each COX isoform have been defined by the therapeutic or adverse effects resulting from NSAIDs. To better characterize the physiological and pathological functions of each COX isoform in vivo, we utilized gene targeting to generate mice deficient in either COX-1 or COX-2 [2,3], or deficient in both COX isoforms [4]. Characteristics of mice deficient in either COX-1 or COX-2 are shown in Table 1. The findings with the COX-deficient mice indicate that COX-1, as well as COX-2, is involved in the development of various pathologies; and that COX-2 has significant roles in perinatal development and in the maintenance of homeostasis. The study of COX-deficient mice has provided valuable insight into the in vivo functions of the COX isoforms.
2. Gastric ulceration Gastric ulceration is a serious complication which has long been associated with the use of NSAIDs. PG synthesis dependent on COX-1 is involved in regulating physiological processes important for maintenance of mucosal integrity [5,6], and thus the inhibition of COX-1 was thought responsible for ulcerative effects of NSAIDs. The reduced gastric toxicity of COX-2-selective inhibition [7] further supported the hypothesis that inhibition
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of COX-1 was responsible for the gastric side effects of NSAIDs. However, our studies have demonstrated that the genetic deficiency of COX-1 does not increase spontaneous gastric ulceration in mice [2]. The gastric mucosa of COX-1-deficient mice appears normal despite a > 99% reduction in gastric PGE2 , which is a level similar to that observed following administration of an ulcerative dose of indomethacin. The gastric pH in COX-1-deficient mice is, however, significantly reduced compared to wild-type and COX-2-deficient mice [8], which is in agreement with a role for COX-1 in the secretion of acid and/or bicarbonate in the stomach. The findings from studies with the COX-1-deficient mice indicate that although the elimination of COX-1 derived PGs increases stomach acidity, this alone is not sufficient to cause gastric lesions. Recent studies using isoform-selective COX inhibitors further support our observation in COX-1-deficient mice that a reduction in the level of PGs derived from COX-1 is not sufficient to induce gastric ulcers. Selective inhibition of COX-1 in healthy rats with SC-560 does not induce gastric ulcers even though gastric prostaglandin synthesis is significantly reduced [9,10]. However, the combined treatment of the COX-1-selective inhibitor with a selective inhibitor of COX-2 does result in gastric damage of comparable severity to a dual COX isoform inhibitor [9,10]. Therefore, genetic and pharmacological studies in rodents suggest that the combined inhibition of both COX-1 and COX-2 may be responsible for the development of gastric ulcers following the administration of NSAIDs.
3. Female reproduction The deficiency of COX-1 in female mice does not impede conception or fetal development, but does significantly prolong gestation and thereby reduce offspring survival [2]. As compared to wild-type mice, the gestation length of COX-1-deficient mice is prolonged 2–3 days due to a delay in the initiation of parturition [11]. Although delayed parturition significantly reduces litter survival following natural delivery, offspring from COX-1-deficient female mice show normal survival when delivered by Cesarean section after a normal gestation length [4]. Therefore, in mice, the maternal expression of COX-1 is not required for fertility or fetal development, but is important for the initiation labor and the generation of viable offspring. In contrast to the COX-1-deficient mice, female mice deficient in COX-2 show numerous reproductive abnormalities with defects in ovulation, implantation and decidualization [12]. It was observed that few eggs were recovered following superovulation of COX-2-deficient mice, and only 2% of the released eggs were fertilized [12]. In preovulatory follicles, COX-2 expression is normally induced by pituitary gonadotropins and this results in the increased synthesis of PGE2 [13]. Reduced PGE2 synthesis in the ovaries of COX-2-deficient mice disrupts follicle expansion and ovulation, which can be restored by treatment with PGE2 [13]. In contrast to the homozygous mutants, female mice heterozygous for the COX-2 gene disruption show normal fertility and offspring survival. The findings with the COX-deficient mice indicate that although both isoforms are essential for normal female reproduction, each isoform generates PGs which function in different reproductive processes.
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4. Renal development and function The kidney has long been known as a tissue rich in PG synthesis and the development of COX-1 or COX-2-deficient mice has contributed to a better understanding of the roles of the individual COX isoforms in renal function. Because of high levels of COX-1 expression in the renal vasculature, PG synthesis dependent on this isoform was thought to be important for regulating fluid and electrolyte balance. However, recent studies with the COX-deficient mice have identified a prominent role for COX-2 in both the development and function of the kidney. Although COX-1 has been thought to be the isoform primarily responsible for the maintenance of physiological processes in the kidney, the genetic deficiency of COX-1 fails to produce an identifiable renal pathology [2]. In contrast to the COX-1-deficient mice, mice deficient in COX-2 show severe developmental defects in the kidney. Kidney development in mice continues after birth and it is during postnatal renal development that COX-2 function appears most critical. In the neonatal mouse kidney, the expression of COX-2 increases during the first 4 days after birth and thereafter declines to undetectable levels [14]. Although the kidneys of COX-2-deficient mice are indistinguishable from wild-type neonatal kidneys at birth, as the kidneys develop postnatally, significant differences in renal morphology are observed [3,14,15]. The initial defects in kidney development in COX-2-deficient mice are observed at 10 days after birth, with the cortex being abnormally thin with regions of small immature glomeruli and tubular atrophy [3,14,15]. Progression of the nephropathy in adult COX-2-deficient mice leads to tubular dilation, interstitial fibrosis and glomerular sclerosis [15]. The kidney pathology observed in adult COX-2-deficient mice is distinct from renal toxicity previously reported following NSAID treatment of adult mice. However, when a COX-2-selective inhibitor is administered to neonatal wild-type mice, the resulting renal pathology is identical to that observed in COX-2-deficient mice [14]. In addition to their role in postnatal renal development, PGs generated via COX-2 also contribute to the modulation of specific functions in the adult kidney. The deficiency of COX-2 disrupts the signaling pathway in the salt sensing region of the kidney, which is important for activation of the renin–angiotensin system. In the renal cortex, COX-2 expression is localized to the macula densa and the adjacent thick ascending limb, a region important for monitoring NaCl levels [16]. COX-2 expression in these regions of the kidney is induced by reductions in the NaCl concentrations. Recent studies have examined the role of COX-2 in the stimulation of renin production and secretion following reduced dietary sodium [17] or the inhibition of angiotensin-converting enzyme [18]. Under conditions where renin expression and activity are induced in wild-type mice, renin production is not stimulated in the kidneys of mice deficient in COX-2 [17,18]. Therefore, COX-2 appears to be the isoform primarily responsible for maintaining renal homeostatic functions involved in the regulation of salt resorption, fluid volume and blood pressure.
5. Ductus arteriosus closure To identify physiological processes dependent on PG synthesis from either COX-1 or COX-2, we developed mice with deficiencies in both COX isoforms and characterized
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their phenotypes. All COX-deficient genotypes, including mice deficient in both COX isoforms (COX-1−/− /COX-2−/− ) are produced in the expected Mendelian ratio, indicating that in mice, embryo implantation and survival in utero does not require fetal PG synthesis [4]. Although COX-1−/− /COX-2−/− mice are born alive, respiratory distress develops after birth and all of these mice die during the first day of life. Pathological analysis of the COX-1−/− /COX-2−/− mice indicates a patent ductus arteriosus, a condition in which the ductus arteriosus fails to close after birth. Patent ductus arteriosus and resulting neonatal mortality is increased in all COX-2-deficient genotypes, however, the incidence varies with the COX-1 genotype. For COX-2-deficient mice that are either wild-type or heterozygous for the COX-1 gene, neonatal mortality is 35 or 79%, respectively. Survival is normal in all mice that express COX-2, including mice deficient in COX-1. Therefore, the expression of COX-2 has a profound effect on neonatal survival. In the ductus arteriosus, COX-2 expression is localized in smooth muscle cells and is likely responsible for contraction and closure of the vessel [4]. These findings indicate that PG synthesis essential for normal closure of the ductus arteriosus depends primarily on COX-2, although COX-1 provides partial compensation in mice deficient in COX-2. The human implications of these studies are that the administration of COX-2-selective inhibitors during pregnancy, which has been suggested for the treatment of premature labor [19–24], may adversely affect ductus arteriosus closure after birth and thereby compromise neonatal health.
6. Cellular proliferation and tissue repair PGs modulate the mitogenic responses of specific growth factors during the proliferation of cells in vitro [25,26] and recent studies utilizing COX-deficient mice have indicated that PGs may also function in the proliferative response during tissue regeneration following injury. During liver regeneration following partial hepatectomy, cellular proliferation is not altered by the deficiency of COX-1, but is significantly reduced when COX-1-deficient mice are treated with a COX-2 inhibitor [27]. Liver regeneration is more significantly attenuated in COX-2 inhibitor treated COX-1deficient mice than COX-2 inhibitor treated wild-type mice, suggesting that during hepatocyte proliferation COX-2 may compensate for the genetic deficiency of COX-1 [27]. In the intestine, cytotoxic damage normally induces proliferation of the stem cell population with the subsequent differentiation of daughter cells into mature epithelial cell types for repopulation of the intestinal lining [28]. However, following ␥-irradiation of COX-1-deficient mice, this process of intestinal epithelial cell repopulation is attenuated, whereas the response to ␥-irradiation is not altered in mice deficient in COX-2 [29]. An explanation for these observations is that the deficiency of COX-1 increases the level of programmed cell death of the intestinal crypt cell population following irradiation and also reduces the clonogenic proliferative response of stem cells which survive irradiation [29]. Therefore, in the intestinal epithelium, COX-1 is the isoform responsible for protecting the stem cell population, which allows for regeneration following radiation-induced injury.
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7. Inflammation To better characterize the contributions of each COX isoform to the development of inflammation, inflammatory responses in either COX-1 or COX-2-deficient mice were compared to wild-type mice. The initial studies with the COX-deficient mice utilized arachidonic acid and the tumor promotor, 12-O-tetradecanoylphorbol-13-acetate (TPA), applied topically to the ear to induce edema [2,3]. Arachidonic acid induces an equivalent inflammatory response in wild-type and COX-2-deficient mice, but edema is reduced by 70% in mice deficient in COX-1. These findings indicate that acute inflammation in response to topically applied arachidonic acid results primarily from COX-1-dependent PG synthesis. The level of edema induced by TPA was not significantly different between wild-type, COX-1-deficient and COX-2-deficient mice. These findings were surprising, particularly for COX-2-deficient mice, because TPA is a potent inducer of COX-2 expression and was expected to mediate, at least in part, the inflammatory response to TPA [30]. The responses observed following TPA treatment suggest that PG synthesis from either COX isoform is sufficient to generate the PGs responsible for TPA-induced edema, or alternatively, TPA-induced edema results from mechanisms independent of PG synthesis. The ear inflammation studies in the COX-deficient mice indicate that COX-1, as well as COX-2, may contribute to inflammatory responses and the isoform responsible for the inflammation may depend on the type of inflammatory stimuli and/or the relative levels of each isoform in the target tissue. The development of inflammation in the COX-deficient mice has also been studied using the subdermal air pouch model [8]. Six hours following carrageenan-induced inflammation, an increase in PGE2 production is observed in the air pouch exudate of all COX genotypes. When compared to wild-type mice, the deficiency of COX-2 reduces the level of PGE2 production by approximately 75%, while the deficiency of COX-1 reduces the PGE2 level by 25%. Studies with the COX-deficient mice also helped to identify isoform-specific functions during the resolution of carrageenan-induced inflammation. Significantly higher numbers of inflammatory cells, including apoptotic neutrophils, are observed in air pouch exudate from COX-2-deficient mice, compared to COX-1-deficient or wild-type mice [8]. Therefore, studies with the COX-deficient mice indicate that both COX isoforms may contribute to PG production during acute inflammation and that COX-2 derived PGs may also be important in the resolution of inflammation.
8. Intestinal tumorigenesis Because the expression of COX-2 is induced in many types of cancers, the majority of research on the role of PG synthesis in the development of cancer has emphasized COX-2, rather than COX-1. The overexpression of COX-2 in cultured intestinal epithelial cells has also been shown to increase cellular adhesion and produce a resistance to apoptosis, both of which are cellular characteristics that may contribute to tumor development [31]. Furthermore, in a mouse model of human familial adenomatous polyposis, intestinal tumor development is reduced by the genetic deficiency of COX-2 [32]. However, by utilizing COX-deficient mice in the study of intestinal tumorigenesis, we have demonstrated
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that the deficiency of COX-1, as well as COX-2, significantly reduces tumor development [33]. For determining the roles of the COX isoforms in the development of intestinal tumorigenesis, the COX-deficient mouse lines were bred into the Min mouse strain. Min mice have a chemically induced mutation in the Apc gene, which results in a 100% incidence of intestinal neoplasia [34]. However, intestinal tumorigenesis is reduced by approximately 80% in Min mice deficient in either COX-1 or COX-2 [33]. Furthermore, the heterozygous deficiency of COX-1 is more effective than the heterozygous deficiency of COX-2 in reducing the number of intestinal polyps in Min mice [33]. These findings of the important role of COX-1 in the development of intestinal tumorigenesis may, in part, explain the mechanism of reduced colon cancer risk for humans using NSAIDs such as aspirin [35,36], which are more effective for the inhibition of COX-1 than of COX-2. References [1] Smith WL, DeWitt DL. Prostaglandin endoperoxide H synthases-1 and-2. Adv Immunol 1996;62:167–215. [2] Langenbach R, Morham SG, Tiano HF, Loftin CD, Ghanayem BI, Chulada PC, et al. Prostaglandin synthase-1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin-induced gastric ulceration. Cell 1995;83:483–92. [3] Morham SG, Langenbach R, Loftin CD, Tiano HF, Vouloumanos N, Jennette JC, et al. Prostaglandin synthase-2 gene disruption causes severe renal pathology in the mouse. Cell 1995;83:473–82. [4] Loftin CD, Trivedi DB, Tiano HF, Clark JA, Lee CA, Epstein JA, et al. Failure of ductus arteriosus closure and remodeling in neonatal mice deficient in cyclooxygenase-1 and cyclooxygenase-2. Proc Natl Acad Sci USA 2001;98:1059–64. [5] Wallace JL, Tigley AW. New insights into prostaglandins and mucosal defence. Aliment Pharmacol Ther 1995;9:227–35. [6] Kargman S, Charleson S, Cartwright M, Frank J, Riendeau D, Mancini J, et al. Characterization of prostaglandin G/H synthase 1 and 2 in rat, dog, monkey, and human gastrointestinal tracts. Gastroenterology 1996;111:445–54. [7] Masferrer JL, Zweifel BS, Manning PT, Hauser SD, Leahy KM, Smith WG, et al. Selective inhibition of inducible cyclooxygenase-2 in vivo is anti-inflammatory and nonulcerogenic. Proc Natl Acad Sci USA 1994;91:3228–32. [8] Langenbach R, Loftin C, Lee C, Tiano H. Cyclooxygenase knockout mice-Models for elucidating isoform-specific functions. Biochem Pharmacol 1999;58:1237–46. [9] Gretzer B, Maricic N, Respondek M, Schuligoi R, Peskar BM. Effects of specific inhibition of cyclooxygenase-1 and cyclooxygenase-2 in the rat stomach with normal mucosa and after acid challenge. Br J Pharmacol 2001;132:1565–73. [10] Wallace JL, Mcknight W, Reuter BK, Vergnolle N. NSAID-induced gastric damage in rats: requirement for inhibition of both cyclooxygenase 1 and 2. Gastroenterology 2000;119:706–14. [11] Gross GA, Imamura T, Luedke C, Vogt SK, Olson LM, Nelson DM, Sadovsky Y, et al. Opposing actions of prostaglandins and oxytocin determine the onset of murine labor. Proc Natl Acad Sci USA 1998;95:11875–9. [12] Lim H, Paria BC, Das SK, Dinchuk JE, Langenbach R, Trzaskos JM, et al. Multiple female reproductive failures in cyclooxygenase 2-deficient mice. Cell 1997;91:197–208. [13] Davis BJ, Lennard DE, Lee CA, Tiano HF, Morham SG, Wetsel WC, et al. Anovulation in cyclooxygenase-2-deficient mice is restored by prostaglandin E-2 and interleukin-1 beta. Endocrinology 1999;140:2685–95. [14] Komhoff M, Wang JL, Cheng HF, Langenbach R, Mckanna JA, Harris RC, et al. Cyclooxygenase-2-selective inhibitors impair glomerulogenesis and renal cortical development. Kidney Int 2000;57:414–22. [15] Norwood VF, Morham SG, Smithies O. Postnatal development and progression of renal dysplasia in cyclooxygenase-2 null mice. Kidney Int 2000;58:2291–300.
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