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W2626800913 abstract "Arachidonic acid is a precursor for biosynthesis of eicosanoids, including prostaglandins, thromboxanes, leukotrienes, and lipoxins. Cytosolic phospholipase A2 (cPLA2) plays a key role in the release of arachidonic acid as the substrate of cyclooxygenase-1 (COX-1) or COX-2. We found that the level of cPLA2 mRNA was markedly elevated in the polyps and correlated with the polyp size in the small intestine of theApc Δ716 knockout mouse, a model for human familial adenomatous polyposis. To determine the role of cPLA2 in intestinal tumorigenesis, we then introduced a cPLA2 gene mutation intoApc Δ716 mice. In the compound mutant mice, the size of the small intestinal polyps was reduced significantly, although the numbers remained unchanged. These results provide direct genetic evidence that cPLA2 plays a key role in the expansion of polyps in the small intestine rather than in the initiation process. In contrast, colonic polyps were not affected in either size or number. Interestingly, group X sPLA2 was constitutively expressed in the colon at much higher levels than in the small intestine. These results suggest that in the colon, group X sPLA2 supplies arachidonic acid in both the normal epithelium and the polyps even in the absence of cPLA2. Arachidonic acid is a precursor for biosynthesis of eicosanoids, including prostaglandins, thromboxanes, leukotrienes, and lipoxins. Cytosolic phospholipase A2 (cPLA2) plays a key role in the release of arachidonic acid as the substrate of cyclooxygenase-1 (COX-1) or COX-2. We found that the level of cPLA2 mRNA was markedly elevated in the polyps and correlated with the polyp size in the small intestine of theApc Δ716 knockout mouse, a model for human familial adenomatous polyposis. To determine the role of cPLA2 in intestinal tumorigenesis, we then introduced a cPLA2 gene mutation intoApc Δ716 mice. In the compound mutant mice, the size of the small intestinal polyps was reduced significantly, although the numbers remained unchanged. These results provide direct genetic evidence that cPLA2 plays a key role in the expansion of polyps in the small intestine rather than in the initiation process. In contrast, colonic polyps were not affected in either size or number. Interestingly, group X sPLA2 was constitutively expressed in the colon at much higher levels than in the small intestine. These results suggest that in the colon, group X sPLA2 supplies arachidonic acid in both the normal epithelium and the polyps even in the absence of cPLA2. arachidonic acid phospholipase A2 cytosolic PLA2 secretory PLA2 cyclooxygenase prostaglandin adenomatous polyposis coli loss of heterozygosity polymerase chain reaction reverse transcription-PCR Arachidonic acid (AA)1is a precursor for biosynthesis of eicosanoids, including prostaglandins (PGs), thromboxanes, leukotrienes, and lipoxins. In the unstimulated mammalian cell membrane, AA is esterified at thesn-2 position of glycerophospholipids (1Holtzman M.J. Am. Rev. Respir. Dis. 1991; 143: 188-203Crossref PubMed Scopus (193) Google Scholar). Free AA is released upon activation of phospholipase A2(PLA2), in response to various extracellular stimuli such as cytokines, hormones, neurotransmitters, mitogens, antigens, or endotoxins (2Dennis E.A. Am. J. Respir. Crit. Care Med. 2000; 161: S32-S35Crossref PubMed Scopus (116) Google Scholar). Mammalian cells contain structurally diverse forms of PLA2, including cytosolic PLA2(cPLA2), secretory PLA2 (sPLA2), and Ca2+-independent PLA2 (iPLA2) (3Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 4Leslie C.C. J. Biol. Chem. 1997; 272: 16709-116712Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, 5Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Furthermore, COX enzymes use free AA released by different phospholipases in certain cell types (6Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 7Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 8Bingham III, C.O. Murakami M. Fujishima H. Hunt J.E. Austen K.F. Arm J.P. J. Biol. Chem. 1996; 271: 25936-25944Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 9Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar). PGH2, the major product of the COX enzymes, is converted further by separate PG synthases to a variety of PGs, which play significant roles in tumorigenesis, including cell proliferation, apoptosis, cell differentiation, and angiogenesis (10Smith W.L. Dewitt D.L. Adv. Immunol. 1996; 62: 167-215Crossref PubMed Google Scholar, 11Tsujii M. Dubois R.N. Cell. 1995; 83: 493-501Abstract Full Text PDF PubMed Scopus (2131) Google Scholar, 12Tsujii M. Kawano S. Tsuji S. Sowaoka H. Hori M. Dubois R.N. Cell. 1998; 93: 705-716Abstract Full Text Full Text PDF PubMed Scopus (2205) Google Scholar).In human familial adenomatous polyposis, the tissue level of PGE2 is increased (13Rigas B. Goldman I.S. Levine L. J. Lab. Clin. Med. 1993; 122: 518-523PubMed Google Scholar, 14Yang V.W. Shields J.M. Hamilton S.R. Spannhake E.W. Hubbard W.C. Hylind L.M. Robinson C.R. Giardiello F.M. Cancer Res. 1998; 58: 1750-1753PubMed Google Scholar). In human colorectal cancer, the amount of COX-2 mRNA correlates with the size of the tumor (15Fujita T. Matsui M. Takaku K. Uetake H. Ichikawa W. Taketo M.M. Sugihara K. Cancer Res. 1998; 58: 4823-4826PubMed Google Scholar). Likewise, COX-2 expression is induced in polyps ofApc Δ716 mice and Minmice, models for human familial adenomatous polyposis (16Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2271) Google Scholar, 17Hull M.A. Booth J.K. Tisbury A. Scott N. Bonifer C. Markham A.F. Coletta P.L. Br. J. Cancer. 1999; 79: 1399-1405Crossref PubMed Scopus (122) Google Scholar, 18Shattuck-Brandt R.L. Varilek G.W. Radhika A. Yang F. Washington M.K. DuBois R.N. Gastroenterology. 2000; 118: 337-345Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 19Taketo M.M. Vane J.R. Botting R.M. Clinical Significance and Potential of Selective COX-2 Inhibitor. William Harvey Press, London1998: 129-139Google Scholar). Interestingly, a COX-2 null mutation significantly reduces the number and size of intestinal polyps inApc Δ716 mice (16Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2271) Google Scholar). Furthermore, treating Apc Δ716 mice with a selective COX-2 inhibitor reduces the polyp number and size more efficiently than treatment with sulindac, which inhibits both COX isoenzymes (16Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2271) Google Scholar). These results indicate that lack or inhibition of COX-2 efficiently suppresses tumorigenesis inApc Δ716 mice.On the other hand, the development of intestinal polyps in theMin mouse is influenced by the genetic background of theMom1 locus on chromosome 4, where a group IIA sPLA2 gene (Pla2g2a) is located (20Dietrich W.F. Lander E.S. Smith J.S. Moser A.R. Gould K.A. Luongo C. Borenstein N Dove W. Cell. 1993; 75: 631-639Abstract Full Text PDF PubMed Scopus (607) Google Scholar, 21MacPhee M. Chepenik K.P. Liddell R.A. Nelson K.K. Siracusa L.D. Buchberg A.M. Cell. 1995; 81: 957-966Abstract Full Text PDF PubMed Scopus (527) Google Scholar). Mouse strains C57BL/6 and 129/Sv carry a homozygous mutation in thePla2g2a gene, but AKR/J and Balb/c are wild type forPla2g2a (21MacPhee M. Chepenik K.P. Liddell R.A. Nelson K.K. Siracusa L.D. Buchberg A.M. Cell. 1995; 81: 957-966Abstract Full Text PDF PubMed Scopus (527) Google Scholar, 22Kennedy B.P. Payette P. Mudgett J. Vadas P. Pruzanski W. Kwan M. Tang C. Rancourt D.E. Cromlish W.A. J. Biol. Chem. 1995; 270: 22378-22385Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). If group IIA sPLA2 plays a major role in the production of PGs in the polyp stromal cells where COX-2 is induced, the Min polyps in the AKR/J background are expected to be larger and more numerous than those in C57BL/6 or 129/Sv. On the contrary, both the number and size are smaller in AKR/J (23Gould K.A. Dietrich W.F. Borenstein N. Lander E.S. Dove W.F. Genetics. 1996; 144: 1769-1776Crossref PubMed Google Scholar). In several human colorectal cancers, it is cPLA2 that is up-regulated (24Dimberg J. Samuelsson A. Hugander A. Soderkvist P. Anticancer Res. 1998; 18: 3283-3287PubMed Google Scholar). In addition to COX-2 induction, the higher levels of cPLA2 and AA may explain why human cancers synthesize increased amounts of PGs (25Soydan A.S. Gaffen J.D. Weech P.K. Tremblay N.M. Kargman S. O'Neill G. Bennett A. Tavares I.A. Eur. J. Cancer. 1997; 33: 1508-1512Abstract Full Text PDF PubMed Scopus (49) Google Scholar). To determine the role of cPLA2 in intestinal tumorigenesis, we constructed compound mutant mice for both cPLA2 (26Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. Miyazaki J. Shimizu T. Nature. 1997; 390: 618-622Crossref PubMed Scopus (636) Google Scholar) andApc (27Oshima M. Oshima H. Kitagawa K. Kobayashi M. Itakura C. Taketo M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4482-4486Crossref PubMed Scopus (494) Google Scholar) genes and investigated their intestinal polyp formation.DISCUSSIONSeveral lines of evidence indicate that cPLA2 is one of the most important PLA2 isozymes, regulating the immediate eicosanoid generation (3Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 6Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In cPLA2 knockout mice, the productions of eicosanoids and platelet-activating factor are markedly decreased in various types of cells (26Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. Miyazaki J. Shimizu T. Nature. 1997; 390: 618-622Crossref PubMed Scopus (636) Google Scholar, 30Bonventre J.V. Haung Z. Taheri M.R. O'Leary E. Li E. Moskowitz M.A. Sapirstein A. Nature. 1997; 390: 622-625Crossref PubMed Scopus (757) Google Scholar, 31Fujishima H. Sanchez Mejia R.O. Bingham III C.O. Lam B.K. Sapirstein A. Bonventre J.V. Austen K.F. Arm J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4803-4807Crossref PubMed Scopus (167) Google Scholar, 32Shindou H. Ishii S. Uozumi N. Shimizu T. Biochem. Cell Biol. 2000; 271: 812-817Google Scholar, 33Gijon M.A. Spencer D.M. Siddiqi A.R. Bonventre J.V. Leslie C.C. J. Biol. Chem. 2000; 275: 20146-20156Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). It has been demonstrated that formation of eicosanoids is regulated by various forms of sPLA2 (34Kuwata H. Nakatani Y. Murakami M Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 35Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 36Reddy S.T. Winstead M.V. Tischfield J.A. Herschman H.R. J. Biol. Chem. 1997; 272: 13591-13596Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) and that a cPLA2activity is essential for induction and activation of sPLA2in mast cells (31Fujishima H. Sanchez Mejia R.O. Bingham III C.O. Lam B.K. Sapirstein A. Bonventre J.V. Austen K.F. Arm J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4803-4807Crossref PubMed Scopus (167) Google Scholar), rat fibroblasts (34Kuwata H. Nakatani Y. Murakami M Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), mouse osteoblasts (37Chen Q. Miyaura C. Higashi S. Murakami M. Kudo I. Saito S. Hiraide T. Shibasaki Y. Suda T. J. Biol. Chem. 1997; 272: 5952-5958Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), macrophages (38Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), and gene-transfected cells (39Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Group V sPLA2 is more widely expressed than group IIA sPLA2 in mouse tissues (40Sawada H. Murakami M. Enomoto A. Shimbara S. Kudo I. Eur. J. Biochem. 1999; 263: 826-833Crossref PubMed Scopus (83) Google Scholar). The C57BL/6 strain, which lacks the group IIA sPLA2 gene (Pla2g2a), does not show any major phenotypes, except that the polyp number and size are increased when the Apc Min mutation is introduced (21MacPhee M. Chepenik K.P. Liddell R.A. Nelson K.K. Siracusa L.D. Buchberg A.M. Cell. 1995; 81: 957-966Abstract Full Text PDF PubMed Scopus (527) Google Scholar, 22Kennedy B.P. Payette P. Mudgett J. Vadas P. Pruzanski W. Kwan M. Tang C. Rancourt D.E. Cromlish W.A. J. Biol. Chem. 1995; 270: 22378-22385Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar).In the present study, we have found that the cPLA2 mRNA is strongly induced in the polyps of theApc Δ716 small intestine, showing an 11-fold increase from the normal intestinal levels (Fig. 1). In addition, the level of cPLA2 mRNA correlates with the size of the polyps in theApc Δ716 small intestine. Because the level of group V sPLA2 mRNA is not affected significantly in intestinal polyps (Fig. 4), cPLA2 and group X sPLA2 appear to be the key regulators of AA supplies to the COX enzymes in the small intestine of the C57BL/6J strain, in which group IIA sPLA2 is mutated.We have demonstrated that the mutation in the cPLA2gene reduces the size but not the number of small intestinal polyps. Thus, cPLA2 appears to play a significant role in polyp expansion rather than in the initiation process. Although the reduction in polyp growth could have been caused by apoptosis in the cPLA2 mutants, there was no significant difference in the apoptotic index of the adenoma epithelium between the cPLA2homozygous and wild-type mice (data not shown). On the other hand, we reported earlier that a mutation in the COX-2 gene (Ptgs2) reduces both the size and the number of small intestinal polyps (16Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2271) Google Scholar,19Taketo M.M. Vane J.R. Botting R.M. Clinical Significance and Potential of Selective COX-2 Inhibitor. William Harvey Press, London1998: 129-139Google Scholar). We also reported that the polyp number correlates with the frequency of LOH in the Apc locus (27Oshima M. Oshima H. Kitagawa K. Kobayashi M. Itakura C. Taketo M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4482-4486Crossref PubMed Scopus (494) Google Scholar). It is therefore possible that the COX-2 mutation may affect not only polyp expansion but also the frequency of Apc LOH.In the colon, on the other hand, the mutation in the cPLA2gene does not affect either the number or size of polyps, which is in clear contrast with the colonic phenotype of the COX-2 mutant mice (16Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2271) Google Scholar,19Taketo M.M. Vane J.R. Botting R.M. Clinical Significance and Potential of Selective COX-2 Inhibitor. William Harvey Press, London1998: 129-139Google Scholar). This difference suggests that AA is supplied in the colon by a source other than cPLA2. In addition to cPLA2and group IIA sPLA2, groups V and X sPLA2s play significant roles in the release of AA in mammalian cells stimulated byA23187 or fetal calf serum/interleukin-1β (29Murakami M. Kambe T. Shimbara S. Higashio K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 41Bezzine S. Koduri R.S. Valentin E. Murakami M. Kudo I. Gromashchi F. Sadilek M. Lambeu G. Gelb M.H. J. Biol. Chem. 2000; 275: 3179-3191Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Our results indicate that group X sPLA2 contributes to the release of AA in colonic polyps as well as in the normal epithelium (Fig. 4). Consistent with this interpretation, the small focal ulcerative lesions developed only in the small intestine, where group X sPLA2 mRNA was not induced in the absence of cPLA2. In addition, COX-2 mRNA was strongly induced also in cPLA2-negative colonic polyps (data not shown). These results collectively suggest that sufficient amounts of PGs are synthesized in the colon to support polyp expansion and to protect the mucosa from ulceration.In conclusion, we have demonstrated that a mutation in the cPLA2 gene causes a reduction in polyp size in the small intestine. These results provide direct genetic evidence that cPLA2 plays a key role in the expansion of intestinal polyps rather than in their initiation process. Arachidonic acid (AA)1is a precursor for biosynthesis of eicosanoids, including prostaglandins (PGs), thromboxanes, leukotrienes, and lipoxins. In the unstimulated mammalian cell membrane, AA is esterified at thesn-2 position of glycerophospholipids (1Holtzman M.J. Am. Rev. Respir. Dis. 1991; 143: 188-203Crossref PubMed Scopus (193) Google Scholar). Free AA is released upon activation of phospholipase A2(PLA2), in response to various extracellular stimuli such as cytokines, hormones, neurotransmitters, mitogens, antigens, or endotoxins (2Dennis E.A. Am. J. Respir. Crit. Care Med. 2000; 161: S32-S35Crossref PubMed Scopus (116) Google Scholar). Mammalian cells contain structurally diverse forms of PLA2, including cytosolic PLA2(cPLA2), secretory PLA2 (sPLA2), and Ca2+-independent PLA2 (iPLA2) (3Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 4Leslie C.C. J. Biol. Chem. 1997; 272: 16709-116712Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, 5Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Furthermore, COX enzymes use free AA released by different phospholipases in certain cell types (6Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 7Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 8Bingham III, C.O. Murakami M. Fujishima H. Hunt J.E. Austen K.F. Arm J.P. J. Biol. Chem. 1996; 271: 25936-25944Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 9Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar). PGH2, the major product of the COX enzymes, is converted further by separate PG synthases to a variety of PGs, which play significant roles in tumorigenesis, including cell proliferation, apoptosis, cell differentiation, and angiogenesis (10Smith W.L. Dewitt D.L. Adv. Immunol. 1996; 62: 167-215Crossref PubMed Google Scholar, 11Tsujii M. Dubois R.N. Cell. 1995; 83: 493-501Abstract Full Text PDF PubMed Scopus (2131) Google Scholar, 12Tsujii M. Kawano S. Tsuji S. Sowaoka H. Hori M. Dubois R.N. Cell. 1998; 93: 705-716Abstract Full Text Full Text PDF PubMed Scopus (2205) Google Scholar). In human familial adenomatous polyposis, the tissue level of PGE2 is increased (13Rigas B. Goldman I.S. Levine L. J. Lab. Clin. Med. 1993; 122: 518-523PubMed Google Scholar, 14Yang V.W. Shields J.M. Hamilton S.R. Spannhake E.W. Hubbard W.C. Hylind L.M. Robinson C.R. Giardiello F.M. Cancer Res. 1998; 58: 1750-1753PubMed Google Scholar). In human colorectal cancer, the amount of COX-2 mRNA correlates with the size of the tumor (15Fujita T. Matsui M. Takaku K. Uetake H. Ichikawa W. Taketo M.M. Sugihara K. Cancer Res. 1998; 58: 4823-4826PubMed Google Scholar). Likewise, COX-2 expression is induced in polyps ofApc Δ716 mice and Minmice, models for human familial adenomatous polyposis (16Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2271) Google Scholar, 17Hull M.A. Booth J.K. Tisbury A. Scott N. Bonifer C. Markham A.F. Coletta P.L. Br. J. Cancer. 1999; 79: 1399-1405Crossref PubMed Scopus (122) Google Scholar, 18Shattuck-Brandt R.L. Varilek G.W. Radhika A. Yang F. Washington M.K. DuBois R.N. Gastroenterology. 2000; 118: 337-345Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 19Taketo M.M. Vane J.R. Botting R.M. Clinical Significance and Potential of Selective COX-2 Inhibitor. William Harvey Press, London1998: 129-139Google Scholar). Interestingly, a COX-2 null mutation significantly reduces the number and size of intestinal polyps inApc Δ716 mice (16Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2271) Google Scholar). Furthermore, treating Apc Δ716 mice with a selective COX-2 inhibitor reduces the polyp number and size more efficiently than treatment with sulindac, which inhibits both COX isoenzymes (16Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2271) Google Scholar). These results indicate that lack or inhibition of COX-2 efficiently suppresses tumorigenesis inApc Δ716 mice. On the other hand, the development of intestinal polyps in theMin mouse is influenced by the genetic background of theMom1 locus on chromosome 4, where a group IIA sPLA2 gene (Pla2g2a) is located (20Dietrich W.F. Lander E.S. Smith J.S. Moser A.R. Gould K.A. Luongo C. Borenstein N Dove W. Cell. 1993; 75: 631-639Abstract Full Text PDF PubMed Scopus (607) Google Scholar, 21MacPhee M. Chepenik K.P. Liddell R.A. Nelson K.K. Siracusa L.D. Buchberg A.M. Cell. 1995; 81: 957-966Abstract Full Text PDF PubMed Scopus (527) Google Scholar). Mouse strains C57BL/6 and 129/Sv carry a homozygous mutation in thePla2g2a gene, but AKR/J and Balb/c are wild type forPla2g2a (21MacPhee M. Chepenik K.P. Liddell R.A. Nelson K.K. Siracusa L.D. Buchberg A.M. Cell. 1995; 81: 957-966Abstract Full Text PDF PubMed Scopus (527) Google Scholar, 22Kennedy B.P. Payette P. Mudgett J. Vadas P. Pruzanski W. Kwan M. Tang C. Rancourt D.E. Cromlish W.A. J. Biol. Chem. 1995; 270: 22378-22385Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). If group IIA sPLA2 plays a major role in the production of PGs in the polyp stromal cells where COX-2 is induced, the Min polyps in the AKR/J background are expected to be larger and more numerous than those in C57BL/6 or 129/Sv. On the contrary, both the number and size are smaller in AKR/J (23Gould K.A. Dietrich W.F. Borenstein N. Lander E.S. Dove W.F. Genetics. 1996; 144: 1769-1776Crossref PubMed Google Scholar). In several human colorectal cancers, it is cPLA2 that is up-regulated (24Dimberg J. Samuelsson A. Hugander A. Soderkvist P. Anticancer Res. 1998; 18: 3283-3287PubMed Google Scholar). In addition to COX-2 induction, the higher levels of cPLA2 and AA may explain why human cancers synthesize increased amounts of PGs (25Soydan A.S. Gaffen J.D. Weech P.K. Tremblay N.M. Kargman S. O'Neill G. Bennett A. Tavares I.A. Eur. J. Cancer. 1997; 33: 1508-1512Abstract Full Text PDF PubMed Scopus (49) Google Scholar). To determine the role of cPLA2 in intestinal tumorigenesis, we constructed compound mutant mice for both cPLA2 (26Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. Miyazaki J. Shimizu T. Nature. 1997; 390: 618-622Crossref PubMed Scopus (636) Google Scholar) andApc (27Oshima M. Oshima H. Kitagawa K. Kobayashi M. Itakura C. Taketo M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4482-4486Crossref PubMed Scopus (494) Google Scholar) genes and investigated their intestinal polyp formation. DISCUSSIONSeveral lines of evidence indicate that cPLA2 is one of the most important PLA2 isozymes, regulating the immediate eicosanoid generation (3Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 6Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In cPLA2 knockout mice, the productions of eicosanoids and platelet-activating factor are markedly decreased in various types of cells (26Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. Miyazaki J. Shimizu T. Nature. 1997; 390: 618-622Crossref PubMed Scopus (636) Google Scholar, 30Bonventre J.V. Haung Z. Taheri M.R. O'Leary E. Li E. Moskowitz M.A. Sapirstein A. Nature. 1997; 390: 622-625Crossref PubMed Scopus (757) Google Scholar, 31Fujishima H. Sanchez Mejia R.O. Bingham III C.O. Lam B.K. Sapirstein A. Bonventre J.V. Austen K.F. Arm J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4803-4807Crossref PubMed Scopus (167) Google Scholar, 32Shindou H. Ishii S. Uozumi N. Shimizu T. Biochem. Cell Biol. 2000; 271: 812-817Google Scholar, 33Gijon M.A. Spencer D.M. Siddiqi A.R. Bonventre J.V. Leslie C.C. J. Biol. Chem. 2000; 275: 20146-20156Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). It has been demonstrated that formation of eicosanoids is regulated by various forms of sPLA2 (34Kuwata H. Nakatani Y. Murakami M Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 35Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 36Reddy S.T. Winstead M.V. Tischfield J.A. Herschman H.R. J. Biol. Chem. 1997; 272: 13591-13596Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) and that a cPLA2activity is essential for induction and activation of sPLA2in mast cells (31Fujishima H. Sanchez Mejia R.O. Bingham III C.O. Lam B.K. Sapirstein A. Bonventre J.V. Austen K.F. Arm J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4803-4807Crossref PubMed Scopus (167) Google Scholar), rat fibroblasts (34Kuwata H. Nakatani Y. Murakami M Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), mouse osteoblasts (37Chen Q. Miyaura C. Higashi S. Murakami M. Kudo I. Saito S. Hiraide T. Shibasaki Y. Suda T. J. Biol. Chem. 1997; 272: 5952-5958Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), macrophages (38Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), and gene-transfected cells (39Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Group V sPLA2 is more widely expressed than group IIA sPLA2 in mouse tissues (40Sawada H. Murakami M. Enomoto A. Shimbara S. Kudo I. Eur. J. Biochem. 1999; 263: 826-833Crossref PubMed Scopus (83) Google Scholar). The C57BL/6 strain, which lacks the group IIA sPLA2 gene (Pla2g2a), does not show any major phenotypes, except that the polyp number and size are increased when the Apc Min mutation is introduced (21MacPhee M. Chepenik K.P. Liddell R.A. Nelson K.K. Siracusa L.D. Buchberg A.M. Cell. 1995; 81: 957-966Abstract Full Text PDF PubMed Scopus (527) Google Scholar, 22Kennedy B.P. Payette P. Mudgett J. Vadas P. Pruzanski W. Kwan M. Tang C. Rancourt D.E. Cromlish W.A. J. Biol. Chem. 1995; 270: 22378-22385Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar).In the present study, we have found that the cPLA2 mRNA is strongly induced in the polyps of theApc Δ716 small intestine, showing an 11-fold increase from the normal intestinal levels (Fig. 1). In addition, the level of cPLA2 mRNA correlates with the size of the polyps in theApc Δ716 small intestine. Because the level of group V sPLA2 mRNA is not affected significantly in intestinal polyps (Fig. 4), cPLA2 and group X sPLA2 appear to be the key regulators of AA supplies to the COX enzymes in the small intestine of the C57BL/6J strain, in which group IIA sPLA2 is mutated.We have demonstrated that the mutation in the cPLA2gene reduces the size but not the number of small intestinal polyps. Thus, cPLA2 appears to play a significant role in polyp expansion rather than in the initiation process. Although the reduction in polyp growth could have been caused by apoptosis in the cPLA2 mutants, there was no significant difference in the apoptotic index of the adenoma epithelium between the cPLA2homozygous and wild-type mice (data not shown). On the other hand, we reported earlier that a mutation in the COX-2 gene (Ptgs2) reduces both the size and the number of small intestinal polyps (16Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2271) Google Scholar,19Taketo M.M. Vane J.R. Botting R.M. Clinical Significance and Potential of Selective COX-2 Inhibitor. William Harvey Press, London1998: 129-139Google Scholar). We also reported that the polyp number correlates with the frequency of LOH in the Apc locus (27Oshima M. Oshima H. Kitagawa K. Kobayashi M. Itakura C. Taketo M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4482-4486Crossref PubMed Scopus (494) Google Scholar). It is therefore possible that the COX-2 mutation may affect not only polyp expansion but also the frequency of Apc LOH.In the colon, on the other hand, the mutation in the cPLA2gene does not affect either the number or size of polyps, which is in clear contrast with the colonic phenotype of the COX-2 mutant mice (16Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2271) Google Scholar,19Taketo M.M. Vane J.R. Botting R.M. Clinical Significance and Potential of Selective COX-2 Inhibitor. William Harvey Press, London1998: 129-139Google Scholar). This difference suggests that AA is supplied in the colon by a source other than cPLA2. In addition to cPLA2and group IIA sPLA2, groups V and X sPLA2s play significant roles in the release of AA in mammalian cells stimulated byA23187 or fetal calf serum/interleukin-1β (29Murakami M. Kambe T. Shimbara S. Higashio K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 41Bezzine S. Koduri R.S. Valentin E. Murakami M. Kudo I. Gromashchi F. Sadilek M. Lambeu G. Gelb M.H. J. Biol. Chem. 2000; 275: 3179-3191Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Our results indicate that group X sPLA2 contributes to the release of AA in colonic polyps as well as in the normal epithelium (Fig. 4). Consistent with this interpretation, the small focal ulcerative lesions developed only in the small intestine, where group X sPLA2 mRNA was not induced in the absence of cPLA2. In addition, COX-2 mRNA was strongly induced also in cPLA2-negative colonic polyps (data not shown). These results collectively suggest that sufficient amounts of PGs are synthesized in the colon to support polyp expansion and to protect the mucosa from ulceration.In conclusion, we have demonstrated that a mutation in the cPLA2 gene causes a reduction in polyp size in the small intestine. These results provide direct genetic evidence that cPLA2 plays a key role in the expansion of intestinal polyps rather than in their initiation process. Several lines of evidence indicate that cPLA2 is one of the most important PLA2 isozymes, regulating the immediate eicosanoid generation (3Tischfield J.A. J. Biol. Chem. 1997; 272: 17247-17250Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 6Reddy S.T. Herschman H.R. J. Biol. Chem. 1997; 272: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In cPLA2 knockout mice, the productions of eicosanoids and platelet-activating factor are markedly decreased in various types of cells (26Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. Miyazaki J. Shimizu T. Nature. 1997; 390: 618-622Crossref PubMed Scopus (636) Google Scholar, 30Bonventre J.V. Haung Z. Taheri M.R. O'Leary E. Li E. Moskowitz M.A. Sapirstein A. Nature. 1997; 390: 622-625Crossref PubMed Scopus (757) Google Scholar, 31Fujishima H. Sanchez Mejia R.O. Bingham III C.O. Lam B.K. Sapirstein A. Bonventre J.V. Austen K.F. Arm J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4803-4807Crossref PubMed Scopus (167) Google Scholar, 32Shindou H. Ishii S. Uozumi N. Shimizu T. Biochem. Cell Biol. 2000; 271: 812-817Google Scholar, 33Gijon M.A. Spencer D.M. Siddiqi A.R. Bonventre J.V. Leslie C.C. J. Biol. Chem. 2000; 275: 20146-20156Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). It has been demonstrated that formation of eicosanoids is regulated by various forms of sPLA2 (34Kuwata H. Nakatani Y. Murakami M Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 35Balboa M.A. Balsinde J. Winstead M.V. Tischfield J.A. Dennis E.A. J. Biol. Chem. 1996; 271: 32381-32384Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 36Reddy S.T. Winstead M.V. Tischfield J.A. Herschman H.R. J. Biol. Chem. 1997; 272: 13591-13596Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar) and that a cPLA2activity is essential for induction and activation of sPLA2in mast cells (31Fujishima H. Sanchez Mejia R.O. Bingham III C.O. Lam B.K. Sapirstein A. Bonventre J.V. Austen K.F. Arm J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4803-4807Crossref PubMed Scopus (167) Google Scholar), rat fibroblasts (34Kuwata H. Nakatani Y. Murakami M Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), mouse osteoblasts (37Chen Q. Miyaura C. Higashi S. Murakami M. Kudo I. Saito S. Hiraide T. Shibasaki Y. Suda T. J. Biol. Chem. 1997; 272: 5952-5958Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), macrophages (38Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), and gene-transfected cells (39Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Group V sPLA2 is more widely expressed than group IIA sPLA2 in mouse tissues (40Sawada H. Murakami M. Enomoto A. Shimbara S. Kudo I. Eur. J. Biochem. 1999; 263: 826-833Crossref PubMed Scopus (83) Google Scholar). The C57BL/6 strain, which lacks the group IIA sPLA2 gene (Pla2g2a), does not show any major phenotypes, except that the polyp number and size are increased when the Apc Min mutation is introduced (21MacPhee M. Chepenik K.P. Liddell R.A. Nelson K.K. Siracusa L.D. Buchberg A.M. Cell. 1995; 81: 957-966Abstract Full Text PDF PubMed Scopus (527) Google Scholar, 22Kennedy B.P. Payette P. Mudgett J. Vadas P. Pruzanski W. Kwan M. Tang C. Rancourt D.E. Cromlish W.A. J. Biol. Chem. 1995; 270: 22378-22385Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). In the present study, we have found that the cPLA2 mRNA is strongly induced in the polyps of theApc Δ716 small intestine, showing an 11-fold increase from the normal intestinal levels (Fig. 1). In addition, the level of cPLA2 mRNA correlates with the size of the polyps in theApc Δ716 small intestine. Because the level of group V sPLA2 mRNA is not affected significantly in intestinal polyps (Fig. 4), cPLA2 and group X sPLA2 appear to be the key regulators of AA supplies to the COX enzymes in the small intestine of the C57BL/6J strain, in which group IIA sPLA2 is mutated. We have demonstrated that the mutation in the cPLA2gene reduces the size but not the number of small intestinal polyps. Thus, cPLA2 appears to play a significant role in polyp expansion rather than in the initiation process. Although the reduction in polyp growth could have been caused by apoptosis in the cPLA2 mutants, there was no significant difference in the apoptotic index of the adenoma epithelium between the cPLA2homozygous and wild-type mice (data not shown). On the other hand, we reported earlier that a mutation in the COX-2 gene (Ptgs2) reduces both the size and the number of small intestinal polyps (16Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2271) Google Scholar,19Taketo M.M. Vane J.R. Botting R.M. Clinical Significance and Potential of Selective COX-2 Inhibitor. William Harvey Press, London1998: 129-139Google Scholar). We also reported that the polyp number correlates with the frequency of LOH in the Apc locus (27Oshima M. Oshima H. Kitagawa K. Kobayashi M. Itakura C. Taketo M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4482-4486Crossref PubMed Scopus (494) Google Scholar). It is therefore possible that the COX-2 mutation may affect not only polyp expansion but also the frequency of Apc LOH. In the colon, on the other hand, the mutation in the cPLA2gene does not affect either the number or size of polyps, which is in clear contrast with the colonic phenotype of the COX-2 mutant mice (16Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2271) Google Scholar,19Taketo M.M. Vane J.R. Botting R.M. Clinical Significance and Potential of Selective COX-2 Inhibitor. William Harvey Press, London1998: 129-139Google Scholar). This difference suggests that AA is supplied in the colon by a source other than cPLA2. In addition to cPLA2and group IIA sPLA2, groups V and X sPLA2s play significant roles in the release of AA in mammalian cells stimulated byA23187 or fetal calf serum/interleukin-1β (29Murakami M. Kambe T. Shimbara S. Higashio K. Hanasaki K. Arita H. Horiguchi M. Arita M. Arai H. Inoue K. Kudo I. J. Biol. Chem. 1999; 274: 31435-31444Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 41Bezzine S. Koduri R.S. Valentin E. Murakami M. Kudo I. Gromashchi F. Sadilek M. Lambeu G. Gelb M.H. J. Biol. Chem. 2000; 275: 3179-3191Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Our results indicate that group X sPLA2 contributes to the release of AA in colonic polyps as well as in the normal epithelium (Fig. 4). Consistent with this interpretation, the small focal ulcerative lesions developed only in the small intestine, where group X sPLA2 mRNA was not induced in the absence of cPLA2. In addition, COX-2 mRNA was strongly induced also in cPLA2-negative colonic polyps (data not shown). These results collectively suggest that sufficient amounts of PGs are synthesized in the colon to support polyp expansion and to protect the mucosa from ulceration. In conclusion, we have demonstrated that a mutation in the cPLA2 gene causes a reduction in polyp size in the small intestine. These results provide direct genetic evidence that cPLA2 plays a key role in the expansion of intestinal polyps rather than in their initiation process. We thank Drs. K. Hanasaki (Shionogi & Co., Ltd.), and M. Oshima (Kyoto University) for discussions, Drs. K. Hioki and N. Shimozawa (Central Institute of Experimental Animals) for the mouse breeding, and S. Iwasaki, S. Hagiwara, and M. Takeda for technical assistance." @default.
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W2626800913 title "Suppression of Intestinal Polyposis inApc Δ716 Knockout Mice by an Additional Mutation in the Cytosolic Phospholipase A2Gene" @default.
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