Suppression of Intestinal Polyposis inApc Δ 716 Knockout Mice by an Additional Mutation in the Cytosolic Phospholipase A2Gene*

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 Δ 716 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 Δ 716 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.


From the ‡Laboratory of Biomedical Genetics, Graduate School of Pharmaceutical Sciences and the §Department of Biochemistry and Molecular Biology, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan
Arachidonic acid is a precursor for biosynthesis of eicosanoids, including prostaglandins, thromboxanes, leukotrienes, and lipoxins. Cytosolic phospholipase A 2 (cPLA 2 ) 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 cPLA 2 mRNA was markedly elevated in the polyps and correlated with the polyp size in the small intestine of the Apc ⌬716 knockout mouse, a model for human familial adenomatous polyposis. To determine the role of cPLA 2 in intestinal tumorigenesis, we then introduced a cPLA 2 gene mutation into Apc ⌬716 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 cPLA 2 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 sPLA 2 was constitutively expressed in the colon at much higher levels than in the small intestine. These results suggest that in the colon, group X sPLA 2 supplies arachidonic acid in both the normal epithelium and the polyps even in the absence of cPLA 2 .
In human familial adenomatous polyposis, the tissue level of PGE 2 is increased (13,14). In human colorectal cancer, the amount of COX-2 mRNA correlates with the size of the tumor (15). Likewise, COX-2 expression is induced in polyps of Apc ⌬716 mice and Min mice, models for human familial adenomatous polyposis (16 -19). Interestingly, a COX-2 null mutation significantly reduces the number and size of intestinal polyps in Apc ⌬716 mice (16). Furthermore, treating Apc ⌬716 mice with a selective COX-2 inhibitor reduces the polyp number and size more efficiently than treatment with sulindac, which inhibits both COX isoenzymes (16). These results indicate that lack or inhibition of COX-2 efficiently suppresses tumorigenesis in Apc ⌬716 mice.
On the other hand, the development of intestinal polyps in the Min mouse is influenced by the genetic background of the Mom1 locus on chromosome 4, where a group IIA sPLA 2 gene (Pla2g2a) is located (20,21). Mouse strains C57BL/6 and 129/Sv carry a homozygous mutation in the Pla2g2a gene, but AKR/J and Balb/c are wild type for Pla2g2a (21,22). If group IIA sPLA 2 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 (23). In several human colorectal cancers, it is cPLA 2 that is up-regulated (24). In addition to COX-2 induction, the higher levels of cPLA 2 and AA may explain why human cancers synthesize increased amounts of PGs (25). To determine the role of cPLA 2 in intestinal tumorigenesis, we constructed compound mutant mice for both cPLA 2 (26) and Apc (27) genes and investigated their intestinal polyp formation.

EXPERIMENTAL PROCEDURES
RT-PCR Analyses for cPLA 2 and sPLA 2 mRNAs-Total RNA was extracted from the normal intestine and polyps in Apc ⌬716 ϩ/Ϫ and Apc ⌬716 ϩ/Ϫ Pla2g4 Ϫ/Ϫ mice. For RT-PCR of the mouse cPLA 2 mRNA (Pla2g4), first-strand cDNA synthesized from mRNA using a kit (Amersham Pharmacia Biotech) was amplified with the following primers: cPLA 2 , forward primer (5Ј-GTGTCTGGGGCAGTGCCTTT-3Ј, codon 391-397) and reverse primer (5Ј-GTTGAAAATGGCGATTCGGG-3Ј, codon 673-678). The PCRs were performed using 25 pmol of each primer under the following conditions: 94°C for 30 s, 65°C for 45 s, and 72°C for 1 min for 32 cycles. For RT-PCR of mouse groups V and X sPLA 2 mRNAs (Pla2g5 and Pla2g10, respectively), the synthesized cDNAs were amplified with the following primers: group V sPLA 2 , * The work was supported in part by the joint research fund between the University of Tokyo and Banyu Pharmaceutical Co.; grants from the Ministry of Education, Science, Culture and Sports; the Organization for Pharmaceutical Safety and Research; the Ministry of Health and Welfare; and the Science and Technology Agency, Japan (CREST). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Northern Blot Analyses for Groups V and X sPLA 2 mRNAs-Twenty micrograms of total RNA was electrophoresed in 1% agarose with formaldehyde as described previously (40). The probes specific for the respective sPLA 2 mRNAs were prepared by random primer labeling of the PCR fragments obtained in the above experiments.
Histological and Immunohistochemical Analyses-Mice were sacrificed by carbon dioxide euthanasia, and tissues were fixed and embedded as described previously (15,27,28).

RESULTS
cPLA 2 mRNA Is Induced in Apc ⌬716 Mouse Polyps-In human colorectal cancer, the level of cPLA 2 mRNA is increased (24). To investigate the role of cPLA 2 in polyp formation, we first determined the mRNA level in intestinal polyps of the Apc ⌬716 knockout mice (27) by RT-PCR. It was markedly increased in the polyps of the small intestine, by 10.6-fold compared with that in the normal intestine (p Ͻ 0.05) (Fig. 1). In colonic polyps, however, the mRNA level was similar to that in the normal colonic epithelium (Fig. 1). These results indicate that the induction of cPLA 2 is associated with polyp formation in the small intestine. cPLA 2 Gene Disruption Reduces Polyp Size in Apc ⌬716 Mice-To directly determine the role of cPLA 2 in polyp formation, we crossed the cPLA 2 gene (Pla2g4) knockout mice (26) with the Apc ⌬716 heterozygotes (27), generating mice that carried compound mutations, Apc ⌬716 ϩ/Ϫ Pla2g4 ϩ/Ϫ or Apc ⌬716 ϩ/Ϫ Pla2g4 Ϫ/Ϫ. Although most Apc ⌬716 ϩ/Ϫ Pla2g4 ϩ/ϩ littermate controls died from anemia caused by intestinal bleeding before reaching the age of 25 weeks, all Apc ⌬716 ϩ/Ϫ Pla2g4 Ϫ/Ϫ mice survived longer than 30 weeks (data not shown). Upon necropsies, the Apc ⌬716 ϩ/Ϫ Pla2g4 ϩ/ϩ controls had 376 Ϯ 144 (S.D.) polyps in the small intestine per mouse at 10 weeks of age. The polyp number was not affected by the cPLA 2 genotype, namely, it was 379 Ϯ 181 in Apc ⌬716 ϩ/Ϫ Pla2g4 ϩ/Ϫ, whereas the number was 319 Ϯ 92 in Apc ⌬716 ϩ/Ϫ Pla2g4 Ϫ/Ϫ (Fig. 2A). The difference between the Pla2g4 wildtype and mutant polyp numbers was not statistically significant. In contrast, the polyp size in the Apc ⌬716 ϩ/Ϫ Pla2g4 Ϫ/Ϫ mice was significantly smaller than that in the Apc ⌬716 ϩ/Ϫ Pla2g4 ϩ/ϩ controls. No polyps were found larger than 2.0 mm in diameter, and most polyps were smaller than 0.5 mm ( Fig.  2A). This tendency was observed even in the Apc ⌬716 ϩ/Ϫ Pla2g4 ϩ/Ϫ mice, although to a less extent ( Fig. 2A).
These results indicate that expression of the cPLA 2 gene promotes the growth but not the initiation of polyps in the small intestine.
Histological Analysis of Intestinal Polyps in Apc ⌬716 ϩ/Ϫ Pla2g4 Ϫ/Ϫ Mice-As described above, the polyp size was much smaller in the Apc ⌬716 ϩ/Ϫ Pla2g4 Ϫ/Ϫ mice (Fig. 3A). We did not find polyps larger than 0.5 mm in diameter in Apc ⌬716 ϩ/Ϫ Pla2g4 Ϫ/Ϫ. Upon histological examinations, the nascent polyps in the Apc ⌬716 ϩ/Ϫ Pla2g4 Ϫ/Ϫ mice (Fig. 3B) showed a similar morphology to that of the Apc ⌬716 ϩ/Ϫ Pla2g4 ϩ/ϩ polyps. Because the polyp size was reduced in the small intestine, it was conceivable that the mutation in the cPLA 2 gene induced apoptosis in the adenomatous epithelium. To The upper panel shows the ratio of the cPLA 2 band intensity relative to that of the normal small intestine, calibrated to the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) band. All analyses were performed three times independently. Error bars show standard deviations. * and †, p Ͻ 0.05 compared with the normal small intestine: *, the difference between the numbers was statistically significant (p Ͻ 0.05); †, the difference between the numbers showed p ϭ 0.28. The lower panels show representative photographs of the agarose gels for cPLA 2 and G3PDH bands, respectively. NI, normal intestine; SmP, small polyps; and LgP, large polyps. investigate such a possibility, we performed TUNEL (terminal deoxynucleotidyltransferase-mediated UTP nick end labeling) assays and scored apoptotic adenoma epithelial cells. However, there was no significant difference between the Apc ⌬716 ϩ/Ϫ Pla2g4 Ϫ/Ϫ or Apc ⌬716 ϩ/Ϫ Pla2g4 ϩ/Ϫ mice and the control Apc ⌬716 heterozygotes (data not shown). Therefore, cPLA 2 does not affect apoptosis in the epithelium of the intestinal polyp adenoma.
Although it was not reported previously, we found in the cPLA 2 gene homozygotes (Pla2g4 Ϫ/Ϫ) numerous small ulcerative lesions of the small intestinal epithelium. Likewise, the Apc ⌬716 ϩ/Ϫ Pla2g4 Ϫ/Ϫ mice also showed similar ulcerative lesions. On average, about 100 small discrete ulcers were observed in the small intestinal epithelium of a 10-week-old Apc ⌬716 ϩ/Ϫ Pla2g4 Ϫ/Ϫ mouse but not in the stomach or colon (Fig. 3C). Histological sections of such lesions showed evidence of mucosal inflammation with neutrophilic and mononuclear cell infiltrations, shortening and thickening of the crypts, and superficial mucosal erosions (Fig. 3D).
Group X sPLA 2 Is Expressed in Colonic Polyps-In the colon, the mutation in the cPLA 2 gene did not affect either the polyp number or size, suggesting a possibility that another phospholipase was releasing AA. In addition to cPLA 2 and group IIA sPLA 2 , it has been reported that groups V (Pla2g5) and X (Pla2g10) sPLA 2 s play important roles in the release of AA (29). Therefore, we determined the mRNA levels for these enzymes in the Apc ⌬716 ϩ/Ϫ and Apc ⌬716 ϩ/Ϫ Pla2g4 Ϫ/Ϫ intestines and polyps. As shown in Fig. 4, the group V sPLA 2 was expressed at low levels, equally in the small intestine and colon, without a significant induction in the polyps of either mutant. In contrast, the group X sPLA 2 mRNA was strongly induced in the polyps of the Apc ⌬716 small intestine, its level correlating with the polyp size. In large polyps, the mRNA level reached 4.7 times of that in the normal mucosa (*p Ͻ 0.01; Fig.  4). In the Apc ⌬716 ϩ/Ϫ Pla2g4 Ϫ/Ϫ mice, however, group X sPLA 2 mRNA was not induced in the polyps of the small intestine but remained unaffected. In the colon, interestingly, the mRNA was expressed constitutively at higher levels than in the small intestine either in the presence or absence of cPLA 2 (p Ͻ 0.05; Fig. 4). These RT-PCR data were verified also by a Northern blot analysis (data not shown). Accordingly, these results suggest that in addition to cPLA 2 , group X sPLA 2 contributes to AA release in both normal colonic epithelium and polyps (see "Discussion").

DISCUSSION
Several lines of evidence indicate that cPLA 2 is one of the most important PLA 2 isozymes, regulating the immediate eicosanoid generation (3,6). In cPLA 2 knockout mice, the productions of eicosanoids and platelet-activating factor are markedly decreased in various types of cells (26, 30 -33). It has been demonstrated that formation of eicosanoids is regulated by various forms of sPLA 2 (34 -36) and that a cPLA 2 activity is essential for induction and activation of sPLA 2 in mast cells (31), rat fibroblasts (34), mouse osteoblasts (37), macrophages (38), and gene-transfected cells (39). Group V sPLA 2 is more widely expressed than group IIA sPLA 2 in mouse tissues (40). The C57BL/6 strain, which lacks the group IIA sPLA 2 gene (Pla2g2a), does not show any major phenotypes, except that the polyp number and size are increased when the Apc Min mutation is introduced (21,22).
In the present study, we have found that the cPLA 2 mRNA is strongly induced in the polyps of the Apc ⌬716 small intestine, showing an 11-fold increase from the normal intestinal levels The upper panel shows the ratios of groups V and X sPLA 2 band intensities relative to those of the normal intestine, calibrated to the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) bands. All determinations were performed three times independently. Error bars show standard deviations. *, †, and #, p Ͻ 0.05 compared with the normal small intestine in the presence (* and †) or absence (#) of cPLA 2 : *, the difference between the numbers showed p Ͻ 0.05; † and #, the differences between the numbers are not statistically significant. The lower panels show representative photographs of the agarose gels for groups V and X sPLA 2 s and the G3PDH bands, respectively. NI, normal intestine; SmP, small polyps; LgP, large polyps. (Fig. 1). In addition, the level of cPLA 2 mRNA correlates with the size of the polyps in the Apc ⌬716 small intestine. Because the level of group V sPLA 2 mRNA is not affected significantly in intestinal polyps (Fig. 4), cPLA 2 and group X sPLA 2 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 sPLA 2 is mutated.
We have demonstrated that the mutation in the cPLA 2 gene reduces the size but not the number of small intestinal polyps. Thus, cPLA 2 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 cPLA 2 mutants, there was no significant difference in the apoptotic index of the adenoma epithelium between the cPLA 2 homozygous 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 (16,19). We also reported that the polyp number correlates with the frequency of LOH in the Apc locus (27). 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 cPLA 2 gene 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 (16,19). This difference suggests that AA is supplied in the colon by a source other than cPLA 2 . In addition to cPLA 2 and group IIA sPLA 2 , groups V and X sPLA 2 s play significant roles in the release of AA in mammalian cells stimulated by A23187 or fetal calf serum/interleukin-1␤ (29,41).
Our results indicate that group X sPLA 2 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 sPLA 2 mRNA was not induced in the absence of cPLA 2 . In addition, COX-2 mRNA was strongly induced also in cPLA 2 -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 cPLA 2 gene causes a reduction in polyp size in the small intestine. These results provide direct genetic evidence that cPLA 2 plays a key role in the expansion of intestinal polyps rather than in their initiation process.