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Originally published In Press as doi:10.1074/jbc.M609930200 on November 16, 2006

J. Biol. Chem., Vol. 282, Issue 2, 1498-1506, January 12, 2007
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Targeted Cyclooxygenase Gene (Ptgs) Exchange Reveals Discriminant Isoform Functionality*

Ying Yu{ddagger}, Jinjin Fan{ddagger}, Yiqun Hui{ddagger}, Carol A. Rouzer§, Lawrence J. Marnett§, Andres J. Klein-Szanto, Garret A. FitzGerald{ddagger}1, and Colin D. Funk{ddagger}||2

From the {ddagger}Institute for Translational Medicine and Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, the §Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, the Department of Pathology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, and the ||Departments of Biochemistry and Physiology, Queen's University, Kingston, Ontario K7L 3N6, Canada

Received for publication, October 23, 2006 , and in revised form, November 14, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The prostaglandin G/H synthase enzymes, commonly termed COX-1 and COX-2, differ markedly in their responses to regulatory stimuli and their tissue expression patterns. COX-1 is the dominant source of "housekeeping" prostaglandins, whereas COX-2 synthesizes prostaglandins of relevance to pain, inflammation, and mitogenesis. Despite these distinctions, the two enzymes are remarkably conserved, and their subcellular distributions overlap considerably. To address the functional interchangeability of the two isozymes, mice in which COX-1 is expressed under COX-2 regulatory elements were created by a gene targeting "knock-in" strategy. In macrophages from these mice, COX-1 was shown to be lipopolysaccharide-inducible in a manner analogous to COX-2 in wild-type macrophages. However, COX-1 failed to substitute effectively for COX-2 in lipopolysaccharide-induced prostaglandin E2 synthesis at low concentrations of substrate and in the metabolism of the endocannabinoid 2-arachidonylglycerol. The marked depression of the major urinary metabolite of prostacyclin in COX-2 null mice was only partially rescued by COX-1 knock-in, whereas the main urinary metabolite of prostaglandin E2 was rescued totally. Replacement with COX-1 partially rescued the impact of COX-2 deletion on reproductive function. The renal pathology consequent to COX-2 deletion was delayed but not prevented, whereas the corresponding peritonitis was unaltered. Insertion of COX-1 under the regulatory sequences that drive COX-2 expression indicated that COX-1 can substitute for some COX-2 actions and rescue only some of the consequences of gene disruption. Manipulation of COX-2 also revealed a preference for coupling with distinct downstream prostaglandin synthases in vivo. These mice will provide a valuable reagent with which to elucidate the distinct roles of the COX enzymes in mammalian biology.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostaglandin H synthase (PGHS)3 exists in two isoforms, COX-1 and COX-2. These enzymes catalyze the oxygenation of arachidonic acid (AA) to PGH2, a key intermediate in the biosynthesis of all prostanoids (1). The discovery of COX-2 15 years ago infused new life and vigorous research into the prostaglandin field and yielded significant new insights into the molecular basis of inflammation, pain, and fever (Ref. 1 and references therein). It also led to the development of COX-2 isoform-specific inhibitors such as the coxibs (2). The reason for the existence of the two COX isoforms is poorly understood. The major functional differences between COX-1 and COX-2 are believed to be related to their differential gene regulation and tissue distribution (3). COX-1 is often referred to as the constitutive isoform responsible for homeostatic prostaglandin production with involvement in renal water and electrolyte balance, gastric cytoprotection, platelet aggregation, and parturition (4-7). COX-2, on the other hand, appears to account for inducible prostaglandin synthesis in inflammation (2) and carcinogenesis (8, 9) and also for regulated kidney development and female reproductive function during ovulation and implantation (10-13). With respect to female infertility observed in COX-2 null mice on a CD1 genetic background, compensatory up-regulation of COX-1 is able to significantly improve female fertility (14). Both isoforms can coordinately contribute to remodeling of the ductus arteriosus (15); the boundaries between distinct isoform functions are not always clear in inflammation and carcinogenesis (7-9).

Although the primary sequences reveal 60% amino acid sequence identity, the crystal structures of both COX-1 and COX-2 reveal homodimeric and heme-containing proteins that are strikingly similar except for a larger "side pocket" for substrate access in COX-2 (1, 2). Moreover, COX-2 oxygenates neutral fatty acid derivatives such as endocannabinoids, which are poor substrates for COX-1 (16). Biochemical studies indicate that each isoform may functionally couple in vitro through independent pathways for PG synthesis (17). Investigators have postulated that the two COX isoforms may act as parts of separate prostaglandin biosynthetic systems that function independently to channel prostaglandins to the extracellular milieu and the nucleus, respectively, as one potential explanation for the two isoforms (18).

We created a gene-targeted "knock-in" of the COX-1 coding sequence into the COX-2 genetic locus under the endogenous COX-2 transcriptional regulatory control elements and 3'-untranslated region (3'-UTR) mRNA stability elements (designated COX-1 > COX-2) in embryonic stem cells and mice to address the apparent redundancy of function between the two COX isoforms and to elucidate their roles in biology. Our results from studying kidney development and function, the gastrointestinal tract, and female reproduction reveal functional redundancy in some biological systems, whereas in others, COX-2 alone suffices.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of Targeting Vector, Gene Targeting, and Germline Transmission—To facilitate Ptgs1 (prostaglandin synthase gene 1) cDNA insertion by the Ptgs2 gene translation initiation site, a BsiWI restriction site (italicized) at both COX-1 (CGT ACG AGT CGA) and COX-2 coding start regions (ATG CTC TTC CGT ACG) was generated by QuikChange mutagenesis (Qiagen). Subsequently, the targeting vector was generated by inserting a combined fragment including a 4.9-kb 5'-Ptgs2 promoter region targeting arm and a 1.8-kb Ptgs1 cDNA sequence downstream of a floxed PGK-Neo gene cassette in pPNT. A 5.4-kb KpnI-HpaI fragment consisting of exon 10 with the 3'-UTR region of the Ptgs2 gene was inserted upstream of the Neo cassette as the 3' targeting arm (Fig. 1A). R1 embryonic stem cells (a gift from A. Nagy, University of Toronto) were electroporated with the linearized targeting construct and selected for G418 and ganciclovir-resistant colonies as described previously (7). 1100 clones were screened by Southern blot analysis after BamHI digestion using a 5' external probe amplified from genomic DNA (forward primer, 5'-AGAACAGCCATGTTGCTGG-3'; reverse primer, 5'-TTTCAAGTCCACCAGCCTG-3'). Correctly targeted clones were verified by both 5' external (Fig. 1B) and specific Ptgs1 cDNA probes (located at exon 11; see Fig. 1C). Two positive clones were injected into C57BL/6 recipient blastocysts. Male chimeric mice were crossed to EIIa Cre transgenic mice (The Jackson Laboratory, Bar Harbor, ME) to obtain COX-1 > COX-2 mice by specific removal of the Neo gene from the Ptgs1 > Ptgs2flox-Neo allele (Fig. 1A), which can be identified by PCR (Fig. 1D) using: primer 1, 5'-TCATAGCCTGAACGAGATC-3'; primer 2, 5'-TCATAGCCTGAAGAACGAGATC-3'; primer 3, 5'-AGAATGGTGCTCCAAGCTCTAC-3'. Subsequent genotyping was performed routinely by specific PCR on tail DNA using three primers (Fig. 1E): primer 4, 5'-CTCACATTG GAGAAGGACTCC-3'; primer 5, 5'-ACCTCTGCGATGCTCTTCC-3'; primer 6, 5'-ACTGGTCAAATCCTGTGCTC-3').

Animal Breeding—COX-1 > COX-2flox-Neo, COX-1 > COX-2, and COX-2 null mice were maintained on a mixed C57BL/6 x 129/Sv genetic background. These mice and WT controls were generated from heterozygous F1 crosses to ensure a similar genetic background. The Institutional Animal Care and Use Committee of the University of Pennsylvania approved these studies.

Isolation of Macrophages and Assay for COX Activity—Peritoneal macrophage isolation and PGE2 generation in vitro have been reported previously (7, 19). Briefly, cold sterile phosphate-buffered saline (5 ml) was injected into the peritoneal cavity, and macrophages were harvested and seeded into 60-mm dishes at 1-2.5 x 107 cells in RPMI 1640 medium supplemented with 3% fetal bovine serum and a 1% mixture of penicillin/streptomycin solution. The cells were allowed to adhere for 2 h with/without aspirin (500 µM) to inactivate endogenous COX-1, washed three times, and then stimulated with LPS (5 µg/ml in RPMI) for 16 h. After the medium was removed, cells were incubated with AA (0.5-30 µM in 3% fetal bovine serum/RPMI medium) for 15 min, and the medium was collected for PG assay by liquid chromatography-positive ion electrospray ionization tandem mass spectrometry (LC/MS/MS). Exogenous 2-arachidonylglycerol metabolism by peritoneal macrophages and analysis of prostaglandin-glycerol from culture medium were performed as described previously (20).

Western Blot Analysis—Protein (10 µg) was loaded into each lane, separated on 4-10% BisTris-NuPAGE gels (Invitrogen) and transferred to Hybond ECL nitrocellulose membranes (Amersham Biosciences). Goat anti-COX-1 polyclonal antibody (Santa Cruz Biotechnology) at a 1:500 dilution, rabbit anti-COX-2 polyclonal antiserum (Cayman Chemical Co.) at a 1:1000 dilution, and mouse anti-beta-actin monoclonal antibody (Sigma) at a 1:5000 dilution were used as primary antibodies. Horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma), horseradish peroxidase-conjugated mouse anti-goat, and horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma), each at a 1:10,000 dilution, were used as secondary antibodies, respectively. Signals were detected by ECL (Amersham Biosciences).

Urinary PG Metabolite Analyses—24-hour urine was collected using metabolic cages, and prostanoid metabolites were extracted and monitored as described (21).

Vascular Permeability Assay—Anesthetized mice (8-10 weeks old) received 200 µl of 2% Evans blue dye in phosphate-buffered saline via the tail vein. Immediately thereafter, the right ear was injected intradermally with 0.1 nmol of bradykinin (Sigma) in 10 µl of phosphate-buffered saline, and the left ear was injected with vehicle. Animals were euthanized after 40 min by CO2 inhalation. An 8-mm ear biopsy (Acu-Punch, Acuderm Inc.) was taken and soaked in 1 ml of formamide overnight at 55 °C, and extracted Evans blue dye was measured by absorbance at 610 nm with a Beckman DU-600 spectrophotometer.

Histopathological Analysis—Kidney and intestine were fixed in 10% buffered formalin for 24 h, processed routinely, and embedded in paraffin for staining with hematoxylin and eosin. For the quantitative analysis, kidney section pictures (x100) were captured with a 3CCD digital camera (Toshiba), and the diameter of the glomeruli was measured perpendicularly to the renal capsule using Image-Pro Plus, version 3.0 (Media Cybernetics). At least three coronal kidney sections from three individual animals from each group were analyzed.

Serum BUN Analysis—Blood collected from the saphenous vein was analyzed for BUN levels by the clinic laboratory of the Veterinary Hospital of University of Pennsylvania.


Figure 1
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  		FIGURE 1.
Targeted knock-in of Ptgs1 cDNA at the Ptgs2 locus. A, schema of Ptgs2 allele, the targeting vector, the locus of Ptgs1 cDNA insertion and homologous recombination (Ptgs1 > Ptgs2flox-Neo), and the deletion of the neomycin cassette (Neo) after Cre-mediated recombination (Ptgs1 > Ptgs2). Red boxes indicate coding exons of the Ptgs2 gene, green boxes indicate the UTR (including promoter region and 3'-UTR), colored arrows indicate primers for genotyping, and P1 and P2 indicate the locations of 5' external and Ptgs1 cDNA probes, respectively. Small black arrows and thick colored arrows represent primers used for genotyping in D and E, respectively. TK, thymidine kinase. Restriction sites: B, BamHI, Bs, BsiWI; H, HindIII; K, KpnI; Hp, HpaI. Panels B and C are Southern blot analyses of BamHI-digested embryonic stem cell DNA using 5' external and COX-1 cDNA probes, respectively. KI (knock-in), Ptgs1 > Ptgs2 mutant allele; WT, wild-type allele. D, detection of Neo and loxP sequences in Ptgs1 > Ptgs2flox-Neo and Ptgs1 > Ptgs2 alleles by specific PCR. E, PCR genotyping of tail biopsies from WT (+/+, 738 bp) and Ptgs1 > Ptgs2 knock-in (-/-, 548 bp) mice.

 


Figure 2
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  		FIGURE 2.
Generation of an LPS-inducible COX-1 in macrophages. A, Western blot analysis of COX isoforms in two representative samples of LPS-stimulated peritoneal macrophages from COX-1 > COX-2flox-Neo mice (left panel) and COX-1 > COX-2 mice (right panel). Flox-Neo, COX-1 > COX-2flox-Neo mice. B, level of COX-1 and COX-2 protein relative to beta-actin. C, PGE2 production by LPS-stimulated peritoneal macrophages in the presence of exogenous AA (20 µM) with aspirin pretreatment and washout to inactivate endogenous COX-1. Arrows indicate near absence of PGE2 production from both COX-1 > COX-2flox-Neo and COX-2 KO groups under these conditions. D, prostaglandin-glycerol (PG-G) synthesis from nonstimulated and LPS-stimulated peritoneal macrophages from WT, COX-1 > COX-2, and COX-2 KO mice in the presence of 1 µM exogenous 2-arachidonylglycerol (2-AG) (12 mice/group, repeated three times).

 
Statistical Analysis—Data are presented as mean ± S.E. Analyses were performed by the Student's t test, one-way analysis of variance test, or Fisher's exact test. A p value of <0.05 was considered significant. Prism 3.0 software (GraphPad) was used for all calculations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Establishment of a Mouse Model COX-1 > COX-2 to Study the Discriminant Functions of COX Isoforms—Ptgs2 (the formal PGHS-2/COX-2 gene nomenclature) is a tightly regulated immediate-early gene with many transcriptional regulatory elementsinthe5'-flankingregion (22) and in the AT-rich 3'-UTR (23). COX-1 > COX-2flox-Neo mice were generated by replacing most of the coding region of Ptgs2 (exons 1-9) with Ptgs1 cDNA and a positive selectable marker neomycin (Neo) cassette flanked by loxP (flox) sites while leaving the Ptgs2 promoter region and 3'-UTR intact through conventional gene targeting technology (Fig. 1A). Targeted embryonic stem cell clones were verified by Southern blot analysis with both 5' external and Ptgs1 cDNA probes (See "Experimental Procedures" and Fig. 1, B and C). Transgenic mice expressing Cre recombinase during early embryogenesis were crossed to COX-1 > COX-2flox-Neo mice to obtain COX-1 > COX-2 mice and eliminate the transcriptional interference from the Neo cassette to the expression of "knocked-in" Ptgs1. Removal of the Neo cassette was distinguished by PCR detection of Neo and loxP sequences (Fig. 1D). Subsequent genotyping of COX-1 > COX-2 mice was carried out by detecting Ptgs1 cDNA within the Ptgs2 genetic locus by PCR using a specific primer set (the forward primer is in the Ptgs 2 promoter region and reverse primer is in the Ptgs 1 cDNA; see "Experimental Procedures" and Fig. 1, A and E), which distinguishes between the endogenous Ptgs1 allele.

Efficacy of Targeting Strategy Demonstrated Using Macrophages from COX-1 > COX-2flox-Neo and COX-1 > COX-2 Mice—An excellent cell system to use for analyzing relative COX-1/COX-2 expression is peritoneal macrophages with and without LPS stimulation (7). COX-1 and COX-2 protein expression in LPS-stimulated macrophages was examined by Western blot analysis to characterize the efficacy of our targeted knock-in strategy in the mice we generated. The results (Fig. 2, A and B) revealed that COX-1 protein was markedly increased ({approx}5-fold) in LPS-stimulated macrophages from COX-1 > COX-2 mice compared with cells from WT mice. In contrast, the level of COX-1 was unchanged in cells from COX-1 > COX-2flox-Neo mice, in which proper transcriptional processing would be expected to be disrupted by the Neo cassette insertion. COX-2 protein was completely abrogated in cells from both COX-1 > COX-2flox-Neo and COX-1 > COX-2 mice, as expected. The level of COX induction in the knock-in mice, however, could not be directly compared because of likely differences in affinity between the epitopes used for generation of the respective COX-1 and COX-2 antibodies. We performed COX enzyme assays to measure prostaglandins using macrophages from COX-1 > COX-2 mice to circumvent this potential difference. Endogenous COX-1 was first inactivated by aspirin treatment prior to LPS challenge, so the level of COX activity should reflect synthetic capacity from induced COX-1 from COX-1 > COX-2 mice and COX-2 from WT mice. As Fig. 2C demonstrates, PGE2 production from either COX-1 > COX-2flox-Neo or COX-2 null macrophages was undetectable, suggesting that COX-1 > COX-2flox-Neo mice without COX-2 protein and COX-2 activity should be equivalent to COX-2 null mice. In stark contrast, macrophage PGE2 production from COX-1 > COX-2 cells was comparable with that from WT cells when incubated with 20 µM AA, suggesting that we had created an LPS-inducible COX-1 system analogous to the endogenous LPS-inducible COX-2 gene in WT mice.

Besides AA, the endocannabinoids 2-arachidonylglycerol and arachidonyl ethanolamide have been shown recently to be selective COX-2 substrates (16). We examined 2-arachidonylglycerol endocannabinoid metabolism to prostaglandin-glycerol products to confirm further our targeted COX-1 gene knock-in strategy in COX-1 > COX-2 mice. Although marked increases in prostaglandin-glycerol formation were seen in WT controls treated with LPS (Fig. 2D), no such increase was apparent in either COX-1 > COX-2 or COX-2 null macrophages with LPS stimulation. Taken together, our results reveal targeted COX-2 gene disruption with concomitant generation of a novel LPS-inducible COX-1 system in COX-1 > COX-2 mice.

Mendelian Inheritance Pattern and Female Reproductive Function in COX-1 > COX-2 Mice—Significant postnatal mortality was reported in COX-2-deficient neonatal mice within the first 2 days of birth due to failure of the ductus arteriosus closure (15). We monitored the neonatal pups from mating pairs of heterozygous COX-1 > COX-2 or COX-1 > COX-2flox-Neo parents. Only 16.5% of COX-1 > COX-2flox-Neo pups (35 of 211; expected Mendelian ratio = 25%; represents a 34% loss) were obtained at genotyping age (10-12 days), whereas the number of live COX-1 > COX-2 mice (95 of 390, 24.3%; 2.8% loss) was in agreement with Mendelian expectations, indicating normal ductus arteriosus remodeling and closure in COX-1 > COX-2 mice. In contrast, we failed to generate any double COX-1 null/COX-1 > COX-2 homozygous mice from numerous double heterozygous parent matings. Because female COX-2-deficient mice display multiple reproductive defects (12), we examined whether COX-1 > COX-2 females display similar phenotypes by comparison with COX-2 null mice. In contrast to the few COX-2 null females (20%) capable of sustaining term pregnancy and the very small litter size, all COX-1 > COX-2 females tested (6-10 weeks old) were fertile with a markedly increased litter size (Table 1). However, when compared with WT mice, COX-1 > COX-2 females still had a somewhat reduced litter size and a longer period between successive pregnancies (data not shown). These results suggest that female reproductive function is in part, but not completely, compensated by COX-1 exchange into the COX-2 genetic locus in COX-1 > COX-2 mice.


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  		TABLE 1
Female reproductive capacity is greatly enhanced in COX-1 > COX-2 mice because of COX-1-targeted exchange, in contrast to COX-2 null mice

Female COX-2 KO, COX-1 > COX-2, and WT mice (6-10 weeks old) were mated with fertile WT males. Copulation plug-positive mice were observed for delivery of pups at term, and the litter size was recorded. Both term pregnancy frequency and litter size in COX-1 > COX-2 were significantly improved compared with COX-2 KO mice.

 
Differential "Rescue" of Total Urinary PG Biosynthesis in COX-1 > COX-2 Mice by COX-1 Exchange for COX-2—COX-1 is highly expressed in mature human platelets and is the major source of thromboxane A2 (TxA2) biosynthesis (7, 24), whereas prostacyclin (PGI2), a dominant product of endothelium (25), is largely derived from COX-2 under physiological conditions in both humans (26) and mice (21). Urinary PGE2 is contributed substantially by both COX-1 and COX-2-derived synthesis (21). We measured the TxA2 metabolite, 2,3-dinor-TxB2, the PGE2 metabolite, 11{alpha}-hydroxy-9,15-dioxo-2,3,4,5-tetranorprostane-1,20-dioic acid (PGEM) (21), and the PGI2 metabolite, 2,3-dinor-6-keto-PGF1{alpha}, to examine the urinary PG profile in COX-1 > COX-2 mice. No significant alterations in the TxA2 metabolite were observed in COX-1 > COX-2 and COX-2 null mice as compared with WT controls (Fig. 3A). Urinary PGEM levels from COX-1 > COX-2 mice (Fig. 3B) were comparable with levels in WT mice (p = 0.69, n = 5-7), which were markedly "rescued" compared with COX-2 null mice (p < 0.05, n = 6-7). Although the PGI2 metabolite (1.23 ± 0.23 pg/mg creatinine) in COX-1 > COX-2 mice was decreased by 55% relative to WT mice (2.74 ± 0.70 pg/mg creatinine, Fig. 3C), COX-1 knock-in partially compensated for PGI2 synthesis when compared with COX-2 null mice. Taken together, PG production can be compensated differentially in COX-1 > COX-2 mice by COX-1 exchange into the COX-2 allele, suggesting that COX-2 coupling to downstream PG synthases in vivo is not the same as for COX-1 or that the activities of the two isoforms are not the same in different cellular environments.

Prostacyclin is the major PG involved in bradykinin-elicited inflammation (27). We performed a bradykinin-induced vascular permeability assay as measured by Evans blue dye extravasation into the ear. Exogenous bradykinin administration elicited an increase in vascular permeability, in all mice tested, after 40 min. Consistent with total PGI2 metabolite data (Fig. 3C), dye extravasation in both COX-1 > COX-2 and COX-2 null mice upon bradykinin challenge fell by 50-60% (n = 7-10; p < 0.05) compared with WT littermates (Fig. 3D).


Figure 3
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  		FIGURE 3.
PG biosynthesis in vivo and vascular permeability in COX-1 > COX-2 mice. A, no alteration of urinary 2,3-dinor-TxB2 occurs in COX-1 > COX-2 mice (8-10 weeks old, n = 5-6). B, urinary PGEM is compensated by COX-1 knock-in in COX-1 > COX-2 mice (8-10 weeks old, n = 5-7; *, p < 0.05). C, decreased urinary 2,3-dinor-6-keto-PGF1{alpha} observed in COX-2 null mice is not rescued by COX-1 knock-in in COX-1 > COX-2 mice (8-10 weeks old, n = 7-10; *, p < 0.05). D, vascular permeability is decreased significantly (n = 7-10; *, p < 0.05) in COX-1 > COX-2 mice (8-10 weeks old) in response to bradykinin as in COX-2 KO mice.

 


Figure 4
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  		FIGURE 4.
Kidney pathology in COX-1 > COX-2 mice differs from that of COX-2-deficient mice. A, representative light photomicrographs of kidneys from WT, COX-1 > COX-2, and COX-2 KO mice (6 months old). Magnification, x200; bar,50 µM. Yellow arrows represent glomeruli. B, mean diameter of glomeruli from WT, COX-1 > COX-2, and COX-2 KO mice. The mean glomerular diameter in COX-1 > COX-2 mice was significantly larger than in WT mice (**, p < 0.001), whereas it was markedly reduced in COX-2 KO mice (#, p < 0.05) C, plasma BUN is significantly increased in mature adult COX-1 > COX-2 mice (6 months old) as in COX-2 KO mice (n = 5-6; *, p < 0.01) compared with WT controls.

 
Effects of COX-1 Knock-in on Renal Pathology—Gene knock-out studies revealed that COX-1 disruption does not yield overt kidney defects under normal conditions (5), whereas COX-2-deficient mice display postnatal renal developmental abnormalities (10, 11, 19). Consistent with nephron hypoplasia, nephropathy in adult COX-2 null mice (>6 weeks old) with mild to marked severity was characterized by abnormal renal cortex composed of small immature glomeruli and tubules that progressively deteriorated with increasing age (6 month age, Fig. 4). We failed to detect remarkable serum BUN elevation and nephron alterations in 2-month-old COX-1 > COX-2 mice (data not shown), suggesting that glomerular developmental defects were rescued in COX-1 > COX-2 mice by COX-1 knock-in. However, in older COX-1 > COX-2 mice (6 months) glomeruli were found to be enlarged significantly because of increased glomerular cellularity, mostly due to inflammatory cells. The mean glomerular cross-sectional diameter in COX-1 > COX-2 mice was increased by about 56% compared with WT mice (62.0 ± 1.8 µm versus WT 39.2 ± 1.0 µm, p < 0.01; Fig. 4, A and B). In contrast, the average glomerular diameter in COX-2 null mice was reduced to 29.4 ± 2.0. µm because of impaired renal cortical development. Likewise, serum BUN levels were elevated correspondingly more than 2-fold in both COX-1 > COX-2 and COX-2 null mice at 6 months (Fig. 4C).

Peritonitis Observed in COX-2 Null Mice Is Not Mitigated in COX-1 > COX-2 Mice by COX-1 Knock-in—Suppurative peritonitis was reported in the original COX-2-deficient mice (10). Similarly, chronic peritonitis was frequently observed in adult COX-1 > COX-2 mice at 5 months of age (Fig. 5, A and B). Gross examination of the peritoneal cavity showed extensive inflammation and multiple adhesions among abdominal organs (intestine, liver) and the abdominal wall. Typical mesenteric abscesses, small bowel adhesions, ileal ulcers, and inflammatory infiltration were observed microscopically (Fig. 5A). The incidence of peritonitis in COX-1 > COX-2 mice was similar to that observed in COX-2 null mice (Fig. 5B). The results imply that the bowel ulceration and peritonitis caused by COX-2 deficiency cannot be rescued by COX-1 gene exchange.


Figure 5
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  		FIGURE 5.
COX-1 knock-in to the COX-2 locus does not rescue the intestinal adhesion and peritonitis phenotype observed in COX-2 null mice. A, histologic analysis of intestinal adhesions and peritonitis of COX-1 > COX-2 mice (5-6 months old). Intestinal adhesions and early abcess (I), two mesenteric abscesses (II), intestinal ulcers (III), and ulcer with inflammatory infiltration penetrating deeper into the bowel wall (IV, high magnification of inset area in III) were observed in peritonitis lesions from COX-1 > COX-2 mice. B, frequency of intestinal adhesions in COX-1 > COX-2 mice (5-6 months old). *, p < 0.001 as determined by Fisher's exact test. Mouse numbers are shown in parentheses over the columns.

 
COX-2 Distinct Metabolism Pattern Is Revealed in COX-1 > COX-2 Mice—We compared AA metabolism in a representative cell type (macrophages) at variable concentrations and measured PGE2 production to investigate the reason for the variable rescue of COX-2 function by targeted COX-1 replacement. No significant difference in PGE2 generation was observed with 1 µM AA without LPS stimulation among cells from WT, COX-1 > COX-2, and COX-2 null mice, as anticipated, because the inducible COX-2 isoform is not expressed under normal culture conditions. A substantial increase in PGE2 was detected in both WT and COX-1 > COX-2 cells, but not in COX-2 null cells, following LPS treatment (Fig. 6A). However, the induced level in COX-1 > COX-2 cells was notably lower than in WT cells, and this became even more apparent following aspirin pretreatment (Fig. 6A, ASA). In our assay system, LPS-stimulated macrophage PGE2 generation from COX-1 > COX-2 mice is comparable with that of WT as tested in the presence of 20-30 µM AA, whereas with endogenous or very low exogenous substrate (AA < 1 µM), LPS-stimulated macrophages from WT mice produced significantly higher PGE2 than that from COX-1 > COX-2 mice (Fig. 6B).


Figure 6
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  		FIGURE 6.
Targeted COX-1 exchange only compensates for macrophage PGE2 synthesis at higher AA concentrations. Shown is PGE2 generation by macrophages from WT, COX-1 > COX-2, and COX-2 KO mice without or with 0.5 mM aspirin (ASA, acetylsalicylic acid) pretreatment in the presence of 1 µM AA (A) or various concentrations of exogenous AA in 3% fetal bovine serum/RPMI 1640 medium without aspirin pretreatment (B). *, p < 0.01 versus WT controls (n = 3-6).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we have described a novel mouse model, COX-1 > COX-2, in which COX-1, exchanged for COX-2, is under COX-2 gene regulatory control. This has allowed us to answer specific questions regarding the biology of the two COX isoforms. Deficits in PG bisosynthesis and in renal and female reproductive function observed in COX-2 null mice cannot be fully compensated by knocked-in COX-1, suggesting that COX-2 possesses unique properties that COX-1 cannot replace in vivo. Notably, low PGI2 biosynthesis and chronic renal pathology in COX-1 > COX-2 mice provide in vivo evidence to support the hypothesis that prostacyclin biosynthesis is coupled preferentially with COX-2. Intestinal ulcers and peritonitis, frequently observed in COX-1 > COX-2 mice, further reflect the distinct role for COX-2 in the maintenance of intestinal mucosa integrity.

COX-2 Is Active at Low AA Concentration and Preferentially Couples to PGI2 Biosynthesis—Biochemical studies demonstrate that COX-1 and COX-2 have almost identical Km values for arachidonate at high substrate/enzyme ratios (28, 29) as well as for other fatty acid substrates (29, 30) and O2 (31). However, COX-1 exhibits a "cooperativity" with AA concentration that is not observed with COX-2 (32-34). This has been explained on the basis of the higher requirement of COX-1 for hydroperoxide for enzyme activation. The lower hydroperoxide requirement for COX-2 may result in a preferential AA oxygenation by COX-2 in intact cells at relatively low endogenous substrate concentrations or low hydroperoxide tone (32-36). Accordingly, we found less PG generation in LPS-induced macrophages from COX-1 > COX-2 (induced COX-1 at COX-2 locus) than from WT (induced COX-2) mice at low AA concentrations tested (≤1 µM, Fig. 6). This may explain partially why biosynthesis of PGs (PGI2 and PGE2) was not fully compensated by knocked-in COX-1 in COX-1 > COX-2 mice. Two additional points are worthy of general consideration: (i) COX-1, expressed in most tissues, is responsible for the majority of urinary PGEM synthesis in vivo (21, 37); and (ii) PGI2 is dominantly generated from COX-2 in healthy individuals and in WT mice (21, 26, 38). The altered PG biosynthesis in COX-1 > COX-2 mice (with COX-2 deletion) might allow a relative rediversion of knocked-in COX-1-derived PGH2 toward PGE2 formation if coupling to PGI2 synthase (PGIS) is affected. Evidence for such rediversion to PGE2 is apparent in PGIS-deficient mice (39).

Although there has been no direct interaction reported between COX isozymes and the downstream PG synthases, accumulating evidence suggests PGIS coupling with COX-2, including physical co-localization (1, 40) and physiological metabolism (21, 37, 40, 41). We did not observe any obvious kidney developmental abnormalities in young COX-1 > COX-2 mice, suggesting that the COX-1 gene at the COX-2 locus is regulated in postnatal COX-1 > COX-2 pups in a manner analogous to the endogenous COX-2 gene in WT mice (42). However, chronic renal pathology was found in adult COX-1 > COX-2 mice (Fig. 4) with low PGI2 biosynthesis, which is somewhat reminiscent of the renal phenotype reported in PGIS-deficient mice (39). These findings with COX-1 > COX-2 mice agree with the notion of PGI2 biosynthesis coupled to COX-2 and provide additional evidence for a COX-2-derived PGI2 role in renal homeostasis. However, no kidney phenotype was reported in PGI2 receptor-deficient mice, and the PGI2 receptor agonist, beraprost, cannot mitigate kidney damage in PGIS-deficient mice (39), implying that COX-2-derived PGI2 might function to maintain kidney homeostasis through pathways other than the PGI2 receptor (39, 40).

COX-2 gene deficiency causes multiple defects in female reproduction (12); COX-2-derived PGI2 is critical for embryo development from oviduct dilation to facilitation of embryo transfer (43), to embryo hatching (44) and implantation and decidualization through a non-PGI2 receptor pathway (45, 46). Reproductive capability in female COX-1 > COX-2 mice was partially, but not completely, rescued by targeted COX-1 exchange, which further confirms that the two isoforms are not completely functionally interchangeable.

A Unique Role for COX-2 in Intestinal Mucosa Integrity Is Identified Using COX-1 > COX-2 Mice—COX-1-deficient mice did not show any intestinal pathology with less than 3% PGE2 synthesis in intestinal tissue (5, 47) and were even more resistant to indomethacin-induced intestinal damage (5) than WT controls, whereas COX-2 deficiency caused intestinal ulceration and perforation resulting in peritonitis despite normal intestinal PGE2 levels (10, 47). Long term administration of a COX-2 inhibitor caused identical intestinal lesions with increased fecal granulocyte marker protein (GMP), a gastrointestinal inflammation marker (47). We found similar spontaneous peritoneal lesions and chronic intestinal inflammation in COX-1 > COX-2 mice as in COX-2 null mice (Fig. 5), suggesting that COX-2 plays a distinct role in mucosal integrity and ulcer healing, which cannot be compensated by COX-1 replacement. COX-2 expression is up-regulated for healing of experimental ulcers, whereas COX-1 expression in mucosa is dramatically reduced in the early phase of ulcer healing, and COX-2 inhibitors delay ulcer healing (48, 49). Moreover, prolonged COX-2 inhibition can induce intestinal perforation (48) and exacerbation of colitis in experimental models (50).

In summary, we have generated a novel mouse model for examining the interchangeability of COX isoforms. Our studies revealed that COX-1 can partially compensate for COX-2 function, but this is limited by the differential ability of these two isoforms to metabolize low concentrations of arachidonate and by the preferential coupling of COX-2 to PGIS.


   FOOTNOTES
 
* This work was supported by Grants GM063130, HL62250, and GM15431 from the National Institutes of Health, Canadian Institutes of Health Research Grant MOP-79459, and American Heart Association National Scientist Development Grant 0730314N. 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. Back

1 The Elmer Bobst Professor of Pharmacology. Back

2 Holds a Tier I Canada Research Chair in Molecular, Cellular, and Physiological Medicine and is a Career Investigator of the Heart and Stroke Foundation of Canada. To whom correspondence should be addressed: Dept. of Physiology, Botterell Hall, Rm. 433 Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-3242; Fax: 613-533-6880; E-mail: funkc{at}post.queensu.ca.

3 The abbreviations used are: PGHS, prostaglandin H synthase; COX, cyclooxygenase; KO mice, knock-out (null) mice; PG, prostaglandin; AA, arachidonic acid; LPS, lipopolysaccharide; Tx, thromboxane; Neo, neomycin resistance gene; PGEM, 11-{alpha}-hydroxy-9,15-dioxo-2,3,4,5-tetranorprostane-1,20-dioic acid; PGI2, prostaglandin I2 (prostacyclin); PGIS, prostacyclin synthase; WT, wild type (control); BUN, blood urea nitrogen; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; UTR, untranslated region. Back


   ACKNOWLEDGMENTS
 
We thank Jean Richa and the Transgenic Core Facilities at the University of Pennsylvania for embryonic stem cell injection and generation of chimeras. We also thank Mary Scotti and the clinical laboratory of the Veterinary Hospital, University of Pennsylvania, for the assay of mouse serum creatinine and BUN.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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