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A novel genetic model of selective COX-2 inhibition: Comparison with COX-2 null mice

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Abstract

Prostaglandin H Synthase (PGHS) is a bi-functional enzyme with a cyclooxygenase (COX) activity and a functionally linked peroxidase (POX) activity that exists in two isoforms (COX-1, COX-2). Non-steroidal anti-inflammatory drugs (NSAIDs), including the selective COX-2 inhibitors, block COX activity while leaving POX activity unscathed. Recently, some selective COX-2 inhibitors were withdrawn from the market due to elevated cardiovascular risk in placebo-controlled trials. Mice deficient for PGHS2 were developed in 1995 and through numerous subsequent studies have revealed significant roles in renal development, ductus arteriosus patency/closure, skin carcinogenesis and cardiovascular function. In this short review, we compare a novel genetic COX-2 selective inhibition mouse model with the originally described COX-2 null mice in these different physiological functions.

Introduction

Prostaglandin H Synthase (PGHS), (known colloquially as cyclooxygenase) catalyzes arachidonic acid conversion by a cyclooxygenase (COX) activity to prostaglandin G2 (PGG2) and a peroxidase (POX) activity in which PGG2 is reduced to PGH2 [1], [2]. Non-steroidal anti-inflammatory drugs (NSAIDs) and aspirin target both PGHS1 and PGHS2 by inhibition of COX activity, leaving POX activities of both enzymes intact. PGHS2 selective inhibitors act similarly but are isoform specific. Some studies have suggested that the isoform-selective COX-2 inhibitors have pharmacological mechanisms in addition to their COX activity inhibition [3], [4], [5], [6], so the effects of NSAIDs may not always reflect the physiological roles of PGHS isoforms.

In general, PGHS1 has been categorized as the “housekeeping” COX isoform since it is constitutively expressed in most tissues, while PGHS2 is highly regulated and can be induced in response to inflammatory stimuli [7], [8]. However, PGHS2 can be expressed constitutively in brain [9], kidney [10], tracheal epithelial cells [11] and some types of endothelial cells [12], [13]. The studies of PGHS-deficient mice [14], [15], [16] have provided valuable insight into various (patho)physiological functions of PGHS isoforms and indicate that PGHS2 has important roles in post-natal development and maintenance of kidney homeostasis.

Tyrosine 385 of PGHS1 is a critical residue for initiation of COX catalysis, and the phenylalanine-385 mutant of PGHS lacks COX activity but retains POX activity [1], [17]. To understand better the biology of PGHS2 containing only a single enzyme activity, we generated a genetic mouse model of selective COX-2 inhibition with a PGHS2 Y385F1 targeted knock-in mutation [18]. In the following sections, the development and general characteristics of PGHS2 Y385F mice and recent studies using this model in skin carcinogenesis and the cardiovascular system are described and comparisons and contrasts with PGHS2 null mice are presented.

Section snippets

Targeting strategy

Disruption of the PGHS2 gene through gene targeting in embryonic stem cells was reported by two different groups over 10 years ago [14], [15]. One group from North Carolina [14] inserted a neomycin resistance gene (Neo) into an exon 8 BstXI-EcoRI deleted segment, as illustrated in Fig. 1 (PGHS2 KO (I)), which leads to an interrupted coding sequence thereby abolishing full length PGHS2 protein translation. The deleted fragment eliminates Tyr 371 (equivalent to Tyr3851) and His 374, which are

General characteristics

The general characteristics of PGHS2 Y385F and PGHS2 null (KO; knockout) mice are displayed in Table 1. PGHS2 KO mice created from previous strategies lack full length mRNA and protein, thus losing both PGHS2 COX and POX activity. However, PGHS2 Y385F mice have normal mutant PGHS2 protein expression in response to inflammatory stimuli, with only COX activity disrupted and POX activity fully intact [18]. Similar to PGHS1 KO or knockdown (KD) mice which did not appear to up-regulate PGHS2

Skin carcinogenesis

During PGHS catalysis, enzyme- and substrate-derived free intermediates are generated which can co-oxidize xenobiotics to biologically reactive intermediates [22], [23], [24]. Thus, the POX activity of PGHS can bioactivate a variety of chemical carcinogens to mutagenic or carcinogenic species in the presence of arachidonic acid or peroxide. One example of a compound among the aromatic amine class, benzidine can be metabolized by PGHS POX activity to a nitrogen-centered free radical which serves

Cardiovasular system

PGs are essential mediators of ductus arteriosus (DA) patency during fetal life and regulators of constriction in the neonatal period [29], [30]. Mice deficient in PGHS2 [18], [20] or the PG receptor EP4 [31], [32] lead to failure of DA remodeling at birth. Surprisingly, patent DA and resulting neonatal mortality were not observed in PGHS2 Y385F mice. Electron microscopy analysis of the DA 4 h post-delivery revealed multiple endothelial cell layers oriented toward the lumen, fragmentation of the

Conclusion

Although PGHS2 deficient mice have been utilized to facilitate investigation of prostaglandin roles in physiological and various pathological conditions for over 10 years, the originally described PGHS2 KO mice [14], [15] generated by traditional strategies render complete absence of PGHS2 expression including both COX and POX activities, which limits dissection of distinct functions for the individual COX and POX components and may mask potential metabolic functions attributable to

Acknowledgments

The study was supported by grants from the National Institutes of Health (GM63130), Canadian Institutes of Health Research (MOP-79459) and Heart and Stroke Foundation of Ontario (NA-5828). We thank A. Klein-Szanto at the Fox Chase Cancer Center for histology assistance. C.D. Funk 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 (CI-5960).

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