David L. DeWitt

Prostaglandin Endoperoxide H Synthases (Cyclooxygenases)-1 and −2

Prostaglandin endoperoxide H synthases (PGHSs) catalyze the conversion of arachidonic acid and O2 to PGH2, the committed step in prostanoid biosynthesis (Fig. 1) (1). Before 1991, only one PGHS had been described, the isozyme now called PGHS-1, COX-1 (for cyclooxygenase-1) or the constitutive enzyme. At that time Simmons and Herschman and their colleagues discovered mRNAs whose expression was induced in chicken and mouse fibroblasts in response to src and tumor-promoting phorbol esters, respectively, and which encoded proteins having 60% amino acid sequence identity with PGHS-1. Subsequent work has shown that the new protein, called PGHS-2, COX-2 or the inducible isoform, is very similar to PGHS-1 in structure but differs substantially from PGHS-1 with respect to its pattern of expression and its biology. The reason for the existence of the two PGHS isozymes is unknown. However, PGHS-1 and -2 are often coexpressed in the same cell and may act as parts of separate prostanoid biosynthetic systems that function somewhat independently to channel prostanoids to the extracellular milieu and the nucleus, respectively. PGHS-1 and -2 are interesting in the context of both structural biology and enzymology in that they are homodimeric, heme-containing, glycosylated proteins with two catalytic sites. Moreover, the enzymes have a novel mechanism for membrane attachment; they are anchored to one leaflet of the lipid bilayer through the hydrophobic surfaces of amphipathic helices rather than through transmembrane motifs typical of many integral membrane proteins. The isozymes are also important pharmacologically as targets of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) (2). For example, aspirin acts via PGHS-1 to inhibit platelet thromboxane A2 formation and as a clinical consequence lowers the relative risk for mortality from cardiovascular disease (3). PGHS-2 is the relevant target of NSAIDs acting to inhibit inflammation (4, 5), fever (5), pain (4), and probably colon cancer (6–8). NSAID therapy may even retard the development of Alzheimer’s disease (9) although it is not clear which PGHS isozyme may be involved. In this review we compare and contrast PGHS-1 and -2 in the context of the regulation of expression of the two enzymes, the mechanisms of enzyme catalysis, and the biological significance of having two PGHSs.

Prostaglandin Endoperoxide H Synthases-1 and -2

Publisher Summary The chapter compares and contrasts the structural and kinetic properties of prostaglandin endoperoxide H synthase-1 (PGHS-1) and -2. It also discusses the description of the interactions of the two isozymes with nonsteroidal anti-inflammatory drugs (NSAIDs). There are three general areas of study important to understanding more about PGHS isozymes: mechanisms of catalysis, regulation of gene expression, and subcellular functional independence. The chapter also explain how is arachidonic acid specifically channeled to PGHS-2 following mobilization in cells in response to TPA and how is the localization of PGHS-2 in the nuclear envelope important for PGHS-2 functioning. The chapter also describes the structures and regulation of expression of the PGHS-1 and -2 genes. The concepts that PGHS-1 and PGHS-2 represent two separate prostaglandin biosynthetic pathways and two separate prostaglandin signaling pathways need to be tested. The chapter also discusses how the two isozymes may act independently in intact cells to mediate the formation of prostanoids destined to act on cell surface and/or nuclear targets to mediate different biological and pathobiological events.

Prostaglandin and thromboxane biosynthesis.

We describe the enzymological regulation of the formation of prostaglandin (PG) D2, PGE2, PGF2 alpha, 9 alpha, 11 beta-PGF2, PGI2 (prostacyclin), and thromboxane (Tx) A2 from arachidonic acid. We discuss the three major steps in prostanoid formation: (a) arachidonate mobilization from monophosphatidylinositol involving phospholipase C, diglyceride lipase, and monoglyceride lipase and from phosphatidylcholine involving phospholipase A2; (b) formation of prostaglandin endoperoxides (PGG2 and PGH2) catalyzed by the cyclooxygenase and peroxidase activities of PGH synthase; and (c) synthesis of PGD2, PGE2, PGF2 alpha, 9 alpha, 11 beta-PGF2, PGI2, and TxA2 from PGH2. We also include information on the roles of aspirin and other nonsteroidal anti-inflammatory drugs, dexamethasone and other anti-inflammatory steroids, platelet-derived growth factor (PDGF), and interleukin-1 in prostaglandin metabolism.

Antioxidant and cyclooxygenase inhibitory phenolic compounds from Ocimum sanctum Linn.

Anti-oxidant bioassay-directed extraction of the fresh leaves and stems of Ocimum sanctum and purification of the extract yielded the following compounds; cirsilineol [1], cirsimaritin [2], isothymusin [3], isothymonin [4], apigenin [5], rosmarinic acid [6], and appreciable quantities of eugenol. The structures of compounds 1-6 were established using spectroscopic methods. Compounds 1 and 5 were isolated previously from O. sanctum whereas compounds 2 and 3 are here identified for the first time from O. sanctum. Eugenol, a major component of the volatile oil, and compounds 1, 3, 4, and 6 demonstrated good antioxidant activity at 10-microM concentrations. Anti-inflammatory activity or cyclooxygenase inhibitory activity of these compounds were observed. Eugenol demonstrated 97% cyclooxygenase-1 inhibitory activity when assayed at 1000-microM concentrations. Compounds 1, 2, and 4-6 displayed 37, 50, 37, 65, and 58% cyclooxygenase-1 inhibitory activity, respectively, when assayed at 1000-microM concentrations. Eugenol and compounds 1, 2, 5, and 6 demonstrated cyclooxygenase-2 inhibitory activity at slightly higher levels when assayed at 1000-microM concentrations. The activities of compounds 1-6 were comparable to ibuprofen, naproxen, and aspirin at 10-, 10-, and 1000-microM concentrations, respectively. These results support traditional uses of O. sanctum and identify the compounds responsible.

PGH synthase isoenzyme selectivity: The potential for safer nonsteroidal antiinflammatory drugs

With the recent cloning of a second gene coding for the prostaglandin endoperoxide (PGH) synthase (cyclooxygenase), it has become obvious that mammalian cells contain two related, but unique, isozymes of PGH synthase. Both of these isozymes catalyze the conversion of arachidonic acid to PGH2, leading to production of biologically active prostaglandins. Although the first of these isozymes, PGH synthase-1 (PGHS-1), has long been thought to be the primary and sole site of action of nonsteroidal antiinflammatory drugs (NSAIDs), it is now known that the second isozyme, PGH synthase-2 (PGHS-2), is also sensitive to NSAIDs. Cloning of complementary DNAs for murine PGHS-1 and PGHS-2 has permitted individual expression of these two isozymes in the cos-1 cell system and comparison of their relative inhibition by several common NSAIDs in vitro. These studies have demonstrated that the two mouse isozymes, PGHS-1 and PGHS-2, are pharmacologically distinct. PGHS-1 is a constitutively expressed enzyme that early observations indicate is the principal enzyme involved in producing prostaglandins that regulate cellular housekeeping functions, such as gastric cytoprotection, vascular homeostasis, and kidney function. In contrast, PGHS-2 appears only to be expressed in inflamed tissue or following exposure to growth factors, lymphokines, or other mediators of inflammation. Expression of PGHS-2 is inhibited by antiinflammatory glucocorticoids, lending further support to the hypothesis that this enzyme produces prostaglandins involved in inflammation. We have identified NSAIDs that preferentially inhibit murine PGHS-1 or PGHS-2 or inhibit both isozymes equally. The finding that the two isozymes can be differentially inhibited provides a possible mechanism for identifying safer, more effective NSAIDs. Screening for drugs that preferentially inhibit PGHS-2 may allow identification of NSAIDs that reduce inflammation, but spare renal and gastric prostaglandin synthesis, thus reducing the untoward side effects commonly associated with most NSAIDs. Thus far, nabumetone is the only NSAID identified that preferentially inhibits murine PGHS-2.

Cytotoxicity, antioxidant and anti-inflammatory activities of Curcumins I–III from Curcuma longa

Curcumin I, curcumin II (monodemethoxycurcumin) and curcumin III (bisdemethoxycurcumin) from Curcuma longa were assayed for their cytotoxicity, antioxidant and anti-inflammatory activities. These compounds showed activity against leukemia, colon, CNS, melanoma, renal, and breast cancer cell lines. The inhibition of liposome peroxidation by curcumins I-III at 100 microg/ml were 58, 40 and 22%, respectively. The inhibition of COX-I and COX-II enzymes by the curcumins was observed. Curcumins I-III were active against COX-I enzyme at 125 microg/ml and showed 32, 38.5 and 39.2% inhibition of the enzyme, respectively. Curcumins I-III also showed good inhibition of the COX-II enzyme at 125 mg/ml with 89.7, 82.5 and 58.9% inhibition of the enzyme, respectively.

Yes, but do they still get headaches?

Much of our present understanding of the action of prostaglandins derives from observing the effects of nonsteroidal anti-inflammatory drugs (NSAIDs). The anti-inflammatory, antipyretic, and analgesic effects resulting from inhibition of prostaglandin synthesis by aspirin, ibuprofen, and other NSAlDs indicate that these lipid-signaling molecules are critical regulators of immune responses, fever, and pain; we now know that prostaglandins are also ubiquitous autocrinelparacrine modulators of cellular responses and likely play roles in mitogenesis and apoptosis. (For a general review, see Smith and Dewitt, 1995). It therefore seems fitting that our latest advance in the understanding of the physiological roles of prostaglandins should again result from inhibition of prostaglandin synthesis, this time as a result of the genetic ablation of the enzymes required for their synthesis. In this issue of Cell, Langenbach et al. (1995) and Morham et al. (1995) describe the construction and phenotypic characterization of transgenic mice deficient in two isozymes central to prostaglandin synthesis, cyclooxygenases 1 and 2 (COX-1 and COX-2), also known as prostaglandin H synthase 1 and 2 (PGHS-1 and PGHSQ). The uniquely different phenotypes of the two knockout strains confirm that the closely related COX-1 and COX-2 isozymes are not redundant, and the genetic pathologies of these mice, or lack thereof, allow us more precisely to assign specific signaling roles to the two prostaglandin biosynthetic pathways defined by these enzymes. In a related article, Tsujii and DuBois (1995 [this issue of Cc//J) describe an unexpected function for the COX-2 pathway and, at the same time, provide a potential solution to a vitally important mystery: how does aspirin reduce the incidence of colon cancer? Separate Prostoglandin Biosynthetic Pathways COX catalyzes the first committed step in the formation of prostaglandins and thromboxanes (Figure 1). Arachidonic acid (AA) released from membrane phospholipids by phospholipase A2 is converted to prostaglandin HP (PGH*) through the action of COX; PGH2 is then converted to PGDz, PGE,, PGF*, PGlz, or thromboxane AZ by cellspecific synthases. Prostaglandins act, at least in part, through specific G protein-linked receptors to modulate the levels of the second messengers CAMP and Ca*+. There are two COX enzymes, referred to as COX-1 and COX-2. The relatively recent and unexpected discovery of the second of these isozymes has stimulated a great deal of research in the field, much of which has been directed at determining the biological rationale for this apparent redundancy. COX-1 and COX-2 are encoded by separate genes, but the enzymes are structurally homologous, and both catalyze the formation of PGH2 from AA with similar kinetic properties. Minireview

Lipopolysaccharide induces prostaglandin H synthase-2 in alveolar macrophages

Prostaglandin H synthase is a key enzyme in the formation of prostaglandins and thromboxane from arachidonic acid. The recent cloning of a second prostaglandin H synthase gene, prostaglandin H synthase-2, which is distinct from the classic prostaglandin H synthase-1 gene, may dramatically alter our concept of how cells regulate prostanoid formation. We have recently shown that the enhanced production of prostanoids by lipopolysaccharide-primed alveolar macrophages involves the induction of a novel prostaglandin H synthase (J. Biol. Chem., (1992), 267, 14547-14550). We report here that the novel PGH synthase induced by lipopolysaccharide in alveolar macrophages is prostaglandin H synthase-2.

Interactions of PGH synthase isozymes-1 and -2 with NSAIDs.

There are two isozymes of prostaglandin endoperoxide (PGH) synthase (cyclooxygenase) called PGH synthase-1 and -2 or COX I and II. Both isozymes catalyze the same two reactions: oxygenation of arachidonate to yield PGG2 and reduction of PGG2 to PGH2. PGH synthase-1 is expressed constitutively and is found in most tissues. PGH synthase-2 is undetectable in most cells but can be induced in fibroblasts, endothelial cells, ovarian follicles, and macrophages by various mitogens, cytokines, and tumor promoters. PGH synthase-1 (PGHS-1) has been presumed to be the site of action of nonsteroidal antiinflammatory drugs (NSAIDs). However, the discovery of the second isozyme, PGH synthase-2 (PGHS-2), and its association with inflammation has suggested that this latter enzyme may be the therapeutic target of NSAIDs functioning in their antiinflammatory capacities. We have cloned cDNAs for murine PGHS-1 and PGHS-2, expressed these enzymes in cos-1 cells, and compared the relative sensitivities of the two isozymes to some common NSAIDs. Indomethacin, piroxicam, and sulindac sulfide were found to preferentially inhibit PGHS-1. Ibuprofen and meclofenamate inhibit both enzymes with comparable potencies. 6-Methoxy-2-naphthylacetic acid, the active metabolite of Relafen, inhibits murine PGHS-2 preferentially. Aspirin irreversibly inhibits PGHS-1, preventing this isozyme from forming PGH2 or any other oxygenated product; in contrast, aspirin treatment of PGHS-2 causes this enzyme to form 15-hydroxy-5c,8c,11c,13t-eicosatetraenoic acid (15-HETE) instead of PGH2. Our results indicate mouse PGHS-1 and PGHS-2 are pharmacologically distinct. Thus, it should be possible to develop agents highly selective for each PGHS isozyme. PGHS-2 is not expressed in stomach but is increased by inflammatory cytokines in cells such as macrophages. Thus, a selective inhibitor of PGHS-2 could be an antiinflammatory agent but without being ulcerogenic.

Regulation of human monocyte matrix metalloproteinases by SPARC

SPARC (secreted protein, acidic and rich in cysteine), also called osteonectin or BM‐40, is a collagen‐binding glycoprotein secreted by a variety of cells and is associated with functional responses involving tissue remodeling, cell movement and proliferation. Because SPARC and monocytes/macrophages are prevalent at sites of inflammation and remodeling in which there is connective tissue turnover, we examined the effect of SPARC on monocyte matrix metalloproteinase (MMP) production. Treatment of human peripheral blood monocytes with SPARC stimulated the production of gelatinase B (MMP‐9) and interstitial collagenase (MMP‐1). Experiments with synthetic peptides indicated that peptide 3.2, belonging to the alpha helical domain III of SPARC, is the major peptide mediating the MMP production by monocytes. SPARC and peptide 3.2 were also shown to induce prostaglandin synthase (PGHS)‐2 as determined by Western and Northern blot analyses. The increase in PGHS‐2 stimulated by SPARC or peptide 3.2 correlated with substantially elevated levels of prostaglandin E2 (PGE2) and other arachidonic acid metabolites as measured by radioimmunoassay and high performance liquid chromatography (HPLC), respectively. Moreover, the synthesis of MMP was dependent on the generation of PGE2 by PGHS‐2, since indomethacin inhibited the production of these enzymes and their synthesis was restored by addition of exogenous PGE2 or dibutyryl cAMP (Bt2cAMP). These results demonstrate that SPARC might play a significant role in the modulation of connective tissue turnover due to its stimulation of PGHS‐2 and the subsequent release of PGE2, a pathway that leads to the production of MMP by monocytes. J. Cell. Physiol. 173:327–334, 1997. Published 1997 Wiley‐Liss, Inc. This article was prepared by a group of United States government employees and non‐United States government employees, and as such is subject to 17 U.S.C. Sec. 105.