Xin-Sheng Chen

Lipoxygenase genes and their targeted disruption

Analysis of the human and mouse genome sequences has enabled a detailed analysis of the structure and organization of the lipoxygenase genes in the respective species. Humans appear to possess six functional genes and at least three pseudogenes while mice have seven functional genes. The arrangement of the genes is quite similar between the species with most of the human lipoxygenase genes appearing on the short arm of chromosome 17 and in mice on the syntenic portion of chromosome 11. The 5-lipoxygenase gene is unique in several respects including its distinct separate chromosomal localization and its size (4-7 x larger than other lipoxygenase genes). Three of the seven murine lipoxygenase genes have been disrupted by gene targeting. While the knockout mice appear outwardly normal, a number of important findings have been discovered using these mice and these will be covered in this review.

The N-terminal “β-Barrel” Domain of 5-Lipoxygenase Is Essential for Nuclear Membrane Translocation

5-Lipoxygenase is the key enzyme in the formation of leukotrienes, which are potent lipid mediators of asthma pathophysiology. This enzyme translocates to the nuclear envelope in a calcium-dependent manner for leukotriene biosynthesis. Eight green fluorescent protein (GFP)-lipoxygenase constructs, representing the major human and mouse enzymes within this family, were constructed and their cDNAs transfected into human embryonic kidney 293 cells. Of these eight lipoxygenases, only the 5-lipoxygenase was clearly nuclear localized and translocated to the nuclear envelope upon stimulation with the calcium ionophore A23187. The N-terminal “β -barrel” domain of 5-lipoxygenase, but not the catalytic domain, was necessary and sufficient for nuclear envelope translocation. The GFP-N-terminal 5-lipoxygenase domain translocated faster than GFP-5-lipoxygenase. β-Barrel/catalytic domain chimeras with 12- and 15-lipoxygenase indicated that only the N-terminal domain of 5-lipoxygenase could carry out this translocation function. Mutations of iron atom binding ligands (His550 or deletion of C-terminal isoleucine) that disrupt nuclear localization do not alter translocation capacity indicating distinct determinants of nuclear localization and translocation. Moreover, data show that GFP-5-lipoxygenase β-barrel containing constructs can translocate to the nuclear membrane whether cytoplasmic or nuclear localized. Thus, the predicted β-barrel domain of 5-lipoxygenase may function like the C2 domain within protein kinase C and cytosolic phospholipase A2 with unique determinants that direct its localization to the nuclear envelope.

Genetic model of selective COX2 inhibition reveals novel heterodimer signaling

Selective inhibitors of cyclooxygenase-2 (COX2) have attracted widespread media attention because of evidence of an elevated risk of cardiovascular complications in placebo-controlled trials, resulting in the market withdrawal of some members of this class. These drugs block the cyclooxygenase activity of prostaglandin H synthase-2 (PGHS2), but do not affect the associated peroxidase function. They were developed with the rationale of conserving the anti-inflammatory and analgesic actions of traditional nonsteroidal anti-inflammatory drugs (tNSAIDs) while sparing the ability of PGHS1-derived prostaglandins to afford gastric cytoprotection. PGHS1 and PGHS2 coexist in the vasculature and in macrophages, and are upregulated together in inflammatory tissues such as rheumatoid synovia and atherosclerotic plaque. They are each believed to function as homodimers. Here, we developed a new genetic mouse model of selective COX2 inhibition using a gene-targeted point mutation, resulting in a Y385F substitution. Structural modeling and biochemical assays showed the ability of PGHS1 and PGHS2 to heterodimerize and form prostaglandins. The heterodimerization of PGHS1-PGHS2 may explain how the ductus arteriosus closes normally at birth in mice expressing PGHS2 Y385F, but not in PGHS2-null mice.

Determinants of 5-Lipoxygenase Nuclear Localization Using Green Fluorescent Protein/5-Lipoxygenase Fusion Proteins

5-Lipoxygenase catalyzes the first two steps in the biosynthesis of leukotrienes, potent extracellular mediators of inflammation and allergic disorders. The unanticipated observation of 5-lipoxygenase in the nucleus of some cell types including bone marrow-derived mast cells (Chen, X. S., Naumann, T. A., Kurre, U., Jenkins, N. A., Copeland, N. G., and Funk, C. D. (1995) J. Biol. Chem. 270, 17993–17999) has raised speculation about intranuclear actions of leukotrienes or the enzyme itself. To explore the entry of 5-lipoxygenase into the nucleus we have transfected various cell types with expression vectors encoding native 5-lipoxygenase and green fluorescent protein/5-lipoxygenase (GFP-5LO) fusion proteins. 5-Lipoxygenase and green fluorescent protein/5-lipoxygenase co-localized with the nuclear DNA stain Hoechst 33258 in each cell type. The three main basic regions of 5-lipoxygenase were incapable of acting as “classical” nuclear localization signal sequences. Mutations that abolished enzyme activity/non-heme iron resulted in proteins that would no longer enter the nucleus. An NH2-terminal 5-lipoxygenase fragment of 80 residues was sufficient for directing nuclear localization of green fluorescent protein but not cytosolic pyruvate kinase. The combined data suggest that 5-lipoxygenase enters the nucleus not by a classical nuclear localization signal but by a non-conventional signal located in the predicted β-barrel domain that may be masked by structural alterations.

Differential impact of prostaglandin H synthase 1 knockdown on platelets and parturition

Platelet activation is a hallmark of severe preeclampsia, and platelet PGH synthase 1-derived (PGHS1-derived) thromboxane A(2) (TxA(2)) has been implicated in its pathogenesis. However, genetic disruption of PGHS1 delays parturition. We created hypomorphic PGHS1 (PGHS1(Neo/Neo)) mice, in which the substantial but tissue-dependent variability in the inhibition of PGHS1-derived eicosanoids achieved by low-dose aspirin treatment is mimicked, to assess the relative impact of this strategy on hemostatic and reproductive function. Depression of platelet TxA(2) by 98% in PGHS1(Neo/Neo) mice decreased platelet aggregation and prevented thrombosis. Similarly, depression of macrophage PGE(2) by 75% was associated with selectively impaired inflammatory responses. PGF(2alpha) at 8% WT levels was sufficient to induce coordinated temporal oxytocin receptor (OTR) expression in uterus and normal ovarian luteolysis in PGHS1(Neo/Neo) mice at late gestation, while absence of PGHS1 expression in null mice delayed OTR induction and the programmed decrease of serum progesterone during parturition. Thus, extensive but tissue-dependent variability in PG suppression, as occurs with low-dose aspirin treatment, prevents thrombosis and impairs the inflammatory response but sustains parturition. PGHS1(Neo/Neo) mice provide a model of low-dose aspirin therapy that elucidates how prevention or delay of preeclampsia might be achieved without compromising reproductive function.

A mutation interfering with 5-lipoxygenase domain interaction leads to increased enzyme activity

5-Lipoxygenase (5-LOX) catalyzes two steps in conversion of arachidonic acid to proinflammatory leukotrienes. Lipoxygenases, including human 5-LOX, consist of an N-terminal C2-like β-sandwich and a catalytic domain. We expressed the 5-LOX domains separately, these were found to interact in the yeast two-hybrid system. The 5-LOX structure suggested association between Arg(101) in the β-sandwich and Asp(166) in the catalytic domain, due to electrostatic interaction as well as hydrogen bonds. Indeed, mutagenic replacements of these residues led to loss of two-hybrid interaction. Interestingly, when Arg(101) was mutated to Asp in intact 5-LOX, enzyme activity was increased. Thus, higher initial velocity of the reaction (vinit) and increased final amount of products were monitored for 5-LOX-R101D, at several different assay conditions. In the 5-LOX crystal structure, helix α2 and adjacent loops (including Asp(166)) of the 5-LOX catalytic domain has been proposed to form a flexible lid controlling access to the active site, and lid movement would be determined by bonding of lid residues to the C2-like β-sandwich. The more efficient catalysis following disruption of the R101-D166 ionic association supports the concept of such a flexible lid in human 5-LOX.

Lipoxygenase Gene Disruption Studies

Mammalian lipoxygenase enzymes (Figure 1) are derived from a multi-gene family, each member consisting of 14 exons (1,2). The 5-lipoxygenase gene is distinct from other lipoxygenase genes in its large size and its location on a separate chromosome (10q1 1.2 in humans and central chromosome 6 in mice) (1-5). A cluster of lipoxygenase genes located on mouse chromosome 11 (5,6) contains genes that encode three distinct 12(S)-lipoxy-genases, referred to as “platelet-type,” “leukocyte-type” and “epidermal-type” (1,7,8). Although substantial information is known about the ability of the various lipoxygenases to oxygenate arachidonic acid at a specific carbon atom position, relatively little is understood regarding the physiological roles of the various metabolites such as 12-hy-dro(pero)xy-eicosatetraenoic acid (12-H(p)ETE) and 15-H(p)ETE. Diverse biological activities have been assigned to lipoxygenase-derived eicosanoids and pathophysiological elevations in their synthesis have been reported (9-11). However, it remains to be determined how relevant these findings are to physiological models in vivo. Thus, we undertook the approach of targeted lipoxygenase gene disruption to elucidate possible functions associated with the various eicosanoid metabolites generated from these pathways. In this review, an effort will be made to emphasize the current status of experimental results using 5-lipoxygenase and 12-lipoxygenase-deficient mice and potential future applications in disease models and other physiological studies.