Leukotrienes constitute a class of potent biological mediators of inflammation and anaphylaxis (for reviews see refs 1 and 2). Their biosynthesis derives from 5-lipoxygenase-catalysed oxygenation of arachidonic acid in granulocytes, macrophages and mast cells. To examine the physiological importance of leukotrienes, we have disrupted the 5-lipoxygenase gene by homologous recombination in embryonic stem cells. 5-Lipoxygenase-deficient (5LX-/-) mice develop normally and are healthy. They show a selective opposition to certain inflammatory insults. Although there is no difference in their reaction to endotoxin shock, the 5LX-/- animals resist the lethal effects of shock induced by platelet-activating factor. Reaction to ear inflammation induced by phorbol ester is normal, whereas inflammation induced by arachidonic acid is markedly reduced. Contrasts were also found in two models of leukocyte chemotaxis in vivo. The phenotype of 5LX-/- mice under injurious insult identifies the role for leukotrienes in the pathophysiology of select inflammatory states.

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5-Lipoxygenase of mouse macrophages and bone marrow-derived mast cells (BMMC) was investigated. Indirect immunocytofluorescence combined with confocal microscopy provided evidence for distinct intracellular expression patterns and trafficking of 5-lipoxygenase upon cellular activation. In resting BMMC, 5-lipoxygenase was found within the nucleus co-localizing with the nuclear stain Yo-Pro-1. When BMMC were IgE/antigen-activated the 5-lipoxygenase immunofluorescence pattern was changed from nuclear to perinuclear. The absence of divalent cations in the incubation medium, or calcium ionophore A23187 challenge, altered the predominantly nuclear expression pattern to new sites both cytosolic and intranuclear. The cDNA for murine macrophage 5-lipoxygenase was cloned by the polymerase chain reaction and would predict a 674 amino acid protein. Using control cells obtained from 5-lipoxygenase-deficient mice it was determined that a single isoform accounts for both soluble and membrane-bound and nuclear and cytosolic-localized enzyme in macrophages and BMMC. A mutation at amino acid 672 (Val-->Met) introduced serendipitously during the cloning process was found to completely abolish 5-lipoxygenase enzyme activity when the enzyme was expressed in human embryonic kidney 293 cells. This subtle change is proposed to affect the ability of the COOH-terminal isoleucine to coordinate the essential non-heme iron atom. In macrophages and BMMC obtained from 5-lipoxygenase-deficient mice, compensatory changes in expression of genes involved in the biosynthesis of leukotriene B4 were investigated. 5-Lipoxygenase-activating protein expression was reduced by 50%, while leukotriene A4 hydrolase expression was unaltered. The 5-lipoxygenase gene was mapped to the central region of mouse chromosome 6 in a region that shares homology with human chromosome 10 by interspecific backcross analysis. These studies provide a global picture of the murine 5-lipoxygenase system and raise questions about the role of 5-lipoxygenase and leukotrienes within the nucleus.

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5-5-Lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) is a non-heme iron enzyme found primarily in white blood cells, macrophages, and mast cells that converts arachidonic acid first to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) 1 and then to leukotriene (LT)A 4 (5,6-oxido-7,9,11,14-eicosatetraenoic acid)(1). Subsequent conversion of leukotriene A 4 by leukotriene A 4 hydrolase yields the potent neutrophil chemoattractant leukotriene B 4 . Alternatively, conjugation of LTA 4 with glutathione by leukotriene C 4 synthase plus downstream metabolism leads to the cysteinyl leukotrienes that influence airway reactivity and mucus secretion especially in asthmatics (1-3).5-Lipoxygenase was isolated originally from the cytosol fraction of human and porcine neutrophils (4, 5). The enzyme was shown subsequently to undergo a calcium-dependent translocation to the nuclear envelope upon ionophore A23187 stimulation and was dependent on the 5-lipoxygenase-activating protein situated in this location for leukotriene biosynthesis (6, 7). More recent work based on immunofluorescence techniques and cellular fractionation demonstrated that certain cell types capable of leukotriene formation (alveolar macrophages, rat basophilic leukemia cells, and mouse bone marrow-derived mast cells) express 5-lipoxygenase completely or partially in the nucleus (8 -11). Additionally, it was shown that cytosolic 5-lipoxygenase in rat neutrophils could enter the nucleus if they were first elicited in vivo with various inflammatory agents or subjected to adherence to glass in vitro (12).The discovery of 5-lipoxygenase in the nucleus was a surprising observation since it is well known that leukotrienes must exit the cell once synthesized to act on cell surface G proteincoupled receptors to exert their actions on neutrophils or bronchiole smooth muscle (13,14). The possibility of 5-lipoxygenase itself or leukotrienes acting in the nucleus was raised. The recent observation that LTB 4 could bind to the nuclear peroxisomal proliferator-activated receptor-␣ indicated that intranuclear actions of leukotrienes are feasible (15).Nothing is known about control of 5-lipoxygenase entry into the nucleus in some cell types but not others. Proteins enter the nucleus by nuclear localization signal (NLS) sequences that are recognized by specific importins, prior to nuclear pore docking, translocation through the pore and release from the pore's inner side (16,17). The NLS is typically a short basic region or bipartite basic sequence (18,19). Increasingly, however, novel NLS sequences are being recognized for import of particular classes of proteins; for example, the 38-amino acid M9 domain of heterogeneous nuclear ribonucleoprotein A1 (20,21). Here, we demonstrate primarily with the use of green fluorescent protein (GFP)/5-lipoxygenase fusion proteins the complexity of events for 5-lipoxygenase nuclear entry. EXPERIMENTAL PROCEDURESConstruction of Expression Vectors-The full-length cDNA encoding human 5-lipoxygenase from pT3-5LO (22) subsequently ...

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.

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Discovery of HIP1 as a fusion partner of ALK in NSCLC is a novel finding. In addition, the HIP1-ALK-rearranged tumor is sensitive to treatment with crizotinib in vivo, implicating HIP1-ALKas an oncogenic driver of lung tumorigenesis. Collectively, our results indicate that HIP1-ALK-positive NSCLC may benefit from clinical applications of crizotinib.