Prostaglandins but not leukotrienes alter extracellular matrix protein deposition and cytokine release in primary human airway smooth muscle cells and fibroblasts.

Eicosanoids are lipid-signaling mediators released by many cells in response to various stimuli. Increasing evidence suggests that eicosanoids such as leukotrienes and prostaglandins (PGs) may directly mediate remodeling. In this study, we assessed whether these substances could alter extracellular matrix (ECM) proteins and the inflammatory profiles of primary human airway smooth muscle cells (ASM) and fibroblasts. PGE(2) decreased both fibronectin and tenascin C in fibroblasts but only fibronectin in ASM. PGD(2) decreased both fibronectin and tenascin C in both ASM and fibroblasts, whereas PGF(2α) had no effect on ECM deposition. The selective PGI(2) analog, MRE-269, decreased fibronectin but not tenascin C in both cell types. All the PGs increased IL-6 and IL-8 release in a dose-dependent manner in ASM and fibroblasts. Changes in ECM deposition and cytokine release induced by prostaglandins in both ASM and fibroblasts were independent of an effect on cell number. Neither the acute nor repeated stimulation with leukotrienes had an effect on the deposition of ECM proteins or cytokine release from ASM or fibroblasts. We concluded that, collectively, these results provide evidence that PGs may contribute to ECM remodeling to a greater extent than leukotrienes in airway cells.

Disruption of leptin receptor-STAT3 signaling enhances leukotriene production and pulmonary host defense against pneumococcal pneumonia.

The adipocyte-derived hormone leptin regulates energy homeostasis and the innate immune response. We previously reported that leptin plays a protective role in bacterial pneumonia, but the mechanisms by which leptin regulates host defense remain poorly understood. Leptin binding to its receptor, LepRb, activates multiple intracellular signaling pathways, including ERK1/2, STAT5, and STAT3. In this study, we compared the responses of wild-type and s/s mice, which possess a mutant LepRb that prevents leptin-induced STAT3 activation, to determine the role of this signaling pathway in pneumococcal pneumonia. Compared with wild-type animals, s/s mice exhibited greater survival and enhanced pulmonary bacterial clearance after an intratracheal challenge with Streptococcus pneumoniae. We also observed enhanced phagocytosis and killing of S. pneumoniae in vitro in alveolar macrophages (AMs) obtained from s/s mice. Notably, the improved host defense and AM antibacterial effector functions in s/s mice were associated with increased cysteinyl-leukotriene production in vivo and in AMs in vitro. Augmentation of phagocytosis in AMs from s/s mice could be blocked using a pharmacologic cysteinyl-leukotriene receptor antagonist. Phosphorylation of ERK1/2 and cytosolic phospholipase A(2) α, known to enhance the release of arachidonic acid for subsequent conversion to leukotrienes, was also increased in AMs from s/s mice stimulated with S. pneumoniae in vitro. These data indicate that ablation of LepRb-mediated STAT3 signaling and the associated augmentation of ERK1/2, cytosolic phospholipase A(2) α, and cysteinyl-leukotriene synthesis confers resistance to s/s mice during pneumococcal pneumonia. These data provide novel insights into the intracellular signaling events by which leptin contributes to host defense against bacterial pneumonia.

Leukotriene B4 is a physiologically relevant endogenous peroxisome proliferator-activated receptor-alpha agonist.

Peroxisome proliferator-activated receptors (PPARs) are nuclear transcription factors that play central roles in metabolism and inflammation. Although a variety of compounds have been shown to activate PPARs, identification of physiologically relevant ligands has proven difficult. In silico studies of lipid derivatives reported here identify specific 5-lipoxygenase products as candidate physiologically relevant PPAR-alpha activators. Subsequent studies show both in vitro and in a murine model of inflammation that 5-lipoxygenase stimulation induces PPAR-alpha signaling and that this results specifically from production of the inflammatory mediator and chemoattractant leukotriene B(4) (LTB(4)). Activation of PPAR-alpha is a direct effect of intracellularly generated LTB(4) binding to the nuclear receptor and not of secreted LTB(4) acting via its cell-surface receptors. Activation of PPAR-alpha reduces secretion of LTB(4) by stimulating degradation of this fatty acid derivative. We also show that the LTB(4) precursors leukotriene A(4) (LTA(4)) and 5-hydroperoxyeicosatetrenoic acid (5-HPETE) activate PPAR-alpha but have no significant endogenous effect independent of conversion to LTB(4). We conclude that LTB(4) is a physiologically relevant PPAR-alpha activator in cells of the immune system. This, together with previous findings, demonstrates that different types of lipids serve as endogenous PPAR-alpha ligands, with the relevant ligand varying between functionally different cell types. Our results also support the suggestion that regulation of inflammation may involve balancing proinflammatory effects of LTB(4), exerted through cell-surface receptors, and anti-inflammatory effects exerted through PPAR-alpha activation.

Two pathways for cyclooxygenase-2 protein degradation in vivo.

COX-2, formally known as prostaglandin endoperoxide H synthase-2 (PGHS-2), catalyzes the committed step in prostaglandin biosynthesis. COX-2 is induced during inflammation and is overexpressed in colon cancer. In vitro, an 18-amino acid segment, residues 595-612, immediately upstream of the C-terminal endoplasmic reticulum targeting sequence is required for N-glycosylation of Asn(594), which permits COX-2 protein to enter the endoplasmic reticulum-associated protein degradation system. To determine the importance of this COX-2 degradation pathway in vivo, we engineered a del595-612 PGHS-2 (Delta 18 COX-2) knock-in mouse lacking this 18-amino acid segment. Delta 18 COX-2 knock-in mice do not exhibit the renal or reproductive abnormalities of COX-2 null mice. Delta 18 COX-2 mice do have elevated urinary prostaglandin E(2) metabolite levels and display a more pronounced and prolonged bacterial endotoxin-induced febrile response than wild type (WT) mice. Normal brain tissue, cultured resident peritoneal macrophages, and cultured skin fibroblasts from Delta 18 COX-2 mice overexpress Delta 18 COX-2 relative to WT COX-2 expression in control mice. These results indicate that COX-2 can be degraded via the endoplasmic reticulum-associated protein degradation pathway in vivo. Treatment of cultured cells from WT or Delta 18 COX-2 mice with flurbiprofen, which blocks substrate-dependent degradation, attenuates COX-2 degradation, and treatment of normal mice with ibuprofen increases the levels of COX-2 in brain tissue. Thus, substrate turnover-dependent COX-2 degradation appears to contribute to COX-2 degradation in vivo. Curiously, WT and Delta 18 COX-2 protein levels are similar in kidneys and spleens from WT and Delta 18 COX-2 mice. There must be compensatory mechanisms to maintain constant COX-2 levels in these tissues.

Phosphorylation of serine 271 on 5-lipoxygenase and its role in nuclear export.

The enzyme 5-lipoxygenase (5-LO) initiates the biosynthesis of leukotrienes, inflammatory mediators involved in immune diseases and defense. The subcellular localization of 5-LO is regulated, with nuclear import commonly leading to increased leukotriene production. We report here that 5-LO is constitutively phosphorylated on Ser-271 in transfected NIH 3T3 cells. This residue is nested in a classical nuclear export sequence, and phosphorylated Ser-271 5-LO was exclusively found in the nucleus by immunofluorescence and by fractionation techniques. Mutation of Ser-271 to Ala allowed nuclear export of 5-LO that was blocked by the specific nuclear export inhibitor leptomycin b, suggesting that phosphorylation of Ser-271 serves to interfere with exportin-1-mediated nuclear export. Consistent with previous reports that purified 5-LO can be phosphorylated on Ser-271 in vitro by MAPK-activated protein kinase 2, the nuclear export of 5-LO was increased by either treatment with the p38 inhibitor SB 203,580 or co-expression of a kinase-deficient p38 MAPK. Nuclear export of 5-LO can also be induced by KN-93, an inhibitor of Ca2+/calmodulin-dependent kinase II, and the effects of SB 203,580 plus KN-93 are additive. Finally, HeLa cells, which lack nuclear 5-LO, also lack constitutive phosphorylation of Ser-271. Taken together, these results indicate that the phosphorylation of Ser-271 serves to inhibit the nuclear export of 5-LO. This action works in concert with nuclear import, which is regulated by phosphorylation on Ser-523, to determine the subcellular distribution of 5-LO, which in turn regulates leukotriene biosynthesis.

Rap1 activation is required for Fc gamma receptor-dependent phagocytosis.

Phagocytosis of IgG-opsonized microbes via the Fc gamma receptor (Fc gammaR) requires the precise coordination of a number of signaling molecules, including the low-molecular mass GTPases. Little is known about the Ras-family GTPase Rap1 in this process. We therefore investigated its importance in mediating Fc gammaR-dependent phagocytosis in NR8383 rat alveolar macrophages. Pulldown of active Rap1 and fluorescence microscopic analysis of GFP-RalGDS (Ral guanine dissociation stimulator)-transfected macrophages revealed that Rap1 is indeed activated by Fc gammaR crosslinking. Inhibition of Rap1 activity, both by Rap1GAP (GTPase-activating protein) expression and liposome-delivered blocking Ab, severely impaired the ability of cells to ingest IgG-opsonized targets. Fc gammaR-induced Rap1 activation was found to be independent of both cAMP and Ca(2+), suggesting a role for the second messenger-independent guanosine exchange factor, C3G. This was supported by the facts that 1) liposome-delivered blocking Ab against C3G inhibited both Fc gammaR-dependent phagocytosis and Rap1 activation, and 2) both active Rap1GTP and C3G were found to translocate to the phagosome. Taken together, our data demonstrate a novel role for Rap1 and its exchange factor C3G in mediating Fc gammaR-dependent phagocytosis.

Misoprostol impairs female reproductive tract innate immunity against Clostridium sordellii.

Fatal cases of acute shock complicating Clostridium sordellii endometritis following medical abortion with mifepristone (also known as RU-486) used with misoprostol were reported. The pathogenesis of this unexpected complication remains enigmatic. Misoprostol is a pharmacomimetic of PGE(2), an endogenous suppressor of innate immunity. Clinical C. sordellii infections were associated with intravaginal misoprostol administration, suggesting that high misoprostol concentrations within the uterus impair immune responses against C. sordellii. We modeled C. sordellii endometritis in rats to test this hypothesis. The intrauterine but not the intragastric delivery of misoprostol significantly worsened mortality from C. sordellii uterine infection, and impaired bacterial clearance in vivo. Misoprostol also reduced TNF-alpha production within the uterus during infection. The intrauterine injection of misoprostol did not enhance mortality from infection by the vaginal commensal bacterium Lactobacillus crispatus. In vitro, misoprostol suppressed macrophage TNF-alpha and chemokine generation following C. sordellii or peptidoglycan challenge, impaired leukocyte phagocytosis of C. sordellii, and inhibited uterine epithelial cell human beta-defensin expression. These immunosuppressive effects of misoprostol, which were not shared by mifepristone, correlated with the activation of the G(s) protein-coupled E prostanoid (EP) receptors EP2 and EP4 (macrophages) or EP4 alone (uterine epithelial cells). Our data provide a novel explanation for postabortion sepsis leading to death and also suggest that PGE(2), in which production is exaggerated within the reproductive tract during pregnancy, might be an important causal determinant in the pathogenesis of more common infections of the gravid uterus.

Capturing proteins that bind polyunsaturated fatty acids: demonstration using arachidonic acid and eicosanoids.

Polyunsaturated fatty acids (PUFA) and their biological derivatives, including the eicosanoids, have numerous roles in physiology and pathology. Although some eicosanoids are known to act through receptors, the molecular actions of many PUFA remain obscure. As the three-dimensional structure of eicosanoids allows them to specifically bind and activate their receptors, we hypothesized that the same structure would allow other proteins to associate with PUFA and eicosanoids. Here, we demonstrate that biotinylation of arachidonic acid and its oxygenated derivatives 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene (LT) B(4) can be used to pull down associated proteins. Separation of proteins by two-dimensional gel electrophoresis indicated that a large number of proteins bound each lipid and that proteins could distinguish between two enantiomers of 5-HETE. Individual proteins, identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry, included proteins that are known to bind lipids, including albumin and phosphatidylethanolamine-binding protein, as well as several novel proteins. These include cytoskeletal proteins, such as actin, moesin, stathmin and coactosin-like protein, and G protein signaling proteins, such as Rho GDP dissociation inhibitor 1 and nucleoside diphosphate kinase B. This method, then, represents a relatively simple and straightforward way to screen for proteins that directly associate with, and are potentially modulated by, PUFA and their derivatives.

Arachidonic acid binds 14-3-3zeta, releases 14-3-3zeta from phosphorylated BAD and induces aggregation of 14-3-3zeta.

Polyunsaturated fatty acids, like arachidonic acid, can bind proteins and affect their function. The 14-3-3 proteins bind phosphorylated sites on a diverse array of client proteins and, in this way, are involved in many intracellular signaling pathways. In this study, we used a novel approach to discover that 14-3-3zeta is able to directly bind arachidonic acid. Furthermore, arachidonic acid, at physiological concentrations, reduced the binding of 14-3-3zeta to phosphorylated BAD, an interaction that is important in regulating apoptosis. In addition, high concentrations of arachidonic acid caused the polymerization of 14-3-3zeta, an event observed in neurodegenerative disorders. Taken together, these results indicate that arachidonic acid directly interacts with 14-3-3zeta and that this interaction may be important in both normal and pathological cellular events. If so, then factors that mediate the release, metabolism and reacylation of arachidonic acid into membranes represent key points of regulation.

Effects of prostaglandin E2 on the subcellular localization of Epac-1 and Rap1 proteins during Fcgamma-receptor-mediated phagocytosis in alveolar macrophages.

Recent studies have demonstrated a central role for the exchange protein activated by cAMP (Epac) in the inhibition of Fcgamma-receptor-mediated phagocytosis and bacterial killing by prostaglandin E(2) (PGE(2)) in macrophages. However, the subcellular localization of Epac, and its primary target Rap1, has yet to be determined in primary macrophages. Therefore, we used immunofluorescent techniques and phagosome isolation to localize Epac-1 and Rap1 in alveolar macrophages. Epac-1 was predominantly expressed on punctate and tubular membranes throughout the cell body; on the plasma membrane; and co-localized with microtubule organizing centers (MTOCs). Rap1 was abundant on punctate membranes, less abundant on plasma membrane, and also found on MTOCs. Following PGE(2) treatment, Epac-1, but not Rap1, accumulated on the nuclear envelope and disappeared from MTOCs. By immunofluorescent microscopy, both Epac-1 and Rap1 were seen to associate with phagosomes containing IgG-opsonized beads, but this association appeared weak, as we failed to observe such interactions in phagosomes isolated from cells at various time points after bead ingestion. Strikingly, however, Epac-1, but not Rap1, appeared to accumulate on maturing phagosomes, but only after PGE(2) treatment (or treatment with a selective Epac-1 agonist). This association was confirmed in isolated phagosome preparations. The changes in Epac-1 localization were too slow to account for the inhibitory effects of PGE(2) on phagocytosis. However, the appearance of Epac-1 on late phagosomes following PGE(2) treatment might be important for suppressing H(2)O(2) production and inhibiting the killing of intraphagosomal pathogens. The absence of Rap1 on late phagosomes suggests that the effect of Epac-1 might not require Rap1.

Activation and regulation of cellular eicosanoid biosynthesis.

There is a growing appreciation for the wide variety of physiological responses that are regulated by lipid messengers. One particular group of lipid messengers, the eicosanoids, plays a central role in regulating immune and inflammatory responses in a receptor-mediated fashion. These mediators are related in that they are all derived from one polyunsaturated fatty acid, arachidonic acid. However, the various eicosanoids are synthesized by a wide variety of cell types by distinct enzymatic pathways, and have diverse roles in immunity and inflammation. In this review, the major pathways involved in the synthesis of eicosanoids, as well as key points of regulation, are presented.

Prolonged exposure to hyperoxia increases perivascular mast cells in rat lungs.

Prolonged hyperoxia, as may be used to treat patients with severe hypoxemia, can lead to lung injury, respiratory failure, and death. Resident mast cells play important roles in regulating the lung response to changing environmental conditions, as evidenced by their roles in asthma and airway hyperresponsiveness. In this study we evaluated the effect of prolonged hyperoxia on the number and distribution of mast cells in the rat lung. In rats maintained in normoxia, mast cells were distributed primarily in the loose connective tissue surrounding large bronchioles and vessels of the lung. In rats exposed to normobaric hyperoxia for 72 hr, mast cell number in lung sections increased significantly, and mast cells were found preferentially accumulated around vessels throughout the lung. Notably, mast cells around smaller vessels were abundant in hyperoxic lungs but rare in normoxic lungs. Also, mast cells were increased in the pleura of lungs exposed to hyperoxia. These changes in mast cell number and distribution in response to hyperoxia were evident in aged (22-month-old) rats as well as young (3-month-old) rats. As mast cell-derived mediators have many effects, e.g., on vascular leak and vascular tone, positioning of increased mast cell numbers throughout the lung vasculature may be an important contributor to changes in lung function subsequent to persistent hyperoxia.

Metabolism of arachidonic acid to eicosanoids within the nucleus.

The eicosanoids are a diverse family of molecules that have powerful effects on cell function. They are best known as intercellular messengers, having autocrine and paracrine effects following their secretion from the cells that synthesize them. Many of the eicosanoids are produced from one polyunsaturated fatty acid, arachidonic acid. The diversity of possible products that can be synthesized from arachidonic acid is due, in part to the variety of enzymes that can act on it. Over the past 15 years, studies have placed many, but not all, of these enzymes at or inside the nucleus. In some cases, the nuclear import or export of arachidonic acid-processing enzymes is highly regulated. Furthermore, nuclear receptors that are activated by specific eicosanoids are known to exist. Taken together, these findings indicate that the enzymatic conversion of arachidonic acid to specific signaling molecules can occur in the nucleus, that it is regulated, and that the synthesized products may act within the nucleus. The objectives of this commentary are to review what is known about the metabolism of arachidonic acid to eicosanoids within the nucleus and to point to important areas for future discovery.

Regulating leukotriene synthesis: the role of nuclear 5-lipoxygenase.

Leukotrienes are lipid messengers involved in autocrine and paracrine cellular signaling. They are synthesized from arachidonic acid by the 5-lipoxygenase pathway. Current models of this enzymatic pathway recognize that a key step in initiating leukotriene synthesis is the calcium-mediated movement of enzymes, including 5-lipoxygenase, to intracellular membranes. However, 5-lipoxygenase can be imported into or exported from the nucleus before calcium activation. As a result, its subcellular localization will affect its ability to be activated by calcium, as well as the membrane to which it binds and its interaction with other enzymes. This commentary focuses on the role of 5-lipoxygenase compartmentation in determining its regulation and, ultimately, leukotriene synthesis.

Phosphorylation by protein kinase a inhibits nuclear import of 5-lipoxygenase.

The enzyme 5-lipoxygenase initiates the synthesis of leukotrienes from arachidonic acid. Protein kinase A phosphorylates 5-lipoxygenase on Ser(523), and this reduces its activity. We report here that phosphorylation of Ser(523) also shifts the subcellular distribution of 5-lipoxygenase from the nucleus to the cytoplasm. Phosphorylation and redistribution of 5-lipoxygenase could be produced by overexpression of the protein kinase A catalytic subunit alpha, by pharmacological activators of protein kinase A, and by prostaglandin E(2). Mimicking phosphorylation by replacing Ser(523) with glutamic acid caused cytoplasmic localization; replacement of Ser(523) with alanine prevented phosphorylation and redistribution in response to protein kinase A activation. Because Ser(523) is positioned within the nuclear localization sequence-518 of 5-lipoxygenase, the ability of protein kinase A to phosphorylate and alter the localization of green fluorescent protein fused to the nuclear localization sequence-518 peptide was also tested. Site-directed replacement of Ser(523) with glutamic acid within the peptide impaired nuclear accumulation; overexpression of the protein kinase A catalytic subunit alpha and pharmacological activation of protein kinase caused phosphorylation of the fusion protein at Ser(523), and the phosphorylated protein was found chiefly in the cytoplasm. Taken together, these results indicate that phosphorylation of Ser(523) inhibits the nuclear import function of a nuclear localization sequence, resulting in the accumulation of 5-lipoxygenase enzyme in the cytoplasm. As cytoplasmic localization can be associated with reduced leukotriene synthetic capacity, phosphorylation of Ser(523) serves to inhibit leukotriene production by both impairing catalytic activity and by placing the enzyme in a site that is unfavorable for action.

Nuclear localization of leukotriene A4 hydrolase in type II alveolar epithelial cells in normal and fibrotic lung.

Leukotriene A4 (LTA4) hydrolase catalyzes the final step in leukotriene B4 (LTB4) synthesis. In addition to its role in LTB4 synthesis, the enzyme possesses aminopeptidase activity. In this study, we sought to define the subcellular distribution of LTA4 hydrolase in alveolar epithelial cells, which lack 5-lipoxygenase and do not synthesize LTA4. Immunohistochemical staining localized LTA4 hydrolase in the nucleus of type II but not type I alveolar epithelial cells of normal mouse, human, and rat lungs. Nuclear localization of LTA4 hydrolase was also demonstrated in proliferating type II-like A549 cells. The apparent redistribution of LTA4 hydrolase from the nucleus to the cytoplasm during type II-to-type I cell differentiation in vivo was recapitulated in vitro. Surprisingly, this change in localization of LTA4 hydrolase did not affect the capacity of isolated cells to convert LTA4 to LTB4. However, proliferation of A549 cells was inhibited by the aminopeptidase inhibitor bestatin. Nuclear accumulation of LTA4 hydrolase was also conspicuous in epithelial cells during alveolar repair following bleomycin-induced acute lung injury in mice, as well as in hyperplastic type II cells associated with fibrotic lung tissues from patients with idiopathic pulmonary fibrosis. These results show for the first time that LTA4 hydrolase can be accumulated in the nucleus of type II alveolar epithelial cells and that redistribution of the enzyme to the cytoplasm occurs with differentiation to the type I phenotype. Furthermore, the aminopeptidase activity of LTA4 hydrolase within the nucleus may play a role in promoting epithelial cell growth.

Structural organization of the regulatory domain of human 5-lipoxygenase.

The enzyme 5-lipoxygenase (5-LO) initiates the synthesis of leukotrienes. For this reason, 5-LO activity is important for immune defense, whereas improper regulation contributes to pathogenesis, including chronic inflammation, asthma and atherosclerosis. Like all lipoxygenases, the 5-LO protein consists of two domains, a regulatory domain and a catalytic domain. Naturally, the regulatory domain determines catalytic activity and controls leukotriene synthesis. This domain shares features with classical C2 domains in that it has a beta-sandwich structure and binds calcium, nucleotides and phospholipids. However, important structural features place this domain in a distinct family, the PLATs (for Polycystin-1, Lipoxygenase, alpha-Toxin). In this review, we summarize our current understanding of the three dimensional organization of this important component of the 5-LO molecule. In addition, we point to findings from structural analyses of related proteins to suggest further details relating 5-LO structure to function.

Expression of 5-lipoxygenase in specialized epithelial cells of nasopharyngeal-associated lymphoid tissue.

Leukotrienes are lipid mediators that are produced primarily by certain types of leukocytes. The synthesis of the leukotriene LTB(4) is initiated by the enzyme 5-lipoxygenase and completed by LTA(4) hydrolase. Epithelial cells constitutively express LTA(4) hydrolase but normally lack 5-lipoxygenase. In this study, we report that the stratified squamous epithelial cells from inflamed or hyperplastic tissues of palatine and pharyngeal tonsils (nasopharyngeal-associated lymphoid tissue) express 5-lipoxygenase protein. The localization of 5-lipoxygenase was indicated by immunohistochemical staining and presence confirmed by immunoblot. Positive staining for 5-lipoxygenase in infiltrating leukocytes in inflamed tissues served as internal positive controls for immunohistochemical staining. Staining for 5-lipoxygenase in appendix tissue was negative for epithelial cells while positive for polymorphonuclear leukocytes, indicating that 5-lipoxygenase expression is not a general feature of epithelial cells in mucosa-associated lymphoid tissue. In tonsils, 5-lipoxygenase staining was pronounced in broad regions but reduced or absent in others, suggesting regional regulation of expression. Epithelial cells of tonsils were also positive for 5-lipoxygenase activating protein and leukotriene A(4) hydrolase, indicating a capacity to produce LTB(4). Taken together, these results suggest that the specialized epithelial cells of the mucosa-associated lymphoid tissue of human tonsils can synthesize LTB(4). This lipid mediator may serve to modulate the function of cells within the lymphoid tissue as well as promote an inflammatory response.

Multiple nuclear localization sequences allow modulation of 5-lipoxygenase nuclear import.

The nuclear import of proteins typically requires the presence of a nuclear localization sequence (NLS). Some proteins have more than one NLS, but the significance of having multiple NLSs is unclear. The enzyme 5-lipoxygenase (5-LO) has three NLSs that, unlike the tight cluster of basic residues of the classical SV40 large T antigen NLS, contain dispersed basic residues. When attached to green fluorescent protein (GFP), individual 5-LO NLSs caused quantitatively and statistically less import than the SV40 NLS. Combined 5-LO NLSs produced nuclear import that was comparable to that of the SV40 NLS. As expected, GFP/NLS proteins displayed relatively uniform import in all cells. However, a fusion protein of GFP plus the 5-LO protein, modified to contain only one functional NLS, produced some cells with import and some cells without import. A GFP/5-LO fusion protein containing two functional NLSs produced four identifiable levels of nuclear import. Quantitative and visual analysis of a population of cells expressing the intact GFP/5-LO protein, with three intact NLSs, indicated five levels of nuclear import. This suggested that the subcellular distribution of 5-LO may vary widely in normal cells of the body. Consistent with this, immunohistochemical staining of lung sections found that individual macrophages, in situ, displayed cell-specific levels of import of 5-LO. Since nuclear accumulation is known to affect 5-LO activity, multiple NLSs may allow graded regulation of activity via controlled import. Multiple NLSs on other proteins may likewise allow fine control of protein action through modulation of the level of import.

Protein kinase A inhibits leukotriene synthesis by phosphorylation of 5-lipoxygenase on serine 523.

Leukotrienes (LTs) are lipid messengers generated by leukocytes that drive inflammation and modulate neighboring cell function. The synthesis of LTs from arachidonic acid is initiated by the enzyme 5-lipoxygenase (5-LO). We report for the first time that LT synthesis is inhibited by the direct action of protein kinase A (PKA) on 5-LO. The catalytic subunit of PKA directly phosphorylated 5-LO in vivo and in vitro and inhibited activity in intact cells and in vitro. Mutation of Ser-523 on human 5-LO prevented phosphorylation by PKA and restored LT synthesis. Treatment with PKA activators inhibited LTB(4) synthesis in 3T3 cells expressing wild type 5-LO but not in cells expressing the S523A mutant of 5-LO. The mechanism of inhibition of LTB(4) synthesis did not involve either reduced membrane association of activated 5-LO or redistribution of 5-LO from the nucleus to the cytoplasm. Instead, PKA phosphorylation of recombinant 5-LO inhibited in vitro activity, as did co-transfection of cells with 5-LO plus the catalytic subunit of PKA. Also, substitution of Ser-523 with glutamic acid, mimicking phosphorylation, resulted in the total loss of 5-LO activity. These results indicate that PKA phosphorylates 5-LO on Ser-523, which inhibits the catalytic activity of 5-LO and reduces cellular LT generation. Thus, PKA activation, as can occur in response to adenosine, prostaglandin E(2), beta-adrenergic agonists, and other mediators, is a means of directly reducing 5-LO activity and LT synthesis that may be important in limiting inflammation and maintaining homeostasis.