Leukotriene C4 Synthase Research Articles

Expression of enzymes and receptors of leukotriene pathway genes in equine endometrium during the estrous cycle and early pregnancy

The aims of the present study were to elucidate the expression profiles of leukotriene (LT) pathway mRNA transcription and to determine the possible interaction of LT and prostaglandin (PTG) pathways genes in equine endometrium during the estrous cycle and early pregnancy. Endometrial biopsies were obtained from mares on the day of ovulation (d0), at late diestrous (LD, n = 4), and after luteolysis in the estrus phase (AL, n = 4) of the cycle. Biopsies were also taken on Days 14 (P14; n = 4), 18 (P18, n = 4), and 22 (P22, n = 4) during early pregnancy that were comparable days to cyclic sampling days. A mixed model was fitted on the normalized relative mRNA levels, quantified by qPCR in duplicate, and least significant difference test was employed to detect significantly different group(s). In addition, to determine the degree of contribution of each gene to separation of treatment groups, the multivariate projection method partial least square regression discriminant analysis was used. The expression of 5-lipoxygenase mRNA was greater on d0 and LD, declined at AL, and was suppressed by early pregnancy. Leukotriene A4 hydrolase mRNA expression increased at LD and during early pregnancy, but was significantly greater at LD compared with P14. The expression of LT C4 synthase mRNA was only induced at LD. Cysteinyl leukotriene receptors (CysLT1 and CysLT2) mRNA expressions were decreased by both cyclic changes and early pregnancy, whereas 5-lipoxygenase-activating protein and B leukotriene receptor mRNA expressions were not affected by early pregnancy or stages of the estrous cycle. Partial least square discriminant analysis suggests that LT and PTG pathway enzymes and receptors appear to behave similarly in terms of mRNA expression. In conclusion, the expression profiles of LT pathway genes are demonstrated in equine endometrium for the first time by the present study, and the present data suggest that LT pathway mRNA transcriptions are tightly regulated during early pregnancy in mares.

The Editor takes a closer look at some of this month's articles

The Editor takes a closer look at some of this month's articles

P.1.008 V1B/CRF1 receptor heterodimerisation as a key mechanism of vasopressin and corticotropin-releasing factor synergism

Leukotrienes (LTs) are biologically active compounds derived from arachidonic acid which have important pathophysiological roles in asthma and inflammation. The cysteinyl leukotriene LTC4 and its metabolites LTD4 and LTE4 stimulate bronchoconstriction, airway mucous formation and generalized edema formation. LTC4 is formed by addition of glutathione to LTA4, catalyzed by the integral membrane protein, LTC4 synthase (LTCS). We now report the use of bioluminescence resonance energy transfer (BRET) to demonstrate that LTCS forms homo-oligomers in living cells. Fusion proteins of LTCS and Renilla luciferase (Rluc) and a variant of green fluorescent protein (GFP), respectively, were prepared. High BRET signals were recorded in transiently transfected human embryonic kidney (HEK 293) cells co-expressing Rluc/LTCS and GFP/LTCS. Homo-oligomer formation in living cells was verified by co-transfection of a plasmid expressing non-chimeric LTCS. This resulted in dose-dependent attenuation of the BRET signal. Additional evidence for oligomer formation was obtained in cell-free assays using glutathione S-transferase (GST) pull-down assay. To map interaction domains for oligomerization, GFP/LTCS fusion proteins were prepared with truncated variants of LTCS. The results obtained identified a C-terminal domain (amino acids 114–150) sufficient for oligomerization of LTCS. Another, centrally located, interaction domain appeared to exist between amino acids 57–88. The functional significance of LTCS homo-oligomer formation is currently being investigated.

Catalytic Characterization of Human Microsomal Glutathione S-Transferase 2: Identification of Rate-Limiting Steps

Microsomal glutathione S-transferase 2 (MGST2) is a 17 kDa trimeric integral membrane protein homologous to leukotriene C4 synthase (LTC4S). MGST2 has been suggested to catalyze the biosynthesis of the pro-inflammatory mediator leukotriene C4 (LTC4) in cells devoid of LTC4S. A detailed biochemical study of MGST2 is critical for the understanding of its cellular function and potential role as an LTC4-producing enzyme. Here we have characterized the substrate specificity and catalytic properties of purified MGST2 by steady-state and pre-steady-state kinetic experiments. In comparison with LTC4S, which has a catalytic efficiency of 8.7 × 10(5) M(-1) s(-1), MGST2, with a catalytic efficiency of 1.8 × 10(4) M(-1) s(-1), is considerably less efficient in producing LTC4. However, the two enzymes display a similar KM(LTA4) of 30-40 μM. While LTC4S has one activated glutathione (GSH) (forming a thiolate) per enzyme monomer, the MGST2 trimer seems to display only third-of-the-sites reactivity for thiolate activation, which in part would explain its lower catalytic efficiency. Furthermore, MGST2 displays GSH-dependent peroxidase activity of ∼0.2 μmol min(-1) mg(-1) toward several lipid hydroperoxides. MGST2, but not LTC4S, is efficient in catalyzing conjugation of the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the lipid peroxidation product 4-hydroxy-2-nonenal with GSH. Using stopped-flow pre-steady-state kinetics, we have characterized the full catalytic reaction of MGST2 with CDNB and GSH as substrates, showing an initial rapid equilibrium binding of GSH followed by thiolate formation. Burst kinetics for the CDNB-GSH conjugation step was observed only at low GSH concentrations (thiolate anion formation becoming rate-limiting under these conditions). Product release is rapid and does not limit the overall reaction. Therefore, in general, the chemical conjugation step is rate-limiting for MGST2 at physiological GSH concentrations. MGST2 and LTC4S exhibit distinct catalytic and mechanistic properties, reflecting adaptation to broad and specific physiological functions, respectively.

A leukotriene C4 synthase inhibitor with the backbone of 5-(5-methylene-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid

The cysteinyl leukotrienes (cys-LTs), leukotriene C4 (LTC4) and its metabolites, LTD4 and LTE4, are proinflammatory lipid mediators in asthma and other inflammatory diseases. They are generated through the 5-lipoxygenase/LTC4 synthase (LTC4S) pathway and act via at least two distinct G protein-coupled receptors. The inhibition of human LTC4S will make a simple way to treat the cys-LT relevant inflammatory diseases. Here, we show that compounds having 5-(5-methylene-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid moiety suppress LTC4 synthesis, glutathione conjugation to the precursor LTA4, in both an enzyme assay and a whole-cell assay. Hierarchical in silico screenings of 6 million compounds provided 300,000 dataset for docking, and after energy minimization based on the crystal structure of LTC4S, 111 compounds were selected as candidates for a competitive inhibitor to glutathione. One of those compounds showed significant inhibitory activity, and subsequently, its derivative 5-((Z)-5-((E)-2-methyl-3-phenylallylidene)-4-oxo-4,5-dihydrothiazol-2-ylamino) isophthalic acid (compound 1) was found to be the most potent inhibitor. The enzyme assay showed the IC50 was 1.9 µM and the corresponding 95% confidence interval was from 1.7 to 2.2 µM. The whole-cell assay showed that compound 1 was cell permeable and inhibited LTC4 synthesis in a concentration dependent manner.

Over-Expression of the LTC4 Synthase Gene in Mice Reproduces Human Aspirin-Induced Asthma

Over-Expression of the LTC4 Synthase Gene in Mice Reproduces Human Aspirin-Induced Asthma

Are Major Basic Protein, Eotaxin-3 and Leukotriene Enzyme Staining Useful for Diagnosing Eosinophilic Esophagitis? (Am J Gastroenterol 2012;107:1503-1511)

The diagnosis of eosinophilic esophagitis (EoE) is based on the clinical symptoms of esophageal dysfunction and histopathological findings of esophageal eosinophilia.1,2 In fact, the clinical, endoscopic, and histopathological characteristics of EoE and gastroesophageal reflux disease (GERD) overlap substantially, and differentiating the two can be difficult.1 Recently, Dellon et al1 suggested that tissue biomarkers of eosinophil activation or inflammation were useful for diagnosing EoE, especially for differentiating it from GERD.1 They performed immunohistochemistry for major basic protein (MBP), eotaxin-3, leukotriene A4 hydrolase (LTA4H) and leukotriene C4 synthase (LTC4S) in esophageal biopsy tissues from EoE and GERD patients and demonstrated that patients with EoE had much higher levels of MBP and eotaxin-3 in the esophageal epithelium than did GERD patients, while there were no differences in LTA4H and LTC4S.1 They evaluated the diagnostic utility of MBP and eotaxin-3 using receiver operating characteristic curves, and found that the combination of MBP and eotaxin-3 staining and the eosinophil numbers nearly completely predicted EoE status without considering other clinical, endoscopic or histopathological characteristics.1 The authors concluded that staining for MBP and eotaxin-3 might improve the current EoE diagnostic standards by adding a measure of eosinophil activity, especially for differentiating it from GERD.1

Association of ALOX5, LTA4H and LTC4S gene polymorphisms with ischemic stroke risk in a cohort of Chinese in east China.

Genetic variations of the 5-lipoxygenase activating protein and leukotriene A4 hydrolase genes that confer an increased risk of ischemic stroke have implicated the family of leukotrienes as potential mediators of ischemic stroke. This study aimed to explore the association of ALOX5, LTA4H and LTC4S gene polymorphisms with ischemic stroke risk in a cohort of Chinese in east China. This case-control study consisted of 690 patients with ischemic stroke and 690 controls. Polymorphisms of ALOX5 rs2029253 A/G, LTA4H rs6538697 T/C, and LTC4S rs730012 A/C were genotyped by the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. The multivariate logistic regression model was used to exclude the effects of conventional risk factors on ischemic stroke. Carriers of C allele in rs730012 were more susceptible to ischemic stroke (OR: 1.37; 95%CI: 1.08-1.73; P=0.009). The rs2029253 GG genotype showed a risk-reducing effect on ischemic stroke (OR: 0.72; 95%CI: 0.55-0.93; P=0.013) while the rs6538697 CC genotype had an increased risk of ischemic stroke (OR: 1.77; 95%CI: 1.09-2.89; P=0.022). The rs730012 variant was not associated with ischemic stroke risk after adjusting confounding factors (P>0.05). The present study suggested that gene polymorphisms in the leukotrienes pathway may exert influences, with independent genetic effects, on ischemic stroke susceptibility in a cohort of Chinese in east China.

Cysteinyl Leukotriene 2 Receptor on Dendritic Cells Negatively Regulates Ligand-Dependent Allergic Pulmonary Inflammation

Cysteinyl leukotrienes (cys-LTs) can mediate Th2 immunity to the house dust mite, Dermatophagoides farinae, via the type 1 receptor CysLT(1)R on dendritic cells (DCs). However, the role of the homologous type 2 receptor CysLT(2)R in Th2 immunity is unknown. D. farinae sensitization and challenge of CysLT(2)R-deficient mice showed a marked augmentation of eosinophilic pulmonary inflammation, serum IgE, and Th2 cytokines. Wild-type (WT) mice sensitized by adoptive transfer of D. farinae-pulsed CysLT(2)R-deficient bone marrow-derived DCs (BMDCs) also had a marked increase in D. farinae-elicited eosinophilic lung inflammation and Th2 cytokines in restimulated hilar nodes. This response was absent in mice sensitized with D. farinae-pulsed BMDCs lacking leukotriene C(4) synthase (LTC(4)S), CysLT(1)R, or both CysLT(2)R/LTC(4)S, suggesting that CysLT(2)R negatively regulates LTC(4)S- and CysLT(1)R-dependent DC-mediated sensitization. CysLT(2)R-deficient BMDCs had increased CysLT(1)R-dependent LTD(4)-induced ERK phosphorylation, whereas N-methyl LTC(4) activation of CysLT(2)R on WT BMDCs reduced such signaling. Activation of endogenously expressed CysLT(1)R and CysLT(2)R occurred over an equimolar range of LTD(4) and N-methyl LTC(4), respectively. Although the baseline expression of cell surface CysLT(1)R was not increased on CysLT(2)R-deficient BMDCs, it was upregulated at 24 h by a pulse of D. farinae, compared with WT or CysLT(2)R/LTC(4)S-deficient BMDCs. Importantly, treatment with N-methyl LTC(4) reduced D. farinae-induced CysLT(1)R expression on WT BMDCs. Thus, CysLT(2)R negatively regulates the development of cys-LT-dependent Th2 pulmonary inflammation by inhibiting both CysLT(1)R signaling and D. farinae-induced LTC(4)S-dependent cell surface expression of CysLT(1)R on DCs. Furthermore, these studies highlight how the biologic activity of cys-LTs can be tightly regulated by competition between these endogenously expressed receptors.

An inhibitor of leukotriene synthesis affects vasopressin secretion following osmotic stimulus in rats

Previous studies revealed the presence of LTC4 synthase in paraventricular vasopressinergic neurons, suggesting a role for leukotrienes (LTs) in certain neuroendocrine system functions. Our aim was to study the effect of an inhibitor of LT synthesis in the release of arginine vasopressin (AVP) following an osmotic stimulus in rats. Male Wistar rats received an intra-cerebroventricular injection of 2μl of the LT synthesis inhibitor MK-886 (1, 2, or 4μg/kg), or vehicle (DMSO 5%), 1h before an intraperitoneal injection of hypertonic saline (NaCl 2M) or isotonic saline (NaCl 0.01M) in a volume corresponding to 1% of body weight. Thirty minutes after the osmotic stimulus, the animals were decapitated and blood was collected for determining hematocrit, plasma osmolality and plasma AVP levels. As expected, the injection of hypertonic saline significantly increased (P<0.05) the hematocrit, plasma osmolality and plasma AVP levels. While inhibiting LT synthesis by central administration of MK-886 did not cause any additional increase in hematocrit or osmolality, plasma AVP levels were augmented (P<0.05). We conclude that central leukotrienes may have a modulatory role in AVP secretion following an osmotic stimulus, this deserving future studies.

Aspirin Exacerbated Respiratory Disease

Aspirin Exacerbated Respiratory Disease

Ischemic preconditioning decreased leukotriene C4 formation by depressing leukotriene C4 synthase expression and activity during hepatic I/R injury in rats

BackgroundExtensive experimental and clinical studies have shown that ischemic preconditioning (IP) can produce protective effects during hepatic ischemia reperfusion (I/R) injury. Our recent studies indicate that rat liver I/R injury is related to an abnormal increase in leukotriene (LT) C4 production. However, the mechanisms underlying IP actions on LTC4 generation during hepatic I/R injury remain to be explored. MethodsWe randomly divided adult male Sprague-Dawley rats into sham (control), I/R, and IP groups (n = 6). We subjected rat liver to 60 min partial hepatic ischemia followed by 5 h reperfusion with saline administered intravenously. We detected protein expression of LTC4 synthase (LTC4S) with Western blot, and measured LTC4 synthesis enzymes' activities and content by reverse-phase high-performance liquid chromatography. We assessed tissue injury using serum aspartate aminotransferase and aspartate aminotransferase activities and histologic changes. We examined liver tissue glutathione levels by a biochemical method. ResultsIschemic preconditioning markedly decreased LTC4 content, reduced LTC4S protein expressions, and inhibited LTC4 synthesis enzymes' activities in rat liver compared with the I/R group (P < 0.05). We also observed a decline in serum alanine aminotransferase and aspartate aminotransferase activities (P < 0.05), together with hepatic tissue glutathione elevation (P < 0.05) in the IP groups. Positive expression of LTC4S on hepatocytes and sinusoidal endothelial cells in the IP group was significantly lower than that in the I/R group. ConclusionsThese findings demonstrate that reduced LTC4 production by IP treatment during hepatic I/R injury could partially result from the down-regulation of LTC4S protein expression and the depression of LTC4 synthesis enzyme activity. They suggest that the beneficial effects of IP may be involved in repression of LTC4 generation during hepatic I/R injury.

The −444A/C Polymorphism in the LTC4S Gene and the Risk of Asthma: A Meta-analysis

The -444A/C polymorphism in the leukotriene C4 synthase (LTC4S) gene has been implicated in susceptibility to asthma, but a large number of studies have reported inconclusive results. The aim of this study was to investigate the association between the -444A/C polymorphism in the LTC4S gene and asthma risk using meta-analysis. We searched Pubmed, Embase, CNKI and Wanfang databases. Statistical analysis was performed using the software Revman4.2 and STATA10.0. A total of 3042 cases and 1902 controls in 13 case-control studies were included in the meta-analysis. The results indicated that the variant C allele carriers (CC+AC) did not have increased/decreased risk of asthma when compared with the homozygote AA (CC+AC vs. AA: OR=1.13, 95% CI=1.00-1.28, p=0.06). In the subgroup analysis by age, ethnicity and aspirin sensitivity, significantly elevated risks were found only in Caucasians (OR=1.21, 95% CI=1.02-1.44, p=0.03) and aspirin-tolerant populations (OR=1.36, 95% CI=1.12-1.65, p=0.002) but not in other subgroups. This meta-analysis suggested that the -444A/C polymorphism in the LTC4S gene would be a risk factor for asthma in Caucasians and aspirin-tolerant populations. Future studies are needed to validate our results.

Diagnostic Utility of Major Basic Protein, Eotaxin-3, and Leukotriene Enzyme Staining in Eosinophilic Esophagitis

Features of eosinophilic esophagitis (EoE) and gastroesophageal reflux disease (GERD) overlap. We aimed to determine whether staining for tissue biomarkers would differentiate EoE from GERD, suggesting utility for diagnosis of EoE. In this case-control study, EoE patients defined by consensus guidelines were compared to GERD patients with eosinophils on esophageal biopsy. Immunohistochemistry was performed for major basic protein (MBP), eotaxin-3, leukotriene A4 hydrolase (LTA4H), and leukotriene C4 synthase (LTC4S). After masking, the maximum staining density (cells per mm(2)) was quantified for each marker and compared between groups. Receiver operator characteristic curves were constructed, and the area under the curve (AUC) calculated to assess the diagnostic utility of each of the biomarkers alone and in combination with eosinophil counts. There were 51 EoE cases (mean age 24; mean 143 eosinophils per high-power field (eos per h.p.f.)) and 54 GERD controls (mean age 34; mean 20 eos per h.p.f.). The MBP density was higher in EoE than in GERD (1479 vs. 59 cells per mm(2); P<0.001), as was the eotaxin-3 density (2219 vs. 479; P<0.001). There were no differences for LTA4H and LTC4S. MBP density and eosinophil count correlated (R=0.81; P<0.001); correlation with eotaxin-3 was weaker (R=0.25; P=0.01). The AUC for diagnosis of EoE was 0.96 for MBP, 0.87 for eotaxin-3, 0.58 for LTA4H, 0.66 for LTC4S, and 0.99 for the combination of MBP, eotaxin-3, and eosinophil count. Patients with EoE had substantially higher levels of MBP and eotaxin-3 staining than GERD patients. These markers may have utility as a diagnostic assay for EoE.

Association of the –1072G/A Polymorphism in the LTC4S Gene with Asthma in an Indian Population

Background: Atopic asthma, the most common chronic disease affecting children and young adults, is a complex disorder with variable phenotypes. Cysteine leukotrienes (Cys-LTs) are powerful bronchoconstrictors and play a critical role in airway inflammation and remodeling that are characteristic of asthma. Objective: To investigate the association of ALOX5, LTC4S and CysLTR2 gene polymorphisms with atopic asthma in an Indian population. Methods: A total of 19 single nucleotide polymorphisms (SNPs) within these genes were genotyped in a family-based cohort (n = 239) and a case-control cohort (139 cases and 194 controls) followed by association analyses. Results: We found a significant association of the –1072G/A (rs3776944) SNP with atopic asthma in the family-based association analysis (p = 0.0004). These results were also replicated in the case-control cohort (p = 0.009). The allele A was negatively associated with atopic asthma. We also noted a significant association in the two-locus (rs3776944G/A and rs730012A/C) haplotypic analysis of this gene both in the family-based (p = 0.03) and the case-control (p = 0.02) analyses. Conclusion: This study supports the role of the LTC4S gene polymorphism in genetic susceptibility to atopic asthma in an Indian population.

Leukotriene C4 Synthase: Upcoming Drug Target For Inflammation

Leukotrienes are important mediators of pain and inflammation and they are produced in the arachidonic acid pathway via 5-lipoxygenase. They have been shown to have important roles in pyresis following antigen attack and in aspirin- intolerant asthma. They promote inflammation processes including eosinophil migration, increase in vascular permeability and bronchoconstriction. Hence, targeting the enzymes involved in the synthesis of these mediators can lead to the development of novel anti-inflammatory drugs. However, no drugs have yet been developed targeting leukotriene C4 synthase, a key enzyme leading to the synthesis of cysteinyl leukotrienes. The recent elucidation of its crystal structure now opens up the possibility of drugs against it. The inhibitors developed for this enzyme until now and the structural features responsible for their activity are discussed in this review. This understanding could lead to the design of new chemical entities.

PTU-186 Exogenous NSAID exposure increases transcriptional expression of the 5-lipoxygenase pathway in normal squamous tissue, but not in Barrett's metaplasia: an ex vivo study of oesophageal biopsy specimens

IntroductionBarrett's oesophagus (BO) is a premalignant condition for oesophageal adenocarcinoma, an increasingly common malignancy with poor survival. The prostanoids and leukotrienes are lipid inflammatory mediators derived from a common precursor...

Cysteinyl leukotriene overproduction in aspirin-exacerbated respiratory disease is driven by platelet-adherent leukocytes

AbstractCysteinyl leukotriene (cysLT) overproduction is a hallmark of aspirin-exacerbated respiratory disease (AERD), but its mechanism is poorly understood. Because adherent platelets can convert the leukocyte-derived precursor leukotriene (LT)A4 to LTC4, the parent cysLT, through the terminal enzyme LTC4 synthase, we investigated the contribution of platelet-dependent transcellular cysLT production in AERD. Nasal polyps from subjects with AERD contained many extravascular platelets that colocalized with leukocytes, and the percentages of circulating neutrophils, eosinophils, and monocytes with adherent platelets were markedly higher in the blood of subjects with AERD than in aspirin-tolerant controls. Platelet-adherent subsets of leukocytes had higher expression of several adhesion markers than did platelet nonadherent subsets. Adherent platelets contributed more than half of the total LTC4 synthase activity of peripheral blood granulocytes, and they accounted for the higher level of LTC4 generation by activated granulocytes from subjects with AERD compared with aspirin-tolerant controls. Urinary LTE4 levels, a measure of systemic cysLT production, correlated strongly with percentages of circulating platelet-adherent granulocytes. Because platelet adherence to leukocytes allows for both firm adhesion to endothelial cells and augmented transcellular conversion of leukotrienes, a disturbance in platelet-leukocyte interactions may be partly responsible for the respiratory tissue inflammation and the overproduction of cysLTs that characterize AERD.

Su1150 Leukotriene C4 Synthase mRNA Levels are Upregulated in Esophageal Biopsies of Patients With Eosinophilic Esophagitis

Su1150 Leukotriene C4 Synthase mRNA Levels are Upregulated in Esophageal Biopsies of Patients With Eosinophilic Esophagitis

Botanical Oil Supplementation Alters Insulin Levels in Diabetic/Metabolic Syndrome Subjects in a Genotypic‐Specific Manner

Dietary botanical seed oils may influence common diseases associated with systemic inflammation such as asthma and diabetes. We have previously determined the optimal dietary concentrations of two botanical seed oils from borage (Borago officinalis) and echium (Echium plantagineum) necessary to reduce ex vivo leukotriene generation in asthmatic subjects. The current study compared the impact of dietary supplementation with borage/echium and corn (placebo) or fish oil on insulin levels in 59 diabetes/metabolic syndrome subjects. Serum PUFA levels changed as expected based on the dietary oil (corn, botanical or fish) provided to subjects. When examined as a group, there was no statistical difference in insulin levels among the dietary groups. A variant LTC4 synthase genotype (−444 LTC4S or rs730012) has been identified that is associated with high levels of mRNA for LTC4S, leukotrienes and responsiveness to leukotriene blockers The borage/echium oil combination, but not placebo or fish oil, markedly increased insulin levels in individuals with the LTC4S variant. These data suggest that dietary supplementation with borage/echium oils alters insulin levels in diabetic/metabolic syndrome subjects in a genotypic‐specific manner. Supported by NIH PO50AT0027820.