Functional characterization of uveal melanoma oncogenes.

Uveal melanoma (UM) is a currently untreatable form of melanoma with a 50% mortality rate. Characterization of the essential signaling pathways driving this cancer is critical to develop target therapies. Activating mutations in the Gαq signaling pathway at the level of GNAQ, GNA11, or rarely CYSLTR2 or PLCß4 are considered alterations driving proliferation in UM and several other neoplastic disorders. Here, we systematically examined the oncogenic signaling output of various mutations recurrently identified in human tumors. We demonstrate that CYSLTR2 → GNAQ/11 → PLCß act in a linear signaling cascade that, via protein kinase C (PKC), activates in parallel the MAP-kinase and FAK/Yes-associated protein pathways. Using genetic ablation and pharmacological inhibition, we show that the PKC/RasGRP3/MAPK signaling branch is the essential component that drives the proliferation of UM. Only inhibition of the MAPK branch but not the FAK branch synergizes with inhibition of the proximal cascade, providing a blueprint for combination therapy. All oncogenic signaling could be extinguished by the novel GNAQ/11 inhibitor YM-254890, in all UM cells with driver mutation in the Gαq subunit or the upstream receptor. Our findings highlight the GNAQ/11 → PLCß â†’ PKC → MAPK pathway as the central signaling axis to be suppressed pharmacologically to treat for neoplastic disorders with Gαq pathway mutations.

Cysteinyl leukotriene receptor 1 modulates autophagic activity in retinal pigment epithelial cells.

The retinal pigment epithelium (RPE), which is among the tissues in the body that are exposed to the highest levels of phagocytosis and oxidative stress, is dependent on autophagy function. Impaired autophagy and continuous cellular stress are associated with various disorders, such as dry age-related macular degeneration (AMD), a disease for which effective therapies are lacking. Cysteinyl leukotriene receptor (CysLTR) 1 is a potential modulator of autophagy; thus, the aim of this study was to investigate the role of CysLTR1 in autophagy regulation in the RPE cell line ARPE-19. The polarized ARPE-19 monolayer exhibited expression of CysLTR1, which was colocalized with ß-tubulin III. In ARPE-19 cells, autophagic activity was rhythmically regulated and was increased upon CysLTR1 inhibition by Zafirlukast (ZK) treatment. H2O2 affected the proautophagic regulatory effect of ZK treatment depending on whether it was applied simultaneously with or prior to ZK treatment. Furthermore, mRNA levels of genes related to the leukotriene system, autophagy and the unfolded protein response were positively correlated. As CysLTR1 is involved in autophagy regulation under basal and oxidative stress conditions, a dysfunctional leukotriene system could negatively affect RPE functions. Therefore, CysLTR1 is a potential target for new treatment approaches for neurodegenerative disorders, such as AMD.

Cysteinyl Leukotriene Receptor 2 is Involved in Inflammation and Neuronal Damage by Mediating Microglia M1/M2 Polarization through NF-κB Pathway.

Microglia activation plays a key role in regulating inflammatory and immune reaction during cerebral ischemia and it exerts pro-inflammatory or anti-inflammatory effect depending on M1/M2 polarization phenotype. Cysteinyl leukotriene 2 receptor (CysLT2R) is a potent inflammatory mediator receptor, and involved in cerebral ischemic injury, but the mechanism of CysLT2R regulating inflammation and neuron damage remains unclear. Here, we found that LPS and CysLT2R agonist NMLTC4 significantly increased microglia proliferation and phagocytosis, up-regulated the mRNA expression of M1 polarization markers (IL-1ß, TNF-α, IFN-γ, CD86 and iNOS), down-regulated the expression of M2 polarization markers (Arg-1, CD206, TGF-ß, IL-10, Ym-1) and increased the release of IL-1ß and TNF-α. CysLT2R selective antagonist HAMI3379 could antagonize these effects. IL-4 significantly up-regulated the mRNA expression of M2 polarization markers, and HAMI3379 further increased IL-4-induced up-regulation of M2 polarization markers expression. Additionally, LPS and NMLTC4 stimulated NF-κB p50 and p65 proteins expression, and promoted p50 transfer to the nucleus. Pre-treatment with HAMI3379 and NF-κB signaling inhibitor Bay 11-7082 could reverse the up-regulation of p50 and p65 proteins expression, and inhibited p50 transfer to the nucleus. The conditional medium of BV-2 cells contained HAMI3379 could inhibit SH-SY5Y cells apoptosis induced by LPS and NMLTC4. These results were further confirmed in primary microglia. The findings indicate that CysLT2R was involved in inflammation and neuronal damage by inducing the activation of microglia M1 polarization and NF-κB pathway, inhibiting microglia M1 polarization and promoting microglia polarization toward M2 phenotype which may exerts neuroprotective effects, and targeting CysLT2R may be a new therapeutic strategy against cerebral ischemia stroke.

Nppb Neurons Are Sensors of Mast Cell-Induced Itch.

Itch is an unpleasant skin sensation that can be triggered by exposure to many chemicals, including those released by mast cells. The natriuretic polypeptide b (Nppb)-expressing class of sensory neurons, when activated, elicits scratching responses in mice, but it is unclear which itch-inducing agents stimulate these cells and the receptors involved. Here, we identify receptors expressed by Nppb neurons and demonstrate the functional importance of these receptors as sensors of endogenous pruritogens released by mast cells. Our search for receptors in Nppb neurons reveals that they express leukotriene, serotonin, and sphingosine-1-phosphate receptors. Targeted cell ablation, calcium imaging of primary sensory neurons, and conditional receptor knockout studies demonstrate that these receptors induce itch by the direct stimulation of Nppb neurons and neurotransmission through the canonical gastrin-releasing peptide (GRP)-dependent spinal cord itch pathway. Together, our results define a molecular and cellular pathway for mast cell-induced itch.

Leukotriene E4 induces airflow obstruction and mast cell activation through the cysteinyl leukotriene type 1 receptor.

BACKGROUND: Leukotriene (LT) E4 is the final active metabolite among the cysteinyl leukotrienes (CysLTs). Animal studies have identified a distinct LTE4 receptor, suggesting that current cysteinyl leukotriene type 1 (CysLT1) receptor antagonists can provide incomplete inhibition of CysLT responses. OBJECTIVE: We tested this hypothesis by assessing the influence of the CysLT1 antagonist montelukast on responses induced by means of inhalation of LTE4 in asthmatic patients. METHODS: Fourteen patients with mild intermittent asthma and 2 patients with aspirin-exacerbated respiratory disease received 20 mg of montelukast twice daily and placebo for 5 to 7 days in a randomized, double-blind, crossover study (NCT01841164). The PD20 value was determined at the end of each treatment period based on an increasing dose challenge. Measurements included lipid mediators in urine and sputum cells 4 hours after LTE4 challenge. RESULTS: Montelukast completely blocked LTE4-induced bronchoconstriction. Despite tolerating an at least 10 times higher dose of LTE4 after montelukast, there was no difference in the percentage of eosinophils in sputum. Urinary excretion of all major lipid mediators increased after LTE4 inhalation. Montelukast blocked release of the mast cell product prostaglandin (PG) D2, as well as release of PGF2α and thromboxane (Tx) A2, but not increased excretion of PGE2 and its metabolites or isoprostanes. CONCLUSION: LTE4 induces airflow obstruction and mast cell activation through the CysLT1 receptor.

Cysteinyl leukotriene receptor 1 regulates glucose-stimulated insulin secretion (GSIS).

Insulin resistance is an important pathological hallmark of type 2 diabetes mellitus. Glucose-stimulated insulin secretion (GSIS) plays a key role in maintaining blood glucose levels within normal range. Impaired GSIS has been associated with type 2 diabetes, however, the underlying molecular mechanisms remain largely unknown. Cysteinyl leukotriene receptor 1 (cysLT1R) is an important G protein-coupled receptor mediating the biological functions of cysteinyl leukotrienes (cys-LTs). Little is known about the effects of cysLT1R in insulin secretion and pathogenesis of T2DM. In the present study, we aimed to define the physiological functions of cysLT1R in GSIS in MIN6 ß-cells. Using reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, we found that cysLT1R was expressed in pancreatic MIN6 ß-cells. We also reported that glucose increased the expression of cysLT1R in MIN6 cells. Additionally, the cysLT1R antagonist montelukast promoted GSIS in a dose dependent manner, however, the cysLT1R agonist LD4 inhibited GSIS, suggesting an antagonistic effect of cysLT1R on GSIS. Silencing of cysLT1R by transfection with cysLT1R siRNA enhanced GSIS while overexpression of cysLT1R reduced GSIS in pancreatic MIN6 ß-cells. Mechanistically, we found that the Arf6/Cdc42/Rac1 pathway was involved in this process. Collectively, our findings highlight the essential role of cysLT1R in suppressing pancreatic insulin secretion, and potentially provided a new insight into understanding the mechanical regulation of glucose homeostasis.

Leukotriene Receptor Antagonists and Antiallergy Drugs.

As one of the candidates of the therapeutic strategy for asthma in addition to inhaled corticosteroids (ICS), leukotriene receptor antagonists (LTRAs) are known to be useful for long-term management of asthma patients complicated by allergic rhinitis (AR) or exercise-induced asthma (EIA). Currently available LTRAs are pranlukast hydrate, zafirlukast, and montelukast. These LTRAs have a bronchodilator action and inhibit airway inflammation, resulting in a significant improvement of asthma symptoms, respiratory function, inhalation frequency of as-needed inhaled ß2-agonist, airway inflammation, airway hyperresponsiveness, dosage of ICSs, asthma exacerbations, and patients' QOL. Although cys-LTs are deeply associated with the pathogenesis of asthma, LTRAs alone are less effective compared with ICS. However, the effects of LTRAs in combination with ICS are the same as those of LABAs in combination with ICS in steroid-naïve asthmatic patients. Concerning antiallergy drugs other than LTRAs, some mediator-release suppressants, H1 histamine receptor antagonists (H1RAs), thromboxane A2 (TXA2) inhibitors/antagonists, and Th2 cytokine inhibitor had been used mainly in Japan until the late 1990s. However, the use of these agents rapidly decreased after ICS/long acting beta agonist (LABA) combination was introduced and recommended for the management of asthma in the early 2000s. The effectiveness of other antiallergic agents on asthma management seems to be quite limited, and the safety of oral antiallergic agents has not been demonstrated in fetuses during pregnancy. Further effectiveness studies are needed to determine the true value of these orally administered agents in combination with ICS as an anti-asthma treatment.

High tumor mast cell density is associated with longer survival of colon cancer patients.

BACKGROUND: Inflammatory cells and inflammatory mediators play an important role in colorectal cancer (CRC). Previous studies have shown that CRC patients with increased expression of cysteinyl leukotriene receptor 1 (CysLTR1) have a poorer prognosis, and Cysltr1-/- mice display fewer intestinal polyps. However, the role of mast cells (MCs) in colon cancer progression remains unclear. The aim of the present study was to explore the relevance of MCs in CRC. MATERIAL AND METHODS: A tissue microarray from 72 CRC patients was stained with MC anti-tryptase and -chymase antibodies. Mouse colon tissue was stained with MC anti-tryptase antibody. Immunohistochemistry was used to identify MCs in patients and mice. RESULTS: Patient colon cancer tissue had in comparison with normal colon tissue a reduced number of MCs, predominantly of chymase-positive cells. Further analysis revealed that patients with a relative high MCD in their cancer tissues showed significantly longer overall survival compared to those with a low MCD [hazard ratio (HR) 0.539; 95% confidence interval (CI), 0.302-0.961]. Similar results were observed in subgroups of patients with either no distant metastasis (p = 0.004), or <75 years (p = 0.015) at time of diagnosis. Multivariate Cox analysis showed that MCD independently correlated with reduced risk of death in colon cancer patients (HR 0.380; 95% CI 0.202-0.713). Additionally, a negative correlation was found between cytoplasmic CysLTR1 expression and number of MCs. In agreement, in the CAC mouse model, Cysltr1-/- mice showed significantly higher MCs in their polyp/tumor areas compared with wild-type mice. CONCLUSION: A high MCD in cancer tissue correlated with longer patient survival independently from other risk factors for CRC. The concept that MCs have an anti-tumor effect in CRC is further supported by the findings of a negative correlation with CysLTR1 expression in patients and a high MCD in colon polyps/tumors from CysLTR1-/- mice.

6-Shogaol has anti-amyloidogenic activity and ameliorates Alzheimer's disease via CysLT1R-mediated inhibition of cathepsin B.

Although 6-shogaol, a constituent of ginger, has been reported to have anti-inflammatory and anti-oxidant effects on neuronal cells, the effects of 6-shogaol on Alzheimer's disease (AD) have not yet been investigated. Here we aimed to determine whether 6-shogaol exerts neuroprotective effects against AD. Specifically, we investigated the effects of 6-shogaol on the cysteinyl leukotriene 1 receptor (CysLT1R), a major factor in AD pathogenesis. Moreover, we clarified the relationship between CysLT1R and cathepsin B, a cysteine protease. We used in vitro and in vivo models to determine whether 6-shogaol inhibits CysLT1R/cathepsin B in an amyloid-beta (Aß; 1-42)-induced model of neurotoxicity. We first confirmed that CysLT1R and cathepsin B are upregulated by Aß (1-42) and that CysLT1R activation induces cathepsin B. In contrast, we found that 6-shogaol-mediated inhibition of CysLT1R downregulates cathepsin B in both in vitro and in vivo models. Furthermore, we found that 6-shogaol-mediated inhibition of CysLT1R/cathepsin B reduces Aß deposition in the brain and ameliorates behavioral deficits in APPSw/PS1-dE9 Tg mice. Our results indicate that 6-shogaol is a CysLT1R/cathepsin B inhibitor and is a novel potential therapeutic agent for the treatment of various neurodegenerative diseases, including AD.

Mechanosensitive Gq/11 Protein-Coupled Receptors Mediate Myogenic Vasoconstriction.

Myogenic vasoconstriction (Bayliss effect) is mediated by vascular smooth muscle cells (VSMCs) of small resistance arteries sensing mechanical forces. During the last three decades, several proteins have been proposed as VSMC mechanosensors. Our previous studies highlighted agonist-independent mechanical activation of Gq/11 protein-coupled receptors (Gq/11 PCRs) in VSMCs of resistance arteries. In particular, angiotensin II AT1 receptors (AT1 Rs) emerged as mechanosensors mediating myogenic tone. Moreover, we found that the AT1B receptor isoform was more mechanosensitive than the AT1A receptor. Interestingly, cysteinyl leukotriene 1 receptors (CysLT1 Rs) were up-regulated in AT1 R-deficient arteries as an essential backup strategy to compensate for the loss of vasoconstrictor receptors. Up-regulation of CysLT1 Rs resulted in increased myogenic tone at low intraluminal pressures resulting in hyperactivity of AT1 R-deficient arteries. Only at high intraluminal pressures myogenic tone was reduced, thus reflecting the loss of AT1 Rs. Further, CysLT1 Rs were involved in myogenic vasoconstriction of wild-type arteries. Simultaneous blockade of AT1 Rs and CysLT1 Rs in wild-type arteries caused reduction in myogenic tone of more than 60% comparable to the application of the selective Gq/11 -protein inhibitor YM-254890. Our findings suggest that AT1 Rs and CysLT1 Rs are crucial mechanosensors in resistance arteries mediating 60% of myogenic vasoconstriction via the Gq/11 -protein pathway without involvement of endogenous agonists.

Expression of CysLTR1 and 2 in Maturating Lymphocytes of Hyperplasic Tonsils Compared to Peripheral Cells in Children.

Cysteinyl-leukotriene receptors 1 and 2 (CysLTR1 and 2) are related to allergic inflammatory responses. Recent studies demonstrated their role in lymphocyte division and maturation in the bone marrow. Few data are available about CysLTRs function in lymphocyte maturation in tonsils. The objectives of this study are to compare CysLTRs expression in peripheral blood lymphocytes with expression in maturating lymphocytes of hyperplasic tonsil and to check the influence of respiratory allergies in this process. Leukocytes of peripheral blood (PL) and hyperplasic tonsils of children were immunostained for CysLTR1, CysLTR2, CD3 (T cells), and CD19 (B cells) and read in flow cytometer. Lymphocyte of tonsils were divided in differentiating small cells (SC) and mitotic large cells (LC); percentage of B and T cells expressing CysLTRs was determined, and comparison was done using ANOVA and Tukey's tests. Data were analyzed as a whole and categorizing patients according the presence of allergies. Sixty children were enrolled in this study. There was a large expression of CysLTR1 and 2 in CD3+ LC, and such expression decreased progressively in SC and PL. In B cells, the highest expression of CysLTR1 and 2 was found in PL while SC showed the lowest and LC showed the intermediate expression. This pattern kept unchanged in groups of allergic and non-allergic individuals. CysLTRs seem to be involved in lymphocyte maturation that occurs in tonsils, without influence of allergies. New studies aiming the clinic treatment of tonsil hyperplasia must be targeted to the development of drugs capable of blocking both CysLTR1 and 2.

Molecular mechanisms of target recognition by lipid GPCRs: relevance for cancer.

Over the past decade the importance of lipids for cancer cell metabolism and cancer-related processes such as proliferation, metastasis and chemotherapy resistance has become more apparent. The mechanisms by which lipid signals are transduced are poorly understood, but frequently involve G-protein Coupled Receptors (GPCRs), which can be explored as druggable targets. Here, we discuss how GPCRs recognize four classes of cancer-relevant lipids (lysophospholipids, phospholipids, fatty acids and eicosanoids). We compare the ligand-binding properties of >50 lipid receptors, we examine how their dysregulation contributes to tumorigenesis and how they may be therapeutically exploited.

[Expression of CysLTR-1 and CysLTR-2 in adenoid tissues from children with adenoid hypertrophy].

OBJECTIVE: To examine the expression of cysteinyl leukotriene receptor-1 (CysLTR-1) and cysteinyl leukotriene receptor-2 (CysLTR-2) in the adenoid tissues from children with adenoid hypertrophy (AH) and to explore the role of leukotrienes in the pathogenesis of AH. METHODS: Sixty children with AH who were treated by adenoidectomy and/or tonsillectomy were classified into two groups: simple AH and AH plus allergic rhinitis (n=30 each). Twenty children who underwent tonsillectomy due to recurrent purulent tonsillitis were selected as the control group. The expression of CysLTR-1 and CysLTR-2 in the excised tonsil and/or adenoid tissues was determined by immunofluorescence histochemical labeling and integrated optical density measurement. RESULTS: The expression of CysLTR-1 and CysLTR-2 in the adenoid and tonsil tissues increased significantly in both the simple AH group and AH plus allergic rhinitis group compared with the control group (P<0.01). The expression of CysLTR-1 and CysLTR-2 in the AH plus allergic rhinitis group increased more significantly compared with the simple AH group (P<0.01). CONCLUSIONS: CysLTR-1 and CysLTR-2 are highly expressed in the adenoid tissues from children with AH, suggesting that leukotrienes are involved in the pathogenesis of AH.

Leukotriene C4 induces bronchoconstriction and airway vascular hyperpermeability via the cysteinyl leukotriene receptor 2 in S-hexyl glutathione-treated guinea pigs.

Cysteinyl leukotrienes act through G-protein-coupled receptors termed cysteinyl leukotriene 1 (CysLT1) and cysteinyl leukotriene 2 (CysLT2) receptors. However, little is known about the pathophysiological role of CysLT2 receptors in asthma. To elucidate the possible involvement of CysLT2 receptors in bronchoconstriction and airway vascular hyperpermeability, we have established a novel guinea pig model of asthma. In vitro study confirmed that CHO-K1 cells, expressing guinea pig CysLT2 and CysLT1 receptors are selectively stimulated by LTC4 and LTD4, respectively. However, when LTC4 was intravenously injected to guinea pigs, the resulting bronchoconstriction was fully abrogated by montelukast, a CysLT1 receptor antagonist, indicating rapid metabolism of LTC4 to LTD4 in the lung. We found that treatment with S-hexyl glutathione (S-hexyl GSH), an inhibitor of gamma-glutamyl transpeptidase, significantly increased LTC4 content and LTC4/(LTD4 plus LTE4) ratio in the lung. Under these circumstances, LTC4-induced bronchoconstriction became resistant to montelukast, but sensitive to Compound A, a CysLT2 receptor antagonist, depending on the dose of S-hexyl GSH. Combination with montelukast and Compound A completely abrogated this spasmogenic response. Additionally, we confirmed that LTC4 elicits airway vascular hyperpermeability via CysLT2 receptors in the presence of high dose of S-hexyl GSH as evidenced by complete inhibition of LTC4-induced hyperpermeability by Compound A, but not montelukast. These results suggest that CysLT2 receptors mediate bronchoconstriction and airway vascular hyperpermeability in guinea pigs and that the animal model used in this study may be useful to elucidate the functional role of CysLT2 receptors in various diseases, including asthma.

House dust mites induce proliferation of severe asthmatic smooth muscle cells via an epithelium-dependent pathway.

RATIONALE: Asthma is a frequent airway disease, and asthma control determinants have been associated with indoor allergen sensitization. The most frequent allergens are house dust mites (HDM), which act in vivo on the bronchial epithelial layer. Severe asthma has also been associated with bronchial remodeling and more specifically with increased mass of bronchial smooth muscle (BSM). However, the relationship between HDM stimulation of the bronchial epithelial layer and BSM remodeling is unknown. OBJECTIVES: To evaluate whether epithelial stimulation with HDM induces BSM cell proliferation in subjects with severe asthma. METHODS: A total of 22 subjects with severe asthma and 27 subjects with no asthma were recruited. We have developed an in vitro culture model combining an epithelium layer in air-liquid interface (ALI) interacting with BSM. We assessed BSM proliferation using BrdU incorporation. We explored the role of epithelium-derived mediators using reverse-transcriptase polymerase chain reaction (RT-PCR) and ELISA in vitro and in vivo. Finally, leukotrienes receptor expression was assessed in vitro by flow cytometry and RT-PCR and ex vivo by laser microdissection and RT-PCR. MEASUREMENTS AND MAIN RESULTS: We found that epithelial stimulation by HDM selectively increased the proliferation of asthmatic BSM cells and not that of nonasthmatic cells. The mechanism involved epithelial protease-activated receptor-2-dependent production of leukotrienes C4 associated with an overexpression of leukotrienes receptor CysLTR1 by asthmatic BSM cells in vitro and ex vivo. CONCLUSIONS: This work demonstrates the selective role of HDM on BSM remodeling in patients with severe asthma and points out different therapeutic targets at epithelial and smooth muscle levels.

Factors driving the aspirin exacerbated respiratory disease phenotype.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is explained in part by overexpression of 5-lipoxygenase and leukotriene C4 synthase (LTC4S), resulting in constitutive overproduction of cysteinyl leukotrienes (CysLTs) and driving the surge in CysLT production that occurs with aspirin ingestion. Similarly, AERD is characterized by the overexpression of CysLT receptors. Increased levels of both interleukin (IL)-4 and interferon (IFN)-γ are present in the tissue of AERD subjects. Previous studies demonstrated that IL-4 is primarily responsible for the up-regulation of LTC4S by mast cells. METHODS: Literature review. RESULTS: Our previous studies demonstrated that IFN-γ, but not IL-4, drives this process in eosinophils. These published studies also extend to both IL-4 and IFN-γ the ability to up-regulate CysLT receptors. Prostaglandin E2 (PGE2) acts to prevent CysLT secretion by inhibiting mast cell and eosinophil activation. PGE2 concentrations are reduced in AERD, and our published studies confirm that this reflects diminished expression of cyclooxygenase (COX)-2. A process again that is driven by IL-4. Thus, IL-4 and IFN-γ together play an important pathogenic role in generating the phenotype of AERD. Finally, induction of LTC4S and CysLT1 receptors by IL-4 reflects in part the IL-4-mediated activation of signal transducer and activator of transcription 6 (STAT6). Our previous studies demonstrated that aspirin blocks trafficking of STAT6 into the nucleus and thereby prevents IL-4-mediated induction of these transcripts, thereby suggesting a modality by which aspirin desensitization could provide therapeutic benefit for AERD patients. CONCLUSION: This review will examine the evidence supporting this model.

Montelukast rescues primary neurons against Aß1-42-induced toxicity through inhibiting CysLT1R-mediated NF-κB signaling.

Amyloid-ß peptide (Aß), which can invoke a cascade of inflammatory responses, is considered to play a causal role in the development and progress of Alzheimer's disease (AD). Montelukast, known as a cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, is currently used for treatment of inflammatory diseases such as asthma. We have previously reported that CysLT1R activation is involved in Aß generation. In this study, we investigated rescuing effect of CysLT1R antagonist montelukast on Aß1-42-induced neurotoxicity in primary neurons. Our data showed that Aß1-42 elicited a marked increase of CysLT1R expression in primary mouse neurons. This increment of CysLT1R expression was accompanied by increases of inflammatory factors such as NF-κB p65, tumor necrosis factor-α (TNFα) and interleukin-1ß (IL-1ß) as well as pro-apoptotic protein Caspase-3 activation and anti-apoptosis protein Bcl-2 reduction. Aß1-42-mediated increase of CysLT1R expression was associated with Aß1-42-induced cytotoxicity as measured by MTT reduction assay and lactate dehydrogenase (LDH) release assay. This observation was confirmed with treatment of montelukast, a selective CysLT1R antagonist, which had significant effect on Aß1-42-induced cytotoxicity. Moreover, blockade of CysLT1R with montelukast reversed Aß1-42-mediated increase of CysLT1R expression, and concomitant changes of the pro-inflammatory factors and the apoptosis-related proteins. The results demonstrate that montelukast rescued neurons against Aß1-42-induced neurotoxicity, neuroinflammation and apoptosis by down-regulating CysLT1R-mediated NF-κB signaling, suggesting that CysLT1R may be a potential target for AD, and its antagonist may have beneficial effects for treatment of AD.

Cysteinyl leukotriene receptor 1 mediates LTD4-induced activation of mouse microglial cells in vitro.

AIM: To investigate the roles of cysteinyl leukotriene receptors CysLT1R and CysLT2R in leukotriene D4 (LTD4)-induced activation of microglial cells in vitro. METHODS: Mouse microglial cell line BV2 was transfected with pcDNA3.1(+)-hCysLT1R or pcDNA3.1(+)-hCysLT2R. The expression of relevant mRNAs and proteins in the cells was detected using RT-PCR and Western blotting, respectively. Phagocytosis was determined with flow cytometry analysis. The release of interleukin-1ß (IL-1ß) from the cells was measured using an ELISA assay. RESULTS: The expression of CysLT1R or CysLT2R was considerably increased in the transfected BV2 cells, and the receptors were mainly distributed in the plasma membrane and cytosol. Treatment of the cells expressing CysLT1R or CysLT2R with CysLT receptor agonist LTD4 (0.1-100 nmol/L) concentration-dependently enhanced the phagocytosis, and increased mRNA expression and release of IL-1ß. Moreover, the responses of hCysLT1R-BV2 cells to LTD4 were significantly larger than those of hCysLT2R-BV2 or WT-BV2 cells. Pretreatment of hCysLT1R-BV2 cells with the selective CysLT1R antagonist montelukast (1 µmol/L) significantly blocked LTD4-induced phagocytosis as well as the mRNA expression and release of IL-1ß, whereas the selective CysLT2R antagonist HAMI 3379 (1 µmol/L) had no such effects. CONCLUSION: CysLT1R mediates LTD4-induced activation of BV2 cells, suggesting that CysLT1R antagonists may exert anti-inflammatory activity in brain diseases.

Involvement of cysteinyl leukotriene receptor 1 in Aß1-42-induced neurotoxicity in vitro and in vivo.

Accumulation of amyloid-ß (Aß) is thought to be associated with the progressive neuronal death observed in Alzheimer's disease, but the mechanisms underlying neurotoxicity triggered by Aß remain elusive. In the current study, we investigated the roles of cysteinyl leukotriene receptor 1 (CysLT1R) in Aß1-42-induced neurotoxicity in vitro or in vivo. In vitro exposure of mouse primary neurons to Aß1-42 caused a gradual increases in CysLT1R expression. In vivo bilateral intrahippocampal injection of Aß1-42 also elicited time-dependent increases of CysLT1R expression in the hippocampus and cortex of mice. The CysLT1R antagonist pranlukast not only reversed Aß1-42-induced upregulation of CysLT1R, but also suppressed Aß1-42-triggered neurotoxicity evidenced by enhanced nuclear factor-kappa B p65, activated caspase-3, decreased B-cell lymphoma-2 and cell viability and impaired memory. Furthermore, chronic treatment with pranlukast produced similar beneficial effects on memory behavior and hippocampal long-term potentiation to memantine or donepezil in intrahippocampal Aß1-42-injected mice. Our data indicate that CysLT1R is involved in Aß1-42-induced neurotoxicity, and that blockade of CysLT1R, such as application of CysLT1R antagonist, could be a novel and promising strategy for the treatment of Alzheimer's disease.

Neutralization of leukotriene C4 and D4 activity by monoclonal and single-chain antibodies.

BACKGROUND: Cysteinyl leukotrienes (LTs) are key mediators in inflammation. To explore the structure of the antigen-recognition site of a monoclonal antibody against LTC4 (mAbLTC), we previously isolated full-length cDNAs for heavy and light chains of the antibody and prepared a single-chain antibody comprising variable regions of these two chains (scFvLTC). METHODS: We examined whether mAbLTC and scFvLTC neutralized the biological activities of LTC4 and LTD4 by competing their binding to their receptors. RESULTS: mAbLTC and scFvLTC inhibited their binding of LTC4 or LTD4 to CysLT1 receptor (CysLT1R) and CysLT2 receptor (CysLT2R) overexpressed in Chinese hamster ovary cells. The induction by LTD4 of monocyte chemoattractant protein-1 and interleukin-8 mRNAs in human monocytic leukemia THP-1 cells expressing CysLT1R was dose-dependently suppressed not only by mAbLTC but also by scFvLTC. LTC4- and LTD4-induced aggregation of mouse platelets expressing CysLT2R was dose-dependently suppressed by either mAbLTC or scFvLTC. Administration of mAbLTC reduced pulmonary eosinophil infiltration and goblet cell hyperplasia observed in a murine model of asthma. Furthermore, mAbLTC bound to CysLT2R antagonists but not to CysLT1R antagonists. CONCLUSIONS: These results indicate that mAbLTC and scFvLTC neutralize the biological activities of LTs by competing their binding to CysLT1R and CysLT2R. Furthermore, the binding of cysteinyl LT receptor antagonists to mAbLTC suggests the structural resemblance of the LT-recognition site of the antibody to that of these receptors. GENERAL SIGNIFICANCE: mAbLTC can be used in the treatment of inflammatory diseases such as asthma.