Dysregulated Leukotriene Metabolism in Patients with COVID-19.

This study aimed to examine the leukotriene metabolism during COVID-19. In total, 180 participants were included in this study, of which 60 were healthy controls, 60 required intensive care units (ICU), and 60 did not require intensive care (non-ICU). The serum levels of 5-lipoxygenase (5-LO), 5-LO activating protein (ALOX5AP), and cysteinyl leukotriene (CYSLT) were measured, and the mRNA expression levels of 5-LO, ALOX5AP, and cysteinyl leukotriene receptor 1 (CYSLTR1) were investigated. Compared with the control group, both the non-ICU and ICU groups had lower levels of 5-LO and mRNA expression. ICU patients had lower levels of 5-LO and mRNA expression than non-ICU patients. CYSLTR1 mRNA expression was highest in the ICU group, followed by the non-ICU group, and healthy controls had the lowest mRNA expression levels. CYSLT levels were higher in the control group than in the non-ICU and ICU groups. CYSLTR1 expression was higher in patients than in controls; therefore, selective leukotriene receptor blockers can be used as treatment options. CYSLTR1 expression was higher in the ICU group than in the non-ICU group. Furthermore, CYSLTR1 mRNA expression may be a promising biomarker of COVID-19 severity.

Leukotriene D4 accelerates antigen-mediated mast cell responses via the cysteinyl leukotriene 1 receptor.

Cysteinyl leukotrienes (CysLTs), released from mast cells (MCs), are important mediators in allergy. Type 1 receptors for CysLTs (CysLT1R) are involved in accelerating IgE-mediated MC activation. In this study, we aimed to elucidate the mechanisms underlying CysLT1R-mediated MC activation. The CysLT1R agonist/antagonist was applied to two types of major MC models-RBL-2H3 cells and bone marrow-derived MCs (BMMCs). The use of CysLT1R and CysLT2R inhibitors revealed that CysLT1R plays a major role in the acceleration of MC activation. The administration of the CysLT1R agonist leukotriene D4 upregulated IgE-mediated Akt and ERK phosphorylation and subsequently enhanced TNF-α expression, suggesting that CysLT1R regulates the downstream pathway of MC activation. However, these observations were not corroborated by CysLT1R knockdown using shRNA, suggesting a differential regulatory mechanism between the temporal and constitutive inhibitions of CysLT. In conclusion, CysLT1R enhances MC activation by accelerating IgE-induced signal transduction, which enables the co-regulation of rapid degranulation and delayed synthesis of inflammatory mediators in MCs.

Antagonism of cysteinyl leukotriene receptors by zafirlukast modulated acute inflammatory reaction in tilapia, Oreochromis niloticus.

To identify activation pathways and effector mechanisms of innate immunity in fish has become relevant for the sanitary management of intensive fish farming. However, little is known about the blocking of cysteinyl leukotrienes receptors (CysLTRs) and their effects in teleost fish. Our study evaluated the anti-inflammatory effect of 250 and 500 µg zafirlukast (antagonist of CysLTRs)/kg b.w., administered orally in the diet, during acute inflammatory reaction induced by Aeromonas hydrophila bacterins in Oreochromis niloticus. 80 tilapia were distributed in 10 aquariums (100L of water each, n = 8) to constitute three treatments: Control (inoculated with A. hydrophila bacterin and untreated); Treated with 250 µg or 500 µg of zafirlukast/kg b.w. and inoculated. To be evaluated in three periods: 6, 24 and 48 h post-inoculation (HPI), totaling nine aquariums. A tenth group was sampled without any stimulus to constitute reference values (Physiological standards). Tilapia treated with zafirlukast demonstrated dose-response effect in the decrease of accumulated inflammatory cells, strongly influenced by granulocytes and macrophages. Zafirlukast treated-tilapia showed decrease in blood leukocyte counts (mainly neutrophils, and monocytes) and reactive oxygen species production. Treatment with zafirlukast resulted in down-regulation of ceruloplasmin, complement 3, alpha2-macroglobulin, transferrin and apolipoprotein A1, as well as up-regulation of haptoglobin. Our study provided convincing results in the pathophysiology of tilapia inflammatory reaction, considering that treatment with zafirlukast, antagonist of cysteinyl leukotriene receptors, resulted in a dose-response effect by suppressing the dynamics between leukocytes in the bloodstream and cell accumulation in the inflamed focus, as well as modulated the leukocyte oxidative burst and the acute phase protein response.

A Dual Role for Cysteinyl Leukotriene Receptors in the Pathogenesis of Corneal Infection.

Cysteinyl leukotrienes (CysLTs) have been defined as central mediators of inflammation. Despite our extensive understanding of these bioactive lipid mediators in the pathogenesis of diseases such as asthma, allergic rhinitis, and even neurological disorders, information regarding the eye is markedly lacking. As a result, this study examined the expression profiles of two major CysLT receptors, CysLT1 and CysLT2, in the cornea using experimental mouse models of Pseudomonas aeruginosa-induced keratitis with contrasting outcomes: susceptible C57BL/6 (B6) and resistant BALB/c. Postinfection, disparate levels of CysLT receptors were accompanied by distinct expression profiles for select proinflammatory and anti-inflammatory cell surface markers detected on macrophages and polymorphonuclear neutrophils between the two strains. Further, inhibition of either CysLT receptor converted the disease response of both strains, where corneal perforation was prevented in B6 mice, and BALB/c mice fared significantly worse. In addition, receptor antagonist studies revealed changes in inflammatory cell infiltrate phenotypes and an influence on downstream CysLT receptor signaling pathways. Although the B6 mouse model highlights the established proinflammatory activities related to CysLT receptor activation, results generated from BALB/c mice indicate a protective mechanism that may be essential to disease resolution. Further, basal expression levels of CysLT1 and CysLT2 were significantly higher in uninfected corneas of both mouse strains as opposed to during infection, suggestive of a novel role in homeostatic maintenance within the eye. In light of these findings, therapeutic targeting of CysLT receptors extends beyond inhibition of proinflammatory activities and may impact inflammation resolution, as well as corneal surface homeostasis.

Discovery of dual-target ligands binding to beta2-adrenoceptor and cysteinyl-leukotriene receptor for the potential treatment of asthma from natural products derived DNA-encoded library.

The design, synthesis, and discovery of dual-target compounds are considered as a promising strategy to develop new drugs with improved safety and efficacy compared with single-target drugs. This necessitates development of the methodologies that enable us to rapidly and accurately achieve the dual-target leads. Applying rosmarinic acid, 18ß-glycyrrhetinic acid, rhein, and ferulic acid as template building blocks, we introduced the self-assembling DNA encoded technique to build the library containing 1,000 compounds. These compounds were screened by receptor chromatography with immobilized beta2-adrenoceptor (ß2-AR) and cysteinyl-leukotriene receptor (CysLT), whereby we obtained a derivative of 18ß-glycyrrhetinic acid (XC267) that specifically binds to the two receptors. In vitro assessment demonstrated the desired binding affinity of 6.57 × 104 M-1 to ß2-AR, 2.82 × 104 M-1 to CysLT, and the dissociation rate constant of 7.52 s-1 to ß2-AR, 17.2 s-1 to CysLT. Pharmacological examination with ovalbumin-induced mice demonstrated that XC267 significantly reduced the levels of IL-4, IL-13, and IgE after oral administration of 10 mg/kg. By Western blot analysis, we observed an up-regulated expression of ß2-AR and a blocked level of CysLT with a dose-dependent manner in pulmonary bronchial. Our results suggest XC627 is a potential candidate to treat asthma by simultaneously regulating the signaling pathway of the two receptors.

Leukotriene C4 synthase is a novel PPARγ target gene, and leukotriene C4 and D4 activate adipogenesis through cysteinyl LT1 receptors in adipocytes.

Leukotriene (LT) C4 synthase (LTC4S) catalyzes the conversion from LTA4 to LTC4, which is a proinflammatory lipid mediator in asthma and other inflammatory diseases. LTC4 is metabolized to LTD4 and LTE4, all of which are known as cysteinyl (Cys) LTs and exert physiological functions through CysLT receptors. LTC4S is expressed in adipocytes. However, the function of CysLTs and the regulatory mechanism in adipocytes remain unclear. In this study, we investigated the expression of LTC4S and production of CysLTs in murine adipocyte 3T3-L1 cells and their underlying regulatory mechanisms. Expression of LTC4S and production of LTC4 and CysLTs increased during adipogenesis, whereas siRNA-mediated suppression of LTC4S expression repressed adipogenesis by reducing adipogenic gene expression. The CysLT1 receptor, one of the two LTC4 receptors, was expressed in adipocytes. LTC4 and LTD4 increased the intracellular triglyceride levels and adipogenic gene expression, and their enhancement was suppressed by co-treatment with pranlukast, a CysLT1 receptor antagonist. Moreover, the expression profiles of LTC4S gene/protein during adipogenesis resembled those of peroxisome proliferator-activated receptor (PPAR) γ. LTC4S expression was further upregulated by treatment with troglitazone, a PPARγ agonist. Promoter-luciferase and chromatin immunoprecipitation assays showed that PPARγ directly bound to the PPAR response element of the LTC4S gene promoter in adipocytes. These results indicate that the LTC4S gene expression was enhanced by PPARγ, and LTC4 and LTD4 activated adipogenesis through CysLT1 receptors in 3T3-L1 cells. Thus, LTC4S and CysLT1 receptors are novel potential targets for the treatment of obesity.

Paired comparisons of mutational profiles before and after brachytherapy in asian uveal melanoma patients.

Uveal melanoma(UM) is the most common primary intraocular malignancy in adults. However, the incidence of UM in Asia is 10 to 20 times less than in Western populations. Therefore, for the first time, we report our whole exome sequencing (WES) data analysis to discover differences in the molecular features of Asian and Western UM, and to determine the disparities between the primary tumor before brachytherapy and enucleated samples after brachytherapy. WES of 19 samples (13 primary tumors, 5 enucleation samples after brachytherapy, and 1 liver metastasis) from 13 patients diagnosed with UM and treated between 2007 and 2019 at the Yonsei University Health System (YUHS) were analyzed using bioinformatics pipelines. We identified significantly altered genes in Asian UM and changes in mutational profiles before and after brachytherapy using various algorithms. GNAQ, BAP1, GNA11, SF3B1 and CYSLTR2 were significantly mutated in Asian UM, which is similar that reported frequently in previous Western-based UM studies. There were also similar copy number alterations (M3, 1p loss, 6p gain, 8q gain) in both groups. In paired comparisons of the same patients, DICER1 and LRP1B were distinctly mutated only in tumor samples obtained after brachytherapy using rare-variant association tests (P = 0.01, 0.01, respectively). The mutational profiles of Asian UM were generally similar to the data from previous Western-based studies. DICER1 and LRP1B were newly mutated genes with statistical significance in the regrowth samples after brachytherapy compared to the primary tumors, which may be related to resistance to brachytherapy.

Bioinformatics analysis of GNAQ, GNA11, BAP1, SF3B1,SRSF2, EIF1AX, PLCB4, and CYSLTR2 genes and their role in the pathogenesis of Uveal Melanoma.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults, and its metastases are known to be fatal. It is critical to identify molecular markers to be used in potential prognostic evaluation for early diagnosis, treatment, and metastasis or to investigate all aspects of known genetic anomalies. Therefore, this study aimed to analyze the eight genes (GNAQ, GNA11, BAP1, SF3B1, SRSF2, EIF1AX, PLCB4, and CYSLTR2) that are associated with the most common genetic anomalies in UM from a molecular perspective. The genome sequences and expression profiles of 108 UM patients were obtained via bioinformatics tools that provide data from TCGA. The overall mutational load and the mutation patterns for eight genes, in particular, were thoroughly determined. Moreover, PolyPhen2 and SNAP2 tools were used to estimate the oncogenic/pathogenic properties of identified mutations for UM. In addition to the mutation profile, the effects of the presence of a mutation on gene expression and survival were determined. Finally, STRING network analysis was performed to better understand the functional relationships of mutated proteins in cellular processes. There were 27 missense mutations, 16 frameshift mutations, six nonsense mutations, and three splice region mutations among the 52 mutations found in eight genes, and 26 of them had pathogenic properties. BAP1 m-RNA expression was significantly lower in tumors with the mutant genotype (p = .001). The impact of gene expression, which has poor prognostic importance, on survival is statistically significant for high-expressed BAP1 (p = .0015) and low-expressed CYSLTR2 (p = .0021). To assess the current state of this potentially devastating disease, a molecular perspective has been evaluated. Defining this molecular perspective can be useful in developing targeted drug therapies and personalized medicine.

The relevance of CYSLTR1 gene polymorphism to the severity of allergic rhinitis and clinical responsiveness of montelukast in children.

PURPOSE: The etiology of allergic rhinitis (AR) is closely correlated with the complex interactions between genetic and environmental factors. This study explored the effect of single-nucleotide polymorphisms (SNPs) of CYSLTR1 gene on the risk of AR and clinical response to montelukast treatment in children. METHODS: A total of 135 children with AR and 100 healthy children were included for subsequent analyses. Genotype and allele distribution of rs321029 SNP of CYSLTR1 gene and inflammatory mediators were detected and compared between AR and healthy children. RESULTS: Genotype and allele frequency of rs321029 SNP of CYSLTR1 gene showed no difference between children with AR and controls or between AR cases with different severity. The total montelukast effective rate of wide-type genotype TT children was significantly higher than variants genotype CC children. CONCLUSION: Polymorphism of rs321029 on CYSLTR1 gene is not related to the susceptibility and severity of AR in children, but it is closely related with the efficacy of montelukast on AR.

Mechanistic insight on the role of leukotriene receptors in ischemic-reperfusion injury.

Leukotrienes (LT) are a class of inflammatory mediators produced by the 5-lipoxygenase (5-LO) enzyme from arachidonic acid (AA). We discussed the various LT inhibitors and downstream pathway modulators, such as Mitogen-Activated Protein Kinases (MAPK), Phosphatidylinositol 3-Kinase/Protein Kinase B (PI3K/Akt), 5'-Adenosine Monophosphate-Activated Protein Kinase (AMPK), Protein Kinase C (PKC), Nitric Oxide (NO), Bradykinin, Early Growth Response-1 (Egr-1), Nuclear Factor-κB (NF-κB), and Tumor Necrosis Factor-Alpha (TNF-α), which in turn regulate various metabolic and physiological processes involving I/R injury. A systematic literature review of Bentham, Scopus, PubMed, Medline, and EMBASE (Elsevier) databases was carried out to understand the nature and mechanistic interventions of the leukotriene receptor modulations in ischemic injury. In the pathophysiology of I/R injuries, LT has been found to play an important role. I/R injury affects most of the vital organs and is characterized by inflammation, oxidative stress, cell death, and apoptosis leading to morbidity and mortality. sThis present review focuses on the various LT receptors, i.e., CysLT, LTC4, LTD4, and LTE4, involved in developing I/R injury in organs, such as the brain, spinal cord, heart, kidney, liver, and intestine.

The CysLT2R receptor mediates leukotriene C4-driven acute and chronic itch.

Acute and chronic itch are burdensome manifestations of skin pathologies including allergic skin diseases and atopic dermatitis, but the underlying molecular mechanisms are not well understood. Cysteinyl leukotrienes (CysLTs), comprising LTC4, LTD4, and LTE4, are produced by immune cells during type 2 inflammation. Here, we uncover a role for LTC4 and its signaling through the CysLT receptor 2 (CysLT2R) in itch. Cysltr2 transcript is highly expressed in dorsal root ganglia (DRG) neurons linked to itch in mice. We also detected CYSLTR2 in a broad population of human DRG neurons. Injection of leukotriene C4 (LTC4) or its nonhydrolyzable form NMLTC4, but neither LTD4 nor LTE4, induced dose-dependent itch but not pain behaviors in mice. LTC4-mediated itch differed in bout duration and kinetics from pruritogens histamine, compound 48/80, and chloroquine. NMLTC4-induced itch was abrogated in mice deficient for Cysltr2 or when deficiency was restricted to radioresistant cells. Itch was unaffected in mice deficient for Cysltr1, Trpv1, or mast cells (WSh mice). CysLT2R played a role in itch in the MC903 mouse model of chronic itch and dermatitis, but not in models of dry skin or compound 48/80- or Alternaria-induced itch. In MC903-treated mice, CysLT levels increased in skin over time, and Cysltr2-/- mice showed decreased itch in the chronic phase of inflammation. Collectively, our study reveals that LTC4 acts through CysLT2R as its physiological receptor to induce itch, and CysLT2R contributes to itch in a model of dermatitis. Therefore, targeting CysLT signaling may be a promising approach to treat inflammatory itch.

Epigenome-wide association study on asthma and chronic obstructive pulmonary disease overlap reveals aberrant DNA methylations related to clinical phenotypes.

We hypothesized that epigenetics is a link between smoking/allergen exposures and the development of Asthma and chronic obstructive pulmonary disease (ACO). A total of 75 of 228 COPD patients were identified as ACO, which was independently associated with increased exacerbations. Microarray analysis identified 404 differentially methylated loci (DML) in ACO patients, and 6575 DML in those with rapid lung function decline in a discovery cohort. In the validation cohort, ACO patients had hypermethylated PDE9A (+ 30,088)/ZNF323 (- 296), and hypomethylated SEPT8 (- 47) genes as compared with either pure COPD patients or healthy non-smokers. Hypermethylated TIGIT (- 173) gene and hypomethylated CYSLTR1 (+ 348)/CCDC88C (+ 125,722)/ADORA2B (+ 1339) were associated with severe airflow limitation, while hypomethylated IFRD1 (- 515) gene with frequent exacerbation in all the COPD patients. Hypermethylated ZNF323 (- 296) / MPV17L (+ 194) and hypomethylated PTPRN2 (+ 10,000) genes were associated with rapid lung function decline. In vitro cigarette smoke extract and ovalbumin concurrent exposure resulted in specific DNA methylation changes of the MPV17L / ZNF323 genes, while 5-aza-2'-deoxycytidine treatment reversed promoter hypermethylation-mediated MPV17L under-expression accompanied with reduced apoptosis and decreased generation of reactive oxygen species. Aberrant DNA methylations may constitute a determinant for ACO, and provide a biomarker of airflow limitation, exacerbation, and lung function decline.

Involvement of mutant and wild-type CYSLTR2 in the development and progression of uveal nevi and melanoma.

BACKGROUND: Activating Gαq signalling mutations are considered an early event in the development of uveal melanoma. Whereas most tumours harbour a mutation in GNAQ or GNA11, CYSLTR2 (encoding G-protein coupled receptor CysLT2R) forms a rare alternative. The role of wild-type CysLT2R in uveal melanoma remains unknown. METHODS: We performed a digital PCR-based molecular analysis of benign choroidal nevi and primary uveal melanomas. Publicly available bulk and single cell sequencing data were mined to further study mutant and wild-type CYSLTR2 in primary and metastatic uveal melanoma. RESULTS: 1/16 nevi and 2/120 melanomas carried the CYSLTR2 mutation. The mutation was found in a subpopulation of the nevus, while being clonal in both melanomas. In the melanomas, secondary, subclonal CYSLTR2 alterations shifted the allelic balance towards the mutant. The resulting genetic heterogeneity was confirmed in distinct areas of both tumours. At the RNA level, further silencing of wild-type and preferential expression of mutant CYSLTR2 was identified, which was also observed in two CYSLTR2 mutant primary melanomas and one metastatic lesion from other cohorts. In CYSLTR2 wild-type melanomas, high expression of CYSLTR2 correlated to tumour inflammation, but expression originated from melanoma cells specifically. CONCLUSIONS: Our findings suggest that CYSLTR2 is involved in both early and late development of uveal melanoma. Whereas the CYSLTR2 p.L129Q mutation is likely to be the initiating oncogenic event, various mechanisms further increase the mutant allele abundance during tumour progression. This makes mutant CysLT2R an attractive therapeutic target in uveal melanoma.

CRISPR/Cas9-mediated CysLT1R deletion reverses synaptic failure, amyloidosis and cognitive impairment in APP/PS1 mice.

As a major pathological hallmark of Alzheimer's disease (AD), amyloid-ß (Aß) is regarded as a causative factor for cognitive impairment. Extensive studies have found Aß induces a series of pathophysiological responses, finally leading to memory loss in AD. Our previous results demonstrated that cysteinyl leukotrienes receptor 1 (CysLT1R) antagonists improved exogenous Aß-induced memory impairment. But the role of CysLT1R in AD and its underlying mechanisms still remain elusive. In this study, we investigated CysLT1R levels in AD patients and APP/PS1 mice. We also generated APP/PS1-CysLT1R-/- mice by clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated CysLT1R deletion in APP/PS1 mice and studied the effect of CysLT1R knockout on amyloidogenesis, synapse structure and plasticity, cognition, neuroinflammation, and kynurenine pathway. These attributes were also studied after lentivirus-mediated knockdown of CysLT1R gene in APP/PS1 mice. We found that CysLT1R knockout or knockdown could conserve synaptic structure and plasticity, and improve cognition in APP/PS1 mice. These effects were associated with concurrent decreases in amyloid processing, reduced neuroinflammation and suppression of the kynurenine pathway. Our study demonstrates that CysLT1R deficiency can mediate several beneficial effects against AD pathogenesis, and genetic/pharmacological ablation of this protein could be a potential therapeutic option for AD.

Montelukast alleviates inflammation in experimental autoimmune encephalomyelitis by altering Th17 differentiation in a mouse model.

Montelukast is a leukotriene receptor antagonist that is known to prevent allergic rhinitis and asthma. Blocking the Cysteinyl leukotriene receptor (CysLTR1), one of the primary receptors of leukotrienes, has been demonstrated to be efficacious in ameliorating experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), through disrupting chemotaxis of infiltrating T cells. However, the role of CysLTR1 in the pathogenesis of MS is not well understood. Here, we show that MS patients had higher expression of CysLTR1 in the circulation and central nervous system (CNS). The majority of CD4+ T cells expressed CysLTR1 in MS lesions. Among T-cell subsets, Th17 cells had the highest expression of CysLTR1, and blocking CysLTR1 signalling abrogated their development in vitro. Inhibition of CysLTR1 by montelukast suppressed EAE development in both a prophylactic and therapeutic manner and inhibited myelin loss in EAE mice. Similarly, the in vivo results showed that montelukast inhibited Th17 response in EAE mice and that Th17 cells treated with montelukast had reduced encephalitogenic in adoptive EAE. Our findings strongly suggest that targeting Th17 response by inhibiting CysLTR1 signalling could be a promising therapeutic strategy for the treatment of MS and CNS inflammatory diseases.

Methylation of cysteinyl leukotriene receptor 1 genes associates with lung function in asthmatics exposed to traffic-related air pollution.

Air pollution is associated with early declines in lung function and increased levels of asthma-related cysteinyl leukotrienes (CysLT) but a biological pathway linking this rapid response has not been delineated. In this randomized controlled diesel exhaust (DE) challenge study of 16 adult asthmatics, increased exposure-attributable urinary leukotriene E4 (uLTE4, a biomarker of cysteinyl leukotriene production) was correlated (p = 0.04) with declines in forced expiratory volume in 1-second (FEV1) within 6 hours of exposure. Exposure-attributable uLTE4 increases were correlated (p = 0.02) with increased CysLT receptor 1 (CysLTR1) methylation in peripheral blood mononuclear cells which, in turn, was marginally correlated (p = 0.06) with decreased CysLTR1 expression. Decreased CysLTR1 expression was, in turn, correlated (p = 0.0007) with FEV1 declines. During the same time period, increased methylation of GPR17 (a negative regulator of CysLTR1) was observed after DE exposure (p = 0.02); this methylation increase was correlated (p = 0.001) with decreased CysLTR1 methylation which, in turn, was marginally correlated (p = 0.06) with increased CysLTR1 expression; increased CysLTR1 expression was correlated (p = 0.0007) with FEV1 increases. Collectively, these data delineate a potential mechanistic pathway linking increased DE exposure-attributable CysLT levels to lung function declines through changes in CysLTR1-related methylation and gene expression.

Direct evidence that the GPCR CysLTR2 mutant causative of uveal melanoma is constitutively active with highly biased signaling.

Uveal melanoma is the most common eye cancer in adults and is clinically and genetically distinct from skin cutaneous melanoma. In a subset of cases, the oncogenic driver is an activating mutation in CYSLTR2, the gene encoding the G protein-coupled receptor cysteinyl-leukotriene receptor 2 (CysLTR2). The mutant CYSLTR2 encodes for the CysLTR2-L129Q receptor, with the substitution of Leu to Gln at position 129 (3.43). The ability of CysLTR2-L129Q to cause malignant transformation has been hypothesized to result from constitutive activity, but how the receptor could escape desensitization is unknown. Here, we characterize the functional properties of CysLTR2-L129Q. We show that CysLTR2-L129Q is a constitutively active mutant that strongly drives Gq/11 signaling pathways. However, CysLTR2-L129Q only poorly recruits ß-arrestin. Using a modified Slack-Hall operational model, we quantified the constitutive activity for both pathways and conclude that CysLTR2-L129Q displays profound signaling bias for Gq/11 signaling pathways while escaping ß-arrestin-mediated downregulation. CYSLTR2 is the first known example of a G protein-coupled receptor driver oncogene that encodes a highly biased constitutively active mutant receptor. These results provide new insights into the mechanism of CysLTR2-L129Q oncoprotein signaling and suggest CYSLTR2 as a promising potential therapeutic target in uveal melanoma.

Identification of cysteinyl-leukotriene-receptor 1 antagonists as ligands for the bile acid receptor GPBAR1.

The cysteinyl leukotrienes (CysLTs), i.e. LTC4, LTD4 and LTE4, are a family of proinflammatory agents synthesized from the arachidonic acid. In target cells, these lipid mediators bind to the cysteinyl leukotriene receptors (CysLTR), a family of seven transmembrane G-protein coupled receptors. The CysLT1R is a validated target for treatment of pulmonary diseases and several selective antagonists for this receptor, including montelukast, zafirlukast and pranlukast, have shown effective in the management of asthma. Nevertheless, others CysLT1R antagonists, such as the alpha-pentyl-3-[2-quinolinylmethoxy] benzyl alcohol (REV5901), have been extensively characterized without reaching sufficient priority for clinical development. Since drug reposition is an efficient approach for maximizing investment in drug discovery, we have investigated whether CysLT1R antagonists might exert off-target effects. In the report we demonstrate that REV5901 interacts with GPBAR1, a well characterized cell membrane receptor for secondary bile acids. REV5901 transactivates GPBAR1 in GPBAR1-transfected cells with an EC50 of 2.5 µM and accommodates the GPBAR1 binding site as shown by in silico analysis. Exposure of macrophages to REV5901 abrogates the inflammatory response elicited by bacterial endotoxin in a GPBAR1-dependent manner. In vivo, in contrast to montelukast, REV5901 attenuates inflammation and immune dysfunction in rodent models of colitis. The beneficial effects exerted by REV5901 in these models were abrogated by GPBAR1 gene ablation, confirming that REV5901, a shelved CysLT1R antagonist, is a GPBAR1 ligand. These data ground the basis for the development of novel hybrid ligands designed for simultaneous modulation of CysTL1R and GPBAR1.

Tumour-suppressive effect of oestrogen receptor ß in colorectal cancer patients, colon cancer cells, and a zebrafish model.

Oestrogen receptor ß (ERß) has been suggested to have anti-proliferative and anti-tumour effects in breast and prostate cancer cells, but other studies have indicated its tumour-promoting effects. Understanding the complex effects of this receptor in different contexts requires further study. We reported that high ERß expression is independently associated with improved prognosis in female colorectal cancer (CRC) patients. Herein, we investigated the possible anti-tumour effect of ERß and its selective agonist. CRC patients with high ERß expression had significantly higher levels of membrane-associated ß-catenin, cysteinyl leukotriene receptor 2 (CysLT2 R), and 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which have anti-tumour effects, but lower levels of nuclear ß-catenin, cysteinyl leukotriene receptor 1 (CysLT1 R), and cyclooxygenase-2 (COX-2), which have tumour-promoting effects. These interesting findings were further supported by two different publicly available CRC mRNA datasets that showed a significant positive correlation between ERß expression and 15-PGDH and CysLT2 R expression and a negative correlation between ERß expression and ß-catenin, CysLT1 R, and COX-2 expression. We next evaluated ERß expression in three different colon cancer mouse models; ERß expression was negatively correlated with tumourigenesis. Furthermore, treatment with the ERß-agonist ERB-041 reduced CysLT1 R, active ß-catenin, and COX-2 levels but increased phospho-ß-catenin, CysLT2 R, and 15-PGDH levels in HCT-116, Caco-2, and SW-480 colon cancer cells compared to vehicle-treated cells. Interestingly, ERB-041-treated cells showed significantly decreased migration, survival, and colonosphere formation and increased apoptotic activity, as indicated by increased CASPASE-3 and apoptotic blebs. Finally, patients with low ERß expression had significantly more distant metastasis at the time of diagnosis than patients with high ERß expression. Therefore, we studied the effects of ERB-041-treated colon cancer cells in a zebrafish xenograft model. We found significantly less distant metastasis of ERB-041-treated cells compared to vehicle-treated cells. These results further support ERß's anti-tumour role in CRC and the possible use of its agonist in CRC patients. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Epigenome-wide analysis of common warts reveals aberrant promoter methylation.

Epigenetic alteration of host DNA is a common occurrence in both low- and high-risk human papillomavirus (HPV) infection. Although changes in promoter methylation have been widely studied in HPV-associated cancers, they have not been the subject of much investigation in HPV-induced warts, which are a temporary manifestation of HPV infection. The present study sought to examine the differences in promoter methylation between warts and normal skin. To achieve this, DNA was extracted from 24 paired wart and normal skin samples and inputted into the Infinium MethylationEPIC BeadChip microarray. Differential methylation analysis revealed a clear pattern of hyper- and hypomethylation in warts compared to normal skin, and the most differentially methylated promoters were found within the EIF3EP2, CYSLTR1, C10orf99, KRT6B, LAMA4, and H3F3B genes as well as the C9orf30 pseudogene. Moreover, pathway analysis showed that the H3F3A, CDKN1A, and MAPK13 genes were the most common regulators among the most differentially methylated promoters. Since the tissue samples were excised from active warts, however, this differential methylation could either be a cellular response to HPV infection or an HPV-driven process to establish the wart and/or promote disease progression. Conclusively, it is apparent that HPV infection alters the methylation status of certain genes to possibly initiate the formation of a wart and maintain its presence.