The cysteinyl leukotriene 2 receptor contributes to all-trans retinoic acid-induced differentiation of colon cancer cells.
ABSTRACT: BACKGROUND: Cysteinyl leukotrienes (CysLTs) are potent pro-inflammatory mediators that are increased in samples from patients with inflammatory bowel diseases (IBDs). Individuals with IBDs have enhanced susceptibility to colon carcinogenesis. In colorectal cancer, the balance between the pro-mitogenic cysteinyl leukotriene 1 receptor (CysLT(1)R) and the differentiation-promoting cysteinyl leukotriene 2 receptor (CysLT(2)R) is lost. Further, our previous data indicate that patients with high CysLT(1)R and low CysLT(2)R expression have a poor prognosis. In this study, we examined whether the balance between CysLT(1)R and CysLT(2)R could be restored by treatment with the cancer chemopreventive agent all-trans retinoic acid (ATRA). METHODS: To determine the effect of ATRA on CysLT(2)R promoter activation, mRNA level, and protein level, we performed luciferase gene reporter assays, real-time polymerase chain reactions, and Western blots in colon cancer cell lines under various conditions. RESULTS: ATRA treatment induces CysLT(2)R mRNA and protein expression without affecting CysLT(1)R levels. Experiments using siRNA and mutant cell lines indicate that the up-regulation is retinoic acid receptor (RAR) dependent. Interestingly, ATRA also up-regulates mRNA expression of leukotriene C4 synthase, the enzyme responsible for the production of the ligand for CysLT(2)R. Importantly, ATRA-induced differentiation of colorectal cancer cells as shown by increased expression of MUC-2 and production of alkaline phosphatase, both of which could be reduced by a CysLT(2)R-specific inhibitor. CONCLUSIONS: This study identifies a novel mechanism of action for ATRA in colorectal cancer cell differentiation and demonstrates that retinoids can have anti-tumorigenic effects through their action on the cysteinyl leukotriene pathway.
Project description:Leukotrienes are pro-inflammatory mediators that are locally produced in coronary atherosclerotic plaques. The response induced by cysteinyl leukotrienes (CysLT) in human coronary arteries may be altered under pathological conditions, such as atherosclerosis. The aim of the present study was to elucidate cysteinyl leukotriene signaling in vascular smooth muscle cells (SMCs) and the effects of inflammation on this process. Immunohistochemical analysis of human carotid endarterectomy samples revealed that the CysLT(1) leukotriene receptor was expressed in areas that also stained positive for α-smooth muscle actin. In human coronary artery smooth muscle cells, lipopolysaccharide significantly upregulated the CysLT(1) receptor and significantly enhanced the changes in intracellular calcium induced by leukotriene C(4) (LTC(4)). In these cells, the CysLT(1) receptor exhibited a perinuclear expression, and LTC(4) stimulation predominantly enhanced nuclear calcium increase, which was significantly inhibited by the CysLT(1) receptor antagonist MK-571. Microarray analysis revealed, among a number of significantly upregulated genes after 24 h stimulation of human coronary artery smooth muscle cells with LTC(4), a 5-fold increase in mRNA levels for plasminogen activator inhibitor (PAI)-2. The LTC(4)-induced increase in PAI-2 expression was confirmed by real-time quantitative PCR and ELISA and was inhibited by the CysLT(1) receptor antagonist MK-571 and by calcium chelators. In summary, pro-inflammatory stimulation of vascular SMCs upregulated a perinuclear CysLT(1) receptor expression coupled to nuclear calcium signaling and changes in gene expression, such as upregulation of PAI-2. Taken together, these findings suggest a role of nuclear CysLT(1) receptor signaling in vascular SMCs inducing gene expression patterns associated with atherosclerosis.
Project description:Atopic dermatitis (AD) skin lesions exhibit epidermal and dermal thickening, eosinophil infiltration, and increased levels of the cysteinyl leukotriene (cys-LT) leukotriene C(4) (LTC(4)). Epicutaneous sensitization with ovalbumin of WT mice but not ?dblGATA mice, the latter of which lack eosinophils, caused skin thickening, collagen deposition, and increased mRNA expression of the cys-LT generating enzyme LTC(4) synthase (LTC(4)S). Skin thickening and collagen deposition were significantly reduced in ovalbumin-sensitized skin of LTC(4)S-deficient and type 2 cys-LT receptor (CysLT(2)R)-deficient mice but not type 1 cys-LT receptor (CysLT(1)R)-deficient mice. Adoptive transfer of bone marrow-derived eosinophils from WT but not LTC(4)S-deficient mice restored skin thickening and collagen deposition in epicutaneous-sensitized skin of ?dblGATA recipients. LTC(4) stimulation caused increased collagen synthesis by human skin fibroblasts, which was blocked by CysLT(2)R antagonism but not CysLT(1)R antagonism. Furthermore, LTC(4) stimulated skin fibroblasts to secrete factors that elicit keratinocyte proliferation. These findings establish a role for eosinophil-derived cys-LTs and the CysLT(2)R in the hyperkeratosis and fibrosis of allergic skin inflammation. Strategies that block eosinophil infiltration, cys-LT production, or the CysLT(2)R might be useful in the treatment of AD.
Project description:Two classes of human G protein-coupled receptors, cysteinyl leukotriene 1 (CysLT(1)) and CysLT(2) receptors, recently have been characterized and cloned. Because the CysLT(1) receptor blockers are effective in treating human bronchial asthma and the mouse is often used to model human diseases, we isolated the mouse CysLT(1) receptor from a mouse lung cDNA library and found two isoforms. A short isoform cDNA containing two exons encodes a polypeptide of 339 aa with 87.3% amino acid identity to the human CysLT(1) receptor. A long isoform has two additional exons and an in-frame upstream start codon resulting in a 13-aa extension at the N terminus. Northern blot analysis revealed that the mouse CysLT(1) receptor mRNA is expressed in lung and skin; and reverse transcription-PCR showed wide expression of the long isoform with the strongest presence in lung and skin. The gene for the mouse CysLT(1) receptor was mapped to band XD. Leukotriene (LT) D(4) induced intracellular calcium mobilization in Chinese hamster ovary cells stably expressing either isoform of the mouse CysLT(1) receptor cDNA. This agonist effect of LTD(4) was fully inhibited by the CysLT(1) receptor antagonist, MK-571. Microsomal membranes from each transformant showed a single class of binding sites for [(3)H]LTD(4); and the binding was blocked by unlabeled LTs, with the rank order of affinities being LTD(4) >> LTE(4) = LTC(4) >> LTB(4). Thus, the dominant mouse isoform with the N-terminal amino acid extension encoded by an additional exon has the same ligand response profile as the spliced form and the human receptor.
Project description:It has been hypothesized that Montelukast, a cysteinyl leukotriene (cysLT) receptor antagonist, with effects of anti-inflammatory, suppress oxidative stress and reduce affect cytokine production, may limited progression of the disease on COVID-19 infection.
Project description:The Influenza A virus (IAV) is a major human pathogen that produces significant morbidity and mortality. To explore the contribution of alveolar macrophages (AlvM?s) in regulating the severity of IAV infection we employed a murine model in which the Core Binding Factor Beta gene is conditionally disrupted in myeloid cells. These mice exhibit a selective deficiency in AlvM?s. Following IAV infection these AlvM? deficient mice developed severe diffuse alveolar damage, lethal respiratory compromise, and consequent lethality. Lethal injury in these mice resulted from increased infection of their Type-1 Alveolar Epithelial Cells (T1AECs) and the subsequent elimination of the infected T1AECs by the adaptive immune T cell response. Further analysis indicated AlvM?-mediated suppression of the cysteinyl leukotriene (cysLT) pathway genes in T1AECs in vivo and in vitro. Inhibition of the cysLT pathway enzymes in a T1AECs cell line reduced the susceptibility of T1AECs to IAV infection, suggesting that AlvM?-mediated suppression of this pathway contributes to the resistance of T1AECs to IAV infection. Furthermore, inhibition of the cysLT pathway enzymes, as well as blockade of the cysteinyl leukotriene receptors in the AlvM? deficient mice reduced the susceptibility of their T1AECs to IAV infection and protected these mice from lethal infection. These results suggest that AlvM?s may utilize a previously unappreciated mechanism to protect T1AECs against IAV infection, and thereby reduce the severity of infection. The findings further suggest that the cysLT pathway and the receptors for cysLT metabolites represent potential therapeutic targets in severe IAV infection.
Project description:Inflammation is an established risk factor for colorectal cancer. We and others have shown that colorectal cancer patients with elevated cysteinyl leukotriene receptor 2 (CysLT2R) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) levels exhibit good prognoses. However, both CysLT2R and 15-PGDH, which act as tumour suppressors, are often suppressed in colorectal cancer. We previously reported that leukotriene C4 (LTC4)-induced differentiation in colon cancer via CysLT2R signalling. Here, we investigated the involvement of Hedgehog (Hh)-GLI1 signalling, which is often hyperactivated in colorectal cancer. We found that the majority of colorectal cancer patients had high-GLI1 expression, which was negatively correlated with CysLT2R, 15-PGDH, and Mucin-2 and overall survival compared with the low-GLI1 group. LTC4-induced 15-PGDH downregulated both the mRNA and protein expression of GLI1 in a protein kinase A (PKA)-dependent manner. Interestingly, the LTC4-induced increase in differentiation markers and reduction in Wnt targets remained unaltered in GLI1-knockdown cells. The restoration of GLI1 in 15-PGDH-knockdown cells did not ameliorate the LTC4-induced effects, indicating the importance of both 15-PGDH and GLI1. LTC4-mediated reduction in the DCLK1 and LGR5 stemness markers in colonospheres was abolished in cells lacking 15-PGDH or GLI1. Both DCLK1 and LGR5 were highly increased in tumour tissue compared with the matched controls. Reduced Mucin-2 levels were observed both in zebrafish xenografts with GLI1-knockdown cells and in the cysltr2-/- colitis-associated colon cancer (CAC) mouse model. Furthermore, GLI1 expression was positively correlated with stemness and negatively correlated with differentiation in CRC patients when comparing tumour and mucosal tissues. In conclusion, restoring 15-PGDH expression via CysLT2R activation might benefit colorectal cancer patients.
Project description:Arachidonic acid can be metabolized to form a group of compounds known as the cysteinyl leukotrienes (CysLT) that bind to one of two receptors to mediate their actions. On circulating cells, expression of the leukotriene receptors is low, but in inflamed tissue the receptor number is dramatically increased. We hypothesized that the cytokine milieu present during inflammation can increase receptor expression on infiltrating immune cells. Various cell populations were purified from peripheral blood and stimulated in vitro with cytokines characteristic of allergic inflammatory disorders, and CysLT receptor expression was measured using quantitative PCR analysis, Western blot, and flow cytometry. IL-4, but not IL-13, was able to significantly induce mRNA and protein levels for both CysLT receptor 1 and 2 from T cells and B cells. CysLT2 receptor expression was also significantly increased in monocytes and eosinophils after IL-4 stimulation. Surprisingly, CysLT2 receptor expression was increased in monocytes, T cells, and B cells when IFN-gamma was used as the stimulus. Factors involved in eosinophil growth and survival were tested for their ability to alter CysLT receptor expression. These results support the concept that cytokines increase expression of both receptors on lymphocytes and granulocytes, allowing these cells to be more responsive to secreted leukotrienes at sites of inflammation.
Project description:BACKGROUND: Leukotriene D(4) (LTD(4)) belongs to the bioactive lipid group known as eicosanoids and has implications in pathological processes such as inflammation and cancer. Leukotriene D(4) exerts its effects mainly through two different G-protein-coupled receptors, CysLT(1) and CysLT(2). The high affinity LTD(4) receptor CysLT(1)R exhibits tumor-promoting properties by triggering cell proliferation, survival, and migration in intestinal epithelial cells. In addition, increased expression and nuclear localization of CysLT(1)R correlates with a poorer prognosis for patients with colon cancer. METHODOLOGY/PRINCIPAL FINDINGS: Using a proximity ligation assay and immunoprecipitation, this study showed that endogenous CysLT(1)R formed heterodimers with its counter-receptor CysLT(2)R under basal conditions and that LTD(4) triggers reduced dimerization of CysLTRs in intestinal epithelial cells. This effect was dependent upon a parallel LTD(4)-induced increase in CysLT(1)R tyrosine phosphorylation. Leukotriene D(4) also led to elevated internalization of CysLT(1)Rs from the plasma membrane and a simultaneous increase at the nucleus. Using sucrose, a clathrin endocytic inhibitor, dominant-negative constructs, and siRNA against arrestin-3, we suggest that a clathrin-, arrestin-3, and Rab-5-dependent process mediated the internalization of CysLT(1)R. Altering the CysLT(1)R internalization process at either the clathrin or the arrestin-3 stage led to disruption of LTD(4)-induced Erk1/2 activation and up-regulation of COX-2 mRNA levels. CONCLUSIONS/SIGNIFICANCE: Our data suggests that upon ligand activation, CysLT(1)R is tyrosine-phosphorylated and released from heterodimers with CysLT(2)R and, subsequently, internalizes from the plasma membrane to the nuclear membrane in a clathrin-, arrestin-3-, and Rab-5-dependent manner, thus, enabling Erk1/2 signaling and downstream transcription of the COX-2 gene.
Project description:Aquaporin-4 (AQP4), the main water channel protein in the brain, plays a critical role in water homeostasis and brain edema. Here, we investigated its role in the inflammatory responses after focal cerebral ischemia.In AQP4-knockout (KO) and wild-type mice, focal cerebral ischemia was induced by 30 min of middle cerebral arterial occlusion (MCAO). Ischemic neuronal injury and cellular inflammatory responses, as well as the expression and localization of cysteinyl leukotriene CysLT(2) and CysLT(1) receptors, were determined at 24 and 72 h after MCAO.AQP4-KO mice showed more neuronal loss, more severe microglial activation and neutrophil infiltration, but less astrocyte proliferation in the brain after MCAO than wild-type mice. In addition, the protein levels of both CysLT(1) and CysLT(2) receptors were up-regulated in the ischemic brain, and the up-regulation was more pronounced in AQP4-KO mice. The CysLT(1) and CysLT(2) receptors were primarily localized in neurons, microglia and neutrophils; those localized in microglia and neutrophils were enhanced in AQP4-KO mice.AQP4 may play an inhibitory role in postischemic inflammation.
Project description:Rationale: Acute exposure to ambient particle matter is associated with increased levels of the cysteinyl leukotriene (CysLT) biomarker, urinary leukotriene E4 (uLTE4), in subjects with asthma. Objectives: The role of CysLTs in mediating asthma worsening after particulate matter exposures was explored. Methods: Daily concentrations of particulate matter of 2.5 ?m and smaller diameter (PM2.5) and repeated measurements of albuterol use over a 5-month period were collected in 44 urban school children with persistent asthma. DNA was analyzed for gene polymorphisms on genes involved in the CysLT pathway to identify gene–environment interactions. An experimental challenge study in 16 adults with mild, nonpersistent asthma was performed to define biological pathways explaining these gene–environment interactions. Subjects in the challenge study were exposed on two different days to filtered air or diesel exhaust (300 ?g PM2.5/m3). FEV1 and CysLT-related gene DNA methylation and messenger RNA expression changes were measured before and 6 hours after exposure challenges. Results: In school children with asthma, associations between PM2.5 and school-time albuterol usage were significantly greater in those with the variant C allele in the CysLT receptor 1 (CysLTR1) rs320995 locus (5.4% increase per interquartile range PM2.5 increase) compared with those homozygous for the wild-type T allele (1.6% decrease; P?=?0.005 for allele?×?PM2.5 interaction). In the challenge study, declines in forced expiratory volume in 1 second after diesel exhaust exposure were associated with lower CysLTR1 expression (r2?=?0.52; P?=?0.01), which, in turn, was associated with decreased leukotriene C4 synthase cg1631890 (P?=?0.02) and increased CysLTR1 cg26848126 (P?=?0.01) methylation, as assessed in a multivariable model (r2?=?0.69). Conclusions: Health effects of acute particulate exposure on asthma are associated with changes in CysLTR1 expression and methylation of CpG sites on CysLTR1 and leukotriene C4 synthase genes.