Background and purpose: Although GPR55 is potently activated by the endogenous lysophospholipid, L-alysophosphatidylinositol (LPI), it is also thought to be sensitive to a number of cannabinoid ligands, including the prototypic CB1 receptor antagonists AM251 and SR141716A (Rimonabant ® ). In this study we have used a range of functional assays to compare the pharmacological activity of selected cannabinoid ligands, AM251, AM281 and SR141716A with LPI in a HEK293 cell line engineered to stably express recombinant, human GPR55. Experimental approach: We evaluated Ca 2+ signalling, stimulation of extracellular signal regulated kinase (ERK1/2) mitogen activated kinase MAP-kinases, induction of transcriptional regulators that are downstream of GPR55, including nuclear factor of activated T cells (NFAT), nuclear factor-kB (NF-kB) and cAMP response element binding protein (CREB), as well as receptor endocytosis. In addition, we assessed the suitability of a novel, label-free assay for GPR55 ligands that involves optical measurement of dynamic mass redistribution following receptor activation. Key results: GPR55 linked to a range of downstream signalling events and that the activity of GPR55 ligands was influenced by the functional assay employed, with differences in potency and efficacy observed. Conclusions and implications:Our data help to resolve some of the issues surrounding the pharmacology of cannabinoid ligands at GPR55 and highlight some differences in effector coupling associated with distinct GPR55 ligands. (2010) AM281, 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide; CREB, cAMP response element binding protein; ERK1/2, extracellular signal regulated kinase 1/2; G13, Ga13 protein; GFP, green fluorescent protein; GPR55, G protein-coupled receptor 55; GPR55-HEK293, stable HEK293 cells expressing 3xHA-GPR55; HA, haemaglutinin; HBS, HEPES-buffered saline; LPI, L-alysophosphatidylinositol; MAP, mitogen activated kinase; NFAT, nuclear factor of activated T British Journal of Pharmacology
The directional migration of neutrophils towards inflammatory mediators, such as chemokines and cannabinoids, occurs via the activation of seven transmembrane G protein coupled receptors (7TM/GPCRs) and is a highly organized process. A crucial role for controlling neutrophil migration has been ascribed to the cannabinoid CB 2 receptor (CB 2 R), but additional modulatory sites distinct from CB 2 R have recently been suggested to impact CB 2 R-mediated effector functions in neutrophils. Here, we provide evidence that the recently de-orphanized 7TM/GPCR GPR55 potently modulates CB 2 R-mediated responses. We show that GPR55 is expressed in human blood neutrophils and its activation augments the migratory response towards the CB 2 R agonist 2-arachidonoylglycerol (2-AG), while inhibiting neutrophil degranulation and reactive oxygen species (ROS) production. Using HEK293 and HL60 cell lines, along with primary neutrophils, we show that GPR55 and CB 2 R interfere with each other's signaling pathways at the level of small GTPases, such as Rac2 and Cdc42. This ultimately leads to cellular polarization and efficient migration as well as abrogation of degranulation and ROS formation in neutrophils. Therefore, GPR55 limits the tissueinjuring inflammatory responses mediated by CB 2 R, while it synergizes with CB 2 R in recruiting neutrophils to sites of inflammation.
Background: Lung eosinophilia is a hallmark of asthma, and eosinophils are believed to play a crucial role in the pathogenesis of allergic inflammatory diseases. Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are produced in high amounts in the gastrointestinal tract by commensal bacteria and can be absorbed into the bloodstream. Although there is recent evidence that SCFAs are beneficial in allergic asthma models, the effect on eosinophils has remained elusive. Objective: The role of SCFAs was investigated in human eosinophil function and a mouse model of allergic asthma. Methods: Eosinophils were purified from self-reported allergic or healthy donors. Migration, adhesion to the endothelium, and
BACKGROUND AND PURPOSEHeteromerization of GPCRs is key to the integration of extracellular signals and the subsequent cell response via several mechanisms including heteromer-selective ligand binding, trafficking and/or downstream signalling. As the lysophosphatidylinositol GPCR 55 (GPR55) has been shown to affect the function of the cannabinoid receptor subtype 2 (CB2 receptor) in human neutrophils, we investigated the possible heteromerization of CB2 receptors with GPR55. EXPERIMENTAL APPROACHThe direct interaction of human GPR55 and CB2 receptors heterologously expressed in HEK293 cells was assessed by co-immunoprecipitation and bioluminescence resonance energy transfer assays. The effect of cross-talk on signalling was investigated at downstream levels by label-free real-time methods (Epic dynamic mass redistribution and CellKey impedance assays), ERK1/2-MAPK activation and gene reporter assays. KEY RESULTSGPR55 and CB2 receptors co-localized on the surface of HEK293 cells, co-precipitated in membrane extracts and formed heteromers in living HEK293 cells. Whereas heteromerization led to a reduction in GPR55-mediated activation of transcription factors (nuclear factor of activated T-cells, NF-κB and cAMP response element), ERK1/2-MAPK activation was potentiated in the presence of CB2 receptors. CB2 receptor-mediated signalling was also affected by co-expression with GPR55. Label-free assays confirmed cross-talk between the two receptors.
Disruption of the blood-air barrier, which is formed by lung microvascular endothelial and alveolar epithelial cells, is a hallmark of acute lung injury. It was shown that alveolar epithelial cells release an unidentified soluble factor that enhances the barrier function of lung microvascular endothelial cells. In this study we reveal that primarily prostaglandin (PG) E2 accounts for this endothelial barrier-promoting activity. Conditioned media from alveolar epithelial cells (primary ATI-like cells) collected from BALB/c mice and A549 cells increased the electrical resistance of pulmonary human microvascular endothelial cells, respectively. This effect was reversed by pretreating alveolar epithelial cells with a cyclooxygenase-2 inhibitor or by blockade of EP4 receptors on endothelial cells, and in A549 cells also by blocking the sphingosine-1-phosphate1 receptor. Cyclooxygenase-2 was constitutively expressed in A549 cells and in primary ATI-like cells, and was upregulated by lipopolysaccharide treatment. This was accompanied by enhanced PGE2 secretion into conditioned media. Therefore, we conclude that epithelium-derived PGE2 is a key regulator of endothelial barrier integrity via EP4 receptors under physiologic and inflammatory conditions. Given that pharmacologic treatment options are still unavailable for diseases with compromised air-blood barrier, like acute lung injury, our data thus support the therapeutic potential of selective EP4 receptor agonists.
Gαi-coupled chemoattractant receptors, such as the 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) receptor (OXE-R), are able to switch on Gαiβγ protein-dependent and β-arrestin–related signaling traits. However, which of these signaling pathways are truly important for the chemoattractant functions in leukocytes is not clarified yet. As we recently reported, Gue1654 is a unique Gβγ-biased OXE-R antagonist having no inhibitory activity on Gαi-related signaling, which makes Gue1654 an unprecedented tool for assessing the involvement of G protein subunits in chemoattractant receptor function. β-arrestin2 recruitment was studied in OXE-R–overexpressing HEK293 cells using bioluminescence resonance energy transfer assays. Activation of leukocytes was assessed by flow cytometric assays and by immunofluorescence microscopy. Leukocyte capture to endothelial cells was addressed under physiological flow conditions. We found that Gue1654 blocks β-arrestin2 recruitment in HEK293 cells overexpressing OXE-R and ERK1/2 phosphorylation in human eosinophils and neutrophils. Furthermore, Gue1654 was able to prevent several 5-oxo-ETE–triggered functional events in eosinophils and neutrophils, such as activation of CD11b/CD18 integrins, oxidative burst, actin polymerization, and interaction with endothelial cells. In addition, Gue1654 completely prevented 5-oxo-ETE–induced Ca2+ flux and chemotaxis of human primary monocytes. All of these leukocyte responses to 5-oxo-ETE, except ERK1/2 phosphorylation and oxidative burst, were likewise prevented by pertussis toxin. Therefore, we conclude that chemoattractant receptors require Gαi subunits only as adaptors to transactivate the Gβγ heteromers, which then act responsible for cell activation. Finally, our data characterize Gue1654 as a non-Gαi–biased antagonist of OXE-R that provides a new basis for therapeutic intervention in inflammatory diseases that involve activation of eosinophils, neutrophils, and monocytes.
BACKGROUND AND PURPOSEPulmonary vascular dysfunction is a key event in acute lung injury. We recently demonstrated that PGE2, via activation of E-prostanoid (EP)4 receptors, strongly enhances microvascular barrier function in vitro. The aim of this study was to investigate the beneficial effects of concomitant EP4 receptor activation in murine models of acute pulmonary inflammation. EXPERIMENTAL APPROACHPulmonary inflammation in male BALB/c mice was induced by LPS (20 μg per mouse intranasally) or oleic acid (0.15 μL·g). In-vitro, endothelial barrier function was determined by measuring electrical impedance. KEY RESULTSPGE2 activation of EP4 receptors reduced neutrophil infiltration, pulmonary vascular leakage and TNF-α concentration in bronchoalveolar lavage fluid from LPS-induced pulmonary inflammation. Similarly, pulmonary vascular hyperpermeability induced by oleic acid was counteracted by EP4 receptor activation. In lung function assays, the EP4 agonist ONO AE1-329 restored the increased resistance and reduced compliance upon methacholine challenge in mice treated with LPS or oleic acid. In agreement with these findings, EP4 receptor activation increased the in vitro vascular barrier function of human and mouse pulmonary microvascular endothelial cells and diminished the barrier disruption induced by LPS. The EP2 agonist ONO AE1-259 likewise reversed LPS-induced lung dysfunction without enhancing vascular barrier function. CONCLUSION AND IMPLICATIONSOur results show that activation of the EP4 receptor strengthens the microvascular barrier function and thereby ameliorates the pathology of acute lung inflammation, including neutrophil infiltration, vascular oedema formation and airway dysfunction. This suggests a potential benefit for EP4 agonists in acute pulmonary inflammation.
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