SUCNR1 Is Expressed in Human Placenta and Mediates Angiogenesis: Significance in Gestational Diabetes.

Placental hypervascularization has been reported in pregnancy-related pathologies such as gestational diabetes mellitus (GDM). Nevertheless, the underlying causes behind this abnormality are not well understood. In this study, we addressed the expression of SUCNR1 (cognate succinate receptor) in human placental endothelial cells and hypothesized that the succinate-SUCNR1 axis might play a role in the placental hypervascularization reported in GDM. We measured significantly higher succinate levels in placental tissue lysates from women with GDM relative to matched controls. In parallel, SUCNR1 protein expression was upregulated in GDM tissue lysates as well as in isolated diabetic fetoplacental arterial endothelial cells (FpECAds). A positive correlation of SUCNR1 and vascular endothelial growth factor (VEGF) protein levels in tissue lysates indicated a potential link between the succinate-SUCNR1 axis and placental angiogenesis. In our in vitro experiments, succinate prompted hallmarks of angiogenesis in human umbilical vein endothelial cells (HUVECs) such as proliferation, migration and spheroid sprouting. These results were further validated in fetoplacental arterial endothelial cells (FpECAs), where succinate induced endothelial tube formation. VEGF gene expression was increased in response to succinate in both HUVECs and FpECAs. Yet, knockdown of SUCNR1 in HUVECs led to suppression of VEGF gene expression and abrogated the migratory ability and wound healing in response to succinate. In conclusion, our data underline SUCNR1 as a promising metabolic target in human placenta and as a potential driver of enhanced placental angiogenesis in GDM.

Prostaglandin D2 strengthens human endothelial barrier by activation of E-type receptor 4.

Life-threatening inflammatory conditions such as acute respiratory distress syndrome or sepsis often go hand in hand with severe vascular leakage. During inflammation, endothelial cell integrity and intact barrier function are crucial to limit leukocyte and plasma extravasation. Prostaglandin D2 (PGD2) is a potent inflammatory lipid mediator with vasoactive properties. Previous studies suggest that PGD2 is involved in the regulation of endothelial barrier function; however, it is unclear whether this is also true for primary human pulmonary microvascular endothelial cells. Furthermore, as PGD2 is a highly promiscuous ligand, we set out to determine which receptors are important in human pulmonary endothelial cells. In the current study, we found that PGD2 and the DP1 agonist BW245c potently strengthened pulmonary and dermal microvascular endothelial cell barrier function and protected against thrombin-induced barrier disruption. Yet surprisingly, these effects were mediated only to a negligible extent via DP1 receptor activation. In contrast, we observed that the EP4 receptor was most important and mediated the barrier enhancement by PGD2 and BW245c. Stimulation with PGE2 or PGD2 reduced AKT phosphorylation which could be reversed by prior blockade of EP4 receptors. These data demonstrate a novel mechanism by which PGD2 may modulate inflammation and emphasizes the role of EP4 receptors in human endothelial cell function.

Butyrate ameliorates allergic airway inflammation by limiting eosinophil trafficking and survival.

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 eosinophil survival were studied in vitro. Ca2+ flux, apoptosis, mitochondrial membrane potential, and expression of surface markers were determined by using flow cytometry and in part by using real-time PCR. Allergic airway inflammation was assessed in vivo in an ovalbumin-induced asthma model by using invasive spirometry. RESULTS: For the first time, we observed that SCFAs were able to attenuate human eosinophils at several functional levels, including (1) adhesion to the endothelium, (2) migration, and (3) survival. These effects were independent from GPR41 and GPR43 but were accompanied by histone acetylation and mimicked by trichostatin A, a pan-histone deacetylase inhibitor. In vivo butyrate ameliorated allergen-induced airway and lung eosinophilia, reduced type 2 cytokine levels in bronchial fluid, and improved airway hyperresponsiveness in mice. CONCLUSION: These in vitro and in vivo findings highlight the importance of SCFAs, especially butyrate as a promising therapeutic agent in allergic inflammatory diseases.

Bile acids increase steroidogenesis in cholemic mice and induce cortisol secretion in adrenocortical H295R cells via S1PR2, ERK and SF-1.

BACKGROUND AND AIMS: Bile acids are now accepted as central signalling molecules for the regulation of glucose, amino acid and lipid metabolism. Adrenal gland cortex cells express the bile acid receptors farnesoid X receptor (FXR), the G protein-coupled bile acid receptor (TGR5) and the sphingosine-1-phosphate receptor 2 (S1PR2). We aimed to determine the effects of cholestasis and more specifically of bile acids on cortisol production. METHODS: FXR and TGR5 knockout mice and controls were subjected to common bile duct ligation (CBDL) or chenodeoxycholic acid (CDCA) feeding to model cholestasis. Human adrenocortical H295R cells were challenged with bile acids for mechanistic studies. RESULTS: We found that CBDL and CDCA feeding increased the levels of corticosterone, the rodent equivalent to human cortisol and mRNA and protein levels of steroidogenesis-related enzymes in adrenals independent of FXR and TGR5. Taurine-conjugated CDCA (TCDCA) significantly stimulated cortisol secretion, phosphorylation of extracellular signal-regulated kinase (ERK) and expression of steroidogenesis-related genes in human adrenocortical H295R cells. FXR and TGR5 agonists failed to induce cortisol secretion in H295R cells. S1PR2 inhibition significantly abolished TCDCA-induced cortisol secretion, lowered phosphorylation of ERK and abrogated enhanced transcription of steroidogenesis-related genes in H295R cells. Likewise, siRNA S1PR2 treatment reduced the phosphorylation of ERK and cortisol secretion. Steroidogenic factor-1 (SF-1) transactivation activity was increased upon TCDCA treatment suggesting that bile acid signalling is linked to SF-1. Treatment with SF-1 inverse agonist AC45594 also reduced TCDCA-induced steroidogenesis. CONCLUSIONS: Our findings indicate that supraphysiological bile acid levels as observed in cholestasis stimulate steroidogenesis via an S1PR2-ERK-SF-1 signalling pathway.

The Role of PGE2 in Alveolar Epithelial and Lung Microvascular Endothelial Crosstalk.

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.

The EP1/EP3 receptor agonist 17-pt-PGE2 acts as an EP4 receptor agonist on endothelial barrier function and in a model of LPS-induced pulmonary inflammation.

Endothelial dysfunction is a hallmark of inflammatory conditions. We recently demonstrated that prostaglandin (PG)E2 enhances the resistance of pulmonary endothelium in vitro and counteracts lipopolysaccharide (LPS)-induced pulmonary inflammation in vivo via EP4 receptors. The aim of this study was to investigate the role of the EP1/EP3 receptor agonist 17-phenyl-trinor-(pt)-PGE2 on acute lung inflammation in a mouse model. In LPS-induced pulmonary inflammation in mice, 17-pt-PGE2 reduced neutrophil infiltration and inhibited vascular leakage. These effects were unaltered by an EP1 antagonist, but reversed by EP4 receptor antagonists. 17-pt-PGE2 increased the resistance of pulmonary microvascular endothelial cells and prevented thrombin-induced disruption of endothelial junctions. Again, these effects were not mediated via EP1 or EP3 but through activation of the EP4 receptor, as demonstrated by the lack of effect of more selective EP1 and EP3 receptor agonists, prevention of these effects by EP4 antagonists and EP4 receptor knock-down by siRNA. In contrast, the aggregation enhancing effect of 17-pt-PGE2 in human platelets was mediated via EP3 receptors. Our results demonstrate that 17-pt-PGE2 enhances the endothelial barrier in vitro on pulmonary microvascular endothelial cells, and accordingly ameliorates the recruitment of neutrophils, via EP4 receptors in vivo. This suggests a beneficial effect of 17-pt-PGE2 on pulmonary inflammatory diseases.

A biased non-Gαi OXE-R antagonist demonstrates that Gαi protein subunit is not directly involved in neutrophil, eosinophil, and monocyte activation by 5-oxo-ETE.

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 Gαißγ protein-dependent and ß-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 Gßγ-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. ß-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 ß-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 Ca(2+) 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 Gßγ 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.

D-type prostanoid receptor enhances the signaling of chemoattractant receptor-homologous molecule expressed on T(H)2 cells.

BACKGROUND: Prostaglandin (PG) D(2) is substantially involved in allergic responses and signals through the 7 transmembrane-spanning/G protein-coupled receptors, chemoattractant receptor-homologous molecule expressed on T(H)2 cells (CRTH2), and D-type prostanoid (DP) receptor. OBJECTIVE: Although the proinflammatory function of CRTH2 is well recognized and CRTH2 is hence considered an important emerging pharmacotherapeutic target, the role of the DP receptor in mediating the biological effects of PGD(2) in patients with allergic inflammation has remained unclear. METHODS: The cross-talk of CRTH2 and DP receptors was investigated by using both a recombinant HEK293 cell model and human eosinophils in Ca(2+) mobilization assays, coimmunoprecipitation, Western blotting, radioligand binding, and immunofluorescence. RESULTS: We show that CRTH2 and DP receptors modulate one another's signaling properties and form CRTH2/DP heteromers without altering their ligand-binding capacities. We find that the DP receptor amplifies the CRTH2-induced Ca(2+) release from intracellular stores and coincidentally forfeits its own signaling potency. Moreover, desensitization or pharmacologic blockade of the DP receptor hinders CRTH2-mediated signal transduction. However, CRTH2 internalization occurs independently of the DP receptor. In cells that express both receptors, pharmacologic blockade of Gα(q/11) proteins abolishes the Ca(2+) response to both CRTH2 and DP agonists, whereas inhibition of Gα(i) proteins selectively attenuates the CRTH2-mediated response but not the DP signal. CONCLUSION: Our data demonstrate the capacity of DP receptors to amplify the biological response to CRTH2 activation. Therefore the CRTH2/DP heteromer might not only represent a functional signaling unit for PGD(2) but also a potential target for the development of heteromer-directed therapies to treat allergic diseases.

GPR55 regulates cannabinoid 2 receptor-mediated responses in human neutrophils.

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 tissue-injuring inflammatory responses mediated by CB(2)R, while it synergizes with CB(2)R in recruiting neutrophils to sites of inflammation.

GPR55 ligands promote receptor coupling to multiple signalling pathways.

BACKGROUND AND PURPOSE: Although GPR55 is potently activated by the endogenous lysophospholipid, L-alpha-lysophosphatidylinositol (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-kappaB (NF-kappaB) 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.

The G-protein coupled receptor associated sorting protein GASP-1 regulates the signalling and trafficking of the viral chemokine receptor US28.

Human cytomegalovirus (HCMV) encodes the seven transmembrane(7TM)/G-protein coupled receptor (GPCR) US28, which signals and endocytoses in a constitutive, ligand-independent manner. Here we show that, following endocytosis, US28 is targeted to the lysosomes for degradation as a consequence of its interaction with the GPCR-associated sorting protein-1 (GASP-1). We find that GASP-1 binds to US28 in vitro and that disruption of the GASP-1/US28 interaction by either (i) overexpression of dominant negative cGASP-1 or by (ii) shRNA knock-down of endogenous GASP-1 is sufficient to inhibit the lysosomal targeting of US28 and slow its post-endocytic degradation. Furthermore, we found that GASP-1 affects US28-mediated signalling. The knock-down of endogenous GASP-1 impairs the US28-mediated Galphaq/PLC/inositol phosphate (IP) accumulation as well as the activation of the transcription factors Nuclear Factor-kappaB (NF-kappaB) and cyclic AMP responsive element binding protein (CREB). Overexpression of GASP-1 enhances both IP accumulation and transcription factor activity. Thus, GASP-1 is an important cellular determinant that not only regulates the post-endocytic trafficking of US28, but also regulates the signalling capacities of US28.