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Sphingosine 1-phosphate (S1P) is an important circulating lipid mediator that is derived from the metabolism of cell membranes. Its diverse homeostatic roles, particularly in immunology and vascular biology, can go awry in numerous diseases, including multiple sclerosis, cardiovascular diseases, and fibrosis. The centrality of S1P signaling has led to the development of several drugs, including two approved for treatment of multiple sclerosis. In a Review, Cartier and Hla discuss the current understanding of how one mediator can carry out so many signaling roles in different tissues, how these become dysregulated in disease, and efforts in drug development to target S1P signaling. Science , this issue p. eaar5551 Sphingosine 1-phosphate (S1P), a metabolic product of cell membrane sphingolipids, is bound to extracellular chaperones, is enriched in circulatory fluids, and binds to G protein–coupled S1P receptors (S1PRs) to regulate embryonic development, postnatal organ function, and disease. S1PRs regulate essential processes such as adaptive immune cell trafficking, vascular development, and homeostasis. Moreover, S1PR signaling is a driver of multiple diseases. The past decade has witnessed an exponential growth in this field, in part because of multidisciplinary research focused on this lipid mediator and the application of S1PR-targeted drugs in clinical medicine. This has revealed fundamental principles of lysophospholipid mediator signaling that not only clarify the complex and wide ranging actions of S1P but also guide the development of therapeutics and translational directions in immunological, cardiovascular, neurological, inflammatory, and fibrotic diseases.

Sphingosine 1-phosphate receptor-1 (S1PR1) is essential for embryonic vascular development and maturation. In the adult, it is a key regulator of vascular barrier function and inflammatory processes. Its roles in tumor angiogenesis, tumor growth, and metastasis are not well understood. In this paper, we show that S1PR1 is expressed and active in tumor vessels. Murine tumor vessels that lack S1PR1 in the vascular endothelium (S1pr1 ECKO) show excessive vascular sprouting and branching, decreased barrier function, and poor perfusion accompanied by loose attachment of pericytes. Compound knockout of S1pr1, 2, and 3 genes further exacerbated these phenotypes, suggesting compensatory function of endothelial S1PR2 and 3 in the absence of S1PR1. On the other hand, tumor vessels with high expression of S1PR1 (S1pr1 ECTG) show less branching, tortuosity, and enhanced pericyte coverage. Larger tumors and enhanced lung metastasis were seen in S1pr1 ECKO, whereas S1pr1 ECTG showed smaller tumors and reduced metastasis. Furthermore, antitumor activity of a chemotherapeutic agent (doxorubicin) and immune checkpoint inhibitor blocker (anti-PD-1 antibody) were more effective in S1pr1 ECTG than in the wild-type counterparts. These data suggest that tumor endothelial S1PR1 induces vascular normalization and influences tumor growth and metastasis, thus enhancing antitumor therapies in mouse models. Strategies to enhance S1PR1 signaling in tumor vessels may be an important adjunct to standard cancer therapy of solid tumors.

Abstract Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease which leads to significant morbidity and mortality from respiratory failure. The two drugs currently approved for clinical use slow the rate of decline in lung function but have not been shown to halt disease progression or reverse established fibrosis. Thus, new therapeutic targets are needed. Endothelial injury and the resultant vascular permeability are critical components in the response to tissue injury and are present in patients with IPF. However, it remains unclear how vascular permeability affects lung repair and fibrosis following injury. Lipid mediators such as sphingosine-1-phosphate (S1P) are known to regulate multiple homeostatic processes in the lung including vascular permeability. We demonstrate that endothelial cell–(EC) specific deletion of the S1P receptor 1 (S1PR1) in mice (EC-S1pr1  −/−) results in increased lung vascular permeability at baseline. Following a low-dose intratracheal bleomycin challenge, EC-S1pr1  −/− mice had increased and persistent vascular permeability compared with wild-type mice, which was strongly correlated with the amount and localization of resulting pulmonary fibrosis. EC-S1pr1  −/− mice also had increased immune cell infiltration and activation of the coagulation cascade within the lung. However, increased circulating S1P ligand in ApoM-overexpressing mice was insufficient to protect against bleomycin-induced pulmonary fibrosis. Overall, these data demonstrate that endothelial cell S1PR1 controls vascular permeability in the lung, is associated with changes in immune cell infiltration and extravascular coagulation, and modulates the fibrotic response to lung injury.

Abstract Respiratory viral infections are frequent causes of acute respiratory distress syndrome (ARDS), a disabling condition with a mortality of up to 46%. The pulmonary endothelium plays an important role in the development of ARDS as well as the pathogenesis of pulmonary fibrosis; however, the therapeutic potential to modulate endothelium-dependent signaling to prevent deleterious consequences has not been well explored. Here, we used a clinically relevant influenza A virus infection model, endothelial cell–specific transgenic gain-of-function and loss-of-function mice as well as pharmacologic approaches and in vitro modeling, to define the mechanism by which S1PR1 expression is dampened during influenza virus infection and determine whether therapeutic augmentation of S1PR1 has the potential to reduce long-term postviral fibrotic complications. We found that the influenza virus–induced inflammatory milieu promoted internalization of S1PR1, which was pharmacologically inhibited with paroxetine, an inhibitor of GRK2. Moreover, genetic overexpression or administration of paroxetine days after influenza virus infection was sufficient to reduce postviral pulmonary fibrosis. Taken together, our data suggest that endothelial S1PR1 signaling provides critical protection against long-term fibrotic complications after pulmonary viral infection. These findings support the development of antifibrotic strategies that augment S1PR1 expression in virus-induced ARDS to improve long-term patient outcomes.

G protein-coupled receptors (GPCRs) represent the largest family of membrane receptors. They relay a great variety of physiological extracellular signals to the interior of the cell. The different classes of GPCRs mediate the activation of numerous signaling pathways by a plethora of second messengers. The cell type, stimulus context and multi-protein complexes influence the specificity of the induced signals. The thromboxane A2 receptor (TP) gene is spliced to produce two isoforms, TPaα and TPβ. Both isoforms are differently regulated at the signaling and desensitizing levels. Therefore, it is important to investigate specific interacting proteins of the two isoforms. We identified the WD repeat-containing WDR36 protein as an interacting partner of the β isoform of thromboxane A2 receptor (TPβ) by yeast two-hybrid screening. We demonstrated that WDR36 directly interacts 'with the C-terminus and the first intracellular loop of TPβ by in vitro GST-pulldown assays. The interaction in a cellular context was observed by co-immunoprecipitation, which was positively modulated by TPβ stimulation. TPβ-WDR36 co-localization was detected by confocal microscopy at the plasma membrane in non-stimulated HEK293 cells but translocated to intracellular vesicles following receptor stimulation. WDR36 co-expression or siRNA-mediated expression knockdown respectively increased and inhibited TPβ-induced Gαq signalling. Interestingly, WDR36 co-immunoprecipitated with Gαq, and promoted the TPβ-Gαq interaction. WDR36 also associated with phospholipase-Cβ (PLCβ) and increased the interaction between Gαq and PLCβ, while preventing sequestration of activated Gαq by GRK2. In addition, the presence of TPβ in PLCβ immunoprecipitates was augmented by expression of WDR36. Finally, disease-associated variants of WDR36 affected its ability to modulate Gαq-mediated signalling by TPβ. We report that WDR36 acts as a new scaffold protein tethering TPβ, Gαq and PLCβ in a signalling complex. Following the first study, we investigated the role for WDR36 in the activation MAPK pathways. We show that WDR36 is a new scaffold of the ERKI/2 cascade, but not of the p38 and JNK MAPKs in HEK293 and HeLa cervical cancer cells. Pharmacological inhibitors revealed that WDR36 enhances ERKI/2 activation by TPβ through a PLCβ-, PKC- and MEKI/2-dependent mechanism. WDR36 forms a complex with endogenous PKC, c-Raf, MEKI/2 and ERKI/2 in basal conditions. These interactions and the WDR36dependent c-Raf-ERKI/2 and MEKI/2-ERKI/2 complex formation are augmented by TPβ stimulation prior to peak ERK1/2 activation. WDR36 also regulates the targeting of ERKI/2 to the nucleus and the induction of c-Fos expression. Furthermore, we report that WDR36 is involved in serum-mediated ERKI/2 activation, as well as TPβ- and serum-induced proliferation of HeLa cells. We propose that WDR36 is a scaffold of the c-RafMEKI/2-ERKI/2 signaling cascade in response to diverse mitogen signals. These two studies support a model whereby WDR36 acts as a scaffold protein in TPβ signaling, tethering the receptor with Gαq and PLCβ, and in assembling protein complexes in the ERKI/2 activation cascade. This is interesting when considering that WDR36 variants were recently associated with various types of cancers. Together, this suggests that WDR36 may constitute a new target of intervention in cancer research. Keywords : WDR36, Thromboxane A2 receptor, GPCR, Cell signaling, ERK 1 /2, Scaffold protein, proliferation.

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) activate G protein-coupled receptors (GPCRs) to regulate key pathobiological processes. Here we report a novel lipid mediator GPCR cross-talk mechanism that modulates lymphatic endothelial junctional architecture in lymph nodes. LPAR1 was identified as an inducer of S1PR1/ ß-arrestin coupling from a genome-wide CRISPR/ Cas9 transcriptional activation screen. LPAR1 activation induced S1PR1 ß-arrestin recruitment while suppressing Gαi protein signaling. Lymphatic endothelial cells from cortical and medullary sinuses of lymph nodes which express LPAR1 and S1PR1, exhibit porous junctional architecture and constitutive S1PR1 coupling to ß-arrestin which was suppressed by the LPAR1 antagonist AM095. In endothelial cells, LPAR1-activation increased trans-endothelial permeability and junctional remodeling from zipper-like structures to puncta of adhesion plaques that terminate at actin-rich stress fibers with abundant intercellular gaps. Cross-talk between LPA and S1P receptors regulates complex junctional architecture of lymphatic sinus endothelial cells, a site of high lymphocyte traffic and lymph flow.

Au cours du développement embryonnaire, les gènes Cdx régulent l'expression de gènes Hox, impliqués dans la formation de l'axe antéro-postérieur. Le facteur de transcription Cdx2/3 est impliqué dans l'arrêt de la prolifération cellulaire et l'induction de la différenciation entérocytaire, entre autres par le contrôle de l'expression d'enzymes digestifs et de protéines de jonctions cellulaires. Son expression est retrouvée au niveau des entérocytes de la villosité du petit intestin ainsi qu'au niveau de l'épithélium du colon proximal. Sa surexpression dans une lignée cellulaire épithéliale intestinale de crypte de rat (IEC-6) induit un début de différenciation cellulaire. Cdx2/3 possède également un rôle de suppresseur de tumeur, puisque son expression inhabituelle est souvent retrouvée dans certains cancers. L'activité transcriptionnelle et la stabilité de Cdx2/3 peuvent être régulées par différentes kinases. Nous avons préalablement démontré que la MAPkinase p38α régule positivement l'activité transcriptionnelle de Cdx2/3 dans les cellules différenciées, que le domaine d'interaction de cette kinase avec Cdx2/3 se situe aux acides aminés 61 à 76 et que la sérine 156 serait une cible de la MAPkinase p38α. Le but de mon projet était d'identifier d'autres domaines candidats à la régulation de Cdx2/3 par la MAPkinase p38α, mais aussi par la MAPkinase ERK1/2. Pour ce faire, plusieurs mutants de sites putatifs, seuls ou combinés, pour la phosphorylation par les MAPKs ont été générés par mutagénèse dirigée. Des essais de phosphorylation in vitro sur ces mutants on été réalisés afin d'étudier leur impact sur la phosphorylation de la protéine. Afin de vérifier si d'autres sites pouvaient être impliqués dans l'interaction de Cdx2/3 avec les MAPKs, des essais de précipitation GST de mutants de délétion de la portion N-terminale Cdx2/3 ont été effectués. L'expression stable dans la lignée IEC-6 des différents mutants a par la suite été effectuée afin d'observer les phénotypes provoqués par ces mutations, notamment la régulation de plusieurs autres cibles, observée par RT-PCR. Nos résultats démontrent que les sérines 33, 60, 99 et 156, de même que le motif 4 sérines situé en C-terminal de la protéine sont des sites de phosphorylation par les MAP kinases p38α et ERK1/2. Nous montrons aussi que les acides aminés 61 à 76 de Cdx2/3 sont nécessaires à l'interaction avec la MAPK p38α, alors que ERK1/2 se lie aux acides aminés 37 à 44 in vitro. Grâce aux populations stables IEC-6-Cdx2/3 sauvage ou mutants, nous avons démontré que certaines mutations affectent le patron de bandes observé en immunobuvardage, le taux de prolifération des cellules, la densité de saturation, le phénotype des cellules à 30 jours post-confluence et l'expression de gènes cibles dans les cellules IEC-6. Ces résultats montrent que la phosphorylation par les MAPKs représenterait un mécanisme de régulation important de l'activité transcriptionnelle de Cdx2/3 et pourrait moduler ses fonctions reliées à la prolifération et à la différenciation des cellules intestinales épithéliales.

Sphingosine 1-phosphate receptor-1 (S1PR1) is essential for embryonic vascular development and maturation. In the adult, it is a key regulator of vascular barrier function and inflammatory processes. Its roles in tumor angiogenesis, tumor growth and metastasis are not well understood. In this report, we show that S1PR1 is expressed and active in tumor vessels. Tumor vessels that lack S1PR1 (S1pr1 ECKO) show excessive vascular sprouting and branching, decreased barrier function, and poor perfusion accompanied by loose attachment of pericytes. Compound knockout of S1pr1, 2 and 3 genes further exacerbated these phenotypes, suggesting compensatory function of endothelial S1PR2 and 3 in the absence of S1PR1. On the other hand, tumor vessels with high expression of S1PR1 (S1pr1 ECTG) show less branching, tortuosity and enhanced pericyte coverage. Larger tumors and enhanced lung metastasis were seen in S1pr1 ECKO whereas S1pr1 ECTG showed smaller tumors and reduced metastasis. Furthermore, anti-tumor activity of doxorubicin was more effective in S1pr1 ECTG than the wild-type counterparts. These data suggest that tumor endothelial S1PR1 induces vascular normalization and influences tumor growth, evolution and spread. Strategies to enhance S1PR1 signaling in tumor vessels may be an important adjunct to standard cancer therapy.

High-density lipoprotein (HDL) particles suppress inflammation-induced tissue injury via vascular and myeloid cell-dependent mechanisms. As such, HDL-associated bioactive lipids such as sphingosine 1-phosphate (S1P) and prostacyclin (PGI2) signal via their respective G protein-coupled receptors on target cells to promote vascular endothelial function and suppress platelet and myeloid-dependent pathophysiology. Here we have constructed a fusion protein of apolipoprotein A1 (ApoA1) and apolipoprotein M (ApoM) (A1M) that forms HDL-like particles and chaperones S1P and Iloprost, stable PGI2 analog. A1M/S1P complex activates S1P receptor-1 (S1PR1) as a Gαi-biased agonist and attenuates the inflammation-induced NFκB pathway while A1M/Iloprost acts via IP receptor to inhibit platelet aggregation and promote endothelial barrier function. In addition to enhancing the endothelial barrier, A1M/S1P suppresses neutrophil influx, oxidative burst and inflammatory mediator secretion in a sterile inflammation model. We propose that A1M could be useful as a therapeutic to induce S1P and PGI2-dependent anti-inflammatory functions and suppress collateral tissue injury. Competing Interest Statement T.H. and S.S. are inventors on patent applications on S1P chaperones (ApoM-Fc and ApoA1-ApoM).