Targeting the SPHK1/S1P/S1PR2 axis ameliorates GH-secreted pituitary adenoma progression.

BACKGROUND: Growth hormone-secreted pituitary adenoma (GHPA) is a prominent subtype of pituitary adenoma (PA) associated with progressive somatic disfigurement, various complications, and elevated mortality rates. Existing treatment options have limited efficacy, highlighting the urgent need for novel pharmacological interventions. Previous studies have revealed that sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P)/S1P receptors (S1PRs) signalling have critical roles in the tumour microenvironment, but their role in GHPA remains unclear. METHODS: We performed integrative analyses including bioinformatics analyses, functional studies, and clinical validation to investigate the pathological roles of SPHK1/S1P and evaluated the effectiveness of the S1P receptor 2 (S1PR2) inhibitor JTE-013 in GHPA treatment. RESULTS: SPHK1/S1P signalling is abnormally expressed in patients with GHPA. Knockdown of SPHK1 suppresses S1P-mediated cell proliferation in GH3 Cells. Mechanistically, S1P inhibits apoptosis and autophagy while promoting the secretion of Growth Hormone (GH) by binding to the S1P receptor subtype 2 (S1PR2) in GH3 cells. Moreover, the function of S1PR2 in GH3 cells is mediated by the downstream Akt-Creb pathway. We then identify the S1PR2 as a novel target for therapeutic intervention in GHPA. Systemic administration of the potent and selective S1PR2 antagonist, JTE-013, significantly reduces both tumour size and GH secretion. Importantly, we identify preoperative serum S1P levels as a biomarker predicting poor prognosis in GHPA patients at follow-up. CONCLUSION: Our study shows that blocking SPHK1/S1P/S1PR2 axis can ameliorate the progression of GHPA, providing evidence of a promising therapeutic target for GHPA.

JTE-013 Alleviates Pulmonary Fibrosis by Affecting the RhoA/YAP Pathway and Mitochondrial Fusion/Fission.

Pulmonary fibrosis may be due to the proliferation of fibroblasts and the aggregation of extracellular matrix, resulting in the stimulation of inflammation damage, destroying lung tissue structure, seriously affecting the patient's respiratory function, and even leading to death. We investigated the role and mechanism of JTE-013 in attenuating bleomycin (BLM)-induced pulmonary fibrosis. BLM-induced pulmonary fibrosis was established in mice. Type 2 alveolar epithelial cells (MLE-12) were stimulated with sphingosine monophosphate (S1P) in vitro. JTE-013, an S1PR2 (sphingosine 1-phosphate receptor 2) antagonist, and Verteporfin were administered in vivo and in vitro. IL-4, IL-5, TNF-α, and IFN-γ were measured by ELISA. IL-4 and IFN-γ positive cells were detected by flow cytometry. Inhibition of S1PR2 with JTE-013 significantly ameliorated BLM-induced pathological changes and inflammatory cytokine levels. JTE-013 also significantly reduced the expression of RHOA/YAP pathway proteins and mitochondrial fission protein Drp1, apoptosis, and the colocalization of α-SMA with YAP, Drp1, and Tom20, as detected by immunohistochemistry, immunofluorescence staining, TUNEL, and Western blot. In vitro, S1PR2 and YAP knockdown downregulated RHOA/YAP pathway protein expression, Drp1 phosphorylation, and Drp1 translocation, promoted YAP phosphorylation and phenotypic transformation of MFN2, and inhibited the up-regulation of mitochondrial membrane potential, reactive oxygen species production, and cell apoptosis (7.13% vs. 18.14%), protecting the integrity of the mitochondrial dynamics. JTE-013 also inhibited the expression of fibrosis markers α-SMA, MMP-9, and COL1A1, and alleviated the symptoms of pulmonary fibrosis. Conclusively, JTE-013 has great anti-pulmonary fibrosis potential by regulating RHOA/YAP and mitochondrial fusion/fission.

Inhibition of Sphingosine-1-Phosphate Receptor 2 by JTE013 Enhanced Alveolar Bone Regeneration by Promoting Angiogenesis.

Sphingosine-1-phosphate receptor 2 (S1PR2) is a G protein-coupled receptor that regulates various immune responses. Herein, we report the effects of a S1PR2 antagonist (JTE013) on bone regeneration. Murine bone marrow stromal cells (BMSCs) were treated with dimethylsulfoxide (DMSO) or JTE013 with or without infection by an oral bacterial pathogen Aggregatibacter actinomycetemcomitans. Treatment with JTE013 enhanced vascular endothelial growth factor A (VEGFA), platelet derived growth factor subunit A (PDGFA), and growth differentiation factor 15 (GDF15) gene expression and increased transforming growth factor beta (TGFß)/Smad and Akt signaling. Eight-week-old male C57BL/6J mice were challenged with ligatures around the left maxillary 2nd molar for 15 days to induce inflammatory bone loss. After ligature removal, mice were treated with diluted DMSO or JTE013 in the periodontal tissues 3 times per week for 3 weeks. Calcein was also injected twice to measure bone regeneration. Micro-CT scanning of maxillary bone tissues and calcein imaging revealed that treatment with JTE013 enhanced alveolar bone regeneration. JTE013 also increased VEGFA, PDGFA, osteocalcin, and osterix gene expressions in the periodontal tissues compared to control. Histological examination of periodontal tissues revealed that JTE013 promoted angiogenesis in the periodontal tissues compared to control. Our findings support that inhibition of S1PR2 by JTE013 increased TGFß/Smad and Akt signaling; enhanced VEGFA, PDGFA, and GDF15 gene expression; and subsequently promoted angiogenesis and alveolar bone regeneration.

JTE-013 mediates RhoA/ROCKl/Drpl signaling axis regulating mitochondrial damage and apoptosis to alleviate allergic rhinitis / 中国药理学通报

Aim To investigate the protective effect and mechanism of JTE-013 on allergic rhinitis (AR) by regulating mitochondrial injury and apoptosis through RhoA/ROCKl/Drpl pathway. Methods AR model was established by ovalbumin (OVA) in mice. Nasal tissue sections were then stained with HE, TUNEL and DHE. Western blot assay. In vitro, human nasal epithelial cells (HNEpCs) were stimulated with human recombinant interleukin-13 (IL-13), and the effects of JTE-013 and Y27632-related protein expression were detected by Western blot. Immunofluorescence was used to observe the effects of JTE-013 and Y 27632 on total ROS, mitochondrial membrane potential and mitochondrial ROS generation, Drpl translocation and Cyt-c expression in cells. Results JTE-013 reduced the frequency of nose rubbing and sneezing, reduced nasal mucosal thickening and decreased eosinophil infiltration in AR mice. TUNEL and DHE staining results suggested that JTE-013 could inhibit apoptosis and reduce ROS expression in mouse nasal epithelial cells. Western blot showed that both JTE-013 and Y 27632 could significantly reduce RhoA, ROCK1, Drpl and p-Drpl(616), inhibit the expression of apoptotic proteins Bax, cleaved-caspase-3, Cyt-c, cleavedcaspase-9 and up-regulate the expression of p-Drpl (637) and Bcl-2. Immunofluorescence showed that inhibitors of JTE-013 or ROCK1 almost blocked IL-13mediated increase in ROS and mtROS production, inhibited decrease in mitochondrial membrane potential, and blocked Cyt-c expression and Drpl translocation in nasal mucosal epithelial cells. Conclusion JTE-013 can regulate the morphology and function of mitochondria by inhibiting RhoA/ROCKl/Drpl signaling axis, thereby alleviating nasal epithelial cell inflammation in mice with allergic rhinitis.

Inhibition of Sphingosine-1-Phosphate Receptor 2 by JTE013 Promoted Osteogenesis by Increasing Vesicle Trafficking, Wnt/Ca2+, and BMP/Smad Signaling.

Sphingosine-1-phosphate receptor 2 (S1PR2) is a G protein-coupled receptor that regulates various immune responses. Herein, we determine the effects of a S1PR2 antagonist (JTE013) or a S1PR2 shRNA on osteogenesis by culturing murine bone marrow stromal cells (BMSCs) in osteogenic media with JTE013, dimethylsulfoxide (DMSO), a S1PR2 shRNA, or a control shRNA. Treatment with JTE013 or the S1PR2 shRNA increased alkaline phosphatase and alizarin red s staining, and enhanced alkaline phosphatase, RUNX2, osteocalcin, and osterix mRNA levels in BMSCs compared with the controls. Protein analysis revealed that a high dose of JTE013 (4 or 8 µM) increased vesicle trafficking-associated proteins (F-actin, clathrin, Early Endosome Antigen 1 (EEA1), and syntaxin 6) and Wnt/Ca2+ signaling. On the other hand, a low dose of JTE013 (1 to 2 µM) increased BMP/Smad signaling. In contrast, the S1PR2 shRNA reduced vesicle trafficking-associated proteins and attenuated Wnts and BMP/Smad signaling, but enhanced p-CaMKII compared with the control, suggesting that the S1PR2 shRNA influenced osteogenesis via different signaling pathways. Moreover, inhibiting protein trafficking by brefeldin A in BMSCs suppressed Wnts and BMPRs expressions. These data supported that enhanced osteogenesis in JTE013-treated BMSCs is associated with increased vesicle trafficking, which promotes the synthesis and transport of osteogenic protein and matrix vesicles and enhances matrix mineralization.

Sphingosine-1-Phosphate Induces ATP Release via Volume-Regulated Anion Channels in Breast Cell Lines.

High interstitial level of ATP and its lysate adenosine in the cancer microenvironment are considered a halo mark of cancer. Adenosine acts as a strong immune suppressor. However, the source of ATP release is unclear. We clarified the release of ATP via volume-regulated anion channels (VRACs) in breast cell lines using an ATP luminescence imaging system. We detected a slowly rising diffuse pattern of ATP release that was only observed in undifferentiated cells, not in differentiated primary cultured cells. This was confirmed by suppression with DCPIB, a blocker of VRACs, and shRNA for LRRC8A, an indispensable subunit of VRACs. We herein demonstrated that the inflammatory mediator sphingosine-1-phosphate (S1P), which exists abundantly in the cancer microenvironment, induced a diffuse pattern of ATP release isovolumetrically. The response was dose-dependent and suppressed by the knock-down of LRRC8A. It was also suppressed by blockers of S1P receptor 1 and 2 (W146 and JTE013, respectively). RTqPCR demonstrated the prominent presence of S1PR1 and S1PR2 mRNAs. We discussed the roles of S1P-induced ATP release in the cancer microenvironment.

JTE-013 Alleviates Inflammatory Injury and Endothelial Dysfunction Induced by Sepsis In Vivo and In Vitro.

BACKGROUND: Nowadays, there is no approved targeted agent for lung injury induced by sepsis. S1PR2 is confirmed to be a promising diagnosis and treatment target. JTE-013 as S1PR2 antagonists may be an agent of great potential. In this research, we sought to determine the functional role of JTE-013 in lung injury induced by sepsis. MATERIALS AND METHODS: Seventy-two rats were assigned into normal group, sepsis model group and JTE-013 group. The animal model of lung injury induced by sepsis was constructed by cecal ligation and puncture. The human pulmonary microvascular endothelial cells (HPMECs) were divided into control, LPS and LPS + JTE-013 group. HPMECs induced by LPS served as the cell model of lung injury induced by sepsis. HE staining assay was performed for assessment of the pathological condition and Evans blue was applied for assessment of pulmonary tissue permeability. Wet/dry ratio was measured as indicators of pulmonary edema degree and neutrophil count was measured as indicators of infection status. The levels of inflammatory factors were detected by corresponding kits, cell survival by CCK-8 assay and protein expression level by western blot. RESULTS: S1PR2 was highly expressed in vivo model of lung injury induced by sepsis. It was observed that JTE-013 as antagonist of S1PR2 alleviated the lung tissue injury, endothelial dysfunction and pulmonary edema induced by sepsis. In addition, JTE-013 reduced neutrophil count and levels of inflammatory factors. Moreover, results confirmed that JTE-013 enhanced cell viability and mitigated inflammatory response in cell model of sepsis. CONCLUSIONS: Overall, JTE-013 as an antagonist of S1PR2 could relieve inflammatory injury and endothelial dysfunction induced by sepsis in vivo and vitro, resulting in attenuation of lung injury. These findings elucidated that JTE-013 may be a promising targeted agent for lung injury induced by sepsis.

Targeting S1PRs as a Therapeutic Strategy for Inflammatory Bone Loss Diseases-Beyond Regulating S1P Signaling.

As G protein coupled receptors, sphingosine-1-phosphate receptors (S1PRs) have recently gained attention for their role in modulating inflammatory bone loss diseases. Notably, in murine studies inhibiting S1PR2 by its specific inhibitor, JTE013, alleviated osteoporosis induced by RANKL and attenuated periodontal alveolar bone loss induced by oral bacterial inflammation. Treatment with a multiple S1PRs modulator, FTY720, also suppressed ovariectomy-induced osteoporosis, collagen or adjuvant-induced arthritis, and apical periodontitis in mice. However, most previous studies and reviews have focused mainly on how S1PRs manipulate S1P signaling pathways, subsequently affecting various diseases. In this review, we summarize the underlying mechanisms associated with JTE013 and FTY720 in modulating inflammatory cytokine release, cell chemotaxis, and osteoclastogenesis, subsequently influencing inflammatory bone loss diseases. Studies from our group and from other labs indicate that S1PRs not only control S1P signaling, they also regulate signaling pathways induced by other stimuli, including bacteria, lipopolysaccharide (LPS), bile acid, receptor activator of nuclear factor κB ligand (RANKL), IL-6, and vitamin D. JTE013 and FTY720 alleviate inflammatory bone loss by decreasing the production of inflammatory cytokines and chemokines, reducing chemotaxis of inflammatory cells from blood circulation to bone and soft tissues, and suppressing RANKL-induced osteoclast formation.

Inhibition of Sphingosine-1-Phosphate Receptor 2 Prevents Thoracic Aortic Dissection and Rupture.

Background: Numerous pieces of evidence have indicated that thoracic aortic dissection (TAD) is an inflammatory disease. Sphingosine-1-phosphate receptor 2 (S1PR2) signaling is a driver in multiple inflammatory diseases. Here, we examined the S1PR2 expression in TAD lesions and explored the effect of interfering with S1PR2 on TAD formation and progression. Methods: Aorta specimens and blood samples were collected from patients with TAD and matched controls. The expression of S1PR1, S1PR2, and S1PR3 was examined. The effect of inhibiting S1PR2 on TAD was evaluated in a TAD mouse model induced by ß-aminopropionitrile fumarate (BAPN) and AngII. The presence of sphingosine kinase 1 (SPHK1), S1P, and neutrophil extracellular traps (NETs) was investigated. Further, the possible association between S1PR2 signaling and NETs in TAD was analyzed. Results: In the aortic tissues of patients with TAD and a mouse model, the S1PR2 expression was significantly up-regulated. In the TAD mouse model, JTE013, a specific S1PR2 antagonist, not only blunted the TAD formation and aortic rupture, but also preserved the elastic fiber architecture, reduced the smooth muscle cells apoptosis level, and mitigated the aortic wall inflammation. Augmented tissue protein expression of SPHK1, citrullinated histone H3 (CitH3, a specific marker of NETs), and serum S1P, CitH3 were detected in TAD patients. Surgical repair normalized the serum S1P and CitH3 levels. Immunofluorescence staining revealed that S1PR2 colocalized with NETs. The protein expression levels of SPHK1 and serum S1P levels positively correlated with the protein expression and serum levels of CitH3, separately. Furthermore, JTE013 treatment reduced NETs accumulation. Conclusion: Inhibiting S1PR2 attenuates TAD formation and prevents aortic rupture. Targeting S1PR2 may provide a promising treatment strategy against TAD.

Increased S1P induces S1PR2 internalization to blunt the sensitivity of colorectal cancer to 5-fluorouracil via promoting intracellular uracil generation.

Sphingosine-1-phosphate (S1P), the backbone of most sphingolipids, activating S1P receptors (S1PRs) and the downstream G protein signaling has been implicated in chemoresistance. In this study we investigated the role of S1PR2 internalization in 5-fluorouracil (5-FU) resistance in human colorectal cancer (CRC). Clinical data of randomly selected 60 CRC specimens showed the correlation between S1PR2 internalization and increased intracellular uracil (P < 0.001). Then we explored the regulatory mechanisms in CRC model of villin-S1PR2-/- mice and CRC cell lines. We showed that co-administration of S1P promoted S1PR2 internalization from plasma membrane (PM) to endoplasmic reticulum (ER), thus blunted 5-FU efficacy against colorectal tumors in WT mice, compared to that in S1PR2-/- mice. In HCT116 and HT-29 cells, application of S1P (10 µM) empowered S1PR2 to internalize from PM to ER, thus inducing 5-FU resistance, whereas the specific S1PR2 inhibitor JTE-013 (10 µM) effectively inhibited S1P-induced S1PR2 internalization. Using Mag-Fluo-AM-labeling [Ca2+]ER and LC-ESI-MS/MS, we revealed that internalized S1PR2 triggered elevating [Ca2+]ER levels to activate PERK-eLF2α-ATF4 signaling in HCT116 cells. The activated ATF4 upregulated RNASET2-mediated uracil generation, which impaired exogenous 5-FU uptake to blunt 5-FU therapy. Overall, this study reveals a previously unrecognized mechanism of 5-FU resistance resulted from S1PR2 internalization-upregulated uracil generation in colorectal cancer, and provides the novel insight into the significance of S1PR2 localization in predicting the benefit of CRC patients from 5-FU-based chemotherapy.

Blocking sphingosine 1-phosphate receptor 2 accelerates hepatocellular carcinoma progression in a mouse model of NASH.

The role of sphingosine 1-phosphate (S1P) and its sphingosine-1-phosphate receptors (S1PRs) in non-alcoholic steatohepatitis (NASH) is unclear. We aimed to analyze the role of S1P/S1PRs in a Melanocortin-4 receptor (Mc4r)-deficient NASH murine model using FTY720, the functional antagonist of S1PR1, S1PR3, S1PR4, and S1PR5, and JTE-013, the antagonist of S1PR2. We observed that, compared to that in the control, the mRNA of S1pr1 tended to decrease, whereas those of S1pr2 and S1pr3 significantly increased in Mc4r-knockout (KO) mice subjected to a Western diet (WD). While the fat area did not differ, fibrosis progression differed significantly between control mice and mice in which liver S1PRs were blocked. Lipidomic and metabolomic analysis of liver tissues showed that JTE-013-administered mice showed elevation of S-adenosyl-l-methionine level, which can induce aberrant methylation due to reduction in glycine N-methyltransferase (GNMT) and elevation in diacylglycerol (DG) and triacylglycerol (TG) levels, leading to increased susceptibility to hepatocellular carcinoma (HCC). These phenotypes are similar to those of Gnmt-KO mice, suggesting that blocking the S1P/S1PR2 axis triggers aberrant methylation, which may increase DG and TG, and hepatocarcinogenesis. Our observations that the S1P/S1PR2 axis averts HCC occurrence may assist in HCC prevention in NASH.

JTE-013 supplementation improves erectile dysfunction in rats with streptozotocin-induced type â diabetes through the inhibition of the rho-kinase pathway, fibrosis, and apoptosis.

BACKGROUND: Erectile dysfunction (ED) is a common complication in patients with diabetes mellitus (DM) that severely affects the patients' quality of life. However, the effectiveness of oral phosphodiesterase type 5 inhibitors in these patients is poor. Sphingosine-1-phosphate (S1P) and S1P receptor 2 (S1PR2) are important factors regulating the Rho-kinase pathway, and understanding these factors may provide ideas for new therapeutic strategies for ED. OBJECTIVES: To investigate whether the S1PR2 receptor antagonist JTE-013 could improve DM-induced ED (DMED) in rats and to explore the potential mechanisms. MATERIALS AND METHODS: We used 50 male Sprague Dawley rats (8 weeks old) for this experiment. Type Ⅰ DM was induced in forty-two rats via streptozotocin administration; the rest of the rats served as controls. Eight weeks after DM induction, rats with ED were selected via an apomorphine test. Eight of them were injected intraperitoneally with JTE-013 each day for 4 weeks. The rest were fed under the same conditions for 4 weeks. Erectile function was measured by cavernous nerve electrostimulation. The expression levels of related signaling pathways were evaluated using Western blotting, real-time PCR, and immunohistochemistry. RESULTS: Erectile function was significantly impaired in the DMED group compared with the control group and was partially improved in the DMED + JTE-013 group. The expression of S1PR2 and the activity of the RhoA/ROCK/phospho-myosin phosphatase target subunit 1 (p-MYPT1) pathway proteins were higher in the DMED group than in the other two groups, and JTE-013 treatment significantly reduced the expression/activity of these proteins. Furthermore, the DMED group showed severe corporal fibrosis, a higher apoptotic index and increased activity in the TGF-ß1/LIMK2/Cofilin pathway compared with the control group. JTE-013 supplementation significantly ameliorated these pathological changes. DISCUSSION AND CONCLUSION: JTE-013 supplementation partially improved erectile function in rats with DMED, likely by inhibiting smooth muscle contraction, corporal fibrosis, and apoptosis.

Inactivation of sphingosine-1-phosphate receptor 2 (S1PR2) decreases demyelination and enhances remyelination in animal models of multiple sclerosis.

Multiple sclerosis is an inflammatory disease of the central nervous system (CNS) in which multiple sites of blood-brain barrier (BBB) disruption, focal inflammation, demyelination and tissue destruction are the hallmarks. Here we show that sphingosine-1-phosphate receptor 2 (S1PR2) has a negative role in myelin repair as well as an important role in demyelination by modulating BBB permeability. In lysolecithin-induced demyelination of adult mouse spinal cord, S1PR2 inactivation by either the pharmacological inhibitor JTE-013 or S1PR2 gene knockout led to enhanced myelin repair as determined by higher numbers of differentiated oligodendrocytes and increased numbers of remyelinated axons at the lesion sites. S1PR2 inactivation in lysolecithin-induced demyelination of the optic chiasm, enhanced oligodendrogenesis and improved the behavioral outcome in an optokinetic reflex test. In order to see the effect of S1PR2 inactivation on demyelination, experimental autoimmune encephalitis (EAE) was induced by MOG-peptide. S1PR2 inhibition or knockout decreased the extent of demyelinated areas as well as the clinical disability in this EAE model. Both toxin induced and EAE models showed decreased BBB leakage and reduced numbers of Iba1+ macrophages following S1PR2 inactivation. Our results suggest that S1PR2 activity impairs remyelination and also enhances BBB leakage and demyelination. The former effect could be mediated by Nogo-A, as antagonism of this factor enhances remyelination and S1PR2 can act as a Nogo-A receptor.

Sphingolipid signaling modulates trans-endothelial cell permeability in dengue virus infected HMEC-1 cells.

Dengue has emerged as a major mosquito-borne disease in the tropics and subtropics. In severe dengue, enhanced microvascular endothelial permeability leads to plasma leakage. Direct dengue virus (DENV) infection in human microvascular endothelial cells (HMEC-1) can enhance trans-endothelial leakage. Using a microarray-based analysis, we identified modulation of key endothelial cell signaling pathways in DENV-infected HMEC-1 cells. One among them was the sphingolipid pathway that regulates vascular barrier function. Sphingosine-1-phosphate receptor 2 (S1PR2) and S1PR5 showed significant up-regulation in the microarray data. In DENV-infected cells, the kinetics of S1PR2 transcript expression and enhanced in vitro trans-endothelial permeability showed a correlation. We also observed an internalization and cytoplasmic translocation of VE-Cadherin, a component of adherens junctions (AJ), upon infection indicating AJ disassembly. Further, inhibition of S1PR2 signaling by a specific pharmacological inhibitor prevented translocation of VE-Cadherin, thus helping AJ maintenance, and abrogated DENV-induced trans-endothelial leakage. Our results show that sphingolipid signaling, especially that involving S1PR2, plays a critical role in vascular leakage in dengue.

Administration of JTE013 abrogates experimental asthma by regulating proinflammatory cytokine production from bronchial epithelial cells.

BACKGROUND: Sphingosine-1-phosphate (S1P) is a bioactive phospholipid that acts as a signal transducer by binding to S1P receptors (S1PR) 1 to 5. The S1P/S1PRs pathway has been associated with remodeling and allergic inflammation in asthma, but the expression pattern of S1PR and its effects on non-immune cells have not been completely clarified. The aim of this study was to examine the contribution of the signaling of S1P and S1PRs expressed in airway epithelial cells (ECs) to asthma responses in mice. METHODS: Bronchial asthma was experimentally induced in BALB/c mice by ovalbumin (OVA) sensitization followed by an OVA inhalation challenge. The effects of S1PR antagonists on the development of asthma were analyzed 24 h after the OVA challenge. RESULTS: Immunohistological analysis revealed S1PR1-3 expression on mouse airway ECs. Quantitative real-time polymerase chain reaction demonstrated that S1P greatly stimulated the induction of CCL3 and TIMP2 mRNA in human airway ECs, i.e., BEAS-2B cells, in a dose-dependent manner. Pretreatment with the S1PR2 antagonist JTE013 inhibited the CCL3 gene expression in BEAS-2B cells. Immunohistological analysis also showed that the expression level of CCL3 was attenuated by JTE013 in asthmatic mice. Furthermore, JTE013 as well as anti-CCL3 antibody attenuated allergic responses. Intratracheal administration of JTE013 also attenuated eosinophilic reactions in bronchoalveolar lavage fluids. S1P induced transcription factor NFκB activation, while JTE013 greatly reduced the NFκB activation. CONCLUSIONS: JTE013 attenuated allergic airway reactions by regulating CCL3 production from bronchial ECs. The intratracheal administration of JTE013 may be a promising therapeutic strategy for bronchial asthma.

TGR5 activation suppressed S1P/S1P2 signaling and resisted high glucose-induced fibrosis in glomerular mesangial cells.

Glucose and lipid metabolism disorders and chronic inflammation in the kidney tissues are largely responsible for causative pathological mechanism of renal fibrosis in diabetic nephropathy (DN). As our previous findings confirmed that, sphingosine 1-phosphate (S1P)/sphingosine 1-phosphate receptor 2 (S1P2) signaling activation promoted renal fibrosis in diabetes. Numerous studies have demonstrated that the G protein-coupled bile acid receptor TGR5 exhibits effective regulation of glucose and lipid metabolism and anti-inflammatory effects. TGR5 is highly expressed in kidney tissues, whether it attenuates the inflammation and renal fibrosis by inhibiting the S1P/S1P2 signaling pathway would be a new insight into the molecular mechanism of DN. Here we investigated the effects of TGR5 on diabetic renal fibrosis, and the underlying mechanism would be also discussed. We found that TGR5 activation significantly decreased the expression of intercellular adhesion molecule-1 (ICAM-1) and transforming growth factor-beta 1 (TGF-ß1), as well as fibronectin (FN) induced by high glucose in glomerular mesangial cells (GMCs), which were pathological features of DN. S1P2 overexpression induced by high glucose was diminished after activation of TGR5, and AP-1 activity, including the phosphorylation of c-Jun/c-Fos and AP-1 transcription activity, was attenuated. As a G protein-coupled receptor, S1P2 interacted with TGR5 in GMCs. Furthermore, INT-777 lowered S1P2 expression and promoted S1P2 internalization. Taken together, TGR5 activation reduced ICAM-1, TGF-ß1 and FN expressions induced by high glucose in GMCs, the mechanism might be through suppressing S1P/S1P2 signaling, thus ameliorating diabetic nephropathy.

Sphingosine-1-phosphate receptor 2.

Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid involved in cell proliferation, angiogenesis, inflammation and malignant transformation among other functions. S1P acts either directly on intracellular targets or activates G protein-coupled receptors, specifically five S1P receptors (S1PRs). The identified S1PRs differ in cellular and tissue distribution, and each is coupled to specific G proteins, which mediate unique functions. Here, we describe functional characteristics of all five receptors, emphasizing S1PR2, which is critical in the immune, nervous, metabolic, cardiovascular, musculoskeletal, and renal systems. This review also describes the role of this receptor in tumor growth and metastasis and suggests potential therapeutic avenues that exploit S1PR2.

Selectivity and specificity of sphingosine-1-phosphate receptor ligands: caveats and critical thinking in characterizing receptor-mediated effects.

Receptors for sphingosine-1-phosphate (S1P) have been identified only recently. Their medicinal chemistry is therefore still in its infancy, and few selective agonists or antagonists are available. Furthermore, the selectivity of S1P receptor agonists or antagonists is not well established. JTE-013 and BML-241 (also known as CAY10444), used extensively as specific S1P(2) and S1P(3) receptors antagonists respectively, are cases in point. When analyzing S1P-induced vasoconstriction in mouse basilar artery, we observed that JTE-013 inhibited not only the effect of S1P, but also the effect of U46619, endothelin-1 or high KCl; JTE-013 strongly inhibited responses to S1P in S1P(2) receptor knockout mice. Similarly, BML-241 has been shown to inhibit increases in intracellular Ca(2+) concentration via P(2) receptor or α(1A)-adrenoceptor stimulation and α(1A)-adrenoceptor-mediated contraction of rat mesenteric artery, while it did not affect S1P(3)-mediated decrease of forskolin-induced cyclic AMP accumulation. Another putative S1P(1/3) receptor antagonist, VPC23019, does not inhibit S1P(3)-mediated vasoconstriction. With these examples in mind, we discuss caveats about relying on available pharmacological tools to characterize receptor subtypes.

Selectivity and specificity of sphingosine 1-phosphate receptor ligands: "off-targets" or complex pharmacology?

A recent perspective published in Frontiers of Pharmacology by Salomone and Waeber (2011) discussed the selectivity and specificity of sphingosine 1-phosphate (S1P) receptor ligands. This perspective surveyed the use of various S1P receptor ligands and attempted to reconcile a number of inconsistencies in the predicted biological outcomes: these were interpreted as "off-target" effects. Therefore the perspective cautioned against the use of these S1P receptor ligands. Here we highlight the complex pharmacology of S1P receptors, which along with "inside-out" signaling might provide an alternative explanation for "off-target" effects.