S1PR2 Signaling in Chronic Glucocorticoid Exposure-Induced Metabolic Disorders
ABSTRACT: Glucocorticoids play a key role in metabolic adaptation during stress, such as fasting and starvation. Excess and/or chronic glucocorticoid exposure, however, cause metabolic disorders that include insulin resistance dyslipidemia and hepatic steatosis. We previously showed that chronic glucocorticoid treatment elevates hepatic production of ceramides and sphingosine-1-phosphate (S1P). Ceramides are converted to sphingosine that is further converted to S1P. We showed that ceramide-initiated signaling in turn inhibits insulin signaling and increases lipogenic program in hepatocytes. However, the role of S1P, a signaling molecule that modulates physiological responses through membrane S1P receptors (S1PRs), in glucocorticoid actions is unclear. Here we found that inhibiting S1PR2 activity, but not S1PR1 and S1PR3, resulted in improved glucocorticoid-induced insulin resistance. Similarly, reducing hepatic S1PR2 expression showed similar results. Interestingly, reducing S1PR2 expression did not affect the ability of glucocorticoids to modify insulin signaling. Instead, glucocorticoids enhanced gluconeogenesis was reduced. Moreover, glucocorticoid-induced dyslipidemia and fatty liver was also compromised in mice with reduced hepatic S1PR2 expression. Indeed, RNA sequencing analysis showed that lipogenic, gluconeogenic and glycolytic genes are significantly lower in glucocorticoid-treated hepatic S1PR2 knockdown mice than those of glucocorticoid-treated hepatic scramble small hairpin RNA (shRNA) expressing mice (Control). Overall, our studies highlight the importance role of S1PR2 signaling in the mediating glucocorticoid regulation on gluconeogenesis and hepatic lipid homeostasis.
Project description:Sphingosine 1-phosphate (S1P)/S1P receptor (S1PR) system has been implicated in the pathological process of liver injury. This study was designed to evaluate the effects of S1P/S1PR on bone marrow-derived monocyte/macrophage (BMM) migration in mouse models of cholestatic liver injury, and identify the signaling pathway underlying this process. S1PR1-3 expression in BMM was characterized by immunofluorescence, RT-PCR and Western blot. Cell migration was determined in Boyden chambers. In vivo, the chimera mice, which received BM transplants from EGFP-transgenic mice, received an operation of bile duct ligation (BDL) to induce liver injury with the administration of S1PR2/3 antagonists. The results showed that S1PR1-3 were all expressed in BMMs. S1P exerted a powerful migratory action on BMMs via S1PR2 and S1PR3. Furthermore, PTX and LY-294002 (PI3K inhibitor) prevented S1PR2/3-mediated BMM migration, and Rac1 activation by S1P was inhibited by JTE-013, CAY-10444 or LY294002. Administration of S1PR2/3 antagonists in vivo significantly reduced BMM recruitment in BDL-treated mice, and attenuated hepatic inflammation and fibrosis. In conclusion, S1P/S1PR2/3 system mediates BMM motility by PTX-PI3K-Rac1 signaling pathway, which provides new compelling information on the role of S1P/S1PR in liver injury and opens new perspectives for the pharmacological treatment of hepatic fibrosis.
Project description:Sphingosine 1-phosphate (S1P) signaling regulates numerous biological processes including neurogenesis, inflammation and neovascularization. However, little is known about the role of S1P signaling in the eye. In this study, we characterize two sphingosine kinases (SPHK1 and SPHK2), which phosphorylate sphingosine to S1P, and three S1P receptors (S1PR1, S1PR2 and S1PR3) in mouse and rat eyes. We evaluated sphingosine kinase and S1P receptor gene expression at the mRNA level in various rat tissues and rat retinas exposed to light-damage, whole mouse eyes, specific eye structures, and in developing retinas. Furthermore, we determined the localization of sphingosine kinases and S1P receptors in whole rat eyes by immunohistochemistry. Our results unveiled unique expression profiles for both sphingosine kinases and each receptor in ocular tissues. Furthermore, these kinases and S1P receptors are expressed in mammalian retinal cells and the expression of SPHK1, S1PR2 and S1PR3 increased immediately after light damage, which suggests a function in apoptosis and/or light stress responses in the eye. These findings have numerous implications for understanding the role of S1P signaling in the mechanisms of ocular diseases such as retinal inflammatory and degenerative diseases, neovascular eye diseases, glaucoma and corneal diseases.
Project description:Sphingosine-1-phosphate (S1P) has been reported to enhance the function of islet ?-cells, providing a potential therapeutic target for diabetes mellitus. In the present study, the effects of S1P on the proliferation and apoptosis of ?-cells in type 2 diabetic mice were investigated. The mice were administered intraperitoneal S1P solution daily at a dose of 20 µg/kg for three weeks. The intraperitoneal glucose tolerance test (IPGTT) and homeostatic model assessment of insulin resistance (HOMA-IR) index determination were carried out. Immunohistochemical staining was used to detect the protein expression of insulin, antigen Ki-67 and S1P receptor isoforms (S1PR1/S1PR2/S1PR3) in pancreatic islets. Compared with the diabetic control (DC) group, the IPGTT results and HOMA-IR index in the S1P treatment group were decreased. The islets in the S1P group exhibited higher insulin immunostaining intensity than the DC group, as well as higher proliferation (P<0.05) and lower apoptosis rates (P<0.05). Positive staining for the S1P receptors S1PR1, S1PR2 and S1PR3 was observed in the cytoplasm and membrane of the islet cells. S1PR1 and S1PR2 proteins showed increased expression in the S1P and DC groups compared with the normal control group (P<0.01 and P<0.05, respectively), whereas no significant difference was observed in the expression of S1PR3 among these groups. In conclusion, extracellular S1P can induce islet ?-cell proliferation and decrease cell apoptosis in diabetic mice. S1P function may be mediated via S1PR1 and S1PR2; therefore, targeting S1P/S1PR signalling pathways may be a novel therapeutic strategy for diabetes mellitus.
Project description:Sphingosine-1-phosphate (S1P) activates a widely expressed family of G protein-coupled receptors, serves as a muscle trophic factor and activates muscle stem cells called satellite cells (SCs) through unknown mechanisms. Here we show that muscle injury induces dynamic changes in S1P signaling and metabolism in vivo. These changes include early and profound induction of the gene encoding the S1P biosynthetic enzyme SphK1, followed by induction of the catabolic enzyme sphingosine phosphate lyase (SPL) 3 days later. These changes correlate with a transient increase in circulating S1P levels after muscle injury. We show a specific requirement for SphK1 to support efficient muscle regeneration and SC proliferation and differentiation. Mdx mice, which serve as a model for muscular dystrophy (MD), were found to be S1P-deficient and exhibited muscle SPL upregulation, suggesting that S1P catabolism is enhanced in dystrophic muscle. Pharmacological SPL inhibition increased muscle S1P levels, improved mdx muscle regeneration and enhanced SC proliferation via S1P receptor 2 (S1PR2)-dependent inhibition of Rac1, thereby activating Signal Transducer and Activator of Transcription 3 (STAT3), a central player in inflammatory signaling. STAT3 activation resulted in p21 and p27 downregulation in a S1PR2-dependent fashion in myoblasts. Our findings suggest that S1P promotes SC progression through the cell cycle by repression of cell cycle inhibitors via S1PR2/STAT3-dependent signaling and that SPL inhibition may provide a therapeutic strategy for MD.
Project description:Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid produced by mast cells (MCs) on cross-linking of their high-affinity receptors for IgE by antigen that can amplify MC responses by binding to its S1P receptors. An acute MC-dependent allergic reaction can lead to systemic shock, but the early events of its development in lung tissues have not been investigated, and S1P functions in the onset of allergic processes remain to be examined.We used a highly specific neutralizing anti-S1P antibody (mAb) and the sphingosine-1-phosphate receptor 2 (S1PR2) antagonist JTE-013 to study the signaling contributions of S1P and S1PR2 to MC- and IgE-dependent airway allergic responses in mice within minutes after antigen challenge.Allergic reaction was triggered by a single intraperitoneal dose of antigen in sensitized mice pretreated intraperitoneally with anti-S1P, isotype control mAb, JTE-013, or vehicle before antigen challenge.Kinetics experiments revealed early pulmonary infiltration of mostly T cells around blood vessels of sensitized mice 20 minutes after antigen exposure. Pretreatment with anti-S1P mAb inhibited in vitro MC activation, as well as in vivo development of airway infiltration and MC activation, reducing serum levels of histamine, cytokines, and the chemokines monocyte chemoattractant protein 1/CCL2, macrophage inflammatory protein 1?/CCL3, and RANTES/CCL5. S1PR2 antagonism or deficiency or MC deficiency recapitulated these results. Both in vitro and in vivo experiments demonstrated MC S1PR2 dependency for chemokine release and the necessity for signal transducer and activator of transcription 3 activation.Activation of S1PR2 by S1P and downstream signal transducer and activator of transcription 3 signaling in MCs regulate early T-cell recruitment to antigen-challenged lungs through chemokine production.
Project description:<b>Background:</b> 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. <b>Methods:</b> 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 β-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. <b>Results:</b> 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. <b>Conclusion:</b> Inhibiting S1PR2 attenuates TAD formation and prevents aortic rupture. Targeting S1PR2 may provide a promising treatment strategy against TAD.
Project description:Production of sphingosine-1-phosphate (S1P) is linked to 17β-estradiol (E2) activity in many estrogen-responsive cells; in bone development, the role of S1P is unclear. We studied effects of S1P on proliferation and differentiation of human osteoblasts (hOB). Ten nM E2, 1 μM S1P, or 1 μM of the S1P receptor 1 (S1PR1) agonist SEW2871 increased hOB proliferation at 24 hours. S1PR 1, 2, and 3 mRNAs are expressed by hOB but not S1PR4 or S1PR5. Expression of S1PR2 was increased at 7 and 14 days of differentiation, in correspondence with osteoblast-related mRNAs. Expression of S1PR1 was increased by E2 or S1P in proliferating hOB, whereas S1PR2 mRNA was unaffected in proliferating cells; S1PR3 was not affected by E2 or S1P. Inhibiting sphingosine kinase (SPHK) activity with sphingosine kinase inhibitor (Ski) greatly reduced the E2 proliferative effect. Both E2 and S1P increased SPHK mRNA at 24 hours in hOB. S1P promoted osteoblast proliferation via activating MAP kinase activity. Either E2 or S1P increased S1P synthesis in a fluorescent S1P assay. Interaction of E2 and S1P signaling was indicated by upregulation of E2 receptor mRNA after S1P treatment. E2 and S1P also promoted alkaline phosphatase expression. During osteoblast differentiation, S1P increased bone-specific mRNAs, similarly to the effects of E2. However, E2 and S1P showed differences in the activation of some osteoblast pathways. Pathway analysis by gene expression arrays was consistent with regulation of pathways of osteoblast differentiation; collagen and cell adhesion proteins centered on Rho/Rac small GTPase signaling and Map kinase or signal transducer and activator of transcription (Stat) intermediates. Transcriptional activation also included significant increases in superoxide dismutase 1 and 2 transcription by either S1P or E2. We demonstrate that the SPHK system is a co-mediator for osteoblast proliferation and differentiation, which is mainly, but not entirely, complementary to E2, whose effects are mediated by S1PR1 and S1PR2.
Project description:NLR family pyrin domain containing 3 (NLRP3) inflammasome accompanies chronic liver injury and is a critical mediator of inflammation-driven liver fibrosis. Sphingosine 1-phosphate (S1P)/S1P Receptor (S1PR) signaling participates in liver fibrogenesis by affecting bone marrow (BM)-derived monocytes/macrophage (BMM) activation. However, the relationship between S1P/S1PR signaling and NLRP3 inflammasome in BMMs remains unclear. Here, we found significantly elevated gene expression of NLRP3 inflammasome components (NLRP3, pro-interleukin-1?, and pro-interleukin-18) and the activation of NLRP3 inflammasome significantly elevated during murine chronic liver injury induced by a bile duct ligation operation, a methionine-choline-deficient and high-fat diet, or carbon tetrachloride intraperitoneal injection. Moreover, the increased expression of sphingosine kinase 1 (SphK1), the rate-limiting synthetic enzyme of S1P, was positively correlated with NLRP3 inflammasome components in both patients and mouse model livers. Flow cytometry analysis and immunofluorescence staining showed BMMs contributed to the significant proportion of NLRP3+ cells in murine inflammatory livers, but not Kupffer cells, dendritic cells, endothelial cells, T cells, and hepatocytes. Focusing on macrophages, S1P promoted NLRP3 inflammasome priming and activation in a dose-dependent manner. Blockade of S1PR2 by JTE-013 (antagonist of S1PR2) or S1PR2-siRNA inhibited S1P-induced NLRP3 inflammasome priming and inflammatory cytokine (interleukin-1? and interleukin-18) secretion, whereas blockade of S1PR1 or S1PR3 had no such effect. in vivo, a ?1,3-d-glucan-encapsulated siRNA particle (GeRP) delivery system is capable of silencing genes in macrophages specifically. Treatment with S1PR2 siRNA-GeRPs markedly reduced NLRP3 inflammasome priming and activation and attenuated liver inflammation and fibrosis. Together, the conclusions indicated that targeting macrophage S1PR2 retarded liver inflammation and fibrogenesis via downregulating NLRP3 inflammasome, which may represent an effective therapeutic strategy for chronic liver injury.
Project description:Mechanisms by which cancer cells communicate with the host organism to regulate lung colonization/metastasis are unclear. We show that this communication occurs via sphingosine 1-phosphate (S1P) generated systemically by sphingosine kinase 1 (SK1), rather than via tumour-derived S1P. Modulation of systemic, but not tumour SK1, prevented S1P elevation, and inhibited TRAMP-induced prostate cancer growth in TRAMP(+/+) SK1(-/-) mice, or lung metastasis of multiple cancer cells in SK1(-/-) animals. Genetic loss of SK1 activated a master metastasis suppressor, Brms1 (breast carcinoma metastasis suppressor 1), via modulation of S1P receptor 2 (S1PR2) in cancer cells. Alterations of S1PR2 using pharmacologic and genetic tools enhanced Brms1. Moreover, Brms1 in S1PR2(-/-) MEFs was modulated by serum S1P alterations. Accordingly, ectopic Brms1 in MB49 bladder cancer cells suppressed lung metastasis, and stable knockdown of Brms1 prevented this process. Importantly, inhibition of systemic S1P signalling using a novel anti-S1P monoclonal antibody (mAb), Sphingomab, attenuated lung metastasis, which was prevented by Brms1 knockdown in MB49 cells. Thus, these data suggest that systemic SK1/S1P regulates metastatic potential via regulation of tumour S1PR2/Brms1 axis.
Project description:Sphingosine kinase 1 (SphK1) and SphK2 are ubiquitous enzymes that generate sphingosine-1-phosphate (S1P), a ligand for a family of G protein-coupled receptors (S1PR1-S1PR5) with important functions in the vascular and immune systems. Here we explore the role of these kinases and receptors in recovery from anaphylaxis in mice. We found that Sphk2-/- mice had a rapid recovery from anaphylaxis. In contrast, Sphk1-/- mice showed poor recovery from anaphylaxis and delayed histamine clearance. Injection of S1P into Sphk1-/- mice increased histamine clearance and promoted recovery from anaphylaxis. Adoptive cell transfer experiments demonstrated that SphK1 activity was required in both the hematopoietic and nonhematopoietic compartments for recovery from anaphylaxis. Mice lacking the S1P receptor S1PR2 also showed a delay in plasma histamine clearance and a poor recovery from anaphylaxis. However, S1P did not promote the recovery of S1pr2-/- mice from anaphylaxis, whereas S1pr2+/- mice showed partial recovery. Unlike Sphk2-/- mice, Sphk1-/- and S1pr2-/- mice had severe hypotension during anaphylaxis. Thus, SphK1-produced S1P regulates blood pressure, histamine clearance, and recovery from anaphylaxis in a manner that involves S1PR2. This suggests that specific S1PR2 agonists may serve to counteract the vasodilation associated with anaphylactic shock.