Sphingosine-1-phosphate enhances satellite cell activation in dystrophic muscles through a S1PR2/STAT3 signaling pathway.
ABSTRACT: 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:Duchenne muscular dystrophy (DMD) is a congenital myopathy caused by mutations in the dystrophin gene. DMD pathology is marked by myositis, muscle fiber degeneration, and eventual muscle replacement by fibrosis and adipose tissue. Satellite cells (SC) are muscle stem cells critical for muscle regeneration. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that promotes SC proliferation, regulates lymphocyte trafficking, and is irreversibly degraded by sphingosine phosphate lyase (SPL). Here, we show that SPL is virtually absent in normal human and murine skeletal muscle but highly expressed in inflammatory infiltrates and degenerating fibers of dystrophic DMD muscle. In mdx mice that model DMD, high SPL expression is correlated with dysregulated S1P metabolism. Perinatal delivery of the SPL inhibitor LX2931 to mdx mice augmented muscle S1P and SC numbers, reduced leukocytes in peripheral blood and skeletal muscle, and attenuated muscle inflammation and degeneration. The effect on SC was also observed in SCID/mdx mice that lack mature T and B lymphocytes. Transcriptional profiling in the skeletal muscles of LX2931-treated vs. control mdx mice demonstrated changes in innate and adaptive immune functions, plasma membrane interactions with the extracellular matrix (ECM), and axon guidance, a known function of SC. Our cumulative findings suggest that by raising muscle S1P and simultaneously disrupting the chemotactic gradient required for lymphocyte egress, SPL inhibition exerts a combination of muscle-intrinsic and systemic effects that are beneficial in the context of muscular dystrophy.
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.
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:Sphingosine 1-phosphate (S1P) is synthesized from sphingosine by sphingosine kinases (SPHK1 and SPHK2) in invertebrates and vertebrates, whereas specific receptors for S1P (S1PRs) selectively appear in vertebrates, suggesting that S1P acquires novel functions in vertebrates. Because the developmental functions of SPHK1 and SPHK2 remain obscure in vertebrates, we generated sphk1 or sphk2 gene-disrupted zebrafish by introducing premature stop codons in their coding regions using transcription activator-like effector nucleases. Both zygotic sphk1 and sphk2 zebrafish mutants exhibited no obvious developmental defects and grew to adults. The maternal-zygotic sphk2 mutant (MZsphk2), but not the maternal-zygotic sphk1 mutant and maternal sphk2 mutant, had a defect in the cardiac progenitor migration and a concomitant decrease in S1P level, leading to a two-heart phenotype (cardia bifida). Cardia bifida in MZsphk2, which was rescued by injecting sphk2 mRNA, was a phenotype identical to that of zygotic mutants of the S1P transporter spns2 and S1P receptor s1pr2, indicating that the Sphk2-Spns2-S1pr2 axis regulates the cardiac progenitor migration in zebrafish. The contribution of maternally supplied lipid mediators during vertebrate organogenesis presents as a requirement for maternal-zygotic Sphk2.
Project description:BACKGROUND: The accumulation of beta amyloid (A?) peptides, a hallmark of Alzheimer's disease (AD) is related to mechanisms leading to neurodegeneration. Among its pleiotropic cellular effects, A? accumulation has been associated with a deregulation of sphingolipid metabolism. Sphingosine 1-phosphate (S1P) derived from sphingosine is emerging as a critical lipid mediator regulating various biological activities including cell proliferation, survival, migration, inflammation, or angiogenesis. S1P tissue level is low and kept under control through equilibrium between its synthesis mostly governed by sphingosine kinase-1 (SphK1) and its degradation by sphingosine 1-phosphate lyase (SPL). We have previously reported that A? peptides were able to decrease the activity of SphK1 in cell culture models, an effect that could be blocked by the prosurvival IGF-1/IGF-1R signaling. RESULTS: Herein, we report for the first time the expression of both SphK1 and SPL by immunohistochemistry in frontal and entorhinal cortices from 56 human AD brains. Immunohistochemical analysis revealed a decreased expression of SphK1 and an increased expression of SPL both correlated to amyloid deposits in the entorhinal cortex. Otherwise, analysis of brain tissue extracts showed a decrease of SphK1 expression in AD brains whereas SPL expression was increased. The content of IGF-1R, an activator of SphK1, was found decreased in AD brains as well as S1P1, the major receptor for S1P. CONCLUSIONS: Collectively, these results highlight the importance of S1P in AD suggesting the existence of a global deregulation of S1P signaling in this disease from its synthesis by SphK1 and degradation by SPL to its signaling by the S1P1 receptor.
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 kinase (SphK)/sphingosine-1-phosphate (S1P)/S1P receptor (S1PR) signaling pathway has been implicated in a variety of pathological processes of ovarian cancer. However, the function of this axis in ovarian cancer angiogenesis remains incompletely defined. Here we provided the first evidence that SphK1/S1P/S1PR1/3 pathway played key roles in ovarian cancer angiogenesis. The expression level of SphK1, but not SphK2, was closely correlated with the microvascular density (MVD) of ovarian cancer tissue. In vitro, the angiogenic potential and angiogenic factor secretion of ovarian cancer cells could be attenuated by SphK1, but not SphK2, blockage and were restored by the addition of S1P. Moreover, in these cells, we found S1P stimulation induced the angiogenic factor secretion via S1PR1 and S1PR3, but not S1PR2. Furthermore, inhibition of S1PR1/3, but not S1PR2, attenuated the angiogenic potential and angiogenic factor secretion of the cells. in vivo, blockage of SphK or S1PR1/3 could attenuate ovarian cancer angiogenesis and inhibit angiogenic factor expression in mouse models. Collectively, the current study showed a novel role of SphK1/S1P/S1PR1/3 axis within the ovarian cancer, suggesting a new target to block ovarian cancer angiogenesis.
Project description:S1P lyase (SPL) catalyzes the irreversible degradation of sphingosine-1-phosphate (S1P), a bioactive lipid whose signaling activities regulate muscle differentiation, homeostasis, and satellite cell (SC) activation. By regulating S1P levels, SPL also controls SC recruitment and muscle regeneration, representing a potential therapeutic target for muscular dystrophy. We found that SPL is induced during myoblast differentiation. To investigate SPL's role in myogenesis at the cellular level, we generated and characterized a murine myoblast SPL-knockdown (SPL-KD) cell line lacking SPL. SPL-KD cells accumulated intracellular and extracellular S1P and failed to form myotubes under conditions that normally stimulate myogenic differentiation. Under differentiation conditions, SPL-KD cells also demonstrated delayed induction of 3 myogenic microRNAs (miRNAs), miR-1, miR-206, and miR-486. SPL-KD cells successfully differentiated when treated with an S1P1 agonist, S1P2 antagonist, and combination treatments, which also increased myogenic miRNA levels. SPL-KD cells transfected with mimics for miR-1 or miR-206 also overcame the differentiation block. Thus, we show for the first time that the S1P/SPL/S1P-receptor axis regulates the expression of a number of miRNAs, thereby contributing to myogenic differentiation.
Project description:Cholangiocarcinoma (CCA) is characterized by a uniquely aggressive behavior and lack of effective targeted therapies. After analyzing the gene expression profiles of seven paired intrahepatic CCA microarrays, a novel sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) pathway and a novel target gene, SPHK1, were identified. We hypothesized that therapeutic targeting of this pathway can be used to kill intrahepatic cholangiocarcinoma (CCA) cells. High levels of SPHK1 protein expression, which was evaluated by immunohistochemical staining of samples from 96 patients with intrahepatic CCA, correlated with poor overall survival. The SPHK1 inhibitor SK1-I demonstrated potent antiproliferative activity in vitro and in vivo. SK1-I modulated the balance of ceramide-sphinogosine-S1P and induced CCA apoptosis. Furthermore, SK1-I combined with JTE013, an antagonist of the predominant S1P receptor S1PR2, inhibited the AKT and ERK signaling pathways in CCA cells. Our preclinical data suggest SPHK1/S1P pathway targeting may be an effective treatment option for patients with CCA.
Project description:Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that activates a family of G protein coupled-receptors (GPCRs) implicated in mammalian development, angiogenesis, immunity and tissue regeneration. S1P functions as a trophic factor for many cell types, including embryonic stem cells (ESCs). Sphingosine phosphate lyase (SPL) is an intracellular enzyme that catalyzes the irreversible degradation of S1P. We found SPL to be highly expressed in murine ESCs (mESCs). To investigate the role of SPL in mESC biology, we silenced SPL in mESCs via stable transfection with a lentiviral SPL-specific short hairpin RNA (shRNA) construct. SPL-knockdown (SPL-KD) mESCs showed a 5-fold increase in cellular S1P levels, increased proliferation rates and high expression of cell surface pluripotency markers SSEA1 and OCT4 compared to vector control cells. Compared to control mESCs, SPL-KD cells showed robust activation of STAT3 and a 10-fold increase in S1P2 expression. Inhibition of S1P2 or STAT3 reversed the proliferation and pluripotency phenotypes of SPL-KD mESCs. Further, inhibition of S1P2 attenuated, in a dose-dependent fashion, the high levels of OCT4 and STAT3 activation observed in SPL-KD mESCs. Finally, we showed that SPL-KD cells are capable of generating embryoid bodies from which muscle stem cells, called satellite cells, can be isolated. These findings demonstrate an important role for SPL in ESC homeostasis and suggest that SPL inhibition could facilitate ex vivo ESC expansion for therapeutic purposes.