Sphingosine-1-phosphate receptor 3 influences cell cycle progression in muscle satellite cells

Skeletal muscle retains a resident stem cell population called satellite cells, which are mitotically quiescent in mature muscle, but can be activated to produce myoblast progeny for muscle homeostasis, hypertrophy and repair. We have previously shown that satellite cell activation is partially controlled by the bioactive phospholipid, sphingosine-1-phosphate, and that S1P biosynthesis is required for muscle regeneration. Here we investigate the role of sphingosine-1-phosphate receptor 3 (S1PR3) in regulating murine satellite cell function. S1PR3 levels were high in quiescent myogenic cells before falling during entry into cell cycle. Retrovirally-mediated constitutive expression of S1PR3 led to suppressed cell cycle progression in satellite cells, but did not overtly affect the myogenic program. Conversely, satellite cells isolated from S1PR3-null mice exhibited enhanced proliferation ex-vivo. In vivo, acute cardiotoxin-induced muscle regeneration was enhanced in S1PR3-null mice, with bigger muscle fibres compared to control mice. Importantly, genetically deleting S1PR3 in the mdx mouse model of Duchenne muscular dystrophy produced a less severe muscle dystrophic phenotype, than when signalling though S1PR3 was operational. In conclusion, signalling though S1PR3 suppresses cell cycle progression to regulate function in muscle satellite cells.     

Accelerated Bone Regeneration by Astragaloside IV through Stimulating the Coupling of Osteogenesis and Angiogenesis

Both osteoblasts and preosteoclasts contribute to the coupling of osteogenesis and angiogenesis, regulating bone regeneration. Astragaloside IV (AS-IV), a glycoside of cycloartane-type triterpene derived from the Chinese herb Astragalus membranaceus, exhibits various biological activities, including stimulating angiogenesis and attenuating ischemic-hypoxic injury. However, the effects and underlying mechanisms of AS-IV in osteogenesis, osteoclastogenesis, and bone regeneration remain poorly understood. In the present study, we found that AS-IV treatment inhibited osteoclastogenesis, preserved preosteoclasts, and enhanced platelet-derived growth factor-BB (PDGF-BB)-induced angiogenesis. Additionally, AS-IV promoted cell viability, osteogenic differentiation, and angiogenic gene expression in bone marrow mesenchymal stem cells (BMSCs). The activation of AKT/GSK-3β/β-catenin signaling was found to contribute to the effects of AS-IV on osteoclastogenesis and osteogenesis. Furthermore, AS-IV accelerated bone regeneration during distraction osteogenesis (DO), as evidenced from the improved radiological and histological manifestations and biomechanical parameters, accompanied by enhanced angiogenesis within the distraction zone. In summary, AS-IV accelerates bone regeneration during DO, by enhancing osteogenesis and preosteoclast-induced angiogenesis simultaneously, partially through AKT/GSK-3β/β-catenin signaling. These findings reveal that AS-IV may serve as a potential bioactive molecule for promoting the coupling of osteogenesis and angiogenesis, and imply that AKT/GSK-3β/β-catenin signaling may be a promising therapeutic target for patients during DO treatment.     

NF-κB and EGFR participate in S1PR3-mediated human renal cell carcinomas progression

The bioactive lipid sphingosine 1-phosphate (S1P) is implicated in many pivotal processes for the physiological and pathological actions via activating five types of G-protein-coupled S1P receptors (S1PR1‐5). The role of S1P in renal cell carcinoma (RCC) and its receptor subtype specific mediating mechanism are poorly studied. So we focus on the regulatory role of S1P in RCC progression and the receptor subtypes involved in S1P-induced actions, intending to further clarify a novel therapeutic target for RCC. Analysis of The Cancer Genome Atlas (TCGA) databases showed that the patients with high expression of S1PR3 had significantly worse overall than with low expression. We further demonstrated that S1P could promote proliferation, migration, and epithelial-mesenchymal transition (EMT) of renal cancer cells in vitro, and the actions were enhanced with the increase of S1PR3 expression. Meanwhile, the results in animal experiments also showed that S1PR3 could accelerate tumorigenesis and metastasis of RCC. Our study also clarified the mechanism for S1P induced cell proliferation is mediated by S1PR3/Gi/p38/Akt/p65/cyclin D1-CDK4 pathway and the main pathway for migration is S1PR3/Gi/q/ERK/p38/p65. In addition, S1PR3 was involved in epidermal growth factor (EGF)-induced actions by enhancing protein expression, not by transactivation of epidermal growth factor receptor (EGFR). These results also further supported our conclusion that the carcinogenic role of S1P/S1PR3 axis. Thus, our findings provide that S1PR3 may be a promising small molecular therapeutic target for S1PR3 expressed cancers.     

High-mobility group box 1 accelerates distraction osteogenesis healing via the recruitment of endogenous stem/progenitor cells

Background aimsWhile distraction osteogenesis (DO) achieves substantial bone regeneration, prolonged fixation may lead to infections. Existing stem cell and physical therapies have limitations, requiring the development of novel therapeutic approaches. Here, we evaluated high-mobility group box 1 (HMGB1) as a novel therapeutic target for DO treatment.MethodsMicro-computed tomography (Micro-CT) analysis and histological staining of samples obtained from tibial DO model mice was performed. Transwell migration, wound healing, and proliferation assays were also performed on cultured human mesenchymal stem cells (hMSCs) and human umbilival vein endothelial cells (HUVECs). Tube formation assay was performed on HUVECs, whereas osteogenic differentiation assay was performed on hMSCs.ResultsMicro-CT analysis and histological staining of mouse samples revealed that HMGB1 promotes bone regeneration during DO via the recruitment of PDGFRα and Sca-1 positve (PαS⁺) cells and endothelial progenitor cells. Furthermore, HMGB1 accelerated angiogenesis during DO, promoted the migration and osteogenic differentiation of hMSCs as well as the proliferation, migration and angiogenesis of HUVECs in vitro.ConclusionsOur findings suggest that HMGB1 has a positive influence on endogenous stem/progenitor cells, representing a novel therapeutic target for the acceleration of DO-driven bone regeneration.     

Moesin Controls Clathrin-Mediated S1PR1 Internalization in T Cells

The lipid mediator sphingosine 1-phosphate (S1P) regulates a wide range of cellular activities, including vascular maturation, angiogenesis, and immune-cell trafficking. Among the five known receptors for S1P (S1PR1-S1PR5), S1PR1 is a critical regulator of lymphocyte trafficking: its signaling is required for lymphocyte egress from lymphoid organs, while its down-modulation by agonist-induced internalization is a prerequisite for lymphocyte entry into lymphoid organs from the bloodstream. Despite the importance of S1PR1 down-regulation in determining lymphocyte behavior, the molecular mechanism of its internalization in lymphocytes has not been defined. Here we show that agonist-induced S1PR1 internalization in T cells occurs via clathrin-mediated endocytosis and is regulated by moesin, an ezrin-radixin-moesin (ERM) family member. In S1P-stimulated T cells, S1PR1 relocalized within clathrin-coated vesicles (CCVs) and early endosomes, and S1PR1 internalization was blocked when clathrin was pharmacologically inhibited. Stimulating moesin-deficient T cells with S1P failed to induce S1PR1 internalization and CCV formation. Furthermore, treating moesin-deficient mice with FTY720, an S1P receptor agonist known to internalize S1PR1, caused delayed lymphopenia, and lymphocytes isolated from FTY720-treated moesin-deficient mice still responded to S1P ex vivo in chemotaxis assays. These results reveal a novel role for moesin in regulating clathrin-dependent S1PR1 internalization through CCV formation.     

Fingolimod inhibits PDGF-B-induced migration of vascular smooth muscle cell by down-regulating the S1PR1/S1PR3 pathway

Platelet Derived Growth Factor (PDGF) and sphingosine-1-phosphate (S1P) pathways play a key role in mural cell recruitment during tumor growth and angiogenesis. Fingolimod, a S1P analogue, has been shown to exert antitumor and antiangiogenic properties. However, molecular targets and modes of action of fingolimod remain unclear. In this study, we confirmed the antagonizing action of S1P and PDGF-B on rat vascular smooth muscle cell (VSMCs) growth and migration. We then compared siRNA and/or fingolimod (100 nM) treatments on PDGFR-β, S1PR1 S1PR2 and S1PR3 expression. Fingolimod induced a 50% reduction in S1PR3 protein expression which was cumulative with that obtained with anti-S1PR3 siRNA. We found that siRNA-induced inhibition of both PDGFR-β and S1PR3 was the most effective means to block VSMC migration induced by PDGF-B. Finally, we observed that fingolimod treatment associated with anti-S1PR1 siRNA principally inhibited VSMC growth while in combination with anti-S1PR3 siRNA it strongly inhibited VSMC migration. These results suggest that for rat VSMCs, the PDGFR-S1PR1 pathway is predominantly dedicated to cell growth while PDGFR-S1PR3 stimulates cell migration. As an S1P analogue, fingolimod is considered a potent activator of S1PR1 and S1PR3. However, its action on the PDGFR-S1PR platform appears to be dependent on S1PR1 and S1PR3 specific downregulation. Considering that the S1P pathway has already been shown to exert various crosstalks with tyrosine kinase pathways, it seems of great interest to evaluate fingolimod potential in combination with the numerous tyrosine kinase inhibitors used in oncology.     

Toxicity and anti-angiogenicity evaluation of Pak1 inhibitor IPA-3 using zebrafish embryo model

p21-activated kinase 1 (Pak1)—a key node protein kinase regulating various cellular process including angiogenesis—has been recognised to be a therapeutic target for multitude of diseases, and hence, various small molecule inhibitors targeting its activity have been tested. However, the direct toxic and anti-angiogenic effects of these pharmacologic agents have not been examined. In this study, we evaluate the translational efficacy of Pak1 inhibitor IPA-3 using zebrafish toxicity model system to stratify its anti-angiogenic potential and off-target effects to streamline the compound for further therapeutic usage. The morphometric analysis has shown explicit delay in hatching, tail bending, pericardial sac oedema and abnormal angiogenesis. We provide novel evidence that Pak1 inhibitor could act as anti-angiogenic agents by impeding the development of sub-intestinal vessel (SIV) and intersegmental vessels (ISVs) by suppressing the expression of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), neurophilin 1 (NRP1) and its downstream genes matrix metalloproteinase (MMP)-2 and MMP-9. Knockdown studies using 2-O-methylated oligoribonucleotides targeting Pak1 also revealed similar phenotypes with inhibition of angiogenesis accompanied with deregulation of major angiogenic factor and cardiac-specific genes. Taken together, our findings indicate that Pak1 signalling facilitates enhanced angiogenesis and also advocated the design and use of small molecule inhibitors of Pak1 as potent anti-angiogenic agents and suggest their utility in combinatorial therapeutic approaches targeting anomalous angiogenesis.     

The outs and the ins of sphingosine-1-phosphate in immunity

The potent lipid mediator sphingosine-1-phosphate (S1P) is produced inside cells by two closely related kinases, sphingosine kinase 1 (SPHK1) and SPHK2, and has emerged as a crucial regulator of immunity. Many of the actions of S1P in innate and adaptive immunity are mediated by its binding to five G protein-coupled receptors, designated S1PR1-5, but recent findings have also identified important roles for S1P as a second messenger during inflammation. In this Review, we discuss recent advances in our understanding of the roles of S1P receptors and describe the newly identified intracellular targets of S1P that are crucial for immune responses. Finally, we discuss the therapeutic potential of new drugs that target S1P signalling and functions.     

鞘氨醇激酶-磷酸鞘氨醇轴在血管生成相关性疾病中的作用

血管生成是指在原有血管的基础上形成新血管的过程.病理性血管生成是癌症、心血管类疾病和视网膜病变等一系列疾病的标志.1-磷酸鞘氨醇(sphingosine-1-phosphate,S1P)是一种信号脂质,由鞘氨醇激酶(sphingosine kinases,SPHK)合成,通过5种G蛋白偶联受体(sphingosine-...     

Sphingosine‐1‐phosphate (S1P) receptors: Promising drug targets for treating bone‐related diseases

Sphingosine‐1‐phosphate (S1P) is a natural bioactive lipid molecule and a common first or second messenger in the cardiovascular and immune systems. By binding with its receptors, S1P can serve as mediator of signalling during cell migration, differentiation, proliferation and apoptosis. Although the predominant role of S1P in bone regeneration has been noted in many studies, this role is not as well‐known as its roles in the cardiovascular and immune systems. In this review, we summarize previous research on the role of S1P receptors (S1PRs) in osteoblasts and osteoclasts. In addition, S1P is regarded as a bridge between bone resorption and formation, which brings hope to patients with bone‐related diseases. Finally, we discuss S1P and its receptors as therapeutic targets for treating osteoporosis, inflammatory osteolysis and bone metastasis based on the biological effects of S1P in osteoclastic/osteoblastic cells, immune cells and tumour cells.     

Follicular fluid high-density lipoprotein-associated sphingosine 1-phosphate (S1P) promotes human granulosa lutein cell migration via S1P receptor type 3 and small G-protein RAC1

Coordinated migration and progesterone production by granulosa cells is critical to the development of the corpus luteum, but the underlying mechanisms remain obscure. Sphingosine 1-phosphate (S1P), which is associated with follicular fluid high-density lipoprotein (FF-HDL), was previously shown to regulate ovarian angiogenesis. We herein examined the effects of S1P and FF-HDL on the function of granulosa lutein cells. Both FF-HDL and S1P induced migration of primary human granulosa lutein cells (hGCs) and the granulosa lutein cell line HGL5. In addition, FF-HDL but not S1P promoted progesterone synthesis, and neither of the two compounds stimulated proliferation of granulosa lutein cells. Polymerase chain reaction and Western blot experiments demonstrated the expression of S1P receptor type 1 (S1PR1), S1PR2, S1PR3, and S1PR5 but not S1PR4 in hGCs and HGL5 cells. The FF-HDL- and S1P-induced granulosa lutein cell migration was emulated by FTY720, an agonist of S1PR1, S1PR3, S1PR4, and S1PR5, and by VPC24191, an agonist of S1PR1 and S1PR3, but not by SEW2871 and phytosphingosine 1-phosphate, agonists of S1PR1 and S1PR4, respectively. In addition, blockade of S1PR3 with CAY1044, suramine, or pertussis toxin inhibited hGC and HGL5 cell migration toward FF-HDL or S1P, while blockade of S1PR1 and S1PR2 with W146 and JTE013, respectively, had no effect. Both FF-HDL and S1P triggered activation of small G-protein RAC1 and actin polymerization in granulosa cells, and RAC1 inhibition with Clostridium difficile toxin B or NSC23766 abolished FF-HDL- and S1P-induced migration. The FF-HDL-associated S1P promotes granulosa lutein cell migration via S1PR3 and RAC1 activation. This may represent a novel mechanism contributing to the development of the corpus luteum.     

The role of Ca2+/Calcineurin/NFAT signalling pathway in osteoblastogenesis

The bone remodelling process is closely related to bone health. Osteoblasts and osteoclasts participate in the bone remodelling process under the regulation of various factors inside and outside. Excessive activation of osteoclasts or lack of function of osteoblasts will cause occurrence and development of multiple bone‐related diseases. Ca²⁺/Calcineurin/NFAT signalling pathway regulates the growth and development of many types of cells, such as cardiomyocyte differentiation, angiogenesis, chondrogenesis, myogenesis, bone development and regeneration, etc. Some evidences indicate that this signalling pathway plays an extremely important role in bone formation and bone pathophysiologic changes. This review discusses the role of Ca²⁺/Calcineurin/NFAT signalling pathway in the process of osteogenic differentiation, as well as the influence of regulating each component in this signalling pathway on the differentiation and function of osteoblasts, whereby the relationship between Ca²⁺/Calcineurin/NFAT signalling pathway and osteoblastogenesis could be deeper understood.     

Sphingosine 1-Phosphate (S1P) Receptors 1 and 2 Coordinately Induce Mesenchymal Cell Migration through S1P Activation of Complementary Kinase Pathways

Normal bone turnover requires tight coupling of bone resorption and bone formation to preserve bone quantity and structure. With aging and during several pathological conditions, this coupling breaks down, leading to either net bone loss or excess bone formation. To preserve or restore normal bone metabolism, it is crucial to determine the mechanisms by which osteoclasts and osteoblast precursors interact and contribute to coupling. We showed that osteoclasts produce the chemokine sphingosine 1-phosphate (S1P), which stimulates osteoblast migration. Thus, osteoclast-derived S1P may recruit osteoblasts to sites of bone resorption as an initial step in replacing lost bone. In this study we investigated the mechanisms by which S1P stimulates mesenchymal (skeletal) cell chemotaxis. S1P treatment of mesenchymal (skeletal) cells activated RhoA GTPase, but this small G protein did not contribute to migration. Rather, two S1P receptors, S1PR1 and S1PR2, coordinately promoted migration through activation of the JAK/STAT3 and FAK/PI3K/AKT signaling pathways, respectively. These data demonstrate that the chemokine S1P couples bone formation to bone resorption through activation of kinase signaling pathways.     

Roles of Wnt signalling in bone growth, remodelling, skeletal disorders and fracture repair

Wnt signalling has an essential role in regulating bone formation and remodelling during embryonic development and throughout postnatal and adult life. Specifically, Wnt signalling regulates bone formation by controlling embryonic cartilage development and postnatal chondrogenesis, osteoblastogenesis, osteoclastogenesis, endochondral bone formation, and bone remodelling. Abnormalities in the function of Wnt genes give rise to or contribute to the development of several pathological bone conditions, including abnormal bone mass, osteosarcomas and bone loss in multiple myeloma. Furthermore, Wnt signalling is activated during bone fracture repair and plays a crucial role in regulating bone regeneration.     

Knock Out of S1P3 Receptor Signaling Attenuates Inflammation and Fibrosis in Bleomycin-Induced Lung Injury Mice Model

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in many critical cellular processes, including proliferation, migration, and angiogenesis, through interaction with a family of five G protein–coupled receptors (S1P1–5). Some reports have implicated S1P as an important inflammatory mediator of the pathogenesis of airway inflammation, but the role of S1P3 in the pathogenesis of lung diseases is not completely understood. We used S1P3-deficient (knockout (KO)) mice to clarify the role of S1P3 receptor signaling in the pathogenesis of pulmonary inflammation and fibrosis using a bleomycin-induced model of lung injury. On the seventh day after bleomycin administration, S1P3 KO mice exhibited significantly less body weight loss and pulmonary inflammation than wild-type (WT) mice. On the 28th day, there was less pulmonary fibrosis in S1P3 KO mice than in WT mice. S1P3 KO mice demonstrated a 56% reduction in total cell count in bronchoalveolar lavage fluid (BALF) collected on the seventh day compared with WT mice; however, the differential white blood cell profiles were similar. BALF analysis on the seventh day showed that connective tissue growth factor (CTGF) levels were significantly decreased in S1P3 KO mice compared with WT mice, although no differences were observed in monocyte chemotactic protein-1 (MCP-1) or transforming growth factor β1 (TGF-β1) levels. Finally, S1P levels in BALF collected on the 7th day after treatment were not significantly different between WT and S1P3 KO mice. Our results indicate that S1P3 receptor signaling plays an important role in pulmonary inflammation and fibrosis and that this signaling occurs via CTGF expression. This suggests that this pathway might be a therapeutic target for pulmonary fibrosis.     

Sphingosine-1-phosphate receptor expression and signaling correlate with uterine prostaglandin-endoperoxide synthase 2 expression and angiogenesis during early pregnancy

Signaling mechanisms coordinating uterine angiogenesis and tissue remodeling during decidualization are not completely understood. Prostanoid signaling is thought to play a functionally important role in each of these events. In the present study, we demonstrate that the subfamily of G-protein-coupled receptors that binds and becomes activated by the terminal signaling lipid in the sphingolipid pathway, sphingosine-1-phosphate (S1P), were expressed during uterine decidualization. Three of the five known S1P receptors, termed endothelial differentiation genes (Edg; Edg1, Edg3, and Edg5) were upregulated in the uterine deciduum from Day of Pregnancy (DOP) 4.5 to 7.5, while Edg6 and Edg8 expression remained unchanged. Consistent with angiogenesis in general during decidualization, we believe EDG1 and EDG5 to be regulated by the embryo because no microvascular expression for these receptors was observed in oil-induced deciduomas. Observed expression of EDG1 and EDG5 showed a similar expression pattern to that previously reported for prostaglandin-endoperoxide synthase 2 (PTGS2), transitioning from the sublumenal stromal compartment in the antimesometrial pole (DOP 5) to the microvasculature of the mesometrial pole (DOP 7). Furthermore, these two receptors colocalized with PTGS2 at three additional sites at the maternal:fetal interface throughout pregnancy. Treatment of cultured predecidualized stromal cells with S1P resulted in upregulation of Ptgs2 mRNA and PTGS2 protein, but not the downstream enzyme prostacyclin synthase. These combined results suggest the existence of a link between the sphingolipid and prostanoid signaling pathways in uterine physiology, and that, based on their expression pattern, S1P receptors function to coordinate uterine mesometrial angiogenesis during the implantation phase of early gestation.