New insights into the role of sphingosine 1-phosphate and lysophosphatidic acid in the regulation of skeletal muscle cell biology

Lysophospholipids are bioactive molecules that are implicated in the control of fundamental biological processes such as proliferation, differentiation, survival and motility in different cell types. Here we review the role of sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) in the regulation of skeletal muscle biology. Indeed, a wealth of experimental data indicate that these molecules are crucial players in the skeletal muscle regeneration process, acting by controllers of activation, proliferation and differentiation not only of muscle-resident satellite cells but also of mesenchymal progenitors that originate outside the skeletal muscle. Moreover, S1P and LPA are clearly involved in the regulation of skeletal muscle metabolism, muscle adaptation to different physiological needs and resistance to muscle fatigue. Notably, studies accomplished so far, have highlighted the complexity of S1P and LPA signaling in skeletal muscle cells that appears to be further complicated by their close dependence on functional cross-talks with growth factors, hormones and cytokines. Our increasing understanding of bioactive lipid signaling can individuate novel molecular targets aimed at enhancing skeletal muscle regeneration and reducing the fibrotic process that impairs full functional recovery of the tissue during aging, after a trauma or skeletal muscle diseases. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Effects of S1P on skeletal muscle repair/regeneration during eccentric contraction

Skeletal muscle regeneration is severely compromised in the case of extended damage. The current challenge is to find factors capable of limiting muscle degeneration and/or potentiating the inherent regenerative program mediated by a specific type of myoblastic cells, the satellite cells. Recent studies from our groups and others have shown that the bioactive lipid, sphingosine 1-phosphate (S1P), promotes myoblast differentiation and exerts a trophic action on denervated skeletal muscle fibres. In the present study, we examined the effects of S1P on eccentric contraction (EC)-injured extensor digitorum longus muscle fibres and resident satellite cells. After EC, skeletal muscle showed evidence of structural and biochemical damage along with significant electrophysiological changes, i.e. reduced plasma membrane resistance and resting membrane potential and altered Na(+) and Ca(2+) current amplitude and kinetics. Treatment with exogenous S1P attenuated the EC-induced tissue damage, protecting skeletal muscle fibre from apoptosis, preserving satellite cell viability and affecting extracellular matrix remodelling, through the up-regulation of matrix metalloproteinase 9 (MMP-9) expression. S1P also promoted satellite cell renewal and differentiation in the damaged muscle. Notably, EC was associated with the activation of sphingosine kinase 1 (SphK1) and with increased endogenous S1P synthesis, further stressing the relevance of S1P in skeletal muscle protection and repair/regeneration. In line with this, the treatment with a selective SphK1 inhibitor during EC, caused an exacerbation of the muscle damage and attenuated MMP-9 expression. Together, these findings are in favour for a role of S1P in skeletal muscle healing and offer new clues for the identification of novel therapeutic approaches to counteract skeletal muscle damage and disease.     

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.     

Sphingosine kinase type 1 induces G12/13-mediated stress fiber formation,yet promotes growth and survival independent of G protein-coupled receptors

Sphingosine 1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors (GPCRs) that regulate a wide variety of important cellular functions, including growth, survival, cytoskeletal rearrangements, and cell motility. However, whether it also has an intracellular function is still a matter of great debate. Overexpression of sphingosine kinase type 1, which generated S1P, induced extensive stress fibers and impaired formation of the Src-focal adhesion kinase signaling complex, with consequent aberrant focal adhesion turnover, leading to inhibition of cell locomotion. We have dissected biological responses dependent on intracellular S1P from those that are receptor-mediated by specifically blocking signaling of Galphaq, Galphai, Galpha12/13, and Gbetagamma subunits, the G proteins that S1P receptors (S1PRs) couple to and signal through. We found that intracellular S1P signaled "inside out" through its cell-surface receptors linked to G12/13-mediated stress fiber formation, important for cell motility. Remarkably, cell growth stimulation and suppression of apoptosis by endogenous S1P were independent of GPCRs and inside-out signaling. Using fibroblasts from embryonic mice devoid of functional S1PRs, we also demonstrated that, in contrast to exogenous S1P, intracellular S1P formed by overexpression of sphingosine kinase type 1 promoted growth and survival independent of its GPCRs. Hence, exogenous and intracellularly generated S1Ps affect cell growth and survival by divergent pathways. Our results demonstrate a receptor-independent intracellular function of S1P, reminiscent of its action in yeast cells that lack S1PRs.     

The role of sphingosine 1‐phosphate and its receptors in cardiovascular diseases

There are many different types of cardiovascular diseases, which impose a huge economic burden due to their extremely high mortality rates, so it is necessary to explore the underlying mechanisms to achieve better supportive and curative care outcomes. Sphingosine 1‐phosphate (S1P) is a bioactive lipid mediator with paracrine and autocrine activities that acts through its cell surface S1P receptors (S1PRs) and intracellular signals. In the circulatory system, S1P is indispensable for both normal and disease conditions; however, there are very different views on its diverse roles, and its specific relevance to cardiovascular pathogenesis remains elusive. Here, we review the synthesis, release and functions of S1P, specifically detail the roles of S1P and S1PRs in some common cardiovascular diseases, and then address several controversial points, finally, we focus on the development of S1P‐based therapeutic approaches in cardiovascular diseases, such as the selective S1PR1 modulator amiselimod (MT‐1303) and the non‐selective S1PR1 and S1PR3 agonist fingolimod, which may provide valuable insights into potential therapeutic strategies for cardiovascular diseases.     

Sphingosine 1-phosphate receptors modulate intracellular Ca2+ homeostasis

Ligation of sphingosine 1-phosphate (S1P) to a set of specific receptors named S1P receptors (S1PRs) regulates important biological processes. Although the ability of S1P to increase cytosolic Ca2+ in various cell types is well known, the role of the individual S1PRs has not been fully characterized. Here, we provide a complete analysis of S1P-dependent intracellular Ca2+ homeostasis in HeLa cells. Overexpression of S1P2, or S1P3, but not S1P1, leads to a significant increase in cytosolic and mitochondrial [Ca2+] in response to S1P challenge. Moreover, cells ectopically expressing S1P2, or S1P3 exhibited an appreciable decrease of the free Ca2+ concentration in the endoplasmic reticulum, dependent on stimulation of receptors by S1P endogenously present in the culture medium which was accompanied by a reduced susceptibility to C2-ceramide-induced cell death. These results demonstrate a differential contribution of individual S1PRs to Ca2+ homeostasis and its possible implication in the regulation of cell survival.     

Involvement of released sphingosine 1-phosphate/sphingosine 1-phosphate receptor axis in skeletal muscle atrophy

Skeletal muscle (SkM) atrophy is caused by several and heterogeneous conditions, such as cancer, neuromuscular disorders and aging. In most types of SkM atrophy overall rates of protein synthesis are suppressed, protein degradation is consistently elevated and atrogenes, such as the ubiquitin ligase Atrogin-1/MAFbx, are up-regulated. The molecular regulators of SkM waste are multiple and only in part known.Sphingolipids represent a class of bioactive molecules capable of modulating the destiny of many cell types, including SkM cells. In particular, we and others have shown that sphingosine 1phosphate (S1P), formed by sphingosine kinase (SphK), is able to act as trophic and morphogenic factor in myoblasts.Here, we report the first evidence that the atrophic phenotype observed in both muscle obtained from mice bearing the C26 adenocarcinoma and C2C12 myotubes treated with dexamethasone was characterized by reduced levels of active phospho-SphK1. The importance of SphK1 activity is also confirmed by the specific pharmacological inhibition of SphK1 able to increase Atrogin-1/MAFbx expression and reduce myotube size and myonuclei number. Furthermore, we found that SkM atrophy was accomplished by significant increase of S1P transporter Spns2 and in changes in the pattern of S1P receptor (S1PRs) subtype expression paralleled by increased Atrogin-1/MAFbx expression, suggesting a role for the released S1P and of specific S1PR-mediated signaling pathways in the control of the ubiquitin ligase. Altogether, these findings provide the first evidence that SphK1/released S1P/S1PR axis acts as a molecular regulator of SkM atrophy, thereby representing a new possible target for therapy in many patho-physiological conditions.     

鞘氨醇-1-磷酸与免疫细胞的调节

鞘氨醇-1-磷酸（sphingosine-1 phosphate,S1P）是来源于鞘脂代谢途径的多效性信号分子,其代谢受到多种因素调控。S1P由细胞内的鞘氨醇激酶（sphingosine kinases,SphKs）催化鞘氨醇的磷酸化而合成,可通过转运蛋白释放至细胞外。S1P可通过在胞外结合其特异性G蛋白偶联受体及胞内作用而调节多种重要生物学效应。作为细胞外介质和细胞内信使,S1P在免疫系统中也发挥重要的调节作用。S1P参与免疫细胞的迁移、增殖、分化及死亡细胞清除等过程。本文对S1P的代谢以及其对于免疫细胞的调节作用进行综述。     

Sphingosine 1-phosphate stimulates proliferation and migration of satellite cells: role of S1P receptors

Satellite cells are resident stem cells of skeletal muscle; they are normally quiescent but upon post-trauma activation start to proliferate and fuse with damaged fibers contributing to muscle regeneration. In this study the effect of the bioactive sphingolipid sphingosine 1-phosphate (S1P) on the proliferative and migratory response of murine satellite cells has been examined. S1P was found to stimulate labeled thymidine incorporation in a phosphatidylinositol 3-kinase-dependent manner. Moreover, by employing selective S1P receptor agonists and antagonists and silencing individual S1P receptors, the mitogenic action of S1P in satellite cells was shown to depend on S1P2 and S1P3. Notably, by using different experimental approaches S1P was found to positively influence satellite cell migration, necessary for their recruitment at the site of muscle damage. Interestingly, the specific silencing of individual S1P receptor subtypes demonstrated the pivotal role of S1P1 and S1P4 in mediating the S1P migratory effect. This latter result demonstrates for the first time that S1P4 receptor has a role in skeletal muscle cells, supporting the notion that this receptor subtype plays a biological action broader than that so far identified in lymphoid tissue. On the contrary, S1P2 was found to negatively regulate cell migration. Collectively, these results are in favour of an important function of S1P in satellite cell biology that could in principle be exploited as novel pharmacological target for improving skeletal muscle regeneration.     

Regulatory role of sphingosine kinase and sphingosine-1-phosphate receptor signaling in progenitor/stem cells

Balanced sphingolipid signaling is important for the maintenance of homeostasis. Sphingolipids were demonstrated to function as structural components, second messengers, and regulators of cell growth and survival in normal and disease-affected tissues. Particularly, sphingosine kinase 1 (SphK1) and its product sphingosine-1-phosphate (S1P) operate as mediators and facilitators of proliferation-linked signaling. Unlimited proliferation (selfrenewal) within the regulated environment is a hallmark of progenitor/stem cells that was recently associated with the S1P signaling network in vasculature, nervous,muscular, and immune systems. S1P was shown to regulate progenitor-related characteristics in normal and cancerstemcells(CSCs) viaG-protein coupled receptorsS1Pn(n=1 to 5). The SphK/S1P axis is crucially involved in the regulation of embryonic development of vasculature and the nervous system, hematopoietic stem cell migration, regeneration of skeletal muscle, and development of multiple sclerosis. The ratio of the S1P receptor expression, localization, and specific S1P receptoractivated downstream effectors influenced the rate of selfrenewal and should be further explored as regeneration related targets. Considering malignant transformation,it is essential to control the level of self-renewal capacity.Proliferation of the progenitor cell should be synchronized with differentiation to provide healthy lifelong function of blood, immune systems, and replacement of damaged ordead cells. The differentiation-related role of SphK/S1P remains poorly assessed. A few pioneering investigations exploredpharmacologicaltoolsthattargetsphingolipid signaling and can potentially confine and direct self-renewal towards normal differentiation. Further investigation is required to test the role of the SphK/S1P axis in regulation of self-renewal and differentiation.     

Ceramide 1-phosphate stimulates proliferation of C2C12 myoblasts

Recent studies have established specific cellular functions for different bioactive sphingolipids in skeletal muscle cells. Ceramide 1-phosphate (C1P) is an important bioactive sphingolipid that has been involved in cell growth and survival. However its possible role in the regulation of muscle cell homeostasis has not been so far investigated. In this study, we show that C1P stimulates myoblast proliferation, as determined by measuring the incorporation of tritiated thymidine into DNA, and progression of the myoblasts through the cell cycle. C1P induced phosphorylation of glycogen synthase kinase-3β and the product of retinoblastoma gene, and enhanced cyclin D1 protein levels. The mitogenic action of C1P also involved activation of phosphatidylinositol 3-kinase/Akt, ERK1/2 and the mammalian target of rapamycin. These effects of C1P were independent of interaction with a putative G(i)-coupled C1P receptor as pertussis toxin, which maintains G(i) protein in the inactive form, did not affect C1P-stimulated myoblast proliferation. By contrast, C1P was unable to inhibit serum starvation- or staurosporine-induced apoptosis in the myoblasts, and did not affect myogenic differentiation. Collectively, these results add up to the current knowledge on cell types targeted by C1P, which so far has been mainly confined to fibroblasts and macrophages, and extend on the mechanisms by which C1P exerts its mitogenic effects. Moreover, the biological activities of C1P described in this report establish that this phosphosphingolipid may be a relevant cue in the regulation of skeletal muscle regeneration, and that C1P-metabolizing enzymes might be important targets for developing cellular therapies for treatment of skeletal muscle degenerative diseases, or tissue injury.     

Roles of sphingosine 1-phosphate on tumorigenesis

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid with a variety of biological activities.It is generated from the conversion of ceramide to sphingosine by ceramidase and the subsequent conversion of sphingosine to S1P,which is catalyzed by sphingosine kinases.Through increasing its intracellular levels by sphingolipid metabolism and binding to its cell surface receptors,S1P regulates several physiological and pathological processes,including cell proliferation,migration,angiogenesis and autophagy.These processes are responsible for tumor growth,metastasis and invasion and promote tumor survival.Since ceramide and S1P have distinct functions in regulating in cell fate decision,the balance between the ceramide/sphingosine/S1P rheostat becomes a potent therapeutic target for cancer cells.Herein,we summarize our current understanding of S1P signaling on tumorigenesis and its potential as a target for cancer therapy.     

Bradykinin mediates myogenic differentiation in murine myoblasts through the involvement of SK1/Spns2/S1P2 axis

Skeletal muscle tissue retains a remarkable regenerative capacity due to the activation of resident stem cells that in pathological conditions or after tissue damage proliferate and commit themselves into myoblasts. These immature myogenic cells undergo differentiation to generate new myofibers or repair the injured ones, giving a strong contribution to muscle regeneration. Cytokines and growth factors, potently released after tissue injury by leukocytes and macrophages, are not only responsible of the induction of the initial inflammatory response, but can also affect skeletal muscle regeneration. Growth factors exploit sphingosine kinase (SK), the enzyme that catalyzes the production of sphingosine 1-phosphate (S1P), to exert their biological effects in skeletal muscle. In this paper we show for the first time that bradykinin (BK), the leading member of kinin/kallikrein system, is able to induce myogenic differentiation in C2C12 myoblasts. Moreover, evidence is provided that SK1, the specific S1P-transporter spinster homolog 2 (Spns2) and S1P₂ receptor are involved in the action exerted by BK, since pharmacological inhibition/antagonism or specific down-regulation significantly alter BK-induced myogenic differentiation. Moreover, the molecular mechanism initiated by BK involves a rapid translocation of SK1 to plasma membrane, analyzed by time-lapse immunofluorescence analysis. The present study highlights the role of SK1/Spns2/S1P receptor 2 signaling axis in BK-induced myogenic differentiation, thus confirming the crucial involvement of this pathway in skeletal muscle cell biology.     

Generation and metabolism of bioactive sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) is a bioactive lysosphingophospholipid that has been implicated in the regulation of vital biological processes. Abundant evidence indicates that S1P acts as both an intracellular messenger and an extracellular ligand for a family of five specific G protein-coupled S1P receptors (S1PRs). Cellular levels of S1P are tightly regulated in a spatio-temporal manner through its synthesis catalyzed by sphingosine kinases (SphKs) and degradation by S1P lyase (SPL) and specific S1P phosphohydrolases. Over the past decade, the identification and cloning of genes encoding S1P metabolizing enzymes has increased rapidly. Overexpression and deletion of these enzymes has provided important insights into the intracellular and the "inside-out" functions of S1P. The purpose of this review is to summarize the current knowledge of S1P metabolizing enzymes, their enzymatic properties, and their roles in the control of cellular functions by S1P.     

Fibro-adipogenic progenitors in skeletal muscle homeostasis,regeneration and diseases

Skeletal muscle possesses a remarkable regenerative capacity that relies on the activity of muscle stem cells, also known as satellite cells. The presence of non-myogenic cells also plays a key role in the coordination of skeletal muscle regeneration. Particularly, fibro-adipogenic progenitors (FAPs) emerged as master regulators of muscle stem cell function and skeletal muscle regeneration. This population of muscle resident mesenchymal stromal cells has been initially characterized based on its bi-potent ability to differentiate into fibroblasts or adipocytes. New technologies such as single-cell RNAseq revealed the cellular heterogeneity of FAPs and their complex regulatory network during muscle regeneration. In acute injury, FAPs rapidly enter the cell cycle and secrete trophic factors that support the myogenic activity of muscle stem cells. Conversely, deregulation of FAP cell activity is associated with the accumulation of fibrofatty tissue in pathological conditions such as muscular dystrophies and ageing. Considering their central role in skeletal muscle pathophysiology, the regulatory mechanisms of FAPs and their cellular and molecular crosstalk with muscle stem cells are highly investigated in the field. In this review, we summarize the current knowledge on FAP cell characteristics, heterogeneity and the cellular crosstalk during skeletal muscle homeostasis and regeneration. We further describe their role in muscular disorders, as well as different therapeutic strategies targeting these cells to restore muscle regeneration.     

Galectin-1 expression in innervated and denervated skeletal muscle

Galectin-1 is a soluble carbohydrate-binding protein with a particularly high expression in skeletal muscle. Galectin-1 has been implicated in skeletal muscle development and in adult muscle regeneration, but also in the degeneration of neuronal processes and/or in peripheral nerve regeneration. Exogenously supplied oxidized galectin-1, which lacks carbohydrate-binding properties, has been shown to promote neurite outgrowth after sciatic nerve sectioning. In this study, we compared the expression of galectin-1 mRNA and immunoreactivity in innervated and denervated mouse and rat hind-limb and hemidiaphragm muscles. The results show that galectin-1 mRNA expression and immunoreactivity are up-regulated following denervation. The galectin-1 mRNA is expressed in the extrasynaptic and perisynaptic regions of the muscle, and its immunoreactivity can be detected in both regions by Western blot analysis. The results are compatible with a role for galectin-1 in facilitating reinnervation of denervated skeletal muscle.