s1p lyase in skeletal muscle regeneration and satellite cell activation: exposing the hidden lyase

9
S1P lyase in skeletal muscle regeneration and satellite cell activation: Exposing the hidden lyase Julie D. Saba , Anabel S. de la Garza-Rodea Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA abstract article info Article history: Received 22 May 2012 Received in revised form 18 June 2012 Accepted 20 June 2012 Available online 28 June 2012 Keywords: Satellite cell Skeletal muscle Sphingosine phosphate lyase Sphingosine-1-phosphate Sphingolipid STAT3 Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid whose actions are essential for many physiological processes including angiogenesis, lymphocyte trafcking and development. In addition, S1P serves as a muscle trophic factor that enables efcient muscle regeneration. This is due in part to S1P's ability to activate quiescent muscle stem cells called satellite cells (SCs) that are needed for muscle repair. However, the molec- ular mechanism by which S1P activates SCs has not been well understood. Further, strategies for harnessing S1P signaling to recruit SCs for therapeutic benet have been lacking. S1P is irreversibly catabolized by S1P lyase (SPL), a highly conserved enzyme that catalyzes the cleavage of S1P at carbon bond C 23 , resulting in for- mation of hexadecenal and ethanolamine-phosphate. SPL enhances apoptosis through substrate- and product-dependent events, thereby regulating cellular responses to chemotherapy, radiation and ischemia. SPL is undetectable in resting murine skeletal muscle. However, we recently found that SPL is dynamically upregulated in skeletal muscle after injury. SPL upregulation occurred in the context of a tightly orchestrated genetic program that resulted in a transient S1P signal in response to muscle injury. S1P activated quiescent SCs via a sphingosine-1-phosphate receptor 2 (S1P2)/signal transducer and activator of transcription 3 (STAT3)-dependent pathway, thereby facilitating skeletal muscle regeneration. Mdx mice, which serve as a model for muscular dystrophy (MD), exhibited skeletal muscle SPL upregulation and S1P deciency. Pharma- cological SPL inhibition raised skeletal muscle S1P levels, enhanced SC recruitment and improved mdx skeletal muscle regeneration. These ndings reveal how S1P can activate SCs and indicate that SPL suppression may provide a therapeutic strategy for myopathies. This article is part of a Special Issue entitled Advances in Lysophospholipid Research. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Sphingosine-1-phosphate (S1P) is a bioactive lipid that binds to a family of ve G protein coupled receptors [1]. Through activation of S1P receptors and their G protein partners, S1P modulates the activities of adenylyl cyclase, the Ras-related C3 botulinum toxin substrate (Ras)/ mitogen activated protein kinase cascade, AKT and STAT3 signaling, phospholipase C and small Ras-homologous (Rho) GTPases. Through these actions, S1P signaling affects cell survival, proliferation, migration and cellcell interactions [2]. Recent studies have revealed the addition- al capability of S1P to modulate the activity of intracellular targets including NFκB, histone deacetylases and mitochondrial proteins through molecular mechanisms that appear to be independent of S1P receptors [3]. S1P signaling is essential for many physiological processes including angiogenesis [4,5], hematopoietic cell trafcking [68], embryonic development [9,10] and skeletal muscle homeostasis [11]. S1P is generated from sphingosine by a phosphorylation reaction cat- alyzed by the sphingosine kinases, SphK1 and SphK2 [12]. Sphingosine can be regenerated from S1P through the actions of S1P-specic phosphatases (Sgpp1 and Sgpp2) [13] and nonspecic lipid phospha- tases (LPPs) [14]. However, only one enzyme, S1P lyase (SPL), is respon- sible for the irreversible catabolism of S1P (Fig. 1) [15,16]. SPL is an integral membrane protein located in the endoplasmic reticulum. SPL degrades S1P in the cytosol by catalyzing its cleavage at the C 23 carbon bond, resulting in the formation of ethanolamine phosphate and the long chain aldehyde, hexadecenal. S1P activity has a major impact on intracel- lular S1P stores as well as tissue and circulating S1P levels, illustrated by the marked accumulation of S1P observed in cells, model organisms and genetically modied mice in which the gene encoding SPL has been Biochimica et Biophysica Acta 1831 (2013) 167175 Abbreviations: Dpl1, dihydrosphingosine phosphate lyase 1; DLMs, dorsal longitudinal muscles; DMD, Duchenne muscular dystrophy; DGC, dystroglycan-associated protein com- plex; ECM, extracellular matrix; HUVECs, human umbilical vein endothelial cells; LPPs, lipid phosphatases; MSCs, mesenchymal stem cells; MD, muscular dystrophy; NTX, notexin; Pax7, paired box protein-7; Rho, Ras-homologous; Rac, Ras-related C3 botulinum toxin substrate; SPL, S1P lyase; SCs, satellite cells; STAT3, signal transducer and activator of tran- scription 3; S1P, sphingosine-1-phosphate; S1P2, sphingosine-1-phosphate receptor 2; THI, tetrahydroxybutylimidazole This article is part of a Special Issue entitled Advances in Lysophospholipid Research. Corresponding author. Tel.: +1 510 450 7690; fax: +1 510 450 7910. E-mail address: [email protected] (J.D. Saba). 1388-1981/$ see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2012.06.009 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip

Upload: julie-d-saba

Post on 24-Nov-2016

214 views

Category:

Documents


1 download

TRANSCRIPT

Page 1: S1P lyase in skeletal muscle regeneration and satellite cell activation: Exposing the hidden lyase

Biochimica et Biophysica Acta 1831 (2013) 167–175

Contents lists available at SciVerse ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r .com/ locate /bba l ip

S1P lyase in skeletal muscle regeneration and satellite cell activation: Exposing thehidden lyase☆

Julie D. Saba ⁎, Anabel S. de la Garza-RodeaChildren's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA

Abbreviations: Dpl1, dihydrosphingosine phosphate lymuscles; DMD, Duchennemuscular dystrophy; DGC, dystroplex; ECM, extracellularmatrix; HUVECs, human umbilicalphosphatases; MSCs, mesenchymal stem cells; MD, musPax7, paired box protein-7; Rho, Ras-homologous; Rac, Rsubstrate; SPL, S1P lyase; SCs, satellite cells; STAT3, signal tscription 3; S1P, sphingosine-1-phosphate; S1P2, sphingositetrahydroxybutylimidazole☆ This article is part of a Special Issue entitled AResearch.⁎ Corresponding author. Tel.: +1 510 450 7690; fax:

E-mail address: [email protected] (J.D. Saba).

1388-1981/$ – see front matter © 2012 Elsevier B.V. Alldoi:10.1016/j.bbalip.2012.06.009

a b s t r a c t

a r t i c l e i n f o

Article history:Received 22 May 2012Received in revised form 18 June 2012Accepted 20 June 2012Available online 28 June 2012

Keywords:Satellite cellSkeletal muscleSphingosine phosphate lyaseSphingosine-1-phosphateSphingolipidSTAT3

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid whose actions are essential for many physiologicalprocesses including angiogenesis, lymphocyte trafficking and development. In addition, S1P serves as amuscletrophic factor that enables efficient muscle regeneration. This is due in part to S1P's ability to activatequiescent muscle stem cells called satellite cells (SCs) that are needed for muscle repair. However, the molec-ular mechanism by which S1P activates SCs has not been well understood. Further, strategies for harnessingS1P signaling to recruit SCs for therapeutic benefit have been lacking. S1P is irreversibly catabolized by S1Plyase (SPL), a highly conserved enzyme that catalyzes the cleavage of S1P at carbon bond C2–3, resulting in for-mation of hexadecenal and ethanolamine-phosphate. SPL enhances apoptosis through substrate- andproduct-dependent events, thereby regulating cellular responses to chemotherapy, radiation and ischemia.SPL is undetectable in resting murine skeletal muscle. However, we recently found that SPL is dynamicallyupregulated in skeletal muscle after injury. SPL upregulation occurred in the context of a tightly orchestratedgenetic program that resulted in a transient S1P signal in response to muscle injury. S1P activated quiescentSCs via a sphingosine-1-phosphate receptor 2 (S1P2)/signal transducer and activator of transcription 3(STAT3)-dependent pathway, thereby facilitating skeletal muscle regeneration. Mdx mice, which serve as amodel for muscular dystrophy (MD), exhibited skeletal muscle SPL upregulation and S1P deficiency. Pharma-cological SPL inhibition raised skeletal muscle S1P levels, enhanced SC recruitment and improvedmdx skeletalmuscle regeneration. These findings reveal how S1P can activate SCs and indicate that SPL suppression mayprovide a therapeutic strategy for myopathies. This article is part of a Special Issue entitled Advances inLysophospholipid Research.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Sphingosine-1-phosphate (S1P) is a bioactive lipid that binds to afamily of five G protein coupled receptors [1]. Through activation ofS1P receptors and their G protein partners, S1Pmodulates the activitiesof adenylyl cyclase, the Ras-related C3 botulinum toxin substrate (Ras)/mitogen activated protein kinase cascade, AKT and STAT3 signaling,phospholipase C and small Ras-homologous (Rho) GTPases. Throughthese actions, S1P signaling affects cell survival, proliferation, migration

ase 1; DLMs, dorsal longitudinalglycan-associated protein com-vein endothelial cells; LPPs, lipidcular dystrophy; NTX, notexin;as-related C3 botulinum toxinransducer and activator of tran-ne-1-phosphate receptor 2; THI,

dvances in Lysophospholipid

+1 510 450 7910.

rights reserved.

and cell–cell interactions [2]. Recent studies have revealed the addition-al capability of S1P to modulate the activity of intracellular targetsincluding NFκB, histone deacetylases and mitochondrial proteinsthrough molecular mechanisms that appear to be independent of S1Preceptors [3]. S1P signaling is essential formany physiological processesincluding angiogenesis [4,5], hematopoietic cell trafficking [6–8],embryonic development [9,10] and skeletal muscle homeostasis [11].

S1P is generated from sphingosine by a phosphorylation reaction cat-alyzed by the sphingosine kinases, SphK1 and SphK2 [12]. Sphingosinecan be regenerated from S1P through the actions of S1P-specificphosphatases (Sgpp1 and Sgpp2) [13] and nonspecific lipid phospha-tases (LPPs) [14]. However, only one enzyme, S1P lyase (SPL), is respon-sible for the irreversible catabolism of S1P (Fig. 1) [15,16]. SPL is anintegral membrane protein located in the endoplasmic reticulum. SPLdegrades S1P in the cytosol by catalyzing its cleavage at the C2–3 carbonbond, resulting in the formation of ethanolamine phosphate and the longchain aldehyde, hexadecenal. S1P activity has amajor impact on intracel-lular S1P stores as well as tissue and circulating S1P levels, illustrated bythe marked accumulation of S1P observed in cells, model organisms andgenetically modified mice in which the gene encoding SPL has been

Page 2: S1P lyase in skeletal muscle regeneration and satellite cell activation: Exposing the hidden lyase

Fig. 1. Formation of sphingosine 1-phosphate. Diagram of the formation of sphingosine1-phosphate starting from ceramide which is a central molecule of sphingolipid metab-olism. Sphingosine kinases (Sph-kinase) catalyze the phosphorylation of sphingosine,generating S1P. S1P lyase (SPL) catalyzes the cleavage of S1P into the long chain alde-hyde hexadecenal and ethanolamine-phosphate.

168 J.D. Saba, A.S. de la Garza-Rodea / Biochimica et Biophysica Acta 1831 (2013) 167–175

knocked down or knocked out [17–19]. By removing S1P in cellular,vascular and tissue compartments, SPL regulates the S1P pools availablefor interactionswith intracellular targets and for autocrine and paracrinereceptor-mediated signaling [20].

Based on the ability of SPL to generate S1P gradients required forlymphocyte trafficking, SPL inhibition via treatment with the drugLX2931 (Lexicon Pharmaceuticals) is now being tested as a strategyfor immunomodulation in the treatment of autoimmune diseasesuch as rheumatoid arthritis [21]. SPL induces apoptosis in responseto stressful conditions including DNA damage and ischemia throughmechanisms involving both substrate depletion and generation ofreactive aldehydes [22,23]. Delivery of a soluble bacterial SPL has alsobeen shown to reduce circulating S1P levels and block the pro-angiogenic effects of S1P receptor signaling [24]. These findings suggestthat modulation of SPL may be useful in the prevention and treatmentof a wide variety of human diseases including autoimmunity, cancer,ischemic tissue damage, fibrosis, inflammation and pathologicalangiogenesis.

The first SPL gene to be identified was the Saccharomyces cerevisiaedihydrosphingosine phosphate lyase 1 (Dpl1) gene [25]. Over the past15 years, the SPL genes of various bacterial, fungal, nematode, plantand mammalian genomes as well as murine and human Sgpl1 genesencoding SPL have been identified and characterized. In many cases,knockout models have been generated, and the mutant phenotypes inthese organisms have revealed critical roles for SPL in cellular function,development and physiology. Interestingly, Drosophila Sply mutantslacking SPL expression exhibit a myopathic phenotype affecting themuscles of the thorax that power the wings and enable flight [26].Based on this observation, we hypothesized that SPL has an essentialand conserved role in skeletal muscle homeostasis. Using a murinemodel of skeletal muscle injury, we have demonstrated that an S1P sig-nal is generated in response to muscle injury and, through activation ofS1P2, leads to downstream events that involve the transcription factorSTAT3 and the recruitment of skeletal muscle stem cells called satellitecells (SCs) that are needed for efficient skeletal muscle regeneration[27]. Further, we found that dystrophic mdx mice, which serve as amodel of Duchenne MD, are S1P-deficient due to chronic injury-inducedupregulation of SPL. Pharmacological inhibition of SPL improved SCrecruitment and muscle regeneration in a STAT3-dependent manner inmdx mice, thereby illustrating the potential utility of targeting SPL fortherapeutic benefit in MD.

This review will highlight our recent findings on the role andmechanism of action of S1P signaling in SC recruitment and muscle

regeneration. These observations will be related to the known functionsof S1P as a skeletal muscle trophic factor and SC activator. We presentsome new findings regarding the potential cellular sources of SPL ininjured muscle and demonstrate the presence of SPL expression inSC-derived myoblasts. We will discuss remaining questions and proposepotential next steps toward further elucidating the biology and clinicalpotential of modulating S1P metabolism and signaling for therapeuticpurposes in human diseases affecting skeletal muscle. We refer readersinterested in learning more about SPL structure, function and regulationto numerous recent reviews describing the biochemical characterizationof SPL, its subcellular localization, tissue distribution, regulation, role indevelopment, function in apoptosis, development of SPL inhibitors,and structure/function relationships predicted by recent crystallizationof a bacterial SPL [16,20,28–31].

2. The muscular dystrophies

MDs are a heterogeneous group of genetic diseases characterizedby the progressive loss of skeletal muscle strength associated withpathological features including pseudohypertrophy, muscle necrosisand fiber splitting, regeneration and centralized nuclei, variation infiber size, and eventual muscle replacement by adipose and fibrotictissues [32]. Together, these effects compromise patient mobilityand quality of life, and in the most severe cases lead to prematuredeath. In 1987, Eric Hoffman identified mutations in the dystrophingene as the cause of the most common and severe form of MD,Duchenne MD (DMD), which affects 1 in 4000 newborn males [33].The dystrophin protein links the plasma membrane to the internalcytoskeleton through interactions with γ-actin and with a plasmamembrane complex called the dystroglycan-associated protein com-plex (DGC). The DGC also interacts with the extracellular matrix(ECM). These connections anchor the plasma membrane internallyand externally, thereby facilitating the distribution of forces generatedupon muscle contraction. The lateral distribution of force stabilizes themyofiber and prevents membrane disruption with each contraction.Subsequent to the cloning of dystrophin, over 30 genes have beenlinked to hereditary MDs [32,34,35]. MD mutations are found in thecomponents of the DGC, DGC-interacting proteins, and enzymes thatregulate the expression, modification, and function of the DGC. In addi-tion, mutations in the ECM proteins laminin and collagen VI also causeMD [35]. Disruption of the normal bridge between ECM, DGC andγ-actin in patients with MD leads to high stress on fragile membranes,resulting in membrane lesions that overwhelm the membrane repairand muscle regeneration systems, ending finally in calcium influx andmuscle cell death [34,36,37]. In addition, there is evidence that modify-ing factors independent of the DGC may influence the severity of dys-trophy in MD [32,38]. Novel treatments for MD focus on reducingmuscle atrophy, inhibiting cell death pathways, and restoring mem-brane protein scaffolding and repair through the use of skeletal musclestem cells [39]. Characterizing the signaling pathways that controlthese processes may reveal new targets for therapeutic interventionin MD and muscle wasting disease. While it remains controversialwhether or not SC reserves and/or functions are reduced/impaired inMD, finding ways to expand endogenous stem cell reserves and/orenhance their myogenic potential may be of therapeutic benefit in MD.

3. S1P signaling, SCs and skeletal muscle regeneration

Adult skeletal muscle is a unique tissue that has the capacity toregenerate after injury [40]. Skeletal muscle regeneration involves theactivation of SCs found between the basal lamina and sarcolemma ofeach myofiber (Fig. 2). After muscle injury, SCs are stimulated to leavetheir quiescent state and reenter the cell cycle. SCs proliferate, andtheir progeny (myoblasts) adhere to and fusewith the damagedmusclecell (multinucleated syncytia or myofiber), stimulating a program ofdifferentiation that leads to regeneration of healthy tissue [41–43].

Page 3: S1P lyase in skeletal muscle regeneration and satellite cell activation: Exposing the hidden lyase

Fig. 2. SC localization. (A) SC stained with Pax7 antibody (red, marked by arrow)in a cross-section of human skeletal muscle. Scale bar 50 μm. (B) Upper panel,co-immunostaining (merged yellow) of SCs with Pax7 (green) and Caveolin-1(red) antibodies in isolated fibers of adult mouse muscle; lower panel, DAPI(blue) counterstained for nuclei. Scale bar 20 μm arrow points to SC.Taken from A.S. de la Garza‐Rodea [85].

Fig. 3. The Drosophila Sply mutant myopathy. Methylene blue and Azure II stain oftransversally sectioned thoraces of a normal fruit fly (A) and Sply mutant (B). The 6pairs of dorsal longitudinal skeletal muscles (asterisks) present in wild type Drosophila(A). Degenerating muscles in a Sply mutant (B). Scale bar 100 μm.

169J.D. Saba, A.S. de la Garza-Rodea / Biochimica et Biophysica Acta 1831 (2013) 167–175

S1P generated from catabolism of muscle membrane sphingolipidswas recently identified as the signal that causes SCs to enter the cellcycle, whereas chemical inhibition of S1P formation prevented skeletalmuscle regeneration [44–46]. This suggests a central role for S1P inskeletal muscle homeostasis. However, the mechanism responsible forthis was not known. Rodent muscles express three of the five knownS1P receptors, and S1P exerts a trophic action on denervated musclefibers [46]. Sphingolipids including S1P have also been implicated inthe regulation of muscle cell signaling, contractile properties, insulinresistance and muscle fiber trophism [47]. These findings suggest thatS1Pmetabolism and signaling contribute tomuscle homeostasis and re-pair, although the fundamental mechanisms responsible for the actionsof S1P in muscle remain poorly understood. The chronic/degenerativephase of MD is caused by a failure of skeletal muscle regeneration tokeep up with the ongoing injury and destruction of muscle fibers [48].This may be accounted for in part by a reduction in SC reserves or SCmyogenic potential. Elucidating the specific roles that S1P signalingplays in SC biology,muscle regeneration, aging andMDmay help devisenew approaches to maintaining muscle function and reducing loss ofmuscle mass in these diseases.

4. The Splymutant as an invertebratemodel formuscular dystrophy

Sply mutants harboring a transposon insertion mutation in the geneencoding theDrosophila homolog of SPL cannot catalyze the degradationof S1P and accumulate sphingolipid intermediates. Sply mutants areflightless due to degeneration of the thoracic flight muscles that powerthe wings [26]. The thoraces of wild type fruit flies exhibit an invariantpattern of six pairs of dorsal longitudinal muscles (DLMs) as shown inFig. 3A. In the Sply mutants, one or more fibers of the DLM are typicallyabsent, and the severity of this myopathy was observed to increasewith age (Fig. 3B). The Sply flightless phenotype can be recapitulatedby knockdown of Sply in myoblast precursor cells using RNA interfer-ence, indicating that the defect is intrinsic to muscle (our unpublishedobservations). Interestingly, we have observed that RNAi-mediatedknockdown of Sply expression in Drosophila S2 cells leads to reductionof dystrophin expression (our unpublished observations). These cumula-tive findings provide dramatic evidence that sphingolipid metabolismplays a crucial and intrinsic role in muscle stability and that tight controlover sphingolipidmetabolism is necessary tomaintainmuscle homeosta-sis. They also specifically implicate SPL in skeletalmuscle homeostasis and

suggest that the Sply mutant may potentially serve as an invertebratemodel of DMD.

5. Murine muscle injury elicits a metabolic response that producesan S1P signal

The genetic and molecular pathways that govern muscle develop-ment are largely conserved between Drosophila and humans [49,50].To explore whether the Sply phenotype accurately predicts a role forS1P signaling in mammalian skeletal muscle homeostasis, we firstexamined the expression of the murine SPL gene Sgpl1 in skeletalmuscle at rest. Our findings indicate that SPL is expressed at low orundetectable levels in resting skeletal muscle [27]. However, we haveobserved that SPL expression is induced in response to various stressfulconditions, such as radiation and ischemia [22,51]. This raised thepossibility that expression of the lyase, while hidden at rest, may be“exposed” in response to injury. Therefore, we examined the effect ofmuscle injury on Sgpl1 aswell as other genes involved in S1Pbiosynthesis,catabolism and signaling. To accomplish this,we employed a commonlyused system for analyzing murine skeletal muscle injury, the notexin(NTX) injury model. NTX is a snake venom phospholipase A2 thatwhen injected into muscle causes small lesions in the sarcolemma,resulting in muscle fiber degeneration. Due to the capacity of skeletalmuscle for regeneration, the muscle recovers from this injury over thecourse of two to four weeks, [37,52,53]. Within 6 h of NTX injectioninto the gastrocnemius muscles of wild type mice, we observed induc-tion of Sphk1 gene expression 100-fold over baseline levels as deter-mined by quantitative RT-PCR. The expression of Sgpl1 was induced20-fold over baseline several days after injury, concomitant withupregulation of the muscle transcription factor, Myf5. Other genesinvolved in S1P signaling including Sphk2, Sgpp1 and genes encodingS1P receptors 1–3 were upregulated after injury, but the changeswere more modest than those of Sphk1 and Sgpl1. Using additional de-tection methods, we confirmed that SPL gene and protein expressionare induced inmuscle fibers after treatmentwith NTX. Not surprisingly,whenwemeasured S1P plasma levels, we found that they are increasedby 50% compared to baseline levels within the first 24 h after injury[27]. Thus, a dynamic and tightly orchestrated genetic programmodulates S1P metabolism and receptor expression in response tomuscle injury. These changes lead to the generation of a plasma S1P“signal.” Based on these findings, it became important to determinethe significance of that signal for skeletal muscle regeneration.

6. SphK1 is essential for efficient skeletal muscle regeneration andSC functions

Two sphingosine kinases (SphK1 and SphK2) contribute to S1Pbiosynthesis in mammals. In mice, the disruption of one SphK geneis not lethal, provided the other is functional. Although SphK1 and

Page 4: S1P lyase in skeletal muscle regeneration and satellite cell activation: Exposing the hidden lyase

170 J.D. Saba, A.S. de la Garza-Rodea / Biochimica et Biophysica Acta 1831 (2013) 167–175

SphK2 knockout mice appear healthy under routine conditions, somephenotypes have been revealed in response to specific stressful stimuli.For example, SphK1knockoutmice fail to achieve cardiac preconditioningand aremore susceptible than controlmice to anaphylactic shock,where-as SphK2 knockout mice are more susceptible to kidney ischemic injury[54–57]. These findings suggest that the developmental functions ofSphKs are redundant, whereas individual SphKs may be responsible forspecific functions in postnatal life.

Therefore, to address whether the profound upregulation of SphK1and corresponding peak in plasma S1P levels we observed withinhours of intramuscular NTX injection signified a specific function forSphK1 in the injured muscle, we compared the biochemical andfunctional consequences of NTX muscle injury in SphK1 global knock-out mice and wild type controls. We found that the skeletal musclesof wild type mice were largely repaired by 10 days after injury. Thepoint of maximal injury exhibited mostly newly regenerated fiberscontaining a centralized nucleus, the hallmark of a regenerated musclefiber. In contrast, at the same time point the SphK1 knockout mousemuscles exhibited many residual necrotic fibers and only partial regen-eration at the site of maximal injury. Importantly, when we measuredSCs at the injury site, SphK1 knockoutmice lacked a robust recruitmentof SCs compared to wild type mice. In addition, plasma S1P levels inSphK1 knockout mice were approximately half the levels found in thecirculation of wild type mice throughout the course of the experiment[27].

To study the requirement for S1P signaling in SC functions in moredetail, we isolated SCs from the injured muscle fibers of wild type andSphK1 knockout mice and characterized them by measuring theircapacity for proliferation and differentiation using in vitro assays. Incomparison to wild type SCs incubated in media supplemented withwild type serum, we observed that SphK1 knockout SCs incubatedin media supplemented with SphK1 knockout serum did not performas well, exhibiting reduced proliferation and differentiation. The func-tions of the SphK1 knockout SCs were enhanced by incubation withwild type serum, although not to the level of wild type SCs. Converse-ly, wild type SCs incubated in medium containing serum from SphK1knockout mice were also functionally compromised, although not asseverely as SphK1 knockout SCs [27]. These experiments revealedthat SphK1 induction and S1P signaling contribute to efficient SCrecruitment and muscle regeneration in response to acute injury.They also suggest that after muscle injury SCs may respond to S1Psignals derived frommultiple sources. S1P production by SphK1with-in the SC itself may be required for autocrine stimulation. In addition,when the sequestered niche of the SC is compromised by injury, SCsare likely exposed to S1P derived from the injured fiber, the vascula-ture or from other source within the muscle fiber microenvironment.It seems likely that maximal SC activation requires S1P signals gener-ated from both sources. However, it is also possible that SphK1 knock-out mice fail to mobilize other circulating factors independent of orcomplementary to S1P that are needed for maximal SC activationand recruitment. Further investigation of the cytokine responses inSphK1 knockout mice may reveal more about the contribution ofSphK1 and S1P signaling to the global inflammatory response toskeletal muscle injury.

7. Dystrophic mice exist in a state of S1P deficiency

Considering the dramatic changes in S1P metabolism and signal-ing that we observed in response to acute muscle injury, we predictedthat chronic, global skeletal muscle injury that occurs in patientssuffering from MD might also have an impact on S1P levels and me-tabolism. If so, this could have potential consequences for muscle re-generation and SC recruitment in these conditions and, conversely,could present opportunities for therapeutic intervention. MaleC57BL/10ScSn-Dmdmdx/J (mdx) mice harbor a single point mutationin the mouse dystrophin gene located on the X chromosome [58].

This mutation is equivalent to the mutation that causes DMD inhumans, making mdx mice a useful model of the disease, althoughthe murine version is milder (due to the presence of revertant fibersand utrophin expression) and has greater capacity for regenerationdue to other genetic factors [59]. When we compared the expressionlevels of the genes by RT-PCR involved in S1P metabolism in themuscles of mdx mice and control mice of the same background, wefound no changes in Sphk1 or Sphk2 levels but a significant increasein Sgpl1 expression. The upregulation of Sgpl1 was confirmed by im-munoblotting, which revealed a very high level of SPL expression inthe dystrophic muscles of mdx mice compared to controls. Skeletalmuscle comprises the largest single organ of the body making upapproximately half of the body's mass. Thus, it was perhaps not toosurprising that the upregulation of SPL in dystrophic skeletal musclecorrelated with a significant reduction of plasma S1P in mdx micecompared to controls. The S1P levels in mdx mouse plasma were aslow as those observed in SphK1 knockout mice [27]. This suggestedthat muscle regeneration and SC recruitment might be compromisedby progressive S1P deficiency in dystrophic muscles. Further, itsuggests that inhibiting SPL might provide a useful way to boostS1P levels and improve efficiency of skeletal muscle regeneration astherapeutic.

8. SPL expression in SCs, myoblasts, myofibers and other cells ofskeletal muscle

The findings above point toward an important role for S1P in SCproliferation and a potential role in muscular dystrophy. However,the role in the muscle regeneration process of intracellular S1P andof SPL, the enzyme responsible for degrading S1P and controllingits intracellular levels, have not been fully explored. Specifically, ourstudy did not investigate the cellular source of SPL responsible for in-creasing SPL levels in skeletal muscle in the aftermath of injury. In ourlaboratory, we have conducted a pilot study in which SC-derivedmyoblasts were isolated from mdx mouse skeletal muscles, culturedfor 24 h, fixed and stained with specific antibodies for SPL and pairedbox protein-7 (Pax7), the latter which serves as a marker for SCs.Fig. 4 shows that majority of cells we isolated express SPL. Further,some of these cells were positive for both Pax7 and SPL. This suggeststhat SPL expression is present in activated SCs and/or in SC progenythat have differentiated to myoblasts. Our observation is consistentwith other results from our laboratory indicating that murine C2C12cells express high levels of SPL and that knockdown of SPL in thiscell line has profound effects on the ability of the cells to differentiateinto myoblasts (our unpublished observations).

Skeletal muscle regeneration involves the complex interaction ofmany cell types, not only the injured fiber and SCs, but also vascularcells, vascular support cells and inflammatory cells that enter the skele-tal muscle interstitium in response to injury. In other studies, we haveobserved that bone marrow-derived blood cells amply express SPL(Borowsky et al., submitted). Thus, inflammatory cells that infiltrate in-jured skeletal muscle likely contribute to the total amount of skeletalmuscle SPL pool after injury. To directly address this possibility,as well as to further explore the presence of SPL in cells within theSC–myoblast–myofiber continuum, cryosections of wild type skeletalmuscle (uninjured and 10 days post NTX injection) were stained forSPL and Pax7. We observed that quiescent SC and muscle fibers at restdo not express detectible amounts of SPL (Fig. 5). In response to injury,many mononucleated infiltrating cells were found to express SPL, con-firming that some of the SPL “upregulation” we observed in injuredmuscle can be explained by inflammatory changes. However, we didnot observe SPL expression in the regenerating muscle fibers or SCs insitu at 10 days after injury. Taken together with our finding of SPLexpression in isolated SCs and SC-derived myoblasts in culture, ourhistological results suggest that SPL upregulationmay largely representan inflammatory response to muscle injury, but that SPL may also be

Page 5: S1P lyase in skeletal muscle regeneration and satellite cell activation: Exposing the hidden lyase

SPL Pax7 Hoescht SPL Pax7 Hoescht

dcba

fe g h

i j k l

Fig. 4. Mdx SC-derived myoblasts express SPL. SC-derived myoblasts were isolated from skeletal muscle of mdx mice. Cells were in culture for 24 h before being fixed and stainedwith specific antibodies for SPL and Pax7. Nuclei were counterstained with Hoechst 33342. Two areas (a to d) and (e to h) show a Pax7 positive cell among several SPL positive cells.(i to l) cells stained with secondary antibodies (Alexa 488 and Alexa 546) and Hoechst only. Scale bar 100 μm.

171J.D. Saba, A.S. de la Garza-Rodea / Biochimica et Biophysica Acta 1831 (2013) 167–175

transiently expressed in activated SCs and/or in the progenyof activatedSCs (Fig. 5). Further imaging studies that follow SPL and Pax7 expres-sion in situ throughout the time course of the first week after injurywill be required to define whether SPL expression within SC-derivedmyoblasts may contribute to localized S1P gradients and affect theS1P levels of regenerating fibers in the aftermath of muscle injury. It isinteresting to note that acute stress responses have been characterizedby rapid and significant lymphopenia. Further, snake venoms, includingnotexin, have been shown to induce lymphopenia in rats [60,61]. Thekinetics of induced lymphopenia after notexin injection, which occurswithin 24 h, is similar to the kinetics of SphK1 upregulation and S1Pincrease that we have observed after notexin-induced injury in mice.Thus, there may be a causal connection between the increased S1P re-lease and retention of cells in peripheral tissues as amechanism leadingto notexin-induced lymphopenia.

9. SPL inhibition improves the response to acute muscle injury inmdx mice

Tetrahydroxybutylimidazole (THI) is a byproduct of sugar car-amelization that is found in many food substances containing caramelfood coloring type III. THI was recently shown to serve as an immuno-modulatory agent by virtue of its ability to inhibit SPL [62]. Chemicalderivatives of THI have subsequently been developed and are undergoingclinical evaluation for therapeutic efficacy in patients with rheumatoidarthritis [21]. To test the effect of SPL inhibition on muscle regenerationin a dystrophicmodel, we compared the short-term effects of THI admin-istration compared to vehicle in mdxmice after acute muscle injury withNTX. We observed a significant improvement in the number ofregenerating fibers after injury when mice received THI. Quantificationof SCs by both flow cytometry and immunostaining revealed that THItreatment improved SC activation as well [27]. These important findingsdemonstrate that SPL inhibition may have potential as a strategy to im-prove SC function and recruitment in the context of muscular dystrophy.Interestingly, T-cells have been shown to promote pathology ofmdxmice

[63]. Further, the immunosuppressant rapamycin ameliorates the dystro-phic phenotype of mdx mice [64]. One of the main effects of THI andgenetic knockout of SPL is retention of lymphocytes in lymphoid organsand preventing them from reaching sites of injury and inflammation.Therefore, it is conceivable that such immunosuppressive activity mayadd to the potential therapeutic efficacy of SPL inhibition in disease statesthat involve muscle injury.

10. THI promotes muscle regeneration and SC recruitment throughSTAT3 signaling

Zammit and colleagues showed that S1P contributes to the activationof SCs [44], but the molecular mechanism underlying this effect was notwell understood. The ability of THI to promote muscle regeneration andSC recruitment gave us a tool with which to investigate this question invivo. Recent studies by the laboratory of Hua Yu demonstrated theexistence of cross-talk between signaling pathways involving S1P andthe transcriptional regulator STAT3 [65]. The interactions between S1Pand STAT3were found to be responsible for the chronic STAT3 activationin some types of cancer. Additional reports provided further evidence forS1P and STAT3 signaling interactions in the context of prostate cancerand cardiomyocyte function [66,67]. When we measured STAT3 activa-tion in the skeletal muscles of mdx mice 5 days after NTX injury, THItreatment appeared to have a positive effect, as shown by increasedphosphorylation on STAT3 Tyrosine 703 [27]. Importantly, inhibition ofSTAT3 signaling using the small molecule WP1066 reversed the abilityof THI to promote muscle regeneration and increase the number of SCsseveral days after injury, indicating the importance of STAT3 activationin this process.

11. S1P activates SCs via S1P2/STAT3-dependent repression of cellcycle inhibitors

Since S1P can act via receptor-dependent as well as intracellular,receptor-independent mechanisms, we next measured the effect of

Page 6: S1P lyase in skeletal muscle regeneration and satellite cell activation: Exposing the hidden lyase

Pax7 Hoescht SPL Pax7 Hoeschtun

inju

red

10d

post

NTX

Pax7 Hoescht SPL Pax7 Hoescht

unin

jure

d10

d po

st N

TXA

a b c d

e f g h

a b c d

e f g h

B

SPL

SPL

Fig. 5. SPL is expressed in inflammatory cells of degenerating/regenerating skeletal muscle. Cross-sections of cryopreserved skeletal muscle of an uninjured wild type (C57BL/6)mouse at rest (upper panels) and 10 days post-NTX-injury (bottom panels). Muscles were excised, sectioned, stained with antibodies specific for SPL and Pax7 and counterstainedwith Hoechst 33342. A: (a to d) resting myofibers do not express SPL; (e to h) strong stain is observed in degenerated/necrotic myofibers undergoing regeneration. Scale bar100 μm. B: SCs (arrows) that were detected in both injured and uninjured muscles did not co-stain for SPL. Scale bar 10 μm.

Fig. 6. Role of S1P in response to skeletalmuscle repair. After skeletalmuscle injury, SphK1generates S1P, which activates S1PR2, leading to RhoA and STAT3 activation, therebyrepressing small cell cycle inhibitor proteins. SCs reenter the cell cycle and contribute tomuscle regeneration. Inhibition of SPL using THI leads to a boost in S1P signaling, therebypromoting SC activation and efficient muscle regeneration. Note that the localization ofSPL is assigned to the myofiber for convenience. However, SPL in SCs and inflammatorycells may also regulate S1P levels in and around the injured muscle.

172 J.D. Saba, A.S. de la Garza-Rodea / Biochimica et Biophysica Acta 1831 (2013) 167–175

inhibitors of the three major S1P receptors in muscle, S1P1-3, onSTAT3 activation in isolated SCs. Since the isolation of SCs from in-jured muscle fibers readily activates them, we were not surprised tofind a significant level of phosphorylated, active STAT3 at baseline.We found that treatment of SCs with S1P2 inhibitor prevented STAT3 ac-tivation in SC-derivedmyoblasts,whereas inhibitors of S1P1 and S1P3 didnot alter STAT3 activation.We found that siRNA-mediated knockdown ofS1P2 also reduced STAT3 activation inmurine SC-derivedmyoblasts [27].Importantly, the inhibition of S1P2 led to the upregulation of two cellcycle inhibitor proteins, p21 and p27. These proteins are known to beunder repressive control by STAT3. We interpreted these findings as re-vealing a plausible mechanism by which S1P recruits SCs into the cellcycle, i.e. by activating STAT3, leading to repression of cell cycle inhibitors,thereby allowing the SC to re-enter the cell cycle and begin proliferatingin response to injury. A schematic diagram of this mechanism is illustrat-ed in Fig. 6. There remained one additional questionwe hoped to answer,i.e., by what mechanism did S1P ligation to S1P2 induce the activation ofSTAT3? We suspected that this process might involve members of theRho family of small GTPases, including Ras-related C3 botulinum toxinsubstrate (Rac) 1, cell division control protein 42 homolog, and RhoA.These proteins function downstream of G protein coupled receptors in-cluding S1P2 and have been shown to promote tyrosine phosphorylationof STAT3 and its translocation to the nucleus in various cellular contextsincluding carcinogenesis. Our results using a combination of Rac1 andRhoA inhibitors and plasmid constructs with which we modulated Rac1

and RhoA expression and activity demonstrated that S1P2 activation in-hibits Rac1 and activates RhoA, thereby promoting STAT3 phosphoryla-tion on Tyrosine 703 in myoblasts (Fig. 6) [27].

Page 7: S1P lyase in skeletal muscle regeneration and satellite cell activation: Exposing the hidden lyase

173J.D. Saba, A.S. de la Garza-Rodea / Biochimica et Biophysica Acta 1831 (2013) 167–175

12. Other recent findings regarding S1P, SCs and skeletal muscleregeneration

Extensive research has been conducted to better understand therole of S1P in the biology of SCs and the process of skeletal muscleregeneration. S1P has been shown to stimulate the proliferation ofquiescent SCs in vivo after cardiotoxin-induced damage [44]. Further,in vitro studies have demonstrated that exogenous S1P increases theintake of labeled thymidine incorporation in a PI3K-dependent man-ner and that the proliferative action of the sphingolipid depends onS1P2 and S1P3 [68]. The involvement of S1P in the migration of SCshas been also explored. Employing S1P receptor agonists and antago-nists as well as specific siRNAs, Calise et al. found that S1P1 and S1P4are involved in mediating the pro-migratory effects in SCs [68].

The C2C12 murine SC line is widely used to study in vitro skeletalmuscle growth and differentiation. Early studies by Bruni and col-leagues demonstrated that addition of S1P to cultured C2C12 cells ledto the activation of phospholipase D [69], monomeric GTPase Rho [70]and increased cytosolic calcium [71]. C2C12 cells do not express S1P5but do have S1P1–S1P4. The most highly expressed S1P receptors inmyoblasts and myotubes are S1P1 and S1P3, respectively. Duringmyogenic differentiation, S1P2 is down-regulated and almost absentin myotubes [72]. The dynamic modulation and critical role of S1P2 inthe myogenic process has been observed in other studies, as well.Danieli-Betto et al. [73] observed an increase in expression of S1P1after muscle damage induced by bupivacaine. S1P2 was noted to beabsent in quiescent cells but transiently expressed in the early phaseof regeneration. The expression patterns of the S1P receptors duringmyogenic differentiation in vitro and in vivo suggest that they exertcomplex and possibly counteracting signals in this process. Futurestudies should elucidate how these receptor signals are synchronizedto coordinately regulate the sequential phases of SC and skeletalmuscleresponse to injury required for efficient muscle regeneration.

13. S1P and cell therapy for skeletal muscle regeneration

The main goal of cell therapy in patients with muscle injury, diseaseor sarcopenia (frailty of aging) is to repair tissue damage and replenishlost myofibers through systemic or local delivery of cells withmyoregenerative properties and thereby restore skeletal muscle func-tion. SCs, recognized as true muscle progenitor cells, have been exten-sively explored for this purpose in laboratory animals and in patients.However, one of the major problems encountered in these studies wasthe limited cell survival of the transplanted SCs [74,75]. In the last fewyears, in addition to SCs and SC-derived myoblasts, other somatic celltypes exhibiting myogenic potential have been identified and character-ized. Someof these cell types (most of themmultipotent stemcells) haveproven capable of contributing to myogenic regeneration (e.g. endothe-lial cells, mesoangioblasts) [76–78], and a few [e.g. pericytes, endothelialcells, mesenchymal stem cells (MSCs)] have demonstrated the abilityto incorporate directly into the SC niche [79–82]. Pericytes and meso-angioblast are blood vessel-associated stem cells that originate fromembryonic and postnatal tissue respectively. Both of these cell typeshold promise for clinical application, because a significant proportion iscapable of reaching skeletal muscles following systemic administration.In mesoangioblasts, S1P strongly stimulates proliferation and protectsfrom apoptosis elicited by different stimuli [83]. Treatment of meso-angioblast ex vivo with S1P enhanced their survival after delivery intra-muscular intomice [78]. This excitingfinding supports the view that S1P-preconditioned allogeneic mesoangioblast cells might enhance theirsurvival, overcoming a critical barrier to cell therapy.

In order to explore the potential impact of S1P/S1PR2 signaling onSTAT3 activation, we applied exogenous S1P to selected cell types:human umbilical vein endothelial cells (HUVECs), the pericyte cellline C3H10T1/2 and M25.2 cells, a clonally-derived population ofmesoangioblasts generated by Suzanne Berry-Miller (University of

Illinois). Of all the cells we tested, SC-derived myoblasts respondedmost robustly to S1P with regard to the activation of STAT3 [27]. Incontrast, HUVECs and M25.2 mesoangioblast cells exhibited minimalSTAT3 phosphorylation in response to S1P, and STAT3 phosphoryla-tion was undetectable in C3H101/2 cells under the conditions weemployed. However, additional experiments will be required toestablish whether endogenous or exogenous S1P signaling plays arole in the biology of these cell types or on their potential for myogen-ic differentiation and in vivo incorporation of these cells into muscle.

MSCs are characterized by their capacity for extensive prolifera-tion ex vivo, relative ease of isolation and ability to differentiate intoadipogenic, osteogenic, chondrogenic and myogenic lineages. S1Phas been shown to stimulate MSCs isolated from adipose tissue todifferentiate into smooth muscle in a dose-dependent manner [84].S1P2 was shown to be a mediator of the differentiation towardsmooth muscle, whereas S1P3 was shown to be less important inthis process. Although these studies are intriguing, the potential ofMSCs to be used for cell therapy in skeletal muscle disease, and the ex-tent to which S1P signaling and metabolism may be used to enhancecell therapy using this source of stem cells remains to be furtherexplored.

14. Summary

The involvement of sphingolipids in many aspects of skeletalmuscle biology is becoming increasingly clear (see Donati and Brunithis issue). Further, the evidence supporting a central role for S1Psignaling and metabolism in SC biology and the coordinated controlof skeletal muscle regeneration is now irrefutable in light of resultsfrom investigations of S1P signaling in myoregeneration by manyresearch groups. These cumulative findings have revealed at least thetheoretical potential for harnessing S1P's actions to modulate theactivation of SCs and enhance muscle repair for therapeutic purposesin a variety of clinical contexts, including muscle wasting diseases,trauma and sarcopenia, wherein reduction of SC numbers contributesto the frailty of aging. Considering the availability of small moleculeS1P receptor agonists and antagonists and SPL inhibitors already inclinical trials for other diseases, it seems likely that these basic researchfindings will be translated into more clinically relevant large animalmodel systems in the near future.

Acknowledgements

This work was funded by NIH grant GM66954, Muscular DystrophyAssociation grantMDA217712 and CIRM fellowship TG2-01164.We aregrateful to Derron Herr and Greg Harris for photomicrographs of Splyflight muscles, Nelle Cronen for expert administrative assistance andEric Hoffman and Suzanne Berry-Miller for many helpful discussions.

References

[1] M. Maceyka, S. Milstien, S. Spiegel, Sphingosine-1-phosphate: the Swiss armyknife of sphingolipid signaling, J. Lipid Res. 50 (2008) S272–S276.

[2] N. Young, J.R. Van Brocklyn, Signal transduction of sphingosine-1-phosphate Gprotein-coupled receptors, Sci. World J. 6 (2006) 946–966.

[3] M. Maceyka, K.B. Harikumar, S. Milstien, S. Spiegel, Sphingosine-1-phosphatesignaling and its role in disease, Trends Cell Biol. 22 (2012) 50–60.

[4] S.C. Su, K.J. Bayless, Utilizing sphingosine-1-phosphate to stimulate sproutingangiogenesis, Methods Mol. Biol. 874 (2012) 201–213.

[5] K.M. Argraves, B.A. Wilkerson, W.S. Argraves, Sphingosine-1-phosphate signalingin vasculogenesis and angiogenesis, World J. Biol. Chem. 1 (2010) 291–297.

[6] B. Bréart, W.D. Ramos-Perez, A. Mendoza, A.K. Salous, M. Gobert, Y. Huang, R.H.Adams, J.J. Lafaille, D. Escalante-Alcalde, A.J. Morris, S.R. Schwab, Lipid phosphatephosphatase 3 enables efficient thymic egress, J. Exp. Med. 208 (2011) 1267–1278.

[7] C.N. Jenne, A. Enders, R. Rivera, S.R. Watson, A.J. Bankovich, J.P. Pereira, Y. Xu, C.M.Roots, J.N. Beilke, A. Banerjee, S.L. Reiner, S.A. Miller, A.S. Weinmann, C.C.Goodnow, L.L. Lanier, J.G. Cyster, J. Chun, T-bet-dependent S1P5 expression inNK cells promotes egress from lymph nodes and bone marrow, J. Exp. Med. 206(2009) 2469–2481.

Page 8: S1P lyase in skeletal muscle regeneration and satellite cell activation: Exposing the hidden lyase

174 J.D. Saba, A.S. de la Garza-Rodea / Biochimica et Biophysica Acta 1831 (2013) 167–175

[8] P. Keul, S. Lucke, K. von Wnuck Lipinski, C. Bode, M. Graler, G. Heusch, B. Levkau,Sphingosine-1-phosphate receptor 3 promotes recruitment ofmonocyte/macrophagesin inflammation and atherosclerosis, Circ. Res. 108 (2011) 314–323.

[9] E. Kupperman, S. An, N. Osborne, S. Waldron, D.Y. Stainier, A sphingosine-1-phosphatereceptor regulates cell migration during vertebrate heart development, Nature 406(2000) 192–195.

[10] K. Mizugishi, T. Yamashita, A. Olivera, G. Miller, S. Spiegel, R. Proia, Essential rolefor sphingosine kinases in neural and vascular development, Mol. Cell. Biol. 25(2005) 11113–11121.

[11] C. Donati, E. Meacci, F. Nuti, L. Becciolini, M. Farnararo, P. Bruni, Sphingosine1-phosphate regulates myogenic differentiation: a major role for S1P2 receptor,FASEB J. 19 (2005) 449–451.

[12] S. Spiegel, S. Milstien, Functions of the multifaceted family of sphingosine kinasesand some close relatives, J. Biol. Chem. 282 (2006) 2125–2129.

[13] S. Pyne, S.C. Lee, J. Long, N.J. Pyne, Role of sphingosine kinases and lipid phosphatephosphatases in regulating spatial sphingosine 1-phosphate signalling in healthand disease, Cell. Signal. 21 (2009) 14–21.

[14] S. Pyne, J. Long, N. Ktistakis, N. Pyne, Lipid phosphate phosphatases and lipidphosphate signalling, Biochem. Soc. Trans. 33 (2005) 1370–1374.

[15] A. Kumar, J.D. Saba, Lyase to live by: sphingosine phosphate lyase as a therapeutictarget, Expert Opin. Ther. Targets 13 (2009) 1013–1025.

[16] F. Bourquin, G. Capitani, M.G. Grutter, PLP-dependent enzymes as entry and exitgates of sphingolipid metabolism, Protein Sci. 20 (2011) 1492–1508.

[17] D. Siow, C. Anderson, E. Berdyshev, A. Skobeleva, S. Pitson, B. Wattenberg, Intra-cellular localization of sphingosine kinase 1 alters access to substrate pools butdoes not affect the degradative fate of sphingosine-1-phosphate, J. Lipid Res. 51(2010) 2546–2559.

[18] P. Vogel, M.S. Donoviel, R. Read, G.M. Hansen, J. Hazlewood, S.J. Anderson, W. Sun, J.Swaffield, T. Oravecz, Incomplete inhibition of sphingosine 1-phosphate lyasemodulates immune system function yet prevents early lethality and non-lymphoid lesions, PLoS One 4 (2009) e4112.

[19] M. Bektas, M.L. Allende, B.G. Lee, W. Chen, M.J. Amar, A.T. Remaley, J.D. Saba, R.L.Proia, Sphingosine 1-phosphate lyase deficiency disrupts lipid homeostasis inliver, J. Biol. Chem. 285 (2010) 10880–10889.

[20] M. Serra, J.D. Saba, Sphingosine 1-phosphate lyase, a key regulator of sphingosine1-phosphate signaling and function, Adv. Enzyme Regul. 50 (2010) 349–362.

[21] J.T. Bagdanoff, M.S. Donoviel, A. Nouraldeen, M. Carlsen, T.C. Jessop, J. Tarver, S.Aleem, L. Dong, H. Zhang, L. Boteju, J. Hazelwood, J. Yan, M. Bednarz, S. Layek,I.B. Owusu, S. Gopinathan, L. Moran, Z. Lai, J. Kramer, S.D. Kimball, P.Yalamanchili, W.E. Heydorn, K.S. Frazier, B. Brooks, P. Brown, A. Wilson, W.K.Sonnenburg, A. Main, K.G. Carson, T. Oravecz, D.J. Augeri, Inhibition of sphingosine1-phosphate lyase for the treatment of rheumatoid arthritis: discovery of(E)-1-(4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)-1H-imidazol-2-yl)ethanone oxime(LX2931) and (1R,2S,3R)-1-(2-(isoxazol-3-yl)-1H-imidazol-4-yl)butane-1,2,3,4-tetraol(LX2932), J. Med. Chem. 53 (2010) 8650–8662.

[22] A. Kumar, B. Oskouian, H. Fyrst, M. Zhang, F. Paris, J.D. Saba, S1P lyase regulatesDNA damage responses through a novel sphingolipid feedback mechanism, CellDeath Dis. 2 (2011) e119.

[23] A. Kumar, H.S. Byun, R. Bittman, J.D. Saba, The sphingolipid degradation producttrans-2-hexadecenal induces cytoskeletal reorganization and apoptosis in aJNK-dependent manner, Cell. Signal. 23 (2011) 11346–11353.

[24] A. Huwiler, F. Bourquin, N. Kotelevets, O. Pastukhov, G. Capitani, M.G. Grutter, U.Zangemeister-Wittke, A prokaryotic S1P lyase degrades extracellular S1P in vitroand in vivo: implication for treating hyperproliferative disorders, PLoS One 6(2011) e22436.

[25] J.D. Saba, F. Nara, A. Bielawska, S. Garrett, Y.A. Hannun, The BST1 gene of Saccharomycescerevisiae is the sphingosine-1-phosphate lyase, J. Biol. Chem. 272 (1997)26087–26090.

[26] D.R. Herr, H. Fyrst, V. Phan, K. Heinecke, R. Georges, G.L. Harris, J.D. Saba, Splyregulation of sphingolipid signaling molecules is essential for Drosophila develop-ment, Development 130 (2003) 2443–2453.

[27] K. Loh, W. Leong, M. Carlson, B. Oskouian, A. Kumar, H. Fyrst, M. Zhang, R. Proia, E.Hoffman, J. Saba, Sphingosine-1-phosphate enhances satellite cell activation indystrophic muscles through a S1PR2/STAT3 signaling pathway, PLoS One 7(2012) e37218.

[28] P.P. Van Veldhoven, Sphingosine-1-phosphate lyase, In: in: A.H. Merrill Jr., Y.A.Hannun (Eds.), Sphingolipid Metabolism and Cell Signaling Part A, vol. 311,Academic Press, New York, 2000, pp. 244–254.

[29] M.H. Graler, Targeting sphingosine 1-phosphate (S1P) levels and S1P receptorfunctions for therapeutic immune interventions, Cell. Physiol. Biochem. 26(2010) 79–86.

[30] S. Alexander, H. Alexander, Lead genetic studies in Dictyostelium discoideum andtranslational studies in human cells demonstrate that sphingolipids are keyregulators of sensitivity to cisplatin and other anticancer drugs, Semin. Cell Dev.Biol. 22 (2010) 97–104.

[31] A. Aguilar, J.D. Saba, Truth and consequences of sphingosine-1-phosphate lyase,Adv. Enzyme Regul. 52 (2012) 17–30.

[32] N. Deconinck, B. Dan, Pathophysiology of Duchenne muscular dystrophy: currenthypotheses, Pediatr. Neurol. 36 (2007) 1–7.

[33] E.P. Hoffman, R.H. Brown Jr., L.M. Kunkel, Dystrophin: the protein product of theDuchenne muscular dystrophy locus, Cell 51 (1987) 919–928.

[34] M. Kanagawa, T. Toda, The genetic and molecular basis of muscular dystrophy:roles of cell-matrix linkage in the pathogenesis, J. Hum. Genet. 51 (2006)915–926.

[35] J. Schessl, Y. Zou, C.G. Bonnemann, Congenital muscular dystrophies and theextracellular matrix, Semin. Pediatr. Neurol. 13 (2006) 80–89.

[36] D. Bansal, K. Miyake, S.S. Vogel, S. Groh, C.C. Chen, R. Williamson, P.L. McNeil, K.P.Campbell, Defective membrane repair in dysferlin-deficient muscular dystrophy,Nature 423 (2003) 168–172.

[37] L. Heslop, J.E. Morgan, T.A. Partridge, Evidence for a myogenic stem cell that isexhausted in dystrophic muscle, J. Cell Sci. 113 (Pt 12) (2000) 2299–2308.

[38] N.P. Evans, S.A. Misyak, J.L. Robertson, J. Bassaganya-Riera, R.W. Grange, Immune-mediated mechanisms potentially regulate the disease time-course of duchennemuscular dystrophy and provide targets for therapeutic intervention, PM R 1(2009) 755–768.

[39] E.P. Hoffman, D. Escolar, Translating mighty mice into neuromuscular therapeutics:is bigger muscle better? Am. J. Pathol. 168 (2006) 1775–1778.

[40] N. Figeac, M. Daczewska, C. Marcelle, K. Jagla, Muscle stem cells and modelsystems for their investigation, Dev. Dyn. 236 (2007) 3332–3342.

[41] J. Ehrhardt, J. Morgan, Regenerative capacity of skeletal muscle, Curr. Opin.Neurol. 18 (2005) 548–553.

[42] S. Grefte, A.M. Kuijpers-Jagtman, R. Torensma, J.W. Von den Hoff, Skeletal muscledevelopment and regeneration, Stem Cells Dev. 16 (2007) 857–868.

[43] S. Kuang, K. Kuroda, F. Le Grand, M.A. Rudnicki, Asymmetric self-renewal andcommitment of satellite stem cells in muscle, Cell 129 (2007) 999–1010.

[44] Y. Nagata, T. Partridge, R. Matsuda, P. Zammit, Entry of muscle satellite cells intothe cell cycle requires sphingolipid signaling, J. Cell Biol. 174 (2006) 245–253.

[45] D. Danieli-Betto, E. Germinario, A. Esposito, A. Megighian, M. Midrio, B. Ravara, E.Damiani, L.D. Libera, R.A. Sabbadini, R. Betto, Sphingosine 1-phosphate protectsmouse extensor digitorum longus skeletal muscle during fatigue, Am. J. Physiol.Cell Physiol. 288 (2005) C1367–C1373.

[46] M. Zanin, E. Germinario, L. Dalla Libera, D. Sandona, R.A. Sabbadini, R. Betto, D.Danieli-Betto, Trophic action of sphingosine 1-phosphate in denervated rat soleusmuscle, Am. J. Physiol. Cell Physiol. 294 (2008) C36–C46.

[47] P. Bruni, C. Donati, Pleiotropic effects of sphingolipids in skeletal muscle, Cell.Mol. Life Sci. 65 (2008) 3725–3736.

[48] X. Shi, D.J. Garry, Muscle stem cells in development, regeneration, and disease,Genes Dev. 20 (2006) 1692–1708.

[49] V. Horsley, G.K. Pavlath, Forming a multinucleated cell: molecules that regulatemyoblast fusion, Cells Tissues Organs 176 (2004) 67–78.

[50] H.R. Shcherbata, A.S. Yatsenko, L. Patterson, V.D. Sood, U. Nudel, D. Yaffe, D. Baker,H. Ruohola-Baker, Dissecting muscle and neuronal disorders in a Drosophilamodel of muscular dystrophy, EMBO J. 26 (2007) 481–493.

[51] P. Bandhuvula, N. Honbo, G.Y. Wang, Z.Q. Jin, H. Fyrst, M. Zhang, A.D. Borowsky, L.Dillard, J.S. Karliner, J.D. Saba, S1P lyase: a novel therapeutic target forischemia-reperfusion injury of the heart, Am. J. Physiol. Heart Circ. Physiol. 300(2011) H1753–H1761.

[52] P. Zhao, S. Iezzi, E. Carver, D. Dressman, T. Gridley, V. Sartorelli, E. Hoffman, Slug isa novel downstream target of MyoD. Temporal profiling in muscle regeneration,J. Biol. Chem. 277 (2002) 30091–30101.

[53] D.R. Plant, F.E. Colarossi, G.S. Lynch, Notexin causes greater myotoxic damage andslower functional repair in mouse skeletal muscles than bupivacaine, MuscleNerve 34 (2006) 577–585.

[54] A. Olivera, C. Eisner, Y. Kitamura, S. Dillahunt, L. Allende, G. Tuymetova, W.Watford, F. Meylan, S.C. Diesner, L. Li, J. Schnermann, R.L. Proia, J. Rivera, Sphingosinekinase 1 and sphingosine-1-phosphate receptor 2 are vital to recovery from anaphy-lactic shock in mice, J. Clin. Invest. 120 (2010) 1429–1440.

[55] J.S. Karliner, Sphingosine kinase and sphingosine 1-phosphate in cardioprotection,J. Cardiovasc. Pharmacol. 53 (2009) 189–197.

[56] S.K. Jo, A. Bajwa, H. Ye, A.L. Vergis, A.S. Awad, Y. Kharel, K.R. Lynch, M.D. Okusa,Divergent roles of sphingosine kinases in kidney ischemia-reperfusion injury,Kidney Int. 75 (2009) 167–175.

[57] S.M. Pitson, Regulation of sphingosine kinase and sphingolipid signaling, TrendsBiochem. Sci. 36 (2011) 97–107.

[58] G. Bulfield, W.G. Siller, P.A. Wight, K.J. Moore, X chromosome-linked musculardystrophy (mdx) in the mouse, Proc. Natl. Acad. Sci. U. S. A. 81 (1984) 1189–1192.

[59] A.M. Connolly, R.M. Keeling, S. Mehta, A. Pestronk, J.R. Sanes, Three mouse modelsof muscular dystrophy: the natural history of strength and fatigue in dystrophin-,dystrophin/utrophin-, and laminin a2-deficient mice, Neuromuscul. Disord. 11(2001) 703–712.

[60] D.F. Cardoso,M. Lopes-Ferreira, E.L. Faquim-Mauro,M.S.Macedo, S.H. Farsky, Role ofcrotoxin, a phospholipase A2 isolated from Crotalus durissus terrificus snake venom,on inflammatory and immune reactions, Mediators Inflamm. 10 (2001) 125–133.

[61] A.M. Emslie-Smith, J.B. Harris, Lymphopaenia in the inflammatory responsecaused by notechis II-5, Toxicon 27 (1989) 499–500.

[62] S. Schwab, J. Pereira, M. Matloubian, Y. Xu, Y. Huang, J. Cyster, Lymphocytesequestration through S1P lyase inhibition an disruption of S1P gradients, Science309 (2005) 1735–1739.

[63] M.J. Spencer, E. Montecino-Rodriguez, K. Dorshkind, J.G. Tidball, Helper (CD4(+))and cytotoxic (CD8(+)) T cells promote the pathology of dystrophin-deficientmuscle, Clin. Immunol. 98 (2001) 235–243.

[64] S. Eghtesad, S. Jhunjhunwala, S.R. Little, P.R. Clemens, Rapamycin amelioratesdystrophic phenotype inmdxmouse skeletalmuscle, Mol. Med. 17 (2011) 917–924.

[65] H. Lee, J. Deng, M. Kujawski, C. Yang, Y. Liu, A. Herrmann, M. Kortylewski, D.Horne, G. Somlo, S. Forman, R. Jove, H. Yu, STAT3-induced S1PR1 expression iscrucial for persistent STAT3 activation in tumors, Nat. Med. 16 (2010) 1421–1428.

[66] Y. Sekine, K. Suzuki, A.T. Remaley, HDL and sphingosine-1-phosphate activatestat3 in prostate cancer DU145 cells via ERK1/2 and S1P receptors, and promotecell migration and invasion, Prostate 71 (2011) 690–699.

[67] M.A. Frias, R.W. James, C. Gerber-Wicht, U. Lang, Native and reconstituted HDL activateStat3 in ventricular cardiomyocytes via ERK1/2: role of sphingosine-1-phosphate,Cardiovasc. Res. 82 (2009) 313–323.

Page 9: S1P lyase in skeletal muscle regeneration and satellite cell activation: Exposing the hidden lyase

175J.D. Saba, A.S. de la Garza-Rodea / Biochimica et Biophysica Acta 1831 (2013) 167–175

[68] S. Calise, S. Blescia, F. Cencetti, C. Bernacchioni, C. Donati, P. Bruni, Sphingosine1-phosphate stimulates proliferation and migration of satellite cells: role of S1Preceptors, Biochim. Biophys. Acta 1823 (2012) 439–450.

[69] E. Meacci, V. Vasta, C. Donati, M. Farnararo, P. Bruni, Receptor-mediated activationof phospholipase D by sphingosine 1-phosphate in skeletal muscle C2C12 cells. Arole for protein kinase C, FEBS Lett. 457 (1999) 184–188.

[70] E. Meacci, C. Donati, F. Cencetti, E. Romiti, P. Bruni, Permissive role of proteinkinase C alpha but not protein kinase C delta in sphingosine 1-phosphate-inducedRho A activation in C2C12 myoblasts, FEBS Lett. 482 (2000) 97–101.

[71] E. Meacci, F. Cencetti, L. Formigli, R. Squecco, C. Donati, B. Tiribilli, F. Quercioli, S.Zecchi Orlandini, F. Francini, P. Bruni, Sphingosine 1-phosphate evokes calciumsignals in C2C12 myoblasts via Edg3 and Edg5 receptors, Biochem. J. 362 (2002)349–357.

[72] E. Meacci, F. Cencetti, C. Donati, F. Nuti, M. Farnararo, T. Kohno, Y. Igarashi, P.Bruni, Down-regulation of EDG5/S1P2 during myogenic differentiation results inthe specific uncoupling of sphingosine 1-phosphate signalling to phospholipase D,Biochim. Biophys. Acta 1633 (2003) 133–142.

[73] D. Danieli-Betto, S. Peron, E. Germinario, M. Zanin, G. Sorci, S. Franzoso, D.Sandona, R. Betto, Sphingosine 1-phosphate signaling is involved in skeletalmuscle regeneration, Am. J. Physiol. Cell Physiol. 298 (2010) C550–C558.

[74] Y. Fan, M. Maley, M. Beilharz, M. Grounds, Rapid death of injected myoblasts inmyoblast transfer therapy, Muscle Nerve 19 (1996) 853–860.

[75] J.R. Beauchamp, C.N. Pagel, T.A. Partridge, A dual-marker system for quantitativestudies of myoblast transplantation in the mouse, Transplantation 63 (1997)1794–1797.

[76] B. Zheng, B. Cao, M. Crisan, B. Sun, G. Li, A. Logar, S. Yap, J.B. Pollett, L. Drowley, T.Cassino, B. Gharaibeh, B.M. Deasy, J. Huard, B. Peault, Prospective identification ofmyogenic endothelial cells in human skeletal muscle, Nat. Biotechnol. 25 (2007)1025–1034.

[77] M. Sampaolesi, S. Blot, G. D'Antona, N. Granger, R. Tonlorenzi, A. Innocenzi, P.Mognol, J.L. Thibaud, B.G. Galvez, I. Barthelemy, L. Perani, S. Mantero, M. Guttinger,O. Pansarasa, C. Rinaldi, M.G. Cusella De Angelis, Y. Torrente, C. Bordignon, R.

Bottinelli, G. Cossu, Mesoangioblast stem cells ameliorate muscle function in dystro-phic dogs, Nature 444 (2006) 574–579.

[78] M. Sampaolesi, Y. Torrente, A. Innocenzi, R. Tonlorenzi, G. D'Antona, M.A. Pellegrino,R. Barresi, N. Bresolin, M.G. De Angelis, K.P. Campbell, R. Bottinelli, G. Cossu, Celltherapy of alpha-sarcoglycan null dystrophic mice through intra-arterial deliveryof mesoangioblasts, Science 301 (2003) 487–492.

[79] M. Crisan, S. Yap, L. Casteilla, C.W. Chen, M. Corselli, T.S. Park, G. Andriolo, B. Sun,B. Zheng, L. Zhang, C. Norotte, P.N. Teng, J. Traas, R. Schugar, B.M. Deasy, S.Badylak, H.J. Buhring, J.P. Giacobino, L. Lazzari, J. Huard, B. Peault, A perivascularorigin for mesenchymal stem cells in multiple human organs, Cell Stem Cell 3(2008) 301–313.

[80] A.M. Rodriguez, D. Pisani, C.A. Dechesne, C. Turc-Carel, J.Y. Kurzenne, B.Wdziekonski, A. Villageois, C. Bagnis, J.P. Breittmayer, H. Groux, G. Ailhaud, C.Dani, Transplantation of a multipotent cell population from human adipose tissueinduces dystrophin expression in the immunocompetent mdx mouse, J. Exp.Med. 201 (2005) 1397–1405.

[81] M. Dezawa, H. Ishikawa, Y. Itokazu, T. Yoshihara, M. Hoshino, S. Takeda, C. Ide, Y.Nabeshima, Bone marrow stromal cells generate muscle cells and repair muscledegeneration, Science 309 (2005) 314–317.

[82] C. De Bari, F. Dell'Accio, F. Vandenabeele, J.R. Vermeesch, J.M. Raymackers, F.P.Luyten, Skeletal muscle repair by adult human mesenchymal stem cells fromsynovial membrane, J. Cell Biol. 160 (2003) 909–918.

[83] C. Donati, F. Cencetti, P. Nincheri, C. Bernacchioni, S. Brunelli, E. Clementi, G.Cossu, P. Bruni, Sphingosine 1-phosphate mediates proliferation and survival ofmesoangioblasts, Stem Cells 25 (2007) 1713–1719.

[84] P. Nincheri, P. Luciani, R. Squecco, C. Donati, C. Bernacchioni, L. Borgognoni, G.Luciani, S. Benvenuti, F. Francini, P. Bruni, Sphingosine 1-phosphate inducesdifferentiation of adipose tissue-derived mesenchymal stem cells towardssmooth muscle cells, Cell. Mol. Life Sci. 66 (2009) 1741–1754.

[85] A.S. de la Garza-Rodea, Mesenchymal stem cell in skeletal muscle regeneration,Doctoral Thesis, Leiden University Medical Center, Leiden University, The Netherlands,(2011) 15.