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Sphingosine phosphate lyase regulates myogenic differentiation via S1P receptor-mediated effects on myogenic microRNA expression Anabel S. de la Garza-Rodea,* Dianna M. Baldwin,*,† Babak Oskouian,* Robert F. Place,* Padmavathi Bandhuvula,* Ashok Kumar,* and Julie D. Saba*,1 *Children’s Hospital Oakland Research Institute, Oakland, California, USA; and †San Francisco State University, San Francisco, California, USA S1P lyase (SPL) catalyzes the irreversible degradation of sphingosine-1-phosphate (S1P), a bioactive lipid whose signaling activities regulate muscle differentiation, homeostasis, and satellite cell (SC) activation. By regulating S1P levels, SPL also controls SC recruitment and muscle regeneration, representing a potential therapeutic target for muscular dystrophy. We found that SPL is induced during myoblast differentiation. To investigate SPL’s role in myogenesis at the cellular level, we generated and characterized a murine myoblast SPL-knockdown (SPL-KD) cell line lacking SPL. SPL-KD cells accumulated intracellular and extracellular S1P and failed to form myotubes under conditions that normally stimulate myogenic differentiation. Under differentiation conditions, SPL-KD cells also demonstrated delayed induction of 3 myogenic microRNAs (miRNAs), miR-1, miR-206, and miR-486. SPL-KD cells successfully differentiated when treated with an S1P1 agonist, S1P2 antagonist, and combination treatments, which also increased myogenic miRNA levels. SPL-KD cells transfected with mimics for miR-1 or miR-206 also overcame the differentiation block. Thus, we show for the first time that the S1P/SPL/S1P-receptor axis regulates the expression of a number of miRNAs, thereby contributing to myogenic differentiation.— de la GarzaRodea, A. S., Baldwin, D. M., Oskouian, B., Place, R. F., Bandhuvula, P., Kumar, A., Saba, J. D. Sphingosine phosphate lyase regulates myogenic differentiation via S1P receptor-mediated effects on myogenic miABSTRACT

Abbreviations: BSA, bovine serum albumin; C17-S1P, d-erythroC17-sphingosine-1-phosphate; C18-S1P, d-erythro-C18sphingosine-1-phosphate; DSHB, Developmental Studies Hybridoma Bank; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HDAC, histone deacetylase; KD, knockdown; KO, knockout; LPF, low-power field; miRNA, microRNA; MHC, myosin heavy chain; PBS, phosphate-buffered saline; PI, propidium iodide; qRT-PCR, quantitative reverse transcriptasepolymerase chain reaction; SC, satellite cell; SphK, sphingosine kinase; S1P, sphingosine-1-phosphate; S1PR, sphingosine-1phosphate receptor; SPL, sphingosine-1-phosphate lyase; SPLKD, sphingosine phosphate lyase knockdown; Spns2, spinster homolog 2 506

croRNA expression. FASEB J. 28, 506 –519 (2014). www.fasebj.org Key Words: myogenesis 䡠 sphingolipid 䡠 C2C12 䡠 Spns2 Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite and signaling molecule generated from the breakdown of membrane sphingolipids. S1P signaling is essential for development, angiogenesis, immune functions, and numerous other physiological and pathological processes (1). S1P serves as a ligand for a family of 5 G-protein-coupled S1P receptors (S1PRs), designated as S1P1–5 (2). In addition to signaling through receptor activation, S1P can act intracellularly and independently of its receptors. For example, S1P has direct roles in the regulation of NF-␬B activation, calcium signaling, and histone deacetylase (HDAC) activity (3–5). Through these diverse mechanisms, S1P regulates multiple cellular functions, including cell proliferation, differentiation, migration, and adhesion (6 –11). S1P is directly synthesized via phosphorylation of the long-chain base sphingosine in a reaction catalyzed by sphingosine kinases (SphKs). SphK activity is required for muscle regeneration in vivo, implicating S1P as a muscle trophic factor (12, 13). Expression of genes involved in S1P signaling and metabolism change dynamically following notexin-induced muscle injury, suggesting an important role for S1P in muscle injury and regeneration (14 –16). S1P signaling has been shown to regulate the activation, proliferation, migration, and differentiation of skeletal muscle stem cells called satellite cells (SCs), SC-derived myoblasts, and reserve cells (14, 17). Notably, S1P treatment increases the number of SCs entering the cell cycle, implicating S1P directly in SC activation (18). 1 Correspondence: Children’s Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, CA 94609, USA. E-mail: [email protected] doi: 10.1096/fj.13-233155 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information.

0892-6638/14/0028-0506 © FASEB

S1P lyase (SPL) is a highly conserved enzyme that irreversibly degrades S1P by catalyzing its cleavage at the C2–3 carbon bond (19, 20). Although a number of specific and nonspecific lipid phosphatases can dephosphorylate S1P, SPL is the major regulator of S1P levels, and loss of SPL function leads to profound accumulation of S1P in cells and tissues (21, 22). Drosophila mutants containing a transposon insertion in the Sply gene that encodes Drosophila SPL demonstrate thoracic flight muscle defects and cannot fly (23, 24). SPL also plays an important role in mammalian muscle homeostasis. For example, SPL expression is increased in injured and dystrophic skeletal muscles, and pharmacological inhibition of SPL leads to increased SC recruitment and muscle regeneration after injury (16). Combined with the Sply myopathy phenotype, these findings suggest that the SPL/S1P signaling axis must be tightly controlled to maintain proper muscle development, homeostasis, and stem cell functioning. MiRNAs are a class of small noncoding RNAs that function as key regulators of gene expression. By targeting complementary sequences in gene transcripts, microRNAs (miRNAs) inhibit translation and/or degrade mRNAs to silence gene expression (25). Mammalian muscle differentiation is under the control of specific myogenic miRNAs whose functions are essential for genetic reprogramming during development. Several miRNAs have been shown to regulate myogenesis, including miR-1, miR-133, miR-206, and miR-486 (26 –28). Myogenic differentiation has been shown to be regulated by miR-1, miR-206, and miR-486, at least in part, through the repression of Pax7, a transcription factor that is specifically expressed in SCs, serves as a SC marker, and is responsible for the specification of progenitor cells to the SC lineage. In this study, we employed a murine muscle stem cell line (C2C12) to address how SPL contributes to skeletal muscle homeostasis by monitoring myotube formation/myogenesis. Our findings suggest that SPL controls myogenic differentiation through S1PR-mediated signaling events that affect myogenic miRNA expression, and that spinster homolog 2 (Spns2) also plays a role in myogenic differentiation. To our knowledge, this is the first report that shows the S1P/SPL/S1PR axis can regulate miRNA expression and the first evidence of a role for Spns2 in myogenesis.

MATERIALS AND METHODS SPL knockdown (KD) in C2C12 cells The lentiviral vector pLKO.1 (Addgene plasmid 10878; Addgene, Cambridge, MA, USA) was used to clone small hairpin RNAs targeting the murine SPL gene Sgpl1, according to pLKO.1 protocol (http://www.addgene.com), as we described for human SPL-KD cells (29). The sequence targeted (ACAATAGGATAAACCATAA) represents the 3= untranslated region of murine SPL. SPL REGULATES MYOGENIC MICRORNAS

Cell proliferation conditions Cells were seeded in 10-cm culture plates and expanded in proliferating medium [Dulbecco’s modified Eagle’s medium (DMEM) low glucose, 2 mM l-glutamine, 100 ␮g/ml streptomycin, 100 U/ml penicillin, 20% fetal bovine serum (FBS) (University of California–San Francisco [UCSF] Cell Culture Facility, San Francisco, CA, USA) and 0.5% of chick embryo extract (US Biological, Swampscott, MA, USA)]. Cultures were grown at 37°C in humidified air with 5% CO2, harvested with 0.25% trypsin/ethylene diamine tetraacetic acid (EDTA), and seeded 1:10. Myogenic differentiation Cells were seeded on 0.1% gelatin (Sigma-Aldrich, St. Louis, MO, USA)-coated plates in complete medium to 80 –90% confluency and induced to differentiate by replacement of FBS with 2% horse serum (UCSF). SPL activity assays and S1P quantification SPL activity was measured as previously described (30). To measure intracellular S1P levels, cells were plated in proliferation medium as described above and propagated for 24 h. Cells were harvested by incubation for 7 min in trypsin/ EDTA, and 106 cells were pelleted and resuspended in 50 ␮l water to extract S1P. S1P extraction was carried out as described previously (1) but with further modifications. Cells were sonicated in 0.25 ml methanol using a tip sonicator (60 Sonic Dismembrator; Thermo Fisher Scientific, Waltham, MA, USA). Homogenate was combined with 100 pmol d-erythro-C17-S1P (C17-S1P; Avanti Polar Lipids, Inc., Alabaster, AL, USA) internal standard and 0.5 ml chloroform: methanol (2:1), and samples were incubated overnight at 48°C. Samples were dried with a flow of nitrogen. Samples were resuspended in 0.5 ml of chloroform:methanol (2:1). From this suspension, 50 ␮l was removed and dried with nitrogen for phospholipid C determination. The extract was made basic by adding 50 ␮l of 1 M KOH in methanol. To accomplish 2-phase separation, 100 ␮l of water was added. The aqueous phase was transferred to a new tube and made acidic by adding 35 ␮l of glacial acetic acid. A second aqueous phase was created by adding 0.2 ml of chloroform:methanol (2:1) and 50 ␮l of water. The organic phase was recovered, dried with a flow of nitrogen, resuspended in 50 ␮l of methanol containing 5 mM ammonium acetate, mixed by vortexing, and placed in a water bath sonication device for 5 min before S1P measurement by mass spectrometry. To measure S1P in medium, 2.5 ⫻ 106 cells were plated in a volume of 9 ml medium in 10-cm culture dishes. Medium was removed after 24 h incubation. A minor experiment was carried out to establish 100 ␮l as the optimum volume of medium that could result in a detectable amount of S1P. S1P extraction was then carried out as we described previously (16) but with further modifications. A volume of 390 ␮l methanol was added to 100 ␮l of medium combined with 100 pmol of C17-S1P as an internal standard. Samples were incubated at 37°C for 30 min. Supernatant was removed after spinning the samples for 5 min at 14,000 rpm, and transferred to a new Eppendorf tube (Eppendorf, Hamburg, Germany). Samples were dried with a flow of nitrogen, and 2-phase separation was performed as described above. The data were acquired on a Micromass Quattro LC mass spectrometer (Waters Corp., Milford, MA, USA) and processed by Masslynx 3.3 (Waters). Lipids were identified based on their specific precursor and product ion pair and quantitated using multiple reactions monitoring (MRM). Data were acquired in 507

Arbor, MI, USA) were administered every 24 h at 10 ␮M in fresh medium. Cells were collected at d 4. Thymidine (SigmaAldrich), mimosine (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and aurora kinase inhibitor II (Calbiochem) were used as described in the figure captions.

electrospray ionization (ESI) positive mode as described previously (31) with some modifications. Samples (10 ␮l) were injected into a high-pressure liquid chromatograph, and lipids were separated on a Luna-RP column (3 ␮m particle size C18, 100 Å pore size, 50 mm length ⫻ 2.00 mm i.d.; Phenomenex, Torrance, CA, USA) at a flow rate of 0.3 ml/min. The mobile phase consists of eluent A (methanol: water:acetic acid in 5 mM ammonium acetate 50:49:1 v/v) and eluent B (methanol:acetic acid in 5 mM ammonium acetate 99:1 v/v). The gradient flow is as follows: t ⫽ 0 to 0.5 min 60% eluent A and 40% eluent B, followed by a linear gradient change to 100% B until t ⫽ 2. Gradient was kept at 100% B for 4.5 min, then a linear gradient change from 100% B to 60% A and 40% B until t ⫽ 7.5. Finally, gradient was kept at 60% A and 40% B for 4.5 min. Primary stock solutions of d-erythro-C18-S1P (C18-S1P; Avanti Polar Lipids) and C17-S1P at a concentration of 1 mM were prepared separately and immediately portioned into aliquots and stored at ⫺20°C. A secondary stock solution with 10 ␮M was prepared for both standards. Calibration curve was prepared by increasing concentration of C18-S1P, by standard-addition method, from secondary stock solution (0.0025– 0.75 ␮M). An equal amount of C17-S1P internal standard was added to each concentration level (2 ␮M). Each standard mixture (10 ␮l) was injected into the machine and analyzed. Calibration curves were prepared by increasing concentrations of C18S1P and equal amounts of the internal standard C17-S1P. Quantitative analysis was based on the following equation: [(C18-S1P peak area)/(C17-S1P peak area)] ⫻ concentration of C17-S1P internal standard. The resulting concentration was further normalized against the total number of cells. Calibration curves were determined using GraphPad Prism 6 software with linear regression analysis using line of best fit (GraphPad, San Diego, CA, USA). P values were calculated by unpaired Student’s t test using Prism 6. Ethanol, methanol, and chloroform (HPLC grade), ammonium acetate (LC/MS grade), and glacial acetic acid (ACS reagent) were from Thermo Fisher Scientific. Potassium hydroxide (ACS reagent) was from Sigma-Aldrich. C18-S1P and internal standard C17-S1P were from Avanti Polar Lipids. Nitrogen gas tanks were purchased from Airgas (Stamford, CT, USA).

Immunoblotting Immunoblotting was performed, as described previously (16) using antibodies: Pax7 [Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA]; myosin heavy chain (MHC) MF20 (DSHB); myogenin FSD (DSHB); SPL (polyclonal antisera described previously; ref. 32); SphK1 (Abcam, Cambridge, MA, USA) and actin (Sigma-Aldrich). MF20 is raised against light meromyosin and reacts with sarcomeric myosin. Immunofluorescence microscopy Cells were fixed in 4% paraformaldehyde for 5 min and washed twice in phosphate buffered saline (PBS). Cells were then permeabilized in 0.4% Triton-X (Sigma-Aldrich) for 5 min. Blocking was performed with 5% bovine serum albumin (BSA; UCSF) in PBS for 1 h. Cells were incubated in 1:100 MHC primary antibody in PBS supplemented with 0.1% Triton-X and 1% BSA followed by 1:500 Alexa Fluor 488 donkey anti-mouse secondary IgG (Molecular Probes, Eugene, OR, USA) in PBS with 1% BSA. After washing, propidium iodide (PI) nuclear stain (Molecular Probes) at concentration of 1 ␮g/ml in PBS was added for 5 min. Finally, cells were washed and imaged in PBS. Images were captured with a fluorescent microscope (Axiovert 25; Carl Zeiss, Oberkochen, Germany) and camera Luca S (Andor Technology, South Windsor, CT, USA) and processed by Kinetic Imaging Komet 6.0 (Andor Technology). Myotubes were counted as cells with ⱖ2 nuclei/tube that were positive for MHC. Three fields of ⫻10 were counted and averaged. Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) for S1PRs

Proliferation assays

Total cellular RNA was extracted using the Aurum total RNA mini kit (Bio-Rad, Hercules, CA, USA). RNA was eluted in 80 ␮l of elution buffer. Concentration was determined with a NanoDrop ND 1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). The cDNA was generated from 2 ␮g of RNA using the iScript cDNA Synthesis kit (Bio-Rad). Quantitative PCR was run on a PTC-240 Tetrad 2 DNA (Bio-Rad). Actin was utilized as internal control. Primers (Table 1) for S1PR qRT-PCR were from Integrated DNA Technologies (IDT; Coralville, IA, USA). Primers for Spns2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from ELIM Biopharmaceuticals (Hayward CA, USA).

Cells were plated at 400, 800, and 1200 cells/well in 96-well plates, and conversion of tetrazolium dye (CellTiter 96 AQ One Solution Cell Proliferation Assay; Promega, Madison, WI, USA) to formazan was quantified using spectrophotometry to measure absorption at 490-nm wavelength. Treatments Cells were plated at 80 –90% confluency and induced to differentiate for 24 h. S1P1–3 modulators (Cayman, Ann

TABLE 1. Primer sequences used for qRT-PCR for quantification of S1PRs Primers Gene

S1P1 S1P2 S1P3 S1P4 S1P5 SPNS2 GAPDH

508

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Forward, 5=–3=

Reverse, 5=–3=

bp

TCATCTGCTGCTTCATCA CTACAATTACACCAAGGAGAC CACCACCATCCTCTTCTT CTCCTGGCTGACATCTTT TCTCTTGCTATTACTGGATGT GCACTTTGGGGTCAAGGA ACCTGCCAAGTATGATGA

CTGCTAATAGGTCCGAGAG CAGCACAAGATGATGATGAA ATTGACCTTGTATGCTATGC TTAATGGCTGAGTTGAACAC TTGGTGAAGGTGTAGATGAT CCCAGGTAGCCAAAGATGG GGAGTTGCTGTTGAAGTC

123 84 163 98 126 93 118

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DE LA GARZA-RODEA ET AL.

proliferating cell cultures and after 48 h incubation in 2% horse serum.

Quantification of miRNA expression Total RNA was extracted using the miRNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Reverse transcription reactions containing 200 ng total RNA were performed using the TaqMan MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) in conjunction with miRNA-specific primers. Real-time PCR was carried out in quadruplicate using proprietary TaqMan MicroRNA assay kits specific to murine miR-1, miR206, or miR-486. Amplification of snU6 served as an endogenous control.

Statistical analysis Values are expressed as means ⫾ sd. Student’s unpaired t test was used to compare 2 data sets. Values of P ⬍ 0.05 were considered significant. All experiments were conducted ⱖ3 times; representative results are shown.

RESULTS MiRNA mimics MiRNA mimics (mirVana; Ambion-Life Technologies, Grand Island, NY, USA) were used to elevate cellular miRNA levels (see Table 2). Proliferating cells were plated in precoated 0.1% gelatin 6-well plates. The following day, Lipofectamine (Invitrogen-Life Technologies, Grand Island, NY, USA) was used to transfect cells with mimics (50 nM) and mock treatments in serum-free Opti-MEM (UCSF). After 24 h, transfection medium was removed, cells were washed twice in phosphate-buffered saline, and differentiation medium was added. Medium was refreshed daily until cells were imaged or harvested. Spns2 silencing To silence Spns2 by siRNA, 2 ⫻ 105 cells were seeded in 0.1% gelatin-coated dishes. After 2 d of incubation in proliferation medium, the medium was replaced with OptiMEM, and control and SPL-KD cells were transfected with Spns2 siRNA (Qiagen) at a dose of 100 pmol administered once using Lipofectamine RNAiMAX (Invitrogen) transfection reagent according to the manufacturer’s instructions. Transfection medium was removed 20 h later and was replaced with proliferation medium. When cells were ⬎80% confluent, they were washed once with PBS, and differentiation medium was added. Myogenic differentiation was monitored over 4 d. Medium was exchanged every other day. Bulk activation and isolation of myofiber-associated SCs and culture of SC-derived myoblasts from S1P2-KO mice S1P2-KO mice in BALB/c background were a gift from Jerold Chun (Scripps Research Institute, La Jolla, CA, USA). Wild type BALB/c mice were obtained from Jackson Laboratories (Sacramento, CA, USA). Animals were utilized following an approved Children’s Hospital Oakland Research Institute Institutional Animal Care and Use Committee protocol. Mice were injected into the gastrocnemius through the skin at 2–5 injury sites with notexin (0.1 ␮g i.m.). SC-derived myoblasts were then isolated and cultured as described previously (1). S1P2 expression was determined by qRT-PCR in proliferating cells. Western blotting and imaging were performed on

SPL is induced during myogenic differentiation of C2C12 cells Numerous studies have implicated S1P and the major SphK, SphK1, in myogenic differentiation (33). By immunoblotting whole-cell extracts, we evaluated expression levels of SphK1 and SPL, the two main regulators of intracellular S1P levels, throughout differentiation. On differentiation d 4, we observed the characteristic morphology of myotube formation by phase-contrast microscopy (data not shown). As shown in Fig. 1A, SphK1 expression was robust at time 0 but diminished during differentiation. In contrast, we observed a pronounced induction of SPL during the differentiation time course (Fig. 1A). Levels of myogenin increased on d 1 of differentiation, and myogenin was robustly up-regulated by d 2. MHC increased on d 3 of differentiation and was robustly up-regulated by d 4 (Fig. 1A). These results were quantified by image analysis (Fig. 1B). SPL silencing in C2C12 cells leads to reduced SPL enzyme activity, increased S1P levels, and altered S1PR expression To elucidate the role of SPL in muscle differentiation, SPL was silenced by lentiviral-mediated KD in C2C12 cells. Morphology of cells transduced with vector control and SPL-KD cells was indistinguishable during exponential growth under standard propagation conditions (Fig. 2A). SPL protein expression was low to undetectable in SPL-KD cells under all growth conditions (Fig. 2B). SPL enzymatic activity in proliferating SPL-KD cells was reduced to a third of control cell levels (P⬍0.05; Fig. 2C). Intracellular S1P levels (measured by lipid extraction and mass spectrometry) were significantly higher in SPL-KD cells than controls (Fig. 2D). Similarly, we observed a significantly higher S1P con-

TABLE 2. mirVana miRNA mimic RNA duplex sequences Sequences Gene

miR-1 miR-206 miR-486

Sense, 5=–3=

Antisense, 5=–3=

ACAUACUUCUUUACAUUCCATT ACACACUUCCUUACAUUCCATT CGGGGCAGCUCAGUACAGGATT

UGGAAUGUAAAGAAGUAUGUAU UGGAAUGUAAGGAAGUGUGUGG UCCUGUACUGAGCUGCCCCGAG

SPL REGULATES MYOGENIC MICRORNAS

509

A

0

1

2

3

4

B

5

Myogenin

MHC

SPL

SphK1

Relative Protein Expression

120 100 80

Myogenin MHC

60

SPL 40

SphK1

20 0 0

GAPDH

1

2

3

4

5

Day of Differentiation

Figure 1. Changes in SphK1 and SPL during differentiation of C2C12 cells. C2C12 cells were propagated under proliferation conditions until time 0, when the medium was replaced with differentiation medium containing 2% horse serum. Cells were harvested at d 0 –5. Whole-cell extracts were evaluated by immunoblotting with antibodies specific for MHC, myogenin, SPL, SphK1 (SK1), and GAPDH loading control. A) Representative immunoblot. B) Quantification of autoradiogram. All bands were normalized to the loading control for the corresponding time point. Experiment is representative of 3 similar experiments.

tent in medium incubated in the presence of SPL-KD cells compared to control medium (Fig. 2E). We then used qRT-PCR to compare the gene expression of the 5 known S1PRs (Fig. 2F). Whereas S1P1 and S1P2 gene expression levels were similar in the two cell lines, S1P3–5 were reduced in SPL-KD cells. SPL is required for the transition from proliferative to differentiation state in muscle stem cells S1P increases proliferation in SCs and decreases proliferation in SC-derived reserve cells, a population of quiescent, undifferentiated cells related to SCs (14). To determine the effect of SPL-KD on C2C12 viability and proliferation, trypan blue counts and proliferation assays were performed. Comparison of SPL-KD cells and controls showed no consistent differences in viability (results not shown) or proliferation (Fig. 3A). However, under differentiation conditions, SPL-KD cells appeared compromised in their ability to undergo differentiation. This was demonstrated by reduced MHC protein levels in SPL-KD cells compared to controls harvested after 4 d in differentiation conditions (Fig. 3B). At d 4, MHC protein levels in SPL-KD cells were 1/4 those of controls (Fig. 3C). To confirm the lack of differentiation, whole cell extracts were harvested on d 4 and analyzed for both MHC and myogenin, an early marker of myogenic differentiation. SPL-KD cells failed to express either myogenic marker, whereas control cells expressed high levels of both markers at d 4 (Fig. 3E, F). The failure of SPL-KD cells to undergo myogenic differentiation was strikingly evident by the lack of myofiber formation observed by immunofluorescence microscopy of cultured cells stained with antibody to MHC followed by Alexa Fluor 488-labeled secondary antibodies, combined with PI labeling of nuclei (Fig. 3G). Whereas many well-developed, MHCpositive, multinucleated myotubes were detected in 510

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control cell cultures, SPL-KD cultures contained fewer myotubes [control 31⫾8.9 vs. SPL-KD 2.3⫾1.2 myotubes per low power field (LPF), respectively], which were characterized by smaller size and fewer nuclei/ myotube (Supplemental Fig. S1). Pax7 is a SC marker and transcription factor whose expression is normally down-regulated during SC activation and subsequent differentiation. Pax7 protein levels decreased under differentiation conditions in control cells as expected (Fig. 3B). In contrast, high levels of Pax7 expression were sustained in SPL-KD cells despite continued culture under differentiation conditions (Fig. 3B). Elevated Pax7 protein expression in SPL-KD cells was found to be 2-fold greater than the levels found in control cells on d 4 of differentiation (Fig. 3D). These cumulative findings demonstrate that SPL silencing results in a failure of myoblasts to fuse and differentiate, revealing that SPL is required for myogenic differentiation. Further, sustained Pax7 and reduced MHC and myogenin expression in SPL-KD cells suggests that global regulation of myogenic differentiation in myoblasts may be disrupted by SPL silencing. SPL is required for basal expression of myogenic miRNAs and their timely induction in response to a differentiation stimulus Three miRNAs have been implicated in the regulation of Pax7 expression during skeletal muscle differentiation: miR-1, miR-206, and miR-486 (26, 34, 35). We considered the possibility that SPL silencing interfered with Pax7 down-regulation during differentiation by affecting the expression of these myogenic miRNAs. We first examined miR-1, miR-206, and miR-486 expression levels during myogenic differentiation in vector control cells by qRTPCR. The 3 miRNAs were found to significantly increase by varying magnitudes over time compared to their control levels, with expression levels beginning to plateau by

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DE LA GARZA-RODEA ET AL.

A

Control

D

SPL-KD

400

Intracellular S1P

*

Control

SPL-KD

[S1P] % control

350 300 250 200 150 50 0

100

E

m

250

Extracellular S1P

*

Control

SPL-KD

B

Proliferation Control SPL-KD

[S1P] % control

200

Differentiation Control

SPL-KD

SPL

0

S1P lyase enzyme activity

50 40 30

*

20 10 0

Control

SPL-KD

1.4

Relative Gene Expression

Specific activity pmol/mg/min

F 60

100

50

Actin

C

150

1.2 1.0 0.8 0.6 0.4 0.2 0

S1P1

S1P2

S1P3

S1P4

S1P5

Figure 2. Generation of SPL-KD and vector control C2C12 lines. A) Phase-contrast images of C2C12 cells after lentiviral transduction to knock down murine SPL. Scale bar ⫽ 100 ␮m. B) SPL knockdown was confirmed by immunoblotting extracts of cells maintained in proliferation or differentiation conditions. Actin was used as a loading control. C) SPL enzyme activity. n ⫽ 3/group. SPL-specific activity is expressed as picomoles product formation per milligram protein per minute. *P ⬍ 0.05. D) Intracellular S1P concentration was determined by mass spectrometry and normalized to phospholipid content (PC). Data are presented as percentage control cell levels, n ⫽ 3/group. *P ⬍ 0.05. E) Extracellular S1P concentration was determined as in D; n ⫽ 4/group. Data are presented as percentage control levels. *P ⬍ 0.05. F) Expression of S1PR1–5 was compared in SPL-KD and control cells by qRT-PCR, with results normalized to GAPDH; n ⫽ 3/group. These experiments were performed ⱖ3 times with similar results. *P ⬍ 0.05.

d 4 (Fig. 4A). The rapid and robust induction of miR-1 suggests that miR-1 may have a more pronounced effect on downstream targets than miR-206 and miR-486 (Fig. 4A). We next compared the expression levels of each miRNA in both control and SPL-KD cell lines. During culture in proliferation medium, expression levels of all 3 miRNAs were found to be significantly reduced in the SPL-KD cells compared to control cells, with the greatest difference occurring with miR-1 (Fig. 4B). During differentiation, expression of all 3 myogenic miRNAs was induced in both cell lines (Fig. 4C). However, the rate of miRNA induction in SPL-KD cells was impaired in contrast to control cells (Fig. 4C). The most pronounced down-regulation of miRNA expression in the SPL-KD was SPL REGULATES MYOGENIC MICRORNAS

observed on d 4, when the highest rate of myogenic fusion is expected to occur in the control cells. Thus, SPL-KD cells express reduced levels of three miRNAs involved in Pax7 regulation and skeletal muscle differentiation. These findings raised the possibility that dysregulation of myogenic miRNA expression at a critical time point in SPL-KD cells interferes with their capacity for myogenic differentiation. Differentiation of SPL-KD cells can be achieved by S1PR modulation S1P1–3 have been shown to regulate SC differentiation (36). However, S1P signaling can also occur through 511

A

B

Control

SPL-KD

Proliferation Assay

1.6

1

4

8

1

4

8

Absorption 490λ

Control 1.2

Day MHC

SPL KD

0.8

PAX7

0.4

SPL Actin

0 1

2

3

4

5

Days in culture

Relative Protein Expression

1.5

D

MHC (day 4)

2.5

Relative Protein Expression

C

1.0

0.5

Control

0.5

Control

F

SPL-KD

10% FBS

2% HS

Myogenin

6

Relative Protein Expression

1.0

KD

E 5

1.5

0

0

7

Pax7 (day 4)

2.0

WT

MHC

SPL-KD

WT

SPL-KD Myogenin

4 3

MHC

2 1

Actin

0

WT FBS

SPL-KD FBS

WT HS

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Figure 3. SPL is required for transition from proliferation to myogenic differentiation. A) Proliferation assays were performed in expoG SPL-KD Control nentially growing SPL-KD and control cells. B) Cells were induced to differentiate and 31±8.9 2.3±1.2 Myotubes/LPF Myotubes/LPF harvested at d 1, 4, and 8. Immunoblotting was performed to detect MHC expression in whole-cell extracts. Pax7 protein expression was detected in the same extracts. C) MHC protein expression on d 4 was quantified and normalized to actin (P⬍0.01). D) Pax7 pro200 m tein expression on d 4 was quantified and normalized to actin (P⬍0.05). E) Myogenin and MHC protein expression on d 4 of differentiation conditions were quantified and normalized to actin. F) Representative immunoblot of control wild-type (WT) and SPL-KD cells in proliferation conditions of 10% FBS and after 4 d in 2% horse serum. G) Representative immunofluorescence images of SPL-KD and control cultures after 4 d under 50 m differentiation conditions. Detection of sarcomeric MHC (green) and PI detection of nuclei (red) revealed well-differentiated, multinucleated myotubes in control cultures, whereas myotubes were not detected in SPL-KD cultures. Mean myotube counts (see Materials and Methods) per low-power field (LPF) are indicated. Scale bars ⫽ 200 ␮m (top panels); 50 ␮m (bottom panels). These results are representative of at ⱖ3 similar experiments.

receptor-independent mechanisms (37, 38). Therefore, to investigate whether the block in differentiation resulting from SPL silencing involves S1PR signaling, 512

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we employed chemical antagonists of S1P1 (W123), S1P2 (JTE013), and S1P3 (BML-241). Inhibition of neither S1P1 nor S1P3 had an appreciable effect on

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Figure 4. Expression of myogenic miRNAs is dysregulated in SPL-KD cells. A) Levels of miR-1, miR-206, miR-486, and snU6 were determined by qRT-PCR at d 1, 4, and 8 of differentiation. Day 0 correlates to miRNA levels in unstimulated cells. Comparative Ct analysis was used to quantify general cellular abundance of each miRNA. Data are plotted relative to snU6 transcript levels at each time point. Data are shown in log scale. For each miRNA species, levels at d 1, 4, and 8 are significantly higher than at d 0; n ⫽ 3/group. *P ⬍ 0.05. B) Relative expression levels of miR-1, miR-206, and miR-486 were determined by qRT-PCR in control and SPL-KD cells in proliferating conditions. Values are given as SPL-KD results relative to control cell results, which are set at normal, with all results being normalized to snU6. *P ⬍ 0.01 (miR-1); 0.02 (miR-206); 0.001 (miR-486). C) miRNA expression was evaluated by qRT-PCR in control and SPL-KD cells at d 1, 4, and 8 following exposure to differentiation medium; n ⫽ 3/group. Values are normalized to snU6 levels (means ⫾ se). *P ⫽ 0.03.

differentiation of control or SPL-KD cells, as determined by MHC protein expression (not shown). Inhibition of S1P2 modestly increased the level of differentiation in SPL-KD cells, as shown by MHC levels in whole-cell extracts obtained on d 4 of differentiation conditions (Fig. 5A, B). We then tested the effects of S1PR agonists on SPL-KD cell differentiation. Treatment with the S1P1 agonist SEW2871 significantly enhanced the differentiation of SPL-KD cells, whereas it had no effect on control cells, as determined by MHC expression on d 4 (Fig. 5A, B). S1P1 agonist treatment also increased the fusion index of SPL-KD myoblasts, as determined by an increase in the number of nuclei per MHC-positive myotube (Supplemental Fig. S1). No other agonist influenced SPL-KD cell differentiation (data not shown). When S1P1 agonist and S1P2 antagonist were administered together, their effect was greater than that of either agent alone, as shown by a significant increase in MHC expression (Fig. 5A, B). To verify the effect of S1P2 inhibition on the ability of SPL-KD cells to undergo differentiation and to avoid potential off-target effects, we conducted a second experiment in which JTE013 was applied to cultures of control and SPL-KD cells for a 24-h time period prior to changing the medium to differentiation conditions. No further inhibitor was added once cells were shifted to differentiation conditions. After 72 h, cells were harvested and evaluated for MHC expression. S1P2 antagSPL REGULATES MYOGENIC MICRORNAS

onism increased MHC expression in SPL-KD cells, as well as in control cells (Supplemental Fig. S2). The ability of S1PR modulation to overcome the block in differentiation induced by SPL silencing was evident by immunofluorescent detection of MHC-expressing myotubes (Fig. 5D). Sustained Pax7 expression in SPL-KD cells is an S1PR-independent phenomenon We suspected that receptor modulation strategies that successfully overcame the block in differentiation induced by SPL silencing would also be accompanied by a reduction in Pax7 levels, since Pax7 down-regulation is generally considered a prerequisite to differentiation. Surprisingly, however, Pax7 protein expression levels were unaffected by treatment with S1P1 agonist combined with S1P2 antagonist, despite the ability of this combined treatment to promote differentiation on d 4 (Fig. 5A, C). These results indicate that S1PR modulation promotes MHC activation and myogenic differentiation through a mechanism that bypasses Pax7 downregulation. Consistent with this notion, Zammit et al. (35) observed that sustained high expression of Pax7 in satellite cell-derived myoblasts is compatible with differentiation and fusion into multinucleated myotubes. The same group showed that YMCA cells (a Pax7deficient clone of C2C12 cells) readily differentiated 513

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Figure 5. Differentiation defect of SPL-KD cells is mediated through S1PR signaling. A) Western blotting to detect MHC protein expression in whole-cell extracts of SPL-KD and control cells after 4 d of myogenic differentiation and treatment with agonist of S1P1, antagonist of S1P2, and a combination of the two. Veh, vehicle; R1 Ag, S1P1 agonist; R2, Ant S1P2 antagonist. B) Immunoblots were quantified for MHC expression in receptor-modulated SPL-KD cells and compared to SPL-KD cells treated only with vehicle, with each band being normalized to actin. Similar quantification was not performed for control cells, as there was no obvious difference in MHC expression levels in response to treatment. C) Immunoblots were quantified for Pax7 expression in receptor-modulated SPL-KD cells and compared to SPL-KD cells treated only with vehicle, with each band being normalized to actin. D) SPL-KD and control cells treated with agonist of S1P1 (R1 Ag), antagonist of S1P2 (R2 Antag), and a combination of both (R1 Ag⫹R2 Antag). Cultures were fixed and stained for immunofluorescence microscopy to visualize multinucleated myotubes by MHC-positive myotubes (green) and PI-positive nuclei (red). Means of myotube counts (see Materials and Methods) per LPF are indicated in each panel. Scale bar ⫽ 100 ␮m.

into large myotubes on serum withdrawal (35). On the basis of these data, we considered whether permanent SPL silencing might promote continued cell proliferation and prevent cell cycle arrest required for myogenic differentiation. Therefore, we employed three different cell cycle inhibitors (mimosine, thymidine, and aurora kinase inhibitor II), as well as serum starvation to artificially arrest SPL-KD cells and evaluate their ability to undergo differentiation. Interestingly, forcing cell cycle arrest with chemical inhibitors or by incubation in serum-free medium induced MHC expression in SPL-KD cells and augmented its expression in control cells (Supplemental Fig. S2). These findings suggest that SPL silencing prevents myogenic differentiation, at least in part, by maintaining myoblasts in a proliferative state. 514

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S1P2 is not required for myogenic differentiation S1P2 has been implicated as a mediator of S1P’s effects on proliferation and/or differentiation in myogenic cells, and silencing of S1P2 has been shown to delay muscle regeneration (16, 39, 40). However, our findings using the S1P2 antagonist JTE013 suggested that S1P2 may be dispensable for myogenic differentiation. To confirm this possibility, we isolated SC-derived myoblasts from S1P2-knockout (KO) mice (BALB/c background) and wild-type BALB/c mice and then compared their ability to differentiate. S1P2 expression was present in proliferating SC-derived myoblasts isolated from wild-type mice but was undetectable in myoblasts from S1P2-KO mice (Supplemental Fig. S3A). After 48 h incubation in differentiation medium, we observed

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sion levels in whole-cell extracts (Fig. 7A, B). In contrast, we did not observe a significant change in MHC levels in response to miR-486 transfection. The effect on Pax7 expression of miR-1 and miR-206 was opposite to that of MHC, leading to a reduction in Pax7 expression (Fig. 7A, B). In contrast, miR-486 had no effect on Pax7 levels in control cells and a slight enhancement of Pax7 levels in SPL-KD cells. Using immunofluorescence detection of MHC-positive myotubes and nuclei PI staining, we confirmed that repletion of miR-1 and miR-206, but not miR-486, promoted myogenic differentiation of SPL-KD cells (Fig. 7C).

robust differentiation in both S1P2-KO and control myoblast cultures. No significant difference in differentiation was observed between the wild-type and S1P2-KO myoblasts, as judged by the expression of myogenic markers detected by immunoblotting (Supplemental Fig. S3B, C) or by myotube formation, detected using immunofluorescence microscopy and quantified by the number of multinucleated myofibers/LPF (Supplemental Fig. S3D). S1PR modulation corrects the deficiency of myogenic miRNAs observed in response to SPL silencing SPL-KD cells fail to differentiate under conditions that normally promote myotube formation (Fig. 3) and also exhibit reduced levels of myogenic miRNAs. To determine whether the deficiency of myogenic miRNAs in cells lacking SPL contributed to their inability to differentiate, we first measured miR-1, miR-206, and miR-486 in SPL-KD cells treated either with vehicle or combined S1P1 agonist/S1P2 antagonist treatments. Receptor modulations that promoted myogenic differentiation also increased expression of all three measured miRNAs in both control and SPL-KD cell lines on d 4 of differentiation (Fig. 6). Treatment of SPL-KD cells restored miR-1 expression levels to those observed in untreated control cells (Fig. 6A), whereas miR-206 and miR-486 expression levels were elevated to levels even greater than those in untreated control cells (Fig. 6B, C).

The S1P transporter Spns2 has a role in myogenic differentiation We were interested to know whether S1P export plays a role in myogenesis. Numerous transporters have been implicated in this process, including ABC transporters and the specific S1P transporter Spns2 (41, 42). Spns2 mutation in zebrafish leads to a cardiac defect, which suggests that it plays an important role in striated muscle cells (42). However, we are not aware of Spns2 expression analysis being reported in C2C12 cells. To characterize Spns2’s role in myogenesis, Spns2 gene expression was measured under baseline proliferation conditions and during differentiation by qRT-PCR. We found that Spns2 is expressed in C2C12 cells, with Spns2 gene expression levels diminishing by more than 50% after d 8 of differentiation conditions (Supplemental Fig. S4A). We then compared Spns2 expression in control and SPL-KD cells. SPL-KD cells expressed significantly less Spns2 in comparison to control cells under baseline conditions (Supplemental Fig. S4B). To evaluate the importance of Spns2 in myogenesis, Spns2 silencing was performed on both control and SPL-KD cells. Spns2 KD using siRNA resulted in ⬃40% suppression compared to cells treated with scrambled siRNA (data not shown). Despite incomplete silencing, Spns2 KD reduced differentiation at d 4 in control cells, as shown by a reduction in myotube formation in culture by phase-contrast microscopy (Supplemental Fig. 4C) and reduced myogenin expression by immunoblotting

Repletion of myogenic miRNAs in SPL-KD cells is sufficient to restore their differentiation capacity To establish whether SPL-deficient myoblasts fail to differentiate specifically because they are lacking in myogenic miRNAs, we boosted the levels of myogenic miRNAs in control and SPL-KD cells by transfection with molecular mimics of murine miR-1, miR-206, or miR-486, followed by incubation in differentiationpromoting conditions for 4 d. Treatment with miR-1 and, to a lesser degree, miR-206 resulted in differentiation of SPL-KD cells, as determined by MHC expres-

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(Supplemental Fig. 4D, E). Spns2 silencing did not correct the SPL-KD defect, which is not surprising, considering the low baseline levels of Spns2 in these cells (Supplemental Fig. S4C–E).

DISCUSSION In this study, we explored the role of the S1P-catabolizing enzyme SPL in myoblast proliferation and differentiation. We observed that SPL is induced during differentiation, whereas SphK1 expression falls during differentiation. These findings are consistent with our previous observations showing SphK1 and SPL gene expression levels change dramatically in injured muscle (16). However, our new findings demonstrate that SPL is induced specifically in myoblasts during differentiation. This suggests that S1P levels are dynamically regulated during myogenic differentiation. To investigate further SPL’s functions in myoblasts, 516

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we explored the biochemical and cellular characteristics of an SPL-KD myoblast line. SPL-KD cells exhibited reduced SPL activity and expression, increased intracellular S1P levels, and higher S1P in conditioned media compared to controls. The source of intracellular S1P accumulation in SPL-KD cells is presumed to be de novo S1P biosynthesis from sphingosine. However, extracellular S1P elevation could derive from export of S1P from SPL-KD cells or their failure to import/catabolize S1P in the growth medium. Our principal observation is that SPL expression is required for myogenic stem cell differentiation. This was revealed by a reduction in cellular MHC and myogenin levels, as well as multinucleated myotube formation, in SPL-KD vs. control cells under differentiation conditions. Both the number of MHC-expressing fibers/well and nuclei/MHC-expressing-fiber were reduced in SPL-KD cells. In addition, sustained Pax7 expression along with the reduced MHC and myogenin expression in SPL-KD cells suggest that regulation of

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myogenic gene expression during differentiation is disrupted by SPL silencing. This is further supported by the dysregulated miRNA expression in SPL-KD cells. Altogether, these data establish an important role for SPL and S1P in the transition from proliferation to differentiation in myogenesis. These findings are consistent with the Sply myopathy (where KD of SPL in fruit flies led to muscle degeneration) and suggest a conserved role for SPL in muscle homeostasis (24). S1P mediates important functions in innate immunity and epigenetic gene regulation by activating intracellular targets in S1PR-independent fashion (5, 43). Intracellular and extracellular S1P levels were influenced by SPL silencing. Thus, we could not immediately infer the site of S1P action on SPL-KD cell phenotype, and it was necessary to examine the potential involvement of S1PRs in the effect of SPL silencing on muscle differentiation. Focusing on S1P1–3, which have been implicated in C2C12 differentiation, we found that a S1P1 agonist and S1P2 antagonist reversed the effect of SPL-KD on MHC expression and myotube formation. Further, treatment of SPL-KD cells with the S1P1 agonist led to an increase in the nuclei/myotube formed. Our results confirm previously reported involvement of S1P1 in myogenic differentiation (14). S1P has been shown to inhibit proliferation and increase differentiation through S1P2 (17). In contrast to these findings, we observed that S1P2 inhibition improved differentiation in the SPL-KD line; an additive effect was observed when the S1P2 antagonist was combined with the S1P1 agonist. This suggests that an absence of SPL either promotes excessive stimulation of S1P2 that interferes with differentiation cues or that there is a context-dependent difference in signaling. SPL silencing also affected expression of S1PR levels, reducing S1P3–5 levels compared to control cell levels. While our results implicate abnormal S1P1 and S1P2 signaling in mediating a differentiation block, further study will be necessary to define the role of S1P3–5 in regulating myoblast differentiation. Our previous findings using an in vivo model system (16) show that SphK1 gene expression is induced within hours after muscle injury, followed by a rapid return to baseline within the first day after injury, followed several days later by sustained SPL up-regulation. In this in vitro study, we show that SPL protein levels are up-regulated during myoblast differentiation, whereas SphK1 levels fall during myogenesis. In the absence of SPL up-regulation, S1P levels remain high intracellularly and extracellularly, in association with a differentiation-impaired phenotype. Taking together our previous and current findings, we suggest that S1P generation is important for SC proliferation in response to injury, and SC recruitment may be enhanced pharmacologically by boosting S1P levels temporarily via SPL inhibition. Whereas high S1P levels may be required for the initial (proliferative) phase of SC response to skeletal muscle injury, SPL up-regulation, metabolism of high local S1P levels, and cell cycle arrest may be required for SC-derived myoblasts to undergo SPL REGULATES MYOGENIC MICRORNAS

fusion and differentiation, thereby assisting in muscle rebuilding. This latter process may additionally require a switch in the predominance of S1PR subtypes involved in signaling. It is likely that the pleiotropic effects of S1P signaling through its multiple receptors are tightly and temporally orchestrated to mediate the multistep process of myogenesis. We and others reported that S1P2 is needed for efficient muscle regeneration (16, 40). Others reported that S1P2 contributes to myogenic differentiation (17, 44, 45). However, our results using SC-derived myoblasts devoid of S1P2 provide clear evidence that S1P2 is dispensable for differentiation of primary myoblasts under standard conditions. We suspect that the role of S1P2 on C2C12 differentiation may reflect an effect on proliferation and expansion of a population of fusogenic myoblasts. Further study will be required to fully elucidate the mechanisms by which S1P2 and other S1PRs influence the complex and multistep process of myogenesis. Chronically high S1P levels in the medium of SPL-KD cells may change the normal response of SPL-KD cells required for differentiation. Whereas our findings are consistent with an inside-out signaling model that involves S1PRs, we cannot exclude the possibility that intracellular (cytoplasmic or nuclear) S1P regulation and signaling also play a role in mediating myogenic differentiation. In addition to controlling S1P levels, SPL generates hexadecenal and ethanolamine phosphate products implicated in the regulation of cell survival and stress responses (46 – 48). However, our results suggest that the major effect of SPL on myoblast differentiation is mediated through S1PRs. SPL silencing results in sustained and even higher Pax7 expression in myoblasts maintained in differentiation conditions (Fig. 3B, D). Pax7 is part of a transcriptional network that governs myogenic differentiation and whose ability to suppress terminal myogenic differentiation appears to be subverted during carcinogenic transformation and the development of rhabdomyosarcoma (49). S1P has been shown to modulate epigenetic gene regulation by serving as an inhibitor of HDAC (50). Thus, it is conceivable that intracellular S1P accumulation in SPL-KD cells promotes Pax7 expression through this function of S1P. To investigate the effect of SPL expression on other critical factors involved in myogenesis, we compared the expression of three myogenic miRNAs implicated in the regulation of Pax7. The miR-1/miR-206 family of miRNAs is a well-established central regulator of myogenesis with numerous established targets important in muscle development, differentiation, chromatin remodeling, and cell fate determination. In contrast, the miR-486 species has been shown primarily to target expression of FoxO1, PTEN, and Pax7 (34, 51). Levels of all 3 miRNAs were reduced in SPL-KD cells compared to controls. Induction of miR-1 in SPL-KD cells was significantly reduced on d 4 of differentiation. Notably, we found that achievement of differentiation in SPL-KD cells by S1PR modulation was accompanied by restoration of myogenic miRNA levels and that 517

miR-1/miR-206 transfection restored differentiation capability to SPL-KD cells. These findings establish that SPL regulates myogenic differentiation through S1PRdependent control of myogenic miRNA expression. Myogenic miRNAs are critical regulators of SC differentiation and growth, have important roles in muscle remodeling, and their dysregulation has been implicated in diseases of cardiac and skeletal muscle. Numerous downstream miR-1/miR-206 targets have been identified in C2C12 cells, but the specific role of miR-1/miR-206 family members in myoblast differentiation remains to be determined (52). Finally, we characterized the expression and function of the S1P transporter Spns2 in myoblast differentiation. Murine Spns2 has been shown to play a role in vascular biology (53). However, a role for Spns2 in skeletal muscle has not been reported. We found that Spns2 is expressed in proliferating myoblasts and downregulated toward the end of differentiation. Spns2 silencing had a negative effect on myogenic differentiation, as shown by reduced myotube numbers and myogenin expression. In the absence of SPL, Spns2 expression was significantly reduced, which suggests that Spns2 is itself regulated in response to S1P metabolism and/or signaling, and that the low Spns2 expression in SPL-KD cells may result from high extracellular S1P levels. Thus, it is not surprising that further KD of Spns2 had no measurable effect on differentiation of SPL-KD cells. It is tempting to speculate that Spns2 may deliver S1P to its cognate membrane receptors in an autocrine fashion, a function that may not be compensated for by S1P presence in the media, where it is conjugated to serum albumins and lipoproteins. To our knowledge, this is the first report indicating that SPL and S1P signaling have an effect on myogenic miRNA regulation. Further studies will be required to establish how SPL affects this critical process, but given our data, we conclude that S1P acts through receptordependent signaling to control miRNA expression. Considering the emerging potential of miRNA-based therapeutics, our findings regarding the effect of pharmacological modulation of SPL and in combination with S1PRs on miRNAs may hold translational relevance for treatment of human disease. The authors are grateful to Abeer Eltanawy, Julia Weisbrod, and Rashed Abu-Alia for technical support and N. Cronen for expert administrative assistance. These studies were supported by U.S. National Institutes of Health grant GM66954, Muscular Dystrophy Association grant MDA217712 and Minority Biomedical Research Support–Research Initiative for Scientific Enhancement (MBRS-RISE) grant 5R25GM59298-12 (to J.D.S.), California Institute of Regenerative Medicine (CIRM) Bridges grant TB1-01194 (to D.B.), and CIRM clinical fellowship TG2-01164 (to A.S.G.R.)

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Sphingosine phosphate lyase regulates myogenic differentiation via S1P receptor-mediated effects on myogenic microRNA expression.

S1P lyase (SPL) catalyzes the irreversible degradation of sphingosine-1-phosphate (S1P), a bioactive lipid whose signaling activities regulate muscle ...
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