Lysophosphatidic acid stimulates cell migration of satellite cells. A role for the sphingosine kinase/sphingosine 1-phosphate axis

Lysophosphatidic acid stimulates cell migration ofsatellite cells. A role for the sphingosine kinase/sphingosine 1-phosphate axisFrancesca Cencetti1,2, Gennaro Bruno1, Sabrina Blescia1, Caterina Bernacchioni1, Paola Bruni1,2

and Chiara Donati1,2

1 Dipartimento di Scienze Biomediche Sperimentali e Cliniche ‘Mario Serio’, Universit�a di Firenze, Italy

2 Istituto Interuniversitario di Miologia, Italy

Keywords

lysophosphatidic acid; satellite cells; skeletal

muscle; sphingosine 1-phosphate;

sphingosine kinase

Correspondence

P. Bruni, Dipartimento di Scienze

Biomediche Sperimentali e Cliniche ‘Mario

Serio’, Universit�a di Firenze, Viale G.B.

Morgagni 50, Firenze 50134, Italy

Fax: +39 05527511351

Tel: +39 0552751204

E-mail: [email protected]

(Received 13 March 2014, revised 4 July

2014, accepted 29 July 2014)

doi:10.1111/febs.12955

Regulation of the motility of skeletal muscle precursor cells, such as satel-

lite cells, is critically important for their proper recruitment at the site of

tissue damage, and ultimately for its correct repair. Here we show that

lysophosphatidic acid (LPA), which is well-recognized as a powerful bioac-

tive agent, strongly stimulates cell migration of activated murine satellite

cells. The biological effect exerted by LPA was found to be induced via

activation of LPA1 and LPA3, being abolished by cell treatment with the

antagonist Ki16425, and severely impaired by siRNA-mediated down-regu-

lation of the two receptor isoforms. In contrast, silencing of LPA2 potenti-

ated the stimulation of cell motility by LPA, suggesting that it is negatively

coupled to cell migration. Pharmacological inhibition of both sphingosine

kinase (SK) isoforms using VPC96047, or the selective blocking of SK1

using VPC96091, abolished cell responsiveness to LPA; in agreement, gene

silencing of SK1 or SK2 significantly reduced the biological effect of LPA.

Moreover, the LPA-dependent stimulation of cell chemotaxis was found to

be impaired by down-regulation of the sphingosine 1-phosphate (S1P)

receptors S1P1 or S1P4 by specific siRNAs. In summary, the results

obtained support the notion that the sphingosine kinase/sphingosine

1-phosphate (SK/S1P) axis is critically involved in the mechanism by which

LPA elicits its pro-migratory action. This study provides compelling new

information on the regulatory mechanisms of satellite cell motility, and

reinforces the view that the SK/S1P signaling pathway plays a crucial role

in the control of skeletal muscle precursor cell biology.

Introduction

Lysophosphatidic acid (LPA) and sphingosine 1-phos-

phate (S1P) are two potent bioactive lysophospholip-

ids that, mainly acting via engagement of multiple

G protein-coupled receptors, are able to regulate key

biological processes in mammalian cells [1]. Impor-

tantly, among the wide variety of cell types respon-

sive to LPA and S1P, a range of adult and

embryonic stem cells and progenitors are included,

suggesting that, by means of regulating their survival,

proliferation, differentiation and motility, these

lysophospholipids play a significant role in the

maintenance, renewal and trafficking of various

populations of stem cells and progenitors in the body

[2].

Abbreviations

BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; LPA, lysophosphatidic acid; S1P, sphingosine 1-phosphate; SCR,

scrambled; SK, sphingosine kinase.

4467FEBS Journal 281 (2014) 4467–4478 ª 2014 FEBS

There is a wealth of experimental results in favor of

a key role of S1P in regulation of the regenerative

potential of skeletal muscle, a tissue that has the ability

to self-repair following trauma or disease due to the

existence of precursor cells, which reside beneath the

basal lamina of each myofiber and are named satellite

cells [3,4]. Indeed, S1P has been shown to act as power-

ful mitogenic cue in quiescent and activated mouse

satellite cells [5,6]. Moreover, this lysosphingolipid has

been reported to be capable of stimulating the motility

of activated satellite cells, which is critical for their cor-

rect recruitment at the site of tissue lesions [6]. Interest-

ingly, these biological effects induced by S1P appear to

be transmitted by ligation to S1P receptors: S1P2 and

S1P3 are involved in transmission of the mitogenic

effect of the sphingolysolipid [6,7], whereas S1P1 and

S1P4 appear to be implicated in the migratory response

elicited by S1P [6]. Interestingly, recent studies per-

formed in mice in which S1P2 was genetically ablated

or pharmacologically blocked have provided experi-

mental evidence that this receptor subtype promotes

skeletal muscle regeneration [8], while altered cell-cycle

progression has been observed in satellite cells of S1P3

knockout mice [9]. Furthermore, comprehensive studies

performed in the C2C12 myoblast cell line, which is

largely used to investigate the biological properties of

skeletal muscle precursor cells, have clearly demon-

strated that S1P and its metabolism play a critical role

in myogenic differentiation, and that the S1P signaling

pathway is used by various growth factors and cyto-

kines to elicit specific biological effects [10–16].Present knowledge of the biological role of LPA in

skeletal muscle precursor cells is more limited. An

early report described the stimulatory effect of LPA

on cell proliferation of C2C12 myoblasts, accompanied

by its inhibitory action on myogenic differentiation

[17]. The mitogenic effect of LPA in myoblasts was

further confirmed in a subsequent study, in which the

LPA action was found to be exerted via ligation of

LPA1/LPA3 receptors and activation of the phosphati-

dylinositol-4,5-bisphosphate 3-kinase pathway [18].

Additionally, a recent study has shown that LPA, act-

ing via Gai2, robustly stimulated hypertrophy of myo-

tubes in a protein kinase C dependent manner [19].

In a previous study, we observed that LPA acts as

mitogenic cue in activated satellite cells [6]. This

prompted us to better characterize the biological

action of LPA in these skeletal muscle precursor cells

and to explore its molecular mechanism of action.

Here we report that LPA significantly enhances migra-

tion of activated satellite cells, and that cross-talk with

sphingosine kinase (SK) and S1P receptors plays a key

role in transmission of this biological effect.

Results

In order to obtain more information on the biological

role performed by LPA in skeletal muscle precursors,

taking into consideration the crucial importance of

regulation of cell motility for skeletal muscle regenera-

tion, we assessed whether it affects this biological

parameter by measuring chemotaxis of activated satel-

lite cells toward LPA using the Boyden chamber assay.

The results shown in Fig. 1A indicate that LPA influ-

enced chemotactic movement, producing a typical

bell-shaped curve. The maximal increase in the number

of migrated cells after 6 h of incubation was observed

using 1 lM LPA. This concentration was therefore

used in subsequent experiments. A scratch wound

healing assay performed at 7 h after cell injury and

treatment with 1 lM LPA confirmed that this lyso-

phospholipid is capable of strongly stimulating cell

motility (Fig. 1B).

To investigate the role of LPA receptor subtypes in

transmitting the biological effect of LPA, their relative

expression at the mRNA level was assessed by real-

time PCR. As shown in Fig. 2A, LPA1 is the domi-

nant receptor subtype expressed, with LPA2 and LPA3

being clearly detectable but significantly less repre-

sented. Cell migration experiments were then per-

formed using cultured satellite cells treated with the

LPA1/LPA3 antagonist Ki16425 (5 lM) 40 min prior

incubation with LPA. The pharmacological blockade

of LPA receptors fully prevented the enhancement

of cell migration exerted by 1 lM LPA (Fig. 2B). To

corroborate the experimental evidence regarding the

involvement of LPA receptor subtypes in the biologi-

cal response elicited by LPA, RNA interference tech-

nology was used. Cell transfection with specific

siRNAs strongly down-regulated individual LPA

receptor subtypes (Fig. 2C), but did not significantly

affect expression levels of the non-targeted LPA recep-

tor subtypes (data not shown). As shown in Fig. 2D,

reduced expression of LPA1 completely abolished the

stimulation of the migratory properties of satellite cells

exerted by 1 lM LPA; similarly, gene silencing of

LPA3 attenuated the biological action of LPA. In

contrast, when LPA2 expression was diminished, the

stimulatory effect of LPA on cell migration was poten-

tiated (Fig. 2D). To further demonstrate the anti-

migratory action mediated by LPA2, the chemotactic

response elicited by LPA was evaluated in cells chal-

lenged with serum in the presence or absence of the

LPA1/LPA3 antagonist Ki16425 (5 lM). As shown in

Fig. 2E, addition of 1 lM LPA strongly enhanced the

cell motility induced by 10% fetal bovine serum,

whereas blockade of LPA1 and LPA3 resulted out in a

4468 FEBS Journal 281 (2014) 4467–4478 ª 2014 FEBS

Role of the S1P axis in LPA-induced cell migration F. Cencetti et al.

dramatic decrease in serum-stimulated migration. As

expected, the chemotactic response induced by 10%

fetal bovine serum was decreased when LPA1 and

LPA3 were inhibited, in line with the key biological

role of endogenous LPA in serum. Together, these

data support the view that LPA enhances cell migra-

tion via engagement of LPA1 and LPA3, whereas liga-

tion to LPA2 inhibits satellite cell motility.

Next, taking into account the key role of the S1P

signaling axis in cultured myoblasts [12–16] and the

robust migratory response elicited by S1P in the cell

type investigated here [6], we examined whether SK

activity is required for the migratory response exerted

by LPA. To address this issue, satellite cell migration

was measured under experimental conditions in which

satellite cells were previously incubated with 1 lMVPC96047, a selective inhibitor of SK that is capable

of blocking both the SK1 and SK2 isoforms, or 1 lMVPC96091, an inhibitor of the SK1 isoform only.

Pharmacological inhibition of both SK isoforms or

selective blockade of SK1 prevented the LPA-induced

increase in satellite cell motility (Fig. 3A), supporting

a key role for SK1 in the mechanism by which LPA

gives rise to a biological response. To confirm the

involvement of SK activity in the mechanism of LPA

action, and to obtain information into the role of

SK2, SK1 or SK2 were specifically silenced by specific

siRNA transfection, and then satellite cells were

probed for responsiveness to LPA in a chemotaxis

assay. Gene silencing of individual SK isoforms effic-

tively reduced their mRNA and protein content

(Fig. 3B) without affecting expression of the non-tar-

geted enzyme isoform (data not shown). Interestingly,

under both experimental conditions, the stimulation of

cell motility by LPA was significantly impaired

(Fig. 3C), indicating that both SK isoforms are impli-

cated in the LPA-induced migration of satellite cells.

Finally, it was determined whether S1P receptor

subtypes are also involved in the signaling axis that

transmits the biological effect exerted by LPA. As

illustrated in Fig. 4A, S1P receptor subtype-specific

siRNAs markedly decreased mRNA levels for individ-

ual receptor isoforms, without significantly altering the

relative expression of the non-targeted isoforms (data

not shown). Satellite cell migration in response to

LPA challenge was then measured under experimental

conditions in which individual S1P receptor subtypes

were specifically down-regulated. The chemotactic

0.00 0.05 0.100

1

2

3

1 2 3 4 5 6 7 8 9 10

μμM LPA

**

Cel

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(Fo

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A

B

Fig. 1. LPA exerts a pro-migratory effect

in activated satellite cells. (A) Cell

migration was measured by a Boyden

chamber assay in murine satellite cells

stimulated with LPA at the indicated

concentrations for 6 h. Values are

means � SEM of three independent

experiments performed in triplicate, and

are reported as the fold change over

control, set as 1. The effect of LPA was

found to be statistically significant by one-

way ANOVA followed by Bonferroni’s

post hoc test (*P < 0.05, **P < 0.01).

(B) A scratch wound healing assay was

performed in cells treated with 1 lM LPA

for 7 h. Left panel: representative

microscopic images at 0 and 7 h. Right

panel: quantitative analysis of cell

migration after 7 h. Values are

means � SEM of at least three

independent experiments performed in

triplicate, and are reported as the fold

change over control at 0 h, set as 1. The

effect of LPA was found to be statistically

significant by Student’s t test (*P < 0.05).


F. Cencetti et al. Role of the S1P axis in LPA-induced cell migration

action of LPA was significantly attenuated when S1P1

or S1P4 were silenced, suggesting that these two recep-

tors are the main transducers of the LPA biological

effect (Fig. 4B). In contrast, down-regulation of S1P2,

which is known to transmit an anti-migratory

response in several cell types, including satellite cells

[1,6,20], did not affect the action exerted by LPA.

Similarly, specific silencing of S1P3 did not influence

the migratory effect of LPA. In agreement with the

results obtained, the chemotactic effect exerted by

LPA was unaltered in satellite cells isolated from S1P3

knockout mice (Fig. 4C). A diagram illustrating the

signaling mechanisms downstream of LPA is provided

in Fig. 5.

Discussion

Directed cell motility of satellite cells is a prerequisite

for proper skeletal muscle repair as these precursor

cells are located at the peripheral region of the skeletal

muscle fibers and must migrate to the site of muscle

damage in order to be recruited for tissue repair.

LPA1 LPA2 LPA3

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Despite its crucial importance, the regulation of

satellite cell motility at present is not clearly character-

ized, and the potential physiological modulators of the

process have not been fully identified.

Here we report that LPA, a lysophospholipid with

powerful biological activity, markedly stimulates migra-

tion of satellite cells isolated from mouse tibialis ante-

rior muscle fibers and maintained in culture. The

described biological action of LPA may be physiologi-

cally relevant, as LPA is present in extracellular fluids

[21] and may become available to skeletal muscle pre-

cursor cells during tissue damage. In this regard, it is

interesting to note that most of the circulating LPA is

generated by the action of autotaxin, a secreted lyso-

phospholipase D that cleaves the choline headgroup

from lysophosphatidylcholine, an abundant plasma

phospholipid [22]. Although there are presently no

reports describing the production of autotaxin by skele-

tal muscle cells, this enzyme shows a broad tissue distri-

bution, and its transcript has been detected in human

skeletal muscle [23]. In addition, it is tempting to specu-

late that the pro-inflammatory environment that favors

skeletal muscle repair may enhance local LPA forma-

tion, in analogy with what has been demonstrated to

occur in lungs [24]. LPA appears to robustly enhance

the chemotaxis of satellite cells acting via LPA1 and

LPA3. This was demonstrated by blockade of the bio-

logical effect by the selective LPA1/LPA3 antagonist

Ki16425, as well as the abolition or reduction of LPA

efficacy following gene silencing of LPA1 or LPA3. The

finding that LPA1 and LPA3 transmit the biological

effect of LPA is in agreement with a recent study in

which these two receptor subtypes were implicated

in transmitting the increase in intracellular calcium in

C2C12 myoblasts [18].

Interestingly, it was found that down-regulation of

LPA2 expression by gene silencing was responsible for

an increased migratory response induced by LPA, sug-

gesting that LPA2 is coupled to anti-migratory signals.

In this respect, previous studies have described anti-

migratory action transmitted by distinct LPA receptors

[25,26], and LPA2 has already been implicated in the

inhibition of migration and invasion of pancreatic

cancer cells [27].

Another notable finding of this study is that the S1P

signaling axis is required for the biological response

elicited by LPA in these cells, consistent with the

notion that a complex cross-talk between LPA and S1P

occurs in these cells. In cultured myoblasts, the SK/S1P

signaling axis was previously implicated in transmission

of important biological effects evoked by growth

factors and cytokines, such as the pro-myogenic effect

of insulin growth factor-1 [15] and tumor necrosis

factor-a at low dose [12], the pro-fibrotic [13] and pro-

apoptotic [16] effects of transforming growth factor-b,and, more pertinent to this study, the pro-migratory

Fig. 2. Role of LPA receptors in the migratory action of LPA in activated satellite cells. (A) Expression of LPA receptors at the mRNA level.

Quantitative mRNA analysis was performed by real-time PCR by concurrent amplification of the target sequence of murine LPA1, LPA2 and

LPA3 genes together with that of 18S rRNA. The results are expressed as fold changes according to the 2�ΔΔCT method, using LPA1 as the

calibrator. Values are means � SEM of three independent experiments performed in triplicate. (B) Effect of LPA1 and LPA3 antagonists on

the migratory action of LPA. Cell migration was assessed using the Boyden chamber apparatus in cells pre-incubated in the presence or not

of 5 lM Ki16425 for 40 min, before being treated with 1 lM LPA, as described in Experimental procedures. Values are means � SEM of

three independent experiments, each performed in triplicate, and are reported as the fold change over the control, set as 1. The effect of

Ki16245 was found to be statistically significant by two-way ANOVA followed by Bonferroni’s post hoc test (*P < 0.05). (C) LPA receptor

down-regulation by gene silencing. Real-time PCR was performed in cells transfected with scrambled siRNA (SCR-siRNA) or with siRNAs

specific for murine LPA1, LPA2 and LPA3, by amplification of the target sequence of murine LPA receptors together with that of 18S rRNA.

The results are expressed as fold changes according to the 2�ΔΔCT method, utilizing as the calibrator each receptor subtype in SCR-siRNA-

transfected myoblasts. Values are means � SEM at least of three independent experiments performed in triplicate. The effect of siRNA

transfection on the mRNA levels of LPA receptors was found to be statistically significant by Student’s t test (*P < 0.05, **P < 0.01). (D)

Effect of down-regulation of LPA receptors on the migratory action of LPA. Cells treated with control SCR-siRNA or with specific siRNA for

individual LPA receptors were used to measure cell migration toward 1 lM LPA for 6 h in a Boyden chamber apparatus. Values are

means � SEM of at least three independent experiments performed in triplicate. The inhibitory effect of LPA1 and LPA3 down-regulation on

LPA-induced cell motility was found to be statistically significant by two-way ANOVA followed by Bonferroni’s post hoc test (#P < 0.05).

The positive effect of LPA2 down-regulation on LPA-induced cell migration was found to be statistically significant by two-way ANOVA

followed by Bonferroni’s post hoc test (**P < 0.01). (E) Effect of LPA1/LPA3 blockade on the migratory action of LPA in the presence of

serum. Cell migration was assessed using a Boyden chamber apparatus in cells pre-incubated with or without 5 lM Ki16425 for 40 min,

before being incubated with 10% fetal bovine serum (FCS) in the lower chamber, in the presence or absence of 1 lM LPA. Values are

means � SEM of three independent experiments, each performed in triplicate, and the results are reported as the fold change over

untreated cells, set as 1. The migration elicited by LPA was found to be statistically significant compared to serum alone by Student’s t test

(**P < 0.01). The reduction of serum-induced cell motility by Ki16425 was found to be statistically significant by Student’s t test

(§§P < 0.01). The inhibitory effect of LPA in the presence of Ki16245 was found to be statistically significant by Student’s t test (#P < 0.05).



effect of platelet-derived growth factor [14]. The pres-

ent results support the notion that transmission of bio-

logical responses evoked by extracellular cues also

relies on S1P inside-out signaling in satellite cells. Inter-

estingly, LPA, here identified as new agonist capable of

stimulating the migratory properties of satellite cells by

exploiting the S1P signaling axis, is an agonist that

shares many biological properties with S1P as well as

an analogous mechanism of action involving ligation to

a panel of G protein-coupled receptors [1,28]. Among

the multiple agents recognized as exerting their biologi-

cal action at least in part via regulation of the S1P sig-

naling pathway, LPA had already been reported to

stimulate migration of gastric cancer cells by means of

SK1 and S1P3 up-regulation through a mechanism that

involves LPA1 engagement and epidermal growth fac-

tor receptor transactivation [29]. In agreement, LPA1

was found to be implicated in this study in the regula-

tion of S1P signaling pathway; however, no significant

change was observed in the mRNA expression or pro-

tein content of SK1 after incubation with LPA within

the time frame adopted for Boyden chamber assays.

0.0

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Fig. 3. Involvement of sphingosine kinase in cell migration induced

by LPA. (A) Effect of SK inhibitors on the migratory action of LPA.

Cell migration across Matrigel-coated membranes was performed

by a Boyden chamber assay in satellite cells treated or not with

1 lM VPC96091 or 1 lM VPC96047, to measure migration toward

1 lM LPA, as described in Experimental procedures. Values are

means � SEM of three independent experiments, each performed

in triplicate, and the results are reported as the fold change over

control, set as 1. The effect of VPC96091 and VPC96047 in LPA-

treated satellite cells was found to be statistically significant by

two-way ANOVA followed by Bonferroni’s post hoc test

(*P < 0.05). (B) SK1 and SK2 down-regulation by gene silencing.

Satellite cells were transfected with non-targeting SCR-siRNA or

with siRNAs specific for SK1 or SK2 as described in Experimental

procedures. Upper panels: real-time PCR was performed by

amplification of the target sequence of murine SK1 or SK2

together with that of 18S rRNA. The results are expressed as fold

changes according to the 2�ΔΔCT method, utilizing as the calibrator

each SK isoform in SCR-siRNA transfected myoblasts. Values are

means � SEM at least of three independent experiments

performed in triplicate. The effect of siRNA transfection on SK1 or

SK2 mRNA levels was found to be statistically significant by

Student’s t test (*P < 0.05). Lower and middle panels: western

blot analysis was performed by using specific antibodies against

SK1 or SK2. The results of densitometric analysis are reported as

means � SEM of three independent experiments. The effect of

siRNA transfection on SK1 or SK2 protein levels was found to be

statistically significant by Student’s t test (*P < 0.05, **P < 0.01).

(C) Effect of SK1 or SK2 down-regulation on the migratory action

of LPA. Cells transfected with control SCR-siRNA or with specific

siRNAs for each SK isoform were used to measure cell migration

toward 1 lM LPA for 6 h in a Boyden chamber apparatus. Values

are means � SEM of at least three independent experiments

performed in triplicate. The inhibitory effect of SK1 and SK2 down-

regulation on LPA-induced cell motility was found to be statistically

significant by two-way ANOVA followed by Bonferroni’s post hoc

test (*P < 0.05; **P < 0.01).



Moreover, siRNA experiments involving SK isoforms

support a role not only for SK1 but also for SK2 in

the stimulatory effect of LPA. This enzyme isoform,

although reported to be involved in biological effects

distinct from those mediated by SK1 [30], in some cases

acts in parallel with SK1, transmitting inside the cell

the biological action exerted by extracellular cues.

Intriguingly, SK2 had already been shown to be

involved in agonist-stimulated cell migration [31], in

agreement with the present results. Another interesting

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Fig. 4. Role of S1P receptors in satellite cell migration induced by LPA. (A) Down-regulation of S1P receptors by gene silencing. Cells

transfected with control SCR-siRNA or with siRNAs specific for murine S1P1, S1P2, S1P3 and S1P4 were subjected to real-time PCR by

amplification of the target sequence of murine S1P receptors together with that of 18S rRNA. Results are expressed as fold changes

according to the 2�ΔΔCT method, utilizing as the calibrator each receptor subtype in SCR-siRNA-transfected myoblasts. Values are

means � SEM at least of three independent experiments performed in triplicate. The effect of siRNA transfection on S1P receptor mRNA

levels was found to be statistically significant by Student’s t test (*P < 0.05; **P < 0.01). (B) Effect of S1P receptor down-regulation on the

migratory action of LPA. Cells transfected with non-targeting SCR-siRNA or with specific siRNAs for individual murine S1P receptors were

used to measure cell migration toward 1 lM LPA for 6 h in a Boyden chamber apparatus. Values are means � SEM of at least three

independent experiments performed in triplicate. The inhibitory effect of S1P1 and S1P4 down-regulation on LPA-induced cell motility was

found to be statistically significant by two-way ANOVA followed by Bonferroni’s post hoc test (**P < 0.01). (C) Chemotactic response to

LPA in S1P3 knockout (KO) satellite cells. Satellite cells isolated from wild-type (wt) and S1P3 KO mice were used to measure cell migration

toward 1 lM LPA for 6 h in a Boyden chamber apparatus. Values are means � SEM of at least three independent experiments performed

in triplicate.



result of this study is the identification of S1P1 and

S1P4 as molecular transducers of the migratory

response exerted by LPA. These two S1P receptor sub-

types were previously identified as being involved in

stimulation of satellite cell migration by exogenous S1P

[6]. Thus, it appears that exogenous as well as endoge-

nous S1P is capable of driving satellite cell chemotaxis

through these two receptor subtypes. In contrast, S1P2

down-regulation by gene silencing did not affect the

migration induced by LPA, whereas it was previously

found to be responsible for the enhancement of satellite

cell motility stimulated by exogenous S1P [6]. This find-

ing supports the view that, in contrast to exogenous

S1P, which is freely available to all the S1P receptors

exposed at plasma membrane, endogenous S1P gener-

ated inside the cells following LPA challenge is avail-

able only at specific membrane domains where S1P2 is

absent, thus making the S1P inside-out signaling selec-

tive for certain receptor subtypes.

Overall, these results shed new light on the molecu-

lar mechanisms that underlie the regulation of directed

cell motility in satellite cells, identifying LPA as a new

player in this process and reinforcing the concept that

the S1P signaling axis is critically involved in the regu-

lation of key biological events in skeletal muscle pre-

cursor cells.

Experimental procedures

Materials

Biochemicals, TRI Reagent, cell culture reagents,

Dulbecco’s modified Eagle’s medium (DMEM), fetal

bovine serum, horse serum, protease inhibitor cocktail,

bovine serum albumin (BSA), collagenase and Ki16425

were obtained from Sigma-Aldrich (Woodlands, TX,

USA). siRNA duplexes corresponding to DNA target

sequences of mouse S1P1 (SASI_Mm02_00312691 and

SASI_Mm02_00312692), mouse S1P2 (SASI_Mm01_00082

880 and SASI_Mm01_00082881), mouse S1P3 (SASI_

Mm01_00145233 and SASI_Mm01_00145234), mouse S1P4

(SASI_Mm01_00094192 and SASI_Mm01_00094193),

mouse LPA1 (SASI_Mm02_00318340), mouse LPA2 (SASI

_Mm01_00165280), mouse LPA3 (SASI_Mm01_00022765),

mouse SK1 (SASI_Mm01_00033983 and SASI_Mm01_

00033984), mouse SK2 (SASI_Mm01_00050883 and SA

SI_Mm01_00050884) and scrambled siRNA (Mission

Universal Negative control #1) were obtained from Sigma-

Proligo (Woodlands, TX, USA). Lipofectamine RNAi-

MAXTM, all reagents required to perform real-time PCR,

and the TaqMan gene expression assay probes for LPA1

(Mm00439145_m1), LPA2 (Mm00469562_m1), LPA3 (Mm0

0469562_m1), S1P1 (Mm02619656_s1), S1P3 (Mm026

20181_s1), S1P4 (Mm00468695_s1), SK1 (Mm00448841_g1)

and SK2 (Mm00445021_m1) were purchased from Life

Technologies (Carlsbad, CA, USA). The gene expression

assay probe for mouse S1P2 (Mm.PT.47.12362698) was

purchased from Integrated DNA Technologies Inc. (Coral-

ville, IA, USA). The enhanced chemiluminescence reagent

was purchased from GE Healthcare Europe (Milan, Italy).

Coomassie Brilliant Blue reagent was obtained from

Bio-Rad (Hercules, CA, USA). Chick embryo extract was

obtained from Sera Laboratories International Ltd

(Haywards Heath, UK). Collagen type I was purchased

from Millipore (Billerica, MA, USA). Matrigel basement

membrane matrix was obtained from BD Biosciences (Bed-

ford, MA, USA). The LPA used in the study was 1-oleoyl

lysophosphatidic acid sodium salt, purchased from Cayman

Chemical (Ann Arbor, MI, USA). Restore western blot

stripping buffer was purchased from ThermoFisher

Scientific (Rockford, IL, USA). Polycarbonate filters (8 lm

Fig. 5. Diagram of the signaling

mechanisms downstream of LPA.



pores) and modified Boyden chambers were obtained from

Neuroprobe (Gaithersburg, MD, USA). Diff-Quick stain-

ing solution was purchased from Dade-Behring (Lieder-

bach, Germany). Pharmacological inhibitors of SK

(VPC96091 and VPC96047) were kindly provided by K.

Lynch and T.L. MacDonald (Department of Pharmacol-

ogy, University of Virginia, Charlottesville, VA, USA).

SK2 (N-terminal region) rabbit polyclonal and SK1 (cen-

tral region) rabbit polyclonal antibodies were purchased

from ECM Biosciences LLC (Versailles, KY, USA). Sec-

ondary antibodies conjugated to horseradish peroxidase

were obtained from Santa Cruz Biotechnology, Inc. (Santa

Cruz, CA, USA).

Animals

Male C57BL/6 mice were obtained from Charles River

Laboratories (Calco, Italy). S1P3 knockout mice were

kindly provided by B. Levkau (Institute of Pathophysiol-

ogy, University of Essen, Germany), with the consent of

Prof. J. Chun, Department of Molecular and Cellular Neu-

roscience, The Scripps Research Institute, La Jolla, CA,

USA. Animals were housed in a specific pathogen-free

facility at a temperature range of 21–24 °C with a light/

dark cycle of 12 h/12 h. Food and water were constantly

available.

Mice (2–7 months old) were killed by rapid cervical dis-

location, following the procedure approved by the Ethical

Committee for Animal Experiments of the University of

Florence and EEC Guidelines for Animal Care (Directive

86/609/EEC). All efforts were made to minimize the num-

ber of the animals used in the study and reduce their suf-

fering.

Primary satellite cell isolation and culture

Individual muscle fibers containing satellite cells were iso-

lated from the tibialis anterior muscle as described previ-

ously [6,32,33]. The muscles were remove from the

hindlimb by microdissection, handling them by their ten-

dons to diminish damage to the fibers, and incubated in

0.2% w/v collagenase type I/DMEM for 1–2 h at 37 °C.Following enzymatic digestion, single myofibers were

detached by repetitively triturating the muscle with a wide-

mouth Pasteur pipette. The digested muscle was subse-

quently pre-plated into 100 mm Petri dishes for 1 h at

37 °C to remove fibroblasts, which adhere more rapidly to

the dish than satellite cells do [34]. The resulting suspension

was then plated into 24-well plates pre-coated with

1 mg�mL�1 Matrigel in a medium comprising DMEM sup-

plemented with 10% horse serum, 0.5% chick embryo

extract, 100 U�mL�1 penicillin, 100 lg�mL�1 streptomycin,

100 lg�mL�1 kanamycin, 2.5 lg�mL�1 amphotericin B and

2 mM L-glutamine (plating medium) at 37 °C in 5% CO2.

Satellite cells were released from the fibers ~ 12–24 h after

plating.

After 3–4 days, the plating medium was replaced with a

medium comprising DMEM supplemented with 20% fetal

bovine serum, 10% horse serum, 1% chick embryo extract,

100 U�mL�1 penicillin, 100 lg�mL�1 streptomycin,

100 lg�mL�1 kanamycin, 2.5 lg�mL�1 amphotericin B and

2 mM L-glutamine), which favors cell proliferation. Satellite

cells were periodically checked for expression of Pax7, a

paired-box transcription factor that is expressed by resting

satellite cells and is involved in the myogenic program [35].

Cell cultures at passage number 1-4 were used.

Cell transfection

Transfection of siRNA duplexes was performed in cells

grown in p60 Petri dishes (45 000 cells per dish), using

Lipofectamine RNAiMAX, as described previously [13].

Lipofectamine RNAiMAX was incubated with siRNA in

DMEM without serum and antibiotics at room tempera-

ture for 20 min, then the lipid/RNA complexes were com-

bined with cells with gentle agitation to a final

concentration of 85 nM in DMEM containing fetal bovine

serum. After 24 h, cells were incubated with DMEM with-

out serum but containing 1 mg�mL�1 BSA, and then used

for experiments within 72 h from the start of transfection.

The efficacy of transfection in down-regulating the expres-

sion of specific molecular targets was evaluated by real-time

RT-PCR and occasionally confirmed by western blot analy-

sis.

Western blot analysis

Activated satellite cells were lysed for 30 min at 4 °C in a

buffer containing 50 mM Tris pH 7.5, 120 mM NaCl, 1 mM

EDTA, 6 mM EGTA, 15 mM Na4P2O7, 20 mM NaF, 1%

Nonidet P-40 and protease inhibitor cocktail (1.04 mM 4-

(2-aminoethyl) benzenesulfonyl fluoride hydrochloride F,

0.08 lM aprotinin, 0.02 mM leupeptin, 0.04 mM bestatin,

15 lM pepstatin A and 14 lM N-[N-(L-3-trans-carboxyir-

ane-2-carbonyl)-L-leucyl]-agmatine; [1-[N-[(L-3-trans-carb

oxyoxirane-2-carbonyl)-L-leucyl]amino]-4-guanidinobutane]).

Cell lysates were prepared by centrifugation of cell extracts

for 15 min at 10 000 g at 4 °C. Total proteins (20 lg) fromlysates were resuspended in Laemmli’s SDS sample buffer.

Samples were subjected to SDS/PAGE for 90 min at

120 mA, before transfer of proteins to poly(vinylidene di-

fluoride) membranes. Then the membranes were washed

(10 min at room temperature in Tris-buffered saline with

0.1% Tween 20 for three times), probed overnight with the

primary antibodies at 4 °C, and subsequently with specific

secondary antibodies for 1 h at room temperature. The

presence of bound antibodies was revealed by chemilumi-

nescence.



Real-time PCR

Total RNA (1 lg), extracted from satellite cells using TRI

Reagent, was reverse-transcribed using a high-capacity

cDNA reverse transcription kit (Applied Biosystems, Foster

City, CA, USA), according to the manufacturer’s instruc-

tions. Quantification of the mRNA level of LPA receptors,

S1P receptors and SK1/SK2 was performed by real-time

PCR using TaqMan gene expression assays with the auto-

mated ABI Prism 7700 sequence detector system (Applied

Biosystems, Foster City, CA, USA). Measurements was

performed in triplicate, as previously described [36], by con-

current amplification of the target sequence together with

that of 18S rRNA, which was chosen as a housekeeping

gene. Relative quantification of mRNA expression was per-

formed using the 2�DDCT method, and data were normal-

ized to 18S rRNA expression [37].

Cell migration

Cell migration was measured using a modified Boyden

chamber apparatus as previously described [20]. Polycar-

bonate filters with 8 lm pores were pre-coated with

Matrigel (250 lg�mL�1) for 60 min at 37 °C. Cells were

detached using 0.05% trypsin containing 0.02% EDTA,

and resuspended in DMEM containing 250 lg�mL�1 heat-

inactivated BSA. An aliquot of the cell suspension

(1.5 9 104) was loaded into the upper chamber. LPA was

dissolved in 2 mL ethanol/water (1 : 1 v/v), and divided

in aliquots into sterile Pyrex tubes, dried under nitrogen

and stored at �20 °C. When necessary, samples were

resuspended by pipetting to a concentration of 1 mM in

sterile NaCl/Pi before sonication (three times for 10 s in a

bath sonicator) and vortexing. Further dilutions were per-

formed in the medium used for the cell migration assay

(DMEM containing 250 lg�mL�1 heat-inactivated BSA).

LPA at the indicated concentrations was added to the

lower chamber, and samples were kept in an incubator at

37 °C with a 5% CO2 atmosphere for 6 h. Polycarbonate

filters were fixed overnight using methanol at 4 °C and

stained using Diff-Quick staining solution. Migration was

evaluated by counting the number of migrated cells in six

random fields per filter. When indicated, satellite cells

were pre-treated with 5 lM Ki16425 for 40 min before

being trypsinized. Treatment with SK inhibitors was

accomplished incubating 1.5 9 104 cells in the presence of

1 lM VPC96047 or 1 lM VPC96091 in the upper well of

a Boyden chamber.

The scratch wound healing assay was performed as

described previously [6]. Satellite cells were seeded in 12-

well tissue culture plates and incubated at 37 °C with 5%

CO2 for 24 h until confluence. Cells were then serum-

starved overnight, and wounded by scratching with a

200 lL standard sterile pipette tip. The cell monolayer was

washed twice with 5 ml DMEM containing 0.1 % BSA at

room temperature for 2 min in order to remove cell debris,

and incubated with or without 1 lM LPA. At least six

scratched areas for each sample were photographed using a

Nikon (Amsterdam, The Netherlands) digital camera con-

nected to a Nikon phase-contrast extra long working

distance 0.3. Migration of satellite cells was quantified by

counting the number of cells that had migrated into the

scratched area after 7 h compared to the number present in

the initial wound.

Statistical analysis

Densitometric analysis of the western blot bands was per-

formed using IMAGEJ software (http://imagej.nih.gov/ij/) and

QUANTITY ONE analysis software (Bio-Rad). Graphical repre-

sentations were created using GRAPHPAD PRISM 5.0 (Graph-

Pad Software, San Diego, CA, USA). Statistical analysis

was performed using Student’s t test, one-way ANOVA

and two-way ANOVA followed by Bonferroni’s post hoc

test. Asterisks and other symbols indicate statistical signifi-

cance at the levels indicated in the legends.

Acknowledgements

This research was supported by grants from the

University of Florence to C.D., and from the Fond-

azione Cassa di Risparmio di Lucca (BRUNICRL12)

to P.B. The authors are indebted to K.L. Lynch and

T.L. MacDonald (University of Virginia, Charlottes-

ville, VA), who kindly provided VPC96047 and

VPC96091, and to J. Chun (Scripps Research Insti-

tute, La Jolla, CA) and B. Levkau (Institute of Path-

ophysiology, University of Essen, Germany), for

generating and providing S1P3 knockout mice,

respectively.

Author contributions

F.C., C.D. and P.B. planned the experiments; F.C.,

G.B., C.B. and S.B. performed the experiments; F.C.,

G.B. and S.B. analyzed the data; P.B. and C.D. wrote

the paper.

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Lysophosphatidic acid stimulates cell migration of satellite cells. A role for the sphingosine kinase/sphingosine 1-phosphate axis