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Sphingosine 1-phosphate regulates myogenic differentiation: a major role for S1P₂ receptor

Chiara Donati^*,, Elisabetta Meacci^*,, Francesca Nuti^*, Laura Becciolini^*, Marta Farnararo^* and Paola Bruni^*,,1

^* Dipartimento di Scienze Biochimiche and
Istituto Interuniversitario di Miologia (IIM), Università degli Studi di Firenze, Florence, Italy

¹Correspondence: Dipartimento di Scienze Biochimiche, Viale G.B. Morgagni 50, Florence 50134, Italy. E-mail: paola.bruni{at}unifi.it

SPECIFIC AIMS

The present study investigated whether the bioactive lipid sphingosine 1-phosphate (S1P) can influence myogenic differentiation, the receptor subtype(s) responsible for the biological response to the bioactive sphingolipid and the molecular mechanisms involved.

PRINCIPAL FINDINGS

1. S1P inhibits serum-induced proliferation and promotes cell cycle exit and myogenic differentiation of C2C12 cells
The effect of S1P on myoblast proliferation was evaluated by labeled thymidine incorporation and MTS dye reduction assay in control and serum-stimulated myoblasts. S1P (1 µM) did not affect basal cell proliferation but reduced the DNA synthesis promoted by 10% fetal calf serum (FCS) evaluated by [³H]thymidine incorporation (Fig. 1 A) and MTS dye reduction assay experiments (Fig. 1B ). The antiproliferative effect of S1P was confirmed by S1P-induced down-regulation of cyclin D1, a mitogen sensor, and by up-regulation of the cyclin-dependent kinase inhibitor p21/Waf1 (Fig. 1C ).

View larger version (36K): [in this window] [in a new window]   Figure 1. S1P inhibits serum-induced proliferation and promotes cell cycle exit and myogenic differentiation of C2C12 myoblasts. A) Incorporation of [3H]thymidine. Myoblasts, (40% confluent), were serum-starved for 6 h and stimulated or not with 10% FCS in the absence (black bar) or in the presence (gray bar) of 1 µM S1P for 16 h. Thirty min after agonist challenge, [3H]thymidine (2 µCi/well) was added to the cells. Data are means ± SE of 4 independent experiments performed in triplicate. *P < 0.05; Student’s t test. B) MTS dye reduction assay. Myoblasts were stimulated or not with 10% FCS in the absence (black bar) or in the presence (gray bar) of 1 µM S1P for 16 h. Values are means ± SE of 3 independent experiments performed in quadruplicate. When not shown, error bars lie within the column. *P < 0.05; Student’s t test. C) Western blot analysis of cyclin D1 and p21/Waf1. Myoblasts were incubated in DMEM containing 1 mg/mL BSA with (+) or without (–) 1 µM S1P. Cell extracts (30 µg) were subjected to immunoblot analysis. Blot representative of 3 independent experiments is shown. Band intensity is reported as % relative to the intensity of the band corresponding to control (1 h, no addition) set as 100. D) Western blot analysis of myogenic marker expression. The content of myogenin, MHC, and caveolin-3 was analyzed by Western blot analysis in lysates of myoblasts treated as above. Equally loaded protein was checked by expression of the nonmuscle specific ß isoform of actin. A blot representative of at least 3 is shown.

Expression of various proteins known to be induced during differentiation was evaluated in confluent, serum-starved C2C12 cells. Expression of the early marker myogenin peaked at 48 h, declining thereafter in control and in agonist-stimulated C2C12 cells. Treatment with 1 µM S1P resulted in markedly enhanced content of the protein starting at 48 h (Fig. 1D ). S1P also potentiated the appearance of myosin heavy chain (MHC) and the progressive increase in amount of protein with respect to control cells. Levels of caveolin-3, whose expression was delayed compared with the other markers, were strikingly augmented by cell challenge with S1P.

2. Role of S1PRs in the antiproliferative and promyogenic effect of S1P
Since the biological action of S1P is largely ascribed to ligation to specific S1P receptors (S1PRs) and Northern blot analysis confirmed that S1P₁, S1P₂, and S1P₃ were expressed in C2C12 myoblasts (Fig. 2 A, inset), their involvement in the S1P-induced inhibition of cell proliferation was examined by measuring [³H]thymidine incorporation in cells loaded with specific antisense oligodeoxyribonucleotides (ODN). Cell treatment with antisense ODN to S1P₁, or S1P₃ did not impair the reduced [³H]thymidine incorporation induced by S1P in cells stimulated with serum (Fig. 2A ), whereas the antiproliferative effect of S1P was fully prevented by antisense ODN to S1P₂, which efficaciously reduced the receptor expression (Fig. 2D ).

View larger version (67K): [in this window] [in a new window]   Figure 2. Role of S1PRs in the antiproliferative and promyogenic action of S1P. A) Effect of down-regulation of S1PRs by antisense ODN treatment on antiproliferative action of S1P. Myoblasts were transfected with scrambled or S1P1, S1P2, S1P3 antisense (AS) ODN and used as described in legend of Fig. 1A . *P < 0.05; Student’s t test. Inset: Northern blot analysis of S1PR expression in C2C12 myoblasts. B) Effect of S1PR overexpression on S1P-induced myogenic marker expression in C2C12 cells. Myoblasts transiently transfected with individual S1PRs or vector alone were analyzed for myogeninic markers after 48 h (see legend, Fig. 1D . Band intensity is reported as % relative to the intensity of the band corresponding to the respective unchallenged control set as 100. C) Effect of down-regulation of S1PRs by AS ODN treatment on S1P-induced myogenic marker expression in C2C12 cells. Myoblasts were transfected as described in Fig. 2A and the experiment performed as described for Fig. 1D . D) AS ODN to S1P2 down-regulates S1P2 in C2C12 cells. Membrane fractions (25 µg) obtained from S1P2-overexpressing myoblasts, loaded with scrambled or S1P2 AS ODN, were subjected to Western blot analysis. Overexpression of S1P2 in C2C12 myoblasts. Membrane fraction (25 µg) obtained from vector- or S1P2-transfected myoblasts were subjected to Western blot analysis. siRNA down-regulates expression of S1P2. Membrane fractions (30 µg) from myoblasts transfected with nonspecific or S1P2-siRNA were subjected to Western blot analysis. E) Effect of S1P2 silencing on S1P myogenic activity. Myoblasts were transfected with nonspecific or S1P2-siRNA and experiments were performed as described in Fig. 1D . F) Effect of S1P2 overexpression on C2C12 myoblast differentiation. Morphological appearance of myoblasts stably transfected with pcDNA3-S1P2 or vector alone incubated with DMEM containing 2% horse serum.

Next, the involvement of S1PRs in the promyogenic action exerted by S1P was investigated in C2C12 myoblasts where individual receptor expression was manipulated. While S1P₁ overexpression was inefficacious, cell transfection with pcDNA3-S1P₂, which significantly increased the receptor content (Fig. 2D ), provoked a significantly higher expression of myogenin and caveolin-3 than mock-transfected cells at 48 h (Fig. 2B ). Addition of 1 µM S1P to S1P₂-overexpressing myoblasts provoked only slight increases of the marker levels. Conversely, the overexpression of S1P₃ attenuated the induction of myogenin and caveolin-3 in control and S1P-treated cells. Employment of antisense ODN to reduce S1P₂ expression blunted the increase of MHC and caveolin-3 content elicited by 1 µM S1P at 48 h of incubation and reduced their basal expression (Fig. 2C ). Lessened S1P₃ levels resulted in appreciable stimulation of the expression of protein markers in cells challenged or not with S1P, whereas antisense ODNs to S1P₁ were inefficacious. The role of S1P₂ was further investigated by short interfering RNA (siRNA) technology. Treatment with S1P₂-siRNA, which down-regulated the receptor (Fig. 2D ), nearly abolished myogenin expression at 24 h and strongly reduced the content of caveolin-3, at 96 h (Fig. 2E ). S1P addition to S1P₂-silenced myoblasts did not affect myogenin content and only slightly increased caveolin-3 expression. Stable overexpression of S1P₂ provoked the onset of a more mature phenotype in myoblasts, which were switched to medium containing 2% horse serum, hastening the formation of multinucleated myotubes (Fig. 2F ).

3. Signaling pathways involved in the biological response to S1P
We next examined the ability of S1P to activate ERK1/ERK2, p38 MAPK, and Akt signaling pathways and their involvement in the biological response to S1P. The sphingolipid was found to induce a rapid and transient phosphorylation of these kinases. Cell treatment with PD98059 (10 µM), or U0126 (10 µM), known inhibitors of ERK1/ERK2 pathway, abolished the inhibitory effect of 1 µM S1P on serum-induced proliferation in thymidine incorporation and MTS dye reduction assay experiments, whereas LY294002 (10 µM) and SB203580 (5 µM) were ineffective.

The prodifferentiating effect of 1 µM S1P was not affected by administration with 10 µM PD98059 or 10 µM LY294002 but was abrogated when p38 MAPK was selectively inhibited by 5 µM SB203580, 2.5 µM SB239063, or overexpression of the kinase-dead, dominant negative p38 mutant.

ERK1/ERK2 and p38 MAPK were found to be coupled to S1P₂, given that the down-regulation of S1P₂ by antisense ODN showed that S1P-induced p38 MAPK activation was strongly reduced and ERK1/ERK2 phosphorylation was attenuated.

CONCLUSIONS AND SIGNIFICANCE

Here the first experimental evidence for a pivotal role of S1P in the regulation of C2C12 myogenic differentiation is presented. It is likely that S1P action is important for muscle cell biology, since it regulates biological processes critical for the control of muscle regeneration, such as proliferation and differentiation.

The principal findings of this study are 1) S1P attenuated serum-induced cell proliferation and promoted cell cycle exit; 2) S1P was an efficacious inducer of myogenic differentiation, up-regulating the expression of various differentiation markers; 3) S1P₂ was identified as the critical receptor for the observed biological responses to S1P; and 4) activation of ERK1/ERK2 and p38 MAPK, both identified as downstream effectors of S1P₂, was required, respectively, for S1P-induced inhibition of cell proliferation and stimulation of myogenic differentiation.

Although p38 MAPK is clearly implicated in myoblast differentiation, here the first evidence is presented for the involvement of this pathway in the prodifferentiating effect of an extracellular cue.

Change in expression of the myogenic markers induced by S1P₂ manipulation in unchallenged cells and the phenotypic appearance of S1P₂-overexpressing myoblasts indicate that S1P₂ stimulates the myogenic program independent of acute S1P treatment. These results support the hypothesis that endogenously produced S1P can act in an autocrine or paracrine fashion.

Experimental evidence has been presented here for a novel biological activity of S1P that can act as morphogenetic factor in skeletal muscle cells, inhibiting cell proliferation and inducing myogenic differentiation by ligation to S1P₂ (Fig. 3 ).

View larger version (13K): [in this window] [in a new window]   Figure 3. Schematic diagram of the promyogenic action of S1P in C2C12 myoblasts.

These findings improve our understanding of the molecular mechanisms implicated in the control of muscle satellite cell functionality. S1P is physiologically released by activated platelets and can easily reach the injured muscle by blood vessels. It is tempting to speculate that its local concentration may be increased by the action of inflammatory cytokines liberated at the lesion site. Further investigation is needed to determine whether S1P contributes to muscle repair in order to individuate novel targets to ameliorate muscle regeneration impaired in degenerative diseases, such as muscular dystrophy.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1780fje;

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This Article Full Text (PDF) All Versions of this Article: 19/3/449 04-1780fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Donati, C. Articles by Bruni, P. Search for Related Content PubMed PubMed Citation Articles by Donati, C. Articles by Bruni, P.