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Sphingosine-1-phosphate enhances satellite cell activation in dystrophic muscles through a S1PR2/STAT3 signaling pathway.

Loh KC, Leong WI, Carlson ME, Oskouian B, Kumar A, Fyrst H, Zhang M, Proia RL, Hoffman EP, Saba JD - PLoS ONE (2012)

Bottom Line: These changes include early and profound induction of the gene encoding the S1P biosynthetic enzyme SphK1, followed by induction of the catabolic enzyme sphingosine phosphate lyase (SPL) 3 days later.STAT3 activation resulted in p21 and p27 downregulation in a S1PR2-dependent fashion in myoblasts.Our findings suggest that S1P promotes SC progression through the cell cycle by repression of cell cycle inhibitors via S1PR2/STAT3-dependent signaling and that SPL inhibition may provide a therapeutic strategy for MD.

View Article: PubMed Central - PubMed

Affiliation: Children's Hospital Oakland Research Institute, Oakland, California, United States of America.

ABSTRACT
Sphingosine-1-phosphate (S1P) activates a widely expressed family of G protein-coupled receptors, serves as a muscle trophic factor and activates muscle stem cells called satellite cells (SCs) through unknown mechanisms. Here we show that muscle injury induces dynamic changes in S1P signaling and metabolism in vivo. These changes include early and profound induction of the gene encoding the S1P biosynthetic enzyme SphK1, followed by induction of the catabolic enzyme sphingosine phosphate lyase (SPL) 3 days later. These changes correlate with a transient increase in circulating S1P levels after muscle injury. We show a specific requirement for SphK1 to support efficient muscle regeneration and SC proliferation and differentiation. Mdx mice, which serve as a model for muscular dystrophy (MD), were found to be S1P-deficient and exhibited muscle SPL upregulation, suggesting that S1P catabolism is enhanced in dystrophic muscle. Pharmacological SPL inhibition increased muscle S1P levels, improved mdx muscle regeneration and enhanced SC proliferation via S1P receptor 2 (S1PR2)-dependent inhibition of Rac1, thereby activating Signal Transducer and Activator of Transcription 3 (STAT3), a central player in inflammatory signaling. STAT3 activation resulted in p21 and p27 downregulation in a S1PR2-dependent fashion in myoblasts. Our findings suggest that S1P promotes SC progression through the cell cycle by repression of cell cycle inhibitors via S1PR2/STAT3-dependent signaling and that SPL inhibition may provide a therapeutic strategy for MD.

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STAT3 and S1PR2 signaling interactions in muscle regeneration of WT and mdx mice.A) Mdx mice received NTX with or without THI treatment. Muscles were harvested on day 5 post-injury, and whole muscle extracts were examined for total STAT3 (STAT3-T) and phosphorylated STAT3 (STAT3-P) protein levels. Actin is used as a loading control. B) STAT3 activation in SC-derived myoblasts treated in vitro with 10 µM S1PR1 (R1i), S1PR2 (R2i), and S1PR3 (R3i) antagonists. S1PR1 is used as a loading control, as its expression is invariant under these conditions. C) WT SC-derived primary myoblasts were treated with S1PR2 antagonist (JTE-013) or vehicle, and whole cell extracts were examined for STAT3 phosphorylation, total STAT3 and p21 and p27 protein levels. Actin is used as a loading control. D) SC-derived primary myoblasts were treated with either 50 nM siRNA against S1PR2 or mock transfection. S1PR2 knockdown was confirmed by qRT-PCR. E) Whole cell extracts of control and S1PR2 knockdown cells were examined for phosphorylated STAT3 and actin protein levels by immunoblotting. p = 0.03 (siRNA vs. mock) for relative densitometric levels of STAT3-P/actin from two experiments performed in duplicates. F) WT mice were treated with NTX plus antagonist of S1PR2 (JTE-013) or vehicle by subcutaneous injection. Muscles were harvested 5 days post injury; regenerating fibers were imaged, and G) regenerating fibers were quantified. H) Flow cytometry analysis of SCs from WT mice treated with JTE-013 or vehicle at 5 days post injury; * indicates p≤0.05, data are means ± SD, n = 5/group.
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pone-0037218-g006: STAT3 and S1PR2 signaling interactions in muscle regeneration of WT and mdx mice.A) Mdx mice received NTX with or without THI treatment. Muscles were harvested on day 5 post-injury, and whole muscle extracts were examined for total STAT3 (STAT3-T) and phosphorylated STAT3 (STAT3-P) protein levels. Actin is used as a loading control. B) STAT3 activation in SC-derived myoblasts treated in vitro with 10 µM S1PR1 (R1i), S1PR2 (R2i), and S1PR3 (R3i) antagonists. S1PR1 is used as a loading control, as its expression is invariant under these conditions. C) WT SC-derived primary myoblasts were treated with S1PR2 antagonist (JTE-013) or vehicle, and whole cell extracts were examined for STAT3 phosphorylation, total STAT3 and p21 and p27 protein levels. Actin is used as a loading control. D) SC-derived primary myoblasts were treated with either 50 nM siRNA against S1PR2 or mock transfection. S1PR2 knockdown was confirmed by qRT-PCR. E) Whole cell extracts of control and S1PR2 knockdown cells were examined for phosphorylated STAT3 and actin protein levels by immunoblotting. p = 0.03 (siRNA vs. mock) for relative densitometric levels of STAT3-P/actin from two experiments performed in duplicates. F) WT mice were treated with NTX plus antagonist of S1PR2 (JTE-013) or vehicle by subcutaneous injection. Muscles were harvested 5 days post injury; regenerating fibers were imaged, and G) regenerating fibers were quantified. H) Flow cytometry analysis of SCs from WT mice treated with JTE-013 or vehicle at 5 days post injury; * indicates p≤0.05, data are means ± SD, n = 5/group.

Mentions: S1P signaling has been implicated in SC activation [20], [26], [40]. Importantly, however, the molecular mechanism responsible for the contribution of S1P signaling to SC activation has not been determined. The STAT3 transcription factor promotes cell proliferation, survival and differentiation, has been implicated in mediating SC functions, and has been linked to S1P receptor signaling in cancer [28], [30], [41]–[46]. Therefore, we investigated the potential impact of THI treatment on STAT3 status in mdx muscles. STAT3 activation as shown by its phosphorylation on residue T705 was higher in injured muscles of THI-treated mdx mice compared to vehicle-treated mdx controls on day 5 post-injury (Figure 6A). To determine the S1PR requirements for STAT3 activation, we evaluated STAT3 phosphorylation in the presence and absence of S1PR antagonists in vitro. STAT3 phosphorylation was significantly reduced in SC-derived primary myoblasts treated with the S1PR2 antagonist JTE-013, whereas the S1PR1 antagonist W123 and the S1PR3 antagonist BML-241 had no impact on STAT3 phosphorylation (Figure 6B). STAT3 has been reported to negatively regulate cell cycle inhibitor proteins p21 and p27, thereby inducing cell cycle progression [41]. Importantly, SC-derived myoblasts treated with the S1PR2 antagonist JTE-013 showed a reduction in STAT3 phosphorylation, concomitant with an increase in p21 and p27 levels (Figure 6C). To further confirm these findings, we employed siRNA to knockdown the expression of S1PR2 and then evaluated the impact on STAT3 activation. As shown in Figure 6D, SC-derived myoblasts transfected with S1PR2 siRNA exhibited a 75% reduction in S1PR2 mRNA expression. S1PR2 knockdown resulted in a marked reduction in STAT3 phosphorylation, similar to the results obtained with JTE-013 (Figure 6E). Given these findings, we analyzed the effects of JTE-013 in vivo 5 days after NTX injury. We found that there was a significant decrease in centrally nucleated myofibers/mm2 after treatment with JTE-013 (Figures 6F and G). Further, JTE-013 treatment was associated with a significant decrease in the number of SCs when evaluated by FACS analysis (Figure 6H). These findings demonstrate that S1PR2 activation and downstream activation of STAT3 are essential for efficient muscle regeneration and SC proliferation.


Sphingosine-1-phosphate enhances satellite cell activation in dystrophic muscles through a S1PR2/STAT3 signaling pathway.

Loh KC, Leong WI, Carlson ME, Oskouian B, Kumar A, Fyrst H, Zhang M, Proia RL, Hoffman EP, Saba JD - PLoS ONE (2012)

STAT3 and S1PR2 signaling interactions in muscle regeneration of WT and mdx mice.A) Mdx mice received NTX with or without THI treatment. Muscles were harvested on day 5 post-injury, and whole muscle extracts were examined for total STAT3 (STAT3-T) and phosphorylated STAT3 (STAT3-P) protein levels. Actin is used as a loading control. B) STAT3 activation in SC-derived myoblasts treated in vitro with 10 µM S1PR1 (R1i), S1PR2 (R2i), and S1PR3 (R3i) antagonists. S1PR1 is used as a loading control, as its expression is invariant under these conditions. C) WT SC-derived primary myoblasts were treated with S1PR2 antagonist (JTE-013) or vehicle, and whole cell extracts were examined for STAT3 phosphorylation, total STAT3 and p21 and p27 protein levels. Actin is used as a loading control. D) SC-derived primary myoblasts were treated with either 50 nM siRNA against S1PR2 or mock transfection. S1PR2 knockdown was confirmed by qRT-PCR. E) Whole cell extracts of control and S1PR2 knockdown cells were examined for phosphorylated STAT3 and actin protein levels by immunoblotting. p = 0.03 (siRNA vs. mock) for relative densitometric levels of STAT3-P/actin from two experiments performed in duplicates. F) WT mice were treated with NTX plus antagonist of S1PR2 (JTE-013) or vehicle by subcutaneous injection. Muscles were harvested 5 days post injury; regenerating fibers were imaged, and G) regenerating fibers were quantified. H) Flow cytometry analysis of SCs from WT mice treated with JTE-013 or vehicle at 5 days post injury; * indicates p≤0.05, data are means ± SD, n = 5/group.
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Related In: Results  -  Collection

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pone-0037218-g006: STAT3 and S1PR2 signaling interactions in muscle regeneration of WT and mdx mice.A) Mdx mice received NTX with or without THI treatment. Muscles were harvested on day 5 post-injury, and whole muscle extracts were examined for total STAT3 (STAT3-T) and phosphorylated STAT3 (STAT3-P) protein levels. Actin is used as a loading control. B) STAT3 activation in SC-derived myoblasts treated in vitro with 10 µM S1PR1 (R1i), S1PR2 (R2i), and S1PR3 (R3i) antagonists. S1PR1 is used as a loading control, as its expression is invariant under these conditions. C) WT SC-derived primary myoblasts were treated with S1PR2 antagonist (JTE-013) or vehicle, and whole cell extracts were examined for STAT3 phosphorylation, total STAT3 and p21 and p27 protein levels. Actin is used as a loading control. D) SC-derived primary myoblasts were treated with either 50 nM siRNA against S1PR2 or mock transfection. S1PR2 knockdown was confirmed by qRT-PCR. E) Whole cell extracts of control and S1PR2 knockdown cells were examined for phosphorylated STAT3 and actin protein levels by immunoblotting. p = 0.03 (siRNA vs. mock) for relative densitometric levels of STAT3-P/actin from two experiments performed in duplicates. F) WT mice were treated with NTX plus antagonist of S1PR2 (JTE-013) or vehicle by subcutaneous injection. Muscles were harvested 5 days post injury; regenerating fibers were imaged, and G) regenerating fibers were quantified. H) Flow cytometry analysis of SCs from WT mice treated with JTE-013 or vehicle at 5 days post injury; * indicates p≤0.05, data are means ± SD, n = 5/group.
Mentions: S1P signaling has been implicated in SC activation [20], [26], [40]. Importantly, however, the molecular mechanism responsible for the contribution of S1P signaling to SC activation has not been determined. The STAT3 transcription factor promotes cell proliferation, survival and differentiation, has been implicated in mediating SC functions, and has been linked to S1P receptor signaling in cancer [28], [30], [41]–[46]. Therefore, we investigated the potential impact of THI treatment on STAT3 status in mdx muscles. STAT3 activation as shown by its phosphorylation on residue T705 was higher in injured muscles of THI-treated mdx mice compared to vehicle-treated mdx controls on day 5 post-injury (Figure 6A). To determine the S1PR requirements for STAT3 activation, we evaluated STAT3 phosphorylation in the presence and absence of S1PR antagonists in vitro. STAT3 phosphorylation was significantly reduced in SC-derived primary myoblasts treated with the S1PR2 antagonist JTE-013, whereas the S1PR1 antagonist W123 and the S1PR3 antagonist BML-241 had no impact on STAT3 phosphorylation (Figure 6B). STAT3 has been reported to negatively regulate cell cycle inhibitor proteins p21 and p27, thereby inducing cell cycle progression [41]. Importantly, SC-derived myoblasts treated with the S1PR2 antagonist JTE-013 showed a reduction in STAT3 phosphorylation, concomitant with an increase in p21 and p27 levels (Figure 6C). To further confirm these findings, we employed siRNA to knockdown the expression of S1PR2 and then evaluated the impact on STAT3 activation. As shown in Figure 6D, SC-derived myoblasts transfected with S1PR2 siRNA exhibited a 75% reduction in S1PR2 mRNA expression. S1PR2 knockdown resulted in a marked reduction in STAT3 phosphorylation, similar to the results obtained with JTE-013 (Figure 6E). Given these findings, we analyzed the effects of JTE-013 in vivo 5 days after NTX injury. We found that there was a significant decrease in centrally nucleated myofibers/mm2 after treatment with JTE-013 (Figures 6F and G). Further, JTE-013 treatment was associated with a significant decrease in the number of SCs when evaluated by FACS analysis (Figure 6H). These findings demonstrate that S1PR2 activation and downstream activation of STAT3 are essential for efficient muscle regeneration and SC proliferation.

Bottom Line: These changes include early and profound induction of the gene encoding the S1P biosynthetic enzyme SphK1, followed by induction of the catabolic enzyme sphingosine phosphate lyase (SPL) 3 days later.STAT3 activation resulted in p21 and p27 downregulation in a S1PR2-dependent fashion in myoblasts.Our findings suggest that S1P promotes SC progression through the cell cycle by repression of cell cycle inhibitors via S1PR2/STAT3-dependent signaling and that SPL inhibition may provide a therapeutic strategy for MD.

View Article: PubMed Central - PubMed

Affiliation: Children's Hospital Oakland Research Institute, Oakland, California, United States of America.

ABSTRACT
Sphingosine-1-phosphate (S1P) activates a widely expressed family of G protein-coupled receptors, serves as a muscle trophic factor and activates muscle stem cells called satellite cells (SCs) through unknown mechanisms. Here we show that muscle injury induces dynamic changes in S1P signaling and metabolism in vivo. These changes include early and profound induction of the gene encoding the S1P biosynthetic enzyme SphK1, followed by induction of the catabolic enzyme sphingosine phosphate lyase (SPL) 3 days later. These changes correlate with a transient increase in circulating S1P levels after muscle injury. We show a specific requirement for SphK1 to support efficient muscle regeneration and SC proliferation and differentiation. Mdx mice, which serve as a model for muscular dystrophy (MD), were found to be S1P-deficient and exhibited muscle SPL upregulation, suggesting that S1P catabolism is enhanced in dystrophic muscle. Pharmacological SPL inhibition increased muscle S1P levels, improved mdx muscle regeneration and enhanced SC proliferation via S1P receptor 2 (S1PR2)-dependent inhibition of Rac1, thereby activating Signal Transducer and Activator of Transcription 3 (STAT3), a central player in inflammatory signaling. STAT3 activation resulted in p21 and p27 downregulation in a S1PR2-dependent fashion in myoblasts. Our findings suggest that S1P promotes SC progression through the cell cycle by repression of cell cycle inhibitors via S1PR2/STAT3-dependent signaling and that SPL inhibition may provide a therapeutic strategy for MD.

Show MeSH
Related in: MedlinePlus