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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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SphK1 disruption and S1P deficiency impairs muscle regeneration and SC recruitment.NTX injury was performed in WT C57BL/6 mice and SphK1 KO mice in the C57BL/6 background. Blood plasma and gastrocnemius muscles were harvested at different time points after injury. A) Plasma levels of WT and KO mice. B) Representative H&E stained frozen muscle sections of WT and SphK1 KO mouse muscles at the point of maximal injury. Scale bar = 50 microns. C) Fiber counts at days 5 and 10. D) Representative immunofluorescence staining on injured C57 BL/6 muscle sections with Hoechst (blue) for nucleus, laminin (red) for basal lamina and Pax-7 (green) or M-cadherin (green) for SCs. Arrow shows merged signals. E) Quantitation of SCs, identified by sublaminar mononucleated cells expressing Pax-7 or M-cadherin in WT and SphK1 KO muscles at baseline (day 0) and 10 days after injury. * p≤0.05 (KO at day 10 compared to WT at day 10 for both markers). F) Gene expression of SC markers (Pax-7, M-cadherin and c-Met) in uninjured (D0) and injured muscles at 5 and 10 days post injury. Data are expressed as means ± SD; n = 4 for immunostained cryosections; n = 3 for Western blot analysis; n = 3–5 for qRT-PCR; * indicates a significant difference between WT and KO at that time point, p≤0.05.
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pone-0037218-g002: SphK1 disruption and S1P deficiency impairs muscle regeneration and SC recruitment.NTX injury was performed in WT C57BL/6 mice and SphK1 KO mice in the C57BL/6 background. Blood plasma and gastrocnemius muscles were harvested at different time points after injury. A) Plasma levels of WT and KO mice. B) Representative H&E stained frozen muscle sections of WT and SphK1 KO mouse muscles at the point of maximal injury. Scale bar = 50 microns. C) Fiber counts at days 5 and 10. D) Representative immunofluorescence staining on injured C57 BL/6 muscle sections with Hoechst (blue) for nucleus, laminin (red) for basal lamina and Pax-7 (green) or M-cadherin (green) for SCs. Arrow shows merged signals. E) Quantitation of SCs, identified by sublaminar mononucleated cells expressing Pax-7 or M-cadherin in WT and SphK1 KO muscles at baseline (day 0) and 10 days after injury. * p≤0.05 (KO at day 10 compared to WT at day 10 for both markers). F) Gene expression of SC markers (Pax-7, M-cadherin and c-Met) in uninjured (D0) and injured muscles at 5 and 10 days post injury. Data are expressed as means ± SD; n = 4 for immunostained cryosections; n = 3 for Western blot analysis; n = 3–5 for qRT-PCR; * indicates a significant difference between WT and KO at that time point, p≤0.05.

Mentions: Use of nonspecific SK inhibitors has implicated a role for S1P in muscle regeneration [23]. We wished to confirm this finding using a genetic model system and to further establish whether SphK1 plays a specific role in muscle regeneration, as implicated by the profound Sphk1 upregulation observed in injured muscle. Toward that end, we compared S1P levels and muscle regeneration capacity in WT mice versus SphK1 KO mice that lack the major enzyme required for S1P biosynthesis, but which are viable and healthy by virtue of an intact Sphk2 gene [36]. S1P levels in SphK1 KO plasma were consistently between 40% to 57% of the circulating S1P levels observed in WT plasma at day 0 and through day 5 post-injury (Figure 2A). Muscle regeneration was compared in WT and SphK1 KO mice by examining the appearance of Hematoxylin & Eosin (H&E)-stained frozen sections and by quantification of nucleated, regenerating muscle fibers found at the point of maximal injury determined by serial sectioning and examination of the entire muscle, as described in Materials and Methods. As shown in Figure 2B, on day 5 after NTX injury both WT and SphK1 KO muscles contained fibers with centralized nuclei, indicating the presence of muscle regeneration at the point of maximal injury. At that time, there was no discernable difference between WT and SphK1 KO muscle fibers. However, by day 10 after injury, a compact assembly of centrally nucleated regenerating fibers was observed at the point of maximal injury in the WT muscles. In contrast, the regenerating fibers in the SphK1 KO muscles at day 10 appeared more sparsely organized, and quantification of regenerating fiber number/mm2 revealed a statistically significant reduction in regenerating fiber numbers in SphK1 KO muscles compared to WT muscles (Figures 2B and C).


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)

SphK1 disruption and S1P deficiency impairs muscle regeneration and SC recruitment.NTX injury was performed in WT C57BL/6 mice and SphK1 KO mice in the C57BL/6 background. Blood plasma and gastrocnemius muscles were harvested at different time points after injury. A) Plasma levels of WT and KO mice. B) Representative H&E stained frozen muscle sections of WT and SphK1 KO mouse muscles at the point of maximal injury. Scale bar = 50 microns. C) Fiber counts at days 5 and 10. D) Representative immunofluorescence staining on injured C57 BL/6 muscle sections with Hoechst (blue) for nucleus, laminin (red) for basal lamina and Pax-7 (green) or M-cadherin (green) for SCs. Arrow shows merged signals. E) Quantitation of SCs, identified by sublaminar mononucleated cells expressing Pax-7 or M-cadherin in WT and SphK1 KO muscles at baseline (day 0) and 10 days after injury. * p≤0.05 (KO at day 10 compared to WT at day 10 for both markers). F) Gene expression of SC markers (Pax-7, M-cadherin and c-Met) in uninjured (D0) and injured muscles at 5 and 10 days post injury. Data are expressed as means ± SD; n = 4 for immunostained cryosections; n = 3 for Western blot analysis; n = 3–5 for qRT-PCR; * indicates a significant difference between WT and KO at that time point, p≤0.05.
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Related In: Results  -  Collection

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pone-0037218-g002: SphK1 disruption and S1P deficiency impairs muscle regeneration and SC recruitment.NTX injury was performed in WT C57BL/6 mice and SphK1 KO mice in the C57BL/6 background. Blood plasma and gastrocnemius muscles were harvested at different time points after injury. A) Plasma levels of WT and KO mice. B) Representative H&E stained frozen muscle sections of WT and SphK1 KO mouse muscles at the point of maximal injury. Scale bar = 50 microns. C) Fiber counts at days 5 and 10. D) Representative immunofluorescence staining on injured C57 BL/6 muscle sections with Hoechst (blue) for nucleus, laminin (red) for basal lamina and Pax-7 (green) or M-cadherin (green) for SCs. Arrow shows merged signals. E) Quantitation of SCs, identified by sublaminar mononucleated cells expressing Pax-7 or M-cadherin in WT and SphK1 KO muscles at baseline (day 0) and 10 days after injury. * p≤0.05 (KO at day 10 compared to WT at day 10 for both markers). F) Gene expression of SC markers (Pax-7, M-cadherin and c-Met) in uninjured (D0) and injured muscles at 5 and 10 days post injury. Data are expressed as means ± SD; n = 4 for immunostained cryosections; n = 3 for Western blot analysis; n = 3–5 for qRT-PCR; * indicates a significant difference between WT and KO at that time point, p≤0.05.
Mentions: Use of nonspecific SK inhibitors has implicated a role for S1P in muscle regeneration [23]. We wished to confirm this finding using a genetic model system and to further establish whether SphK1 plays a specific role in muscle regeneration, as implicated by the profound Sphk1 upregulation observed in injured muscle. Toward that end, we compared S1P levels and muscle regeneration capacity in WT mice versus SphK1 KO mice that lack the major enzyme required for S1P biosynthesis, but which are viable and healthy by virtue of an intact Sphk2 gene [36]. S1P levels in SphK1 KO plasma were consistently between 40% to 57% of the circulating S1P levels observed in WT plasma at day 0 and through day 5 post-injury (Figure 2A). Muscle regeneration was compared in WT and SphK1 KO mice by examining the appearance of Hematoxylin & Eosin (H&E)-stained frozen sections and by quantification of nucleated, regenerating muscle fibers found at the point of maximal injury determined by serial sectioning and examination of the entire muscle, as described in Materials and Methods. As shown in Figure 2B, on day 5 after NTX injury both WT and SphK1 KO muscles contained fibers with centralized nuclei, indicating the presence of muscle regeneration at the point of maximal injury. At that time, there was no discernable difference between WT and SphK1 KO muscle fibers. However, by day 10 after injury, a compact assembly of centrally nucleated regenerating fibers was observed at the point of maximal injury in the WT muscles. In contrast, the regenerating fibers in the SphK1 KO muscles at day 10 appeared more sparsely organized, and quantification of regenerating fiber number/mm2 revealed a statistically significant reduction in regenerating fiber numbers in SphK1 KO muscles compared to WT muscles (Figures 2B and C).

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