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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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Dynamic changes in S1P signaling after muscle injury.A) Heat map of S1P receptors, S1P metabolic genes and other markers in WT C57BL/6 mouse skeletal muscle from day 0–40 after NTX injection. Red is induced, green is repressed relative to the mean. Note: two probes were available for some genes. Lox = lysyl oxidase; S1pr1–5 = S1PRs; Sgpl1 = SPL. Sgpp = S1P phosphatases; Sphk = sphingosine kinases. B) SPL protein (Sgpl1p) expression is induced after muscle injury, as determined by immunoblotting (above) and imageJ quantification relative to day 0 (uninjured muscle) and normalized to actin (below). C) Relative gene expression of Sphk1, Sphk2, Sgpl1 and Sgpp1 (normalized to Gapdh) were evaluated in whole muscle over time after injury by qRT-PCR. Gene levels are depicted relative to their own baseline levels (day 0 set as 1 for all genes). D) Corresponding plasma S1P levels were measured by LC-MS, E) Relative gene expression of S1PRs was determined as in “C” and is depicted relative to S1pr1 (day 0 set as 1 for S1pr1 only). F) S1PR induction is shown relative to baseline levels for each receptor (day 0 set as 1 for all genes). * indicates significant difference compared to day 0, p≤0.05. Data are means ± SD; n = 3–5/group.
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pone-0037218-g001: Dynamic changes in S1P signaling after muscle injury.A) Heat map of S1P receptors, S1P metabolic genes and other markers in WT C57BL/6 mouse skeletal muscle from day 0–40 after NTX injection. Red is induced, green is repressed relative to the mean. Note: two probes were available for some genes. Lox = lysyl oxidase; S1pr1–5 = S1PRs; Sgpl1 = SPL. Sgpp = S1P phosphatases; Sphk = sphingosine kinases. B) SPL protein (Sgpl1p) expression is induced after muscle injury, as determined by immunoblotting (above) and imageJ quantification relative to day 0 (uninjured muscle) and normalized to actin (below). C) Relative gene expression of Sphk1, Sphk2, Sgpl1 and Sgpp1 (normalized to Gapdh) were evaluated in whole muscle over time after injury by qRT-PCR. Gene levels are depicted relative to their own baseline levels (day 0 set as 1 for all genes). D) Corresponding plasma S1P levels were measured by LC-MS, E) Relative gene expression of S1PRs was determined as in “C” and is depicted relative to S1pr1 (day 0 set as 1 for S1pr1 only). F) S1PR induction is shown relative to baseline levels for each receptor (day 0 set as 1 for all genes). * indicates significant difference compared to day 0, p≤0.05. Data are means ± SD; n = 3–5/group.

Mentions: S1P signaling has been implicated in various aspects of muscle biology [25]. However, the global effect of muscle injury on S1P signaling and metabolism has not previously been characterized in vivo. We previously performed temporal expression profiling on whole muscle tissue in the mouse notexin (NTX) muscle regeneration model in order to characterize myogenic genetic reprogramming after injury [32]. A search of this microarray database revealed that the expression levels of several genes involved in S1P metabolism and receptor signaling change over time after muscle injury (Figure 1A). The SPL gene (Sgpl1) was significantly upregulated on day 3.5, concomitant with transcriptional upregulation of the Myf5 transcription factor, the ECM enzyme lysyl oxidase (Lox), the major SK Sphk1, and S1PR genes s1pr2 and s1pr3. In contrast, s1pr1, s1pr4, s1pr5, and the gene encoding S1P phosphatase 1 (Sgpp1) were downregulated or unchanged at the time points interrogated in this array, and Sphk2 expression results were inconsistent using two different probes. To confirm these findings, we first administered a single NTX intramuscular (i.m.) injection into the gastrocnemius muscles of C57BL/6 male mice (as described in Materials and Methods) and evaluated SPL gene and protein expression at different time points from day 0 (untreated) to day 10 after injury. Immunoblotting confirmed that muscle SPL protein expression increased over baseline levels by day 1 and reached maximal expression levels 5 days after injury (Figure 1B). To comprehensively characterize genetic changes affecting S1P metabolism and signaling in the aftermath of skeletal muscle injury, we administered a single NTX injection into the gastrocnemius muscles of C57BL/6 male mice as described above and followed the gene expression of S1PRs and major genes of S1P metabolism over time from 6 hours to 20 days in injured muscle by quantitative real time polymerase chain reaction (qRT-PCR). Within 6 hours after injury, we observed a 100-fold induction of Sphk1, followed by upregulation of Sphk2, Sgpl1 and Sgpp1 on days 3–5 or beyond after injury (Figure 1C). Measurement of S1P in the plasma of C57BL/6 mice under baseline conditions by liquid chromatography mass spectrometry (LC-MS) revealed circulating S1P levels of approximately 2 µM, consistent with our previous findings [33], [34]. In response to focal muscle injury, plasma S1P levels were found to increase by 50%, (Figure 1D), a perturbation that is known to exert physiological effects in other contexts [33]–[35]. We next characterized S1PR expression at baseline and in injured muscle. The s1pr1 gene expression levels in whole muscle exceeded those of the other four S1PR subtypes at rest and after injury (Figure 1E). From 6 hours through day 3, s1pr1 expression increased 5-fold and decreased thereafter, diminishing to near baseline levels by day 5. In contrast, a 25-fold increase in s1pr2 expression was observed by day 3 and remained high through day 5 (Figure 1F). The expression levels of genes encoding S1PR3-5 also increased over time but remained low compared to those encoding S1PR1 and S1PR2 (Figures 1E and F). These findings establish that dynamic changes in S1P metabolism and signaling occur in the skeletal muscle fiber and microenvironment in response to injury.


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)

Dynamic changes in S1P signaling after muscle injury.A) Heat map of S1P receptors, S1P metabolic genes and other markers in WT C57BL/6 mouse skeletal muscle from day 0–40 after NTX injection. Red is induced, green is repressed relative to the mean. Note: two probes were available for some genes. Lox = lysyl oxidase; S1pr1–5 = S1PRs; Sgpl1 = SPL. Sgpp = S1P phosphatases; Sphk = sphingosine kinases. B) SPL protein (Sgpl1p) expression is induced after muscle injury, as determined by immunoblotting (above) and imageJ quantification relative to day 0 (uninjured muscle) and normalized to actin (below). C) Relative gene expression of Sphk1, Sphk2, Sgpl1 and Sgpp1 (normalized to Gapdh) were evaluated in whole muscle over time after injury by qRT-PCR. Gene levels are depicted relative to their own baseline levels (day 0 set as 1 for all genes). D) Corresponding plasma S1P levels were measured by LC-MS, E) Relative gene expression of S1PRs was determined as in “C” and is depicted relative to S1pr1 (day 0 set as 1 for S1pr1 only). F) S1PR induction is shown relative to baseline levels for each receptor (day 0 set as 1 for all genes). * indicates significant difference compared to day 0, p≤0.05. Data are means ± SD; n = 3–5/group.
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Related In: Results  -  Collection

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pone-0037218-g001: Dynamic changes in S1P signaling after muscle injury.A) Heat map of S1P receptors, S1P metabolic genes and other markers in WT C57BL/6 mouse skeletal muscle from day 0–40 after NTX injection. Red is induced, green is repressed relative to the mean. Note: two probes were available for some genes. Lox = lysyl oxidase; S1pr1–5 = S1PRs; Sgpl1 = SPL. Sgpp = S1P phosphatases; Sphk = sphingosine kinases. B) SPL protein (Sgpl1p) expression is induced after muscle injury, as determined by immunoblotting (above) and imageJ quantification relative to day 0 (uninjured muscle) and normalized to actin (below). C) Relative gene expression of Sphk1, Sphk2, Sgpl1 and Sgpp1 (normalized to Gapdh) were evaluated in whole muscle over time after injury by qRT-PCR. Gene levels are depicted relative to their own baseline levels (day 0 set as 1 for all genes). D) Corresponding plasma S1P levels were measured by LC-MS, E) Relative gene expression of S1PRs was determined as in “C” and is depicted relative to S1pr1 (day 0 set as 1 for S1pr1 only). F) S1PR induction is shown relative to baseline levels for each receptor (day 0 set as 1 for all genes). * indicates significant difference compared to day 0, p≤0.05. Data are means ± SD; n = 3–5/group.
Mentions: S1P signaling has been implicated in various aspects of muscle biology [25]. However, the global effect of muscle injury on S1P signaling and metabolism has not previously been characterized in vivo. We previously performed temporal expression profiling on whole muscle tissue in the mouse notexin (NTX) muscle regeneration model in order to characterize myogenic genetic reprogramming after injury [32]. A search of this microarray database revealed that the expression levels of several genes involved in S1P metabolism and receptor signaling change over time after muscle injury (Figure 1A). The SPL gene (Sgpl1) was significantly upregulated on day 3.5, concomitant with transcriptional upregulation of the Myf5 transcription factor, the ECM enzyme lysyl oxidase (Lox), the major SK Sphk1, and S1PR genes s1pr2 and s1pr3. In contrast, s1pr1, s1pr4, s1pr5, and the gene encoding S1P phosphatase 1 (Sgpp1) were downregulated or unchanged at the time points interrogated in this array, and Sphk2 expression results were inconsistent using two different probes. To confirm these findings, we first administered a single NTX intramuscular (i.m.) injection into the gastrocnemius muscles of C57BL/6 male mice (as described in Materials and Methods) and evaluated SPL gene and protein expression at different time points from day 0 (untreated) to day 10 after injury. Immunoblotting confirmed that muscle SPL protein expression increased over baseline levels by day 1 and reached maximal expression levels 5 days after injury (Figure 1B). To comprehensively characterize genetic changes affecting S1P metabolism and signaling in the aftermath of skeletal muscle injury, we administered a single NTX injection into the gastrocnemius muscles of C57BL/6 male mice as described above and followed the gene expression of S1PRs and major genes of S1P metabolism over time from 6 hours to 20 days in injured muscle by quantitative real time polymerase chain reaction (qRT-PCR). Within 6 hours after injury, we observed a 100-fold induction of Sphk1, followed by upregulation of Sphk2, Sgpl1 and Sgpp1 on days 3–5 or beyond after injury (Figure 1C). Measurement of S1P in the plasma of C57BL/6 mice under baseline conditions by liquid chromatography mass spectrometry (LC-MS) revealed circulating S1P levels of approximately 2 µM, consistent with our previous findings [33], [34]. In response to focal muscle injury, plasma S1P levels were found to increase by 50%, (Figure 1D), a perturbation that is known to exert physiological effects in other contexts [33]–[35]. We next characterized S1PR expression at baseline and in injured muscle. The s1pr1 gene expression levels in whole muscle exceeded those of the other four S1PR subtypes at rest and after injury (Figure 1E). From 6 hours through day 3, s1pr1 expression increased 5-fold and decreased thereafter, diminishing to near baseline levels by day 5. In contrast, a 25-fold increase in s1pr2 expression was observed by day 3 and remained high through day 5 (Figure 1F). The expression levels of genes encoding S1PR3-5 also increased over time but remained low compared to those encoding S1PR1 and S1PR2 (Figures 1E and F). These findings establish that dynamic changes in S1P metabolism and signaling occur in the skeletal muscle fiber and microenvironment in response to injury.

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