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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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SPL inhibition improves muscle regeneration and SC recruitment in MD.A) Treatment of WT mice with THI for 2 days (2D) or 5 days (5D) results in elevated muscle S1P levels compared to vehicle-treated controls at 5 days post injury (5DPI); * indicates p≤0.05. Data are means ± SD, n = 4–5/group. B) Mdx mice received NTX+THI or vehicle daily on days −1 to 5. On day 5, mice were euthanized and gastrocnemius muscles were harvested. Ten µm tissue cryosections were obtained every 200 µm in sets of 4. H&E stained sections were examined for point of maximal injury. Representative photographs are shown. Scale bar = 50 microns. Recovering WT control and resting mdx muscles are shown for comparison. C) Fiber quantification of experiment in “B”. Centrally nucleated myofibers were counted in a field containing only regenerating fibers and normalized to 1 mm2 area. * p≤0.05. D) Quantification of SC in situ in mdx mouse muscles identified by M-cadherin or Pax-7 expression and localization beneath the basal lamina. * indicates p≤0.05. E) Flow cytometry analysis of freshly isolated muscle cells from mdx mice treated with THI or vehicle at 5 days post injury. SCs were identified as CD45-, CD31-, Sca1-, integrin alpha-7+ and CD34+ cells; * indicates p≤0.05, data are means ± SD, n  =  5/group.
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pone-0037218-g005: SPL inhibition improves muscle regeneration and SC recruitment in MD.A) Treatment of WT mice with THI for 2 days (2D) or 5 days (5D) results in elevated muscle S1P levels compared to vehicle-treated controls at 5 days post injury (5DPI); * indicates p≤0.05. Data are means ± SD, n = 4–5/group. B) Mdx mice received NTX+THI or vehicle daily on days −1 to 5. On day 5, mice were euthanized and gastrocnemius muscles were harvested. Ten µm tissue cryosections were obtained every 200 µm in sets of 4. H&E stained sections were examined for point of maximal injury. Representative photographs are shown. Scale bar = 50 microns. Recovering WT control and resting mdx muscles are shown for comparison. C) Fiber quantification of experiment in “B”. Centrally nucleated myofibers were counted in a field containing only regenerating fibers and normalized to 1 mm2 area. * p≤0.05. D) Quantification of SC in situ in mdx mouse muscles identified by M-cadherin or Pax-7 expression and localization beneath the basal lamina. * indicates p≤0.05. E) Flow cytometry analysis of freshly isolated muscle cells from mdx mice treated with THI or vehicle at 5 days post injury. SCs were identified as CD45-, CD31-, Sca1-, integrin alpha-7+ and CD34+ cells; * indicates p≤0.05, data are means ± SD, n  =  5/group.

Mentions: We hypothesized that, like WT muscles injured with NTX, dystrophic muscles may also upregulate SPL in response to muscle injury. However, in the case of mdx mice, the injury results from intrinsic muscle fiber instability. We further hypothesized that the resulting chronically S1P-deficient state of dystrophic mice contributes to MD pathology by preventing efficient muscle repair. To address whether transient SPL inhibition could enhance muscle regeneration in mdx mice, we employed 2-acetyl-5-tetrahydroxybutylimidazole (THI), a nontoxic FDA-approved component of caramel food coloring that functions as an SPL inhibitor in vivo[38]. We have shown previously that THI treatment suppresses SPL activity and raises circulating plasma S1P levels by approximately 25–30% [33]. To assess the effect of SPL inhibition on muscle S1P levels, THI was administered in water for either 2 or 5 days to WT mice after NTX injury, and gastrocnemius muscle S1P levels were measured on day 5 post injury by LC-MS. THI treatment raised muscle S1P levels by 3–5-fold (Figure 5A). Mdx mice then received NTX injections in combination with orally administered THI or vehicle for 5 days after injury. Areas of uninjured mdx muscles showed the expected pathological findings including heterogeneous fiber size, the presence of large, necrotic fibers, small centrally nucleated fibers, and extensive inflammatory cellular infiltrates (Figure 5B). Five days after NTX injury, mdx muscles showed many areas lacking regenerating fibers at the point of maximal injury. In contrast, muscles from THI-treated mdx mice exhibited regenerating muscle architecture indistinguishable from regenerating WT muscle. THI-treated mdx muscles contained 2-fold more regenerating fibers than vehicle-treated mdx controls (Figure 5C). Quantification of SC numbers in regenerating mdx muscle using either M-cadherin or Pax-7 immunostaining and fluorescence microscopy demonstrated improvement in SC activation within the muscle fibers of THI-treated mdx mice compared to control mdx mice (Figure 5D). Flow cytometry approaches represent an unbiased method to identify SCs based on the presence or absence of specific cell surface markers. This approach was employed to confirm the effect of THI on SC proliferation that we had observed using histological methods as described above. Toward that end, CD45-negative, CD31-negative, Sca1-negative, alpha-integrin7-positive, CD34-positive SCs were quantified using flow cytometry of disaggregated muscle cells as described [39]. As shown in Figure 5E, THI treatment was associated with a significant increase in the number of SCs present in injured mdx muscle. These findings demonstrate that SPL inhibition enhances muscle regeneration and SC proliferation in a rodent model of MD.


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)

SPL inhibition improves muscle regeneration and SC recruitment in MD.A) Treatment of WT mice with THI for 2 days (2D) or 5 days (5D) results in elevated muscle S1P levels compared to vehicle-treated controls at 5 days post injury (5DPI); * indicates p≤0.05. Data are means ± SD, n = 4–5/group. B) Mdx mice received NTX+THI or vehicle daily on days −1 to 5. On day 5, mice were euthanized and gastrocnemius muscles were harvested. Ten µm tissue cryosections were obtained every 200 µm in sets of 4. H&E stained sections were examined for point of maximal injury. Representative photographs are shown. Scale bar = 50 microns. Recovering WT control and resting mdx muscles are shown for comparison. C) Fiber quantification of experiment in “B”. Centrally nucleated myofibers were counted in a field containing only regenerating fibers and normalized to 1 mm2 area. * p≤0.05. D) Quantification of SC in situ in mdx mouse muscles identified by M-cadherin or Pax-7 expression and localization beneath the basal lamina. * indicates p≤0.05. E) Flow cytometry analysis of freshly isolated muscle cells from mdx mice treated with THI or vehicle at 5 days post injury. SCs were identified as CD45-, CD31-, Sca1-, integrin alpha-7+ and CD34+ cells; * 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-g005: SPL inhibition improves muscle regeneration and SC recruitment in MD.A) Treatment of WT mice with THI for 2 days (2D) or 5 days (5D) results in elevated muscle S1P levels compared to vehicle-treated controls at 5 days post injury (5DPI); * indicates p≤0.05. Data are means ± SD, n = 4–5/group. B) Mdx mice received NTX+THI or vehicle daily on days −1 to 5. On day 5, mice were euthanized and gastrocnemius muscles were harvested. Ten µm tissue cryosections were obtained every 200 µm in sets of 4. H&E stained sections were examined for point of maximal injury. Representative photographs are shown. Scale bar = 50 microns. Recovering WT control and resting mdx muscles are shown for comparison. C) Fiber quantification of experiment in “B”. Centrally nucleated myofibers were counted in a field containing only regenerating fibers and normalized to 1 mm2 area. * p≤0.05. D) Quantification of SC in situ in mdx mouse muscles identified by M-cadherin or Pax-7 expression and localization beneath the basal lamina. * indicates p≤0.05. E) Flow cytometry analysis of freshly isolated muscle cells from mdx mice treated with THI or vehicle at 5 days post injury. SCs were identified as CD45-, CD31-, Sca1-, integrin alpha-7+ and CD34+ cells; * indicates p≤0.05, data are means ± SD, n  =  5/group.
Mentions: We hypothesized that, like WT muscles injured with NTX, dystrophic muscles may also upregulate SPL in response to muscle injury. However, in the case of mdx mice, the injury results from intrinsic muscle fiber instability. We further hypothesized that the resulting chronically S1P-deficient state of dystrophic mice contributes to MD pathology by preventing efficient muscle repair. To address whether transient SPL inhibition could enhance muscle regeneration in mdx mice, we employed 2-acetyl-5-tetrahydroxybutylimidazole (THI), a nontoxic FDA-approved component of caramel food coloring that functions as an SPL inhibitor in vivo[38]. We have shown previously that THI treatment suppresses SPL activity and raises circulating plasma S1P levels by approximately 25–30% [33]. To assess the effect of SPL inhibition on muscle S1P levels, THI was administered in water for either 2 or 5 days to WT mice after NTX injury, and gastrocnemius muscle S1P levels were measured on day 5 post injury by LC-MS. THI treatment raised muscle S1P levels by 3–5-fold (Figure 5A). Mdx mice then received NTX injections in combination with orally administered THI or vehicle for 5 days after injury. Areas of uninjured mdx muscles showed the expected pathological findings including heterogeneous fiber size, the presence of large, necrotic fibers, small centrally nucleated fibers, and extensive inflammatory cellular infiltrates (Figure 5B). Five days after NTX injury, mdx muscles showed many areas lacking regenerating fibers at the point of maximal injury. In contrast, muscles from THI-treated mdx mice exhibited regenerating muscle architecture indistinguishable from regenerating WT muscle. THI-treated mdx muscles contained 2-fold more regenerating fibers than vehicle-treated mdx controls (Figure 5C). Quantification of SC numbers in regenerating mdx muscle using either M-cadherin or Pax-7 immunostaining and fluorescence microscopy demonstrated improvement in SC activation within the muscle fibers of THI-treated mdx mice compared to control mdx mice (Figure 5D). Flow cytometry approaches represent an unbiased method to identify SCs based on the presence or absence of specific cell surface markers. This approach was employed to confirm the effect of THI on SC proliferation that we had observed using histological methods as described above. Toward that end, CD45-negative, CD31-negative, Sca1-negative, alpha-integrin7-positive, CD34-positive SCs were quantified using flow cytometry of disaggregated muscle cells as described [39]. As shown in Figure 5E, THI treatment was associated with a significant increase in the number of SCs present in injured mdx muscle. These findings demonstrate that SPL inhibition enhances muscle regeneration and SC proliferation in a rodent model of MD.

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