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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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Function of SCs isolated from sphingolipid mutant mice.A) Proliferation of WT and SphK1 KO primary SCs isolated 3 days after NTX injury, measured by BrdU uptake of desmin-positive cells incubated with either SphK1 WT or KO serum. B) Representative immunofluorescence images for experiment in “A”. Desmin-stained cells, green; BrdU-stained nuclei, orange. C) Differentiation of SC-derived myoblasts as shown by myotube formation. D) Representative immunofluorescence images for experiment in “C”. Hoechst-stained nuclei, blue; eMHC-stained fibers, green. E) Proliferation of SPL heterozygous  (+/−) and littermate control (+/+) mouse primary SC isolated 3 days after injury. F) Representative images for experiment in “E”. Desmin-stained cells, green; BrdU-stained nuclei, orange. G) Differentiation of heterozygous  (+/−) and littermate control (+/+) mouse SC. H) Representative immunofluorescence images for experiment in “G”. Hoechst-stained nuclei, blue; eMHC-stained fibers, green. * indicates p≤0.05.
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pone-0037218-g003: Function of SCs isolated from sphingolipid mutant mice.A) Proliferation of WT and SphK1 KO primary SCs isolated 3 days after NTX injury, measured by BrdU uptake of desmin-positive cells incubated with either SphK1 WT or KO serum. B) Representative immunofluorescence images for experiment in “A”. Desmin-stained cells, green; BrdU-stained nuclei, orange. C) Differentiation of SC-derived myoblasts as shown by myotube formation. D) Representative immunofluorescence images for experiment in “C”. Hoechst-stained nuclei, blue; eMHC-stained fibers, green. E) Proliferation of SPL heterozygous (+/−) and littermate control (+/+) mouse primary SC isolated 3 days after injury. F) Representative images for experiment in “E”. Desmin-stained cells, green; BrdU-stained nuclei, orange. G) Differentiation of heterozygous (+/−) and littermate control (+/+) mouse SC. H) Representative immunofluorescence images for experiment in “G”. Hoechst-stained nuclei, blue; eMHC-stained fibers, green. * indicates p≤0.05.

Mentions: S1P metabolism influences primary SC responses in vitro. Considering the importance of SphK1 in muscle regeneration and SC recruitment, we next examined the role of SphK1 in mediating SC functions in response to injury using a cell culture system. We wished to examine the intrinsic role of SphK1 in mediating SC functions in autocrine fashion, as well as the potential indirect contribution of SphK1 to regulating SC functions by influencing the muscle microenvironment. Toward that end, we isolated activated SCs from injured SphK1 KO and WT mice, cultured them in SphK1 WT or KO serum, and measured their ability to proliferate and differentiate. The combination of SphK1 KO cells and KO serum resulted in reduced proliferation compared to controls, as determined by BrdU uptake by desmin-positive cells (Figures 3A and B). A global (non-autonomous) requirement for SphK1 in mediating activated SC proliferation was identified, as shown by reduced proliferation of WT SCs in KO serum, whereas KO SC proliferation was increased in the presence of WT serum. Activated SCs also appear to have an intrinsic requirement for SphK1 to promote maximal proliferation, as WT serum did not restore KO SC proliferation to WT levels. Similar studies established that SCs also require SphK1 in non-autonomous fashion to support myotube formation, as determined by eMHC-positive myotubes after 48 hours in differentiation media (Figures 3C and D). SPL plays a major role in regulating S1P levels in cells and tissues. Mice homozygous for a gene-trap KO Sgpl1 allele are unable to survive the neonatal period. In contrast, mice heterozygous for this allele exhibit a 30–40% increase in circulating S1P levels over WT levels and live normal lifespans [33]. In contrast to SCs derived from SphK1 KO mice, SCs isolated from SPL heterozygous mice showed more robust proliferation (Figures 3E and F) and differentiation responses (Figures 3G and H) than littermate WT control SCs when incubated in the presence of their own serum. These studies establish that both biosynthetic and catabolic enzymes involved in S1P metabolism play an important role in the regulation of primary SC proliferation and differentiation.


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)

Function of SCs isolated from sphingolipid mutant mice.A) Proliferation of WT and SphK1 KO primary SCs isolated 3 days after NTX injury, measured by BrdU uptake of desmin-positive cells incubated with either SphK1 WT or KO serum. B) Representative immunofluorescence images for experiment in “A”. Desmin-stained cells, green; BrdU-stained nuclei, orange. C) Differentiation of SC-derived myoblasts as shown by myotube formation. D) Representative immunofluorescence images for experiment in “C”. Hoechst-stained nuclei, blue; eMHC-stained fibers, green. E) Proliferation of SPL heterozygous  (+/−) and littermate control (+/+) mouse primary SC isolated 3 days after injury. F) Representative images for experiment in “E”. Desmin-stained cells, green; BrdU-stained nuclei, orange. G) Differentiation of heterozygous  (+/−) and littermate control (+/+) mouse SC. H) Representative immunofluorescence images for experiment in “G”. Hoechst-stained nuclei, blue; eMHC-stained fibers, green. * indicates p≤0.05.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351440&req=5

pone-0037218-g003: Function of SCs isolated from sphingolipid mutant mice.A) Proliferation of WT and SphK1 KO primary SCs isolated 3 days after NTX injury, measured by BrdU uptake of desmin-positive cells incubated with either SphK1 WT or KO serum. B) Representative immunofluorescence images for experiment in “A”. Desmin-stained cells, green; BrdU-stained nuclei, orange. C) Differentiation of SC-derived myoblasts as shown by myotube formation. D) Representative immunofluorescence images for experiment in “C”. Hoechst-stained nuclei, blue; eMHC-stained fibers, green. E) Proliferation of SPL heterozygous (+/−) and littermate control (+/+) mouse primary SC isolated 3 days after injury. F) Representative images for experiment in “E”. Desmin-stained cells, green; BrdU-stained nuclei, orange. G) Differentiation of heterozygous (+/−) and littermate control (+/+) mouse SC. H) Representative immunofluorescence images for experiment in “G”. Hoechst-stained nuclei, blue; eMHC-stained fibers, green. * indicates p≤0.05.
Mentions: S1P metabolism influences primary SC responses in vitro. Considering the importance of SphK1 in muscle regeneration and SC recruitment, we next examined the role of SphK1 in mediating SC functions in response to injury using a cell culture system. We wished to examine the intrinsic role of SphK1 in mediating SC functions in autocrine fashion, as well as the potential indirect contribution of SphK1 to regulating SC functions by influencing the muscle microenvironment. Toward that end, we isolated activated SCs from injured SphK1 KO and WT mice, cultured them in SphK1 WT or KO serum, and measured their ability to proliferate and differentiate. The combination of SphK1 KO cells and KO serum resulted in reduced proliferation compared to controls, as determined by BrdU uptake by desmin-positive cells (Figures 3A and B). A global (non-autonomous) requirement for SphK1 in mediating activated SC proliferation was identified, as shown by reduced proliferation of WT SCs in KO serum, whereas KO SC proliferation was increased in the presence of WT serum. Activated SCs also appear to have an intrinsic requirement for SphK1 to promote maximal proliferation, as WT serum did not restore KO SC proliferation to WT levels. Similar studies established that SCs also require SphK1 in non-autonomous fashion to support myotube formation, as determined by eMHC-positive myotubes after 48 hours in differentiation media (Figures 3C and D). SPL plays a major role in regulating S1P levels in cells and tissues. Mice homozygous for a gene-trap KO Sgpl1 allele are unable to survive the neonatal period. In contrast, mice heterozygous for this allele exhibit a 30–40% increase in circulating S1P levels over WT levels and live normal lifespans [33]. In contrast to SCs derived from SphK1 KO mice, SCs isolated from SPL heterozygous mice showed more robust proliferation (Figures 3E and F) and differentiation responses (Figures 3G and H) than littermate WT control SCs when incubated in the presence of their own serum. These studies establish that both biosynthetic and catabolic enzymes involved in S1P metabolism play an important role in the regulation of primary SC proliferation and differentiation.

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