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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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S1P activation of STAT3 is mediated via an S1PR2/RhoGTPase pathway.SC-derived myoblasts were treated with indicated dose of: A) Rac1 inhibitor, or B) RhoA inhibitor for 24 hours in complete media, and cell lysates were immunoblotted with indicated antibodies. Levels of total STAT3 (STAT3-T) and phosphorylated STAT3 (STAT3-P) were measured and compared to actin loading control. C) SC-derived myoblasts were treated with 10 µM S1PR2 antagonist JTE-013 (R2i) for 20 hours, cells were incubated in fresh media for 5 minutes with or without R2i, and activated Rac1 was pulled down with GST-PAK1 fusion protein and immunoblotted with Rac1 antibody. STAT3 activation (STAT3-P) also shown for this experiment. D) Myoblasts were transfected with GFP, dominant negative Rac1 (Rac1 DN) or wild type RhoA (RhoA WT) plasmid constructs. Forty-eight hours later, cell lysates were immunoblotted to detect total and phosphorylated STAT3, GFP, Rac1 and RhoA, with GAPDH used as a loading control. E) Representative micrographs (10×) of H&E stained 10 µm frozen sections taken from mdx gastrocnemius muscles 5 days post NTX-injury. Groups were treated with vehicle, THI, STAT3 inhibitor WP1066, or THI+WP1066. F) Quantification of regenerating myofibers/mm2 (n = 3/group). Scale bar = 50 microns. G) Analysis of SCs by flow cytometry as described in Figure 5F. Data are means ± SD, n = 5/group, * indicates p≤0.05.
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pone-0037218-g007: S1P activation of STAT3 is mediated via an S1PR2/RhoGTPase pathway.SC-derived myoblasts were treated with indicated dose of: A) Rac1 inhibitor, or B) RhoA inhibitor for 24 hours in complete media, and cell lysates were immunoblotted with indicated antibodies. Levels of total STAT3 (STAT3-T) and phosphorylated STAT3 (STAT3-P) were measured and compared to actin loading control. C) SC-derived myoblasts were treated with 10 µM S1PR2 antagonist JTE-013 (R2i) for 20 hours, cells were incubated in fresh media for 5 minutes with or without R2i, and activated Rac1 was pulled down with GST-PAK1 fusion protein and immunoblotted with Rac1 antibody. STAT3 activation (STAT3-P) also shown for this experiment. D) Myoblasts were transfected with GFP, dominant negative Rac1 (Rac1 DN) or wild type RhoA (RhoA WT) plasmid constructs. Forty-eight hours later, cell lysates were immunoblotted to detect total and phosphorylated STAT3, GFP, Rac1 and RhoA, with GAPDH used as a loading control. E) Representative micrographs (10×) of H&E stained 10 µm frozen sections taken from mdx gastrocnemius muscles 5 days post NTX-injury. Groups were treated with vehicle, THI, STAT3 inhibitor WP1066, or THI+WP1066. F) Quantification of regenerating myofibers/mm2 (n = 3/group). Scale bar = 50 microns. G) Analysis of SCs by flow cytometry as described in Figure 5F. Data are means ± SD, n = 5/group, * indicates p≤0.05.

Mentions: We next sought to define the link between S1PR2 signaling and STAT3 activation. STAT3 activation is mediated by the ligation of various cytokines to gp130 and its associated receptors, which are then activated by JAK kinases, forming a docking site for STAT3 [28]. STAT signaling pathways are responsive to many intrinsic and environmental stimuli, including MAP kinases and Rho GTPases, known downstream targets of S1PR signaling [51]–[53]. We observed no effect of MAP kinase inhibitors on STAT3 phosphorylation in SC-derived primary myoblasts (data not shown). The Rho GTPase Rac1 has been shown to induce STAT3 activation in response to growth factors [54]. Surprisingly, SC-derived primary myoblasts treated with Rac1 inhibitors exhibited dose-dependent and significant STAT3 activation in comparison to vehicle-treated controls (Figure 7A). In contrast, RhoA inhibitors reduced myoblast STAT3 activation (Figure 7B). Importantly, when myoblasts were treated with an S1PR2 antagonist, Rac1 activation was enhanced, concomitant with STAT3 inactivation, as shown by reduced cellular levels of phosphorylated STAT3 (Figure 7C). Myoblasts were found to contain abundant Rac1 and low levels of RhoA and Cdc42 (data not shown). Therefore, to confirm the influence of Rac1 and RhoA on myoblast STAT3 activation, myoblasts were transfected with either a dominant negative construct of Rac1, a wild type RhoA construct or a GFP vector control, followed by measurement of Rac1, RhoA, total and phosphorylated STAT3 protein levels, with GAPDH serving as a loading control. As shown in Figure 7D, the expression in myoblasts of either dominant negative Rac1 or wild type RhoA resulted in higher activation of STAT3 in comparison to GFP control-transfected cells. These cumulative results indicate that in myoblasts, as in other cell types, S1P signaling through S1PR2 negatively regulates Rac1. However, in contrast to other cell types, inhibition of myoblast Rac1 releases STAT3 from suppression, thereby allowing it to repress cell cycle inhibitors and recruit SCs into the cell cycle.


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)

S1P activation of STAT3 is mediated via an S1PR2/RhoGTPase pathway.SC-derived myoblasts were treated with indicated dose of: A) Rac1 inhibitor, or B) RhoA inhibitor for 24 hours in complete media, and cell lysates were immunoblotted with indicated antibodies. Levels of total STAT3 (STAT3-T) and phosphorylated STAT3 (STAT3-P) were measured and compared to actin loading control. C) SC-derived myoblasts were treated with 10 µM S1PR2 antagonist JTE-013 (R2i) for 20 hours, cells were incubated in fresh media for 5 minutes with or without R2i, and activated Rac1 was pulled down with GST-PAK1 fusion protein and immunoblotted with Rac1 antibody. STAT3 activation (STAT3-P) also shown for this experiment. D) Myoblasts were transfected with GFP, dominant negative Rac1 (Rac1 DN) or wild type RhoA (RhoA WT) plasmid constructs. Forty-eight hours later, cell lysates were immunoblotted to detect total and phosphorylated STAT3, GFP, Rac1 and RhoA, with GAPDH used as a loading control. E) Representative micrographs (10×) of H&E stained 10 µm frozen sections taken from mdx gastrocnemius muscles 5 days post NTX-injury. Groups were treated with vehicle, THI, STAT3 inhibitor WP1066, or THI+WP1066. F) Quantification of regenerating myofibers/mm2 (n = 3/group). Scale bar = 50 microns. G) Analysis of SCs by flow cytometry as described in Figure 5F. Data are means ± SD, n = 5/group, * indicates p≤0.05.
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pone-0037218-g007: S1P activation of STAT3 is mediated via an S1PR2/RhoGTPase pathway.SC-derived myoblasts were treated with indicated dose of: A) Rac1 inhibitor, or B) RhoA inhibitor for 24 hours in complete media, and cell lysates were immunoblotted with indicated antibodies. Levels of total STAT3 (STAT3-T) and phosphorylated STAT3 (STAT3-P) were measured and compared to actin loading control. C) SC-derived myoblasts were treated with 10 µM S1PR2 antagonist JTE-013 (R2i) for 20 hours, cells were incubated in fresh media for 5 minutes with or without R2i, and activated Rac1 was pulled down with GST-PAK1 fusion protein and immunoblotted with Rac1 antibody. STAT3 activation (STAT3-P) also shown for this experiment. D) Myoblasts were transfected with GFP, dominant negative Rac1 (Rac1 DN) or wild type RhoA (RhoA WT) plasmid constructs. Forty-eight hours later, cell lysates were immunoblotted to detect total and phosphorylated STAT3, GFP, Rac1 and RhoA, with GAPDH used as a loading control. E) Representative micrographs (10×) of H&E stained 10 µm frozen sections taken from mdx gastrocnemius muscles 5 days post NTX-injury. Groups were treated with vehicle, THI, STAT3 inhibitor WP1066, or THI+WP1066. F) Quantification of regenerating myofibers/mm2 (n = 3/group). Scale bar = 50 microns. G) Analysis of SCs by flow cytometry as described in Figure 5F. Data are means ± SD, n = 5/group, * indicates p≤0.05.
Mentions: We next sought to define the link between S1PR2 signaling and STAT3 activation. STAT3 activation is mediated by the ligation of various cytokines to gp130 and its associated receptors, which are then activated by JAK kinases, forming a docking site for STAT3 [28]. STAT signaling pathways are responsive to many intrinsic and environmental stimuli, including MAP kinases and Rho GTPases, known downstream targets of S1PR signaling [51]–[53]. We observed no effect of MAP kinase inhibitors on STAT3 phosphorylation in SC-derived primary myoblasts (data not shown). The Rho GTPase Rac1 has been shown to induce STAT3 activation in response to growth factors [54]. Surprisingly, SC-derived primary myoblasts treated with Rac1 inhibitors exhibited dose-dependent and significant STAT3 activation in comparison to vehicle-treated controls (Figure 7A). In contrast, RhoA inhibitors reduced myoblast STAT3 activation (Figure 7B). Importantly, when myoblasts were treated with an S1PR2 antagonist, Rac1 activation was enhanced, concomitant with STAT3 inactivation, as shown by reduced cellular levels of phosphorylated STAT3 (Figure 7C). Myoblasts were found to contain abundant Rac1 and low levels of RhoA and Cdc42 (data not shown). Therefore, to confirm the influence of Rac1 and RhoA on myoblast STAT3 activation, myoblasts were transfected with either a dominant negative construct of Rac1, a wild type RhoA construct or a GFP vector control, followed by measurement of Rac1, RhoA, total and phosphorylated STAT3 protein levels, with GAPDH serving as a loading control. As shown in Figure 7D, the expression in myoblasts of either dominant negative Rac1 or wild type RhoA resulted in higher activation of STAT3 in comparison to GFP control-transfected cells. These cumulative results indicate that in myoblasts, as in other cell types, S1P signaling through S1PR2 negatively regulates Rac1. However, in contrast to other cell types, inhibition of myoblast Rac1 releases STAT3 from suppression, thereby allowing it to repress cell cycle inhibitors and recruit SCs into the cell cycle.

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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Related in: MedlinePlus