In this study, we have explored the function of the S1P signaling pathway within the context of muscle injury using the well-characterized NTX injury model. Initial exploratory studies characterized the impact of muscle injury on S1P signaling in WT murine muscle. Our findings demonstrate that, subsequent to acute muscle injury, dynamic changes occur in the expression of S1P biosynthetic and catabolic enzymes in whole muscle tissue. Marked Sphk1 induction was observed, followed by elevation of plasma S1P levels. Importantly, circulating S1P level elevations of the magnitude observed in our study have been implicated in mediating physiologically relevant effects such as radioprotection and ischemic cardioprotection 
. Plasma S1P gradually returned to baseline levels concomitant with upregulation of SPL, the enzyme responsible for irreversible S1P degradation and the major regulator of S1P levels in cells and tissues. S1PR expression also changed over time after muscle injury, with S1PR1 and S1PR2 expression exhibiting the most significant changes.
To establish the physiological relevance of Sphk1 induction after muscle injury, we tested the efficiency of muscle regeneration in SphK1 KO mice. Our studies in this model demonstrate a specific requirement for SphK1 in both muscle regeneration and endogenous SC recruitment. Consistent with these findings, myoblast-derived SCs isolated from SphK1 KO mice exhibited a reduced capacity to proliferate and differentiate in vitro. Further, the S1P-deficient serum from SphK1 KO mice did not sustain optimal proliferation and differentiation of WT myoblast-derived SCs. These findings suggest that SphK1-derived S1P likely acts through a combination of autocrine mechanisms and effects on the muscle niche to promote SC functions and thereby facilitate regeneration after muscle injury.
We observed that SPL gene and protein expression rise after injury in WT mice and are also elevated under baseline conditions in the dystrophic muscles characteristic of mdx mice. Our results corroborate a previous study that found Sgpl1 upregulation as a component of a “chronic inflammatory response” pattern in mdx mice 
. SPL is upregulated under a variety of stressful conditions including ischemia, infection, injury and DNA damage 
. In contrast, the expression of enzymes required for S1P biosynthesis were not found to be elevated in mdx muscles. We propose that chronic muscle injury in MD may induce genetic changes that favor S1P catabolism and result in S1P deficiency, thereby impairing SC activation and muscle regeneration. The effect of chronic S1P depletion in dystrophic muscles, SCs and/or the muscle niche could in part explain the failure of genetic and cellular therapies to restore muscle health in MD.
Our findings in the mdx mouse model suggested the possibility that SPL inhibition could serve as a strategy to boost S1P levels, enhance endogenous SC functions and thereby promote muscle regeneration in MD. This notion is supported by our finding that myoblast-derived SCs isolated from SPL KO mice have greater capacity to proliferate and differentiate in vitro
than SCs isolated from littermate controls. Genetic or pharmacological SPL suppression protects against other forms of injury including ischemia, DNA damage, and autoimmune and infection/inflammatory insults 
. Our results in mdx mice demonstrate that regeneration after injury of dystrophic muscles is improved by pharmacological SPL inhibition using the nontoxic food additive, THI. SPL inhibition by THI results in an expansion of endogenous SC pools, as determined by SC quantification using histological methods and analysis by flow cytometry. We speculate that the expansion of SCs with THI treatment results in a larger reserve population of SCs capable of activating with subsequent injury. It remains to be tested whether SPL inhibition using THI or other SPL inhibitors in recipients of cellular therapy will facilitate donor SC proliferation and incorporation into dystrophic muscles.
We have shown that STAT3 activation plays a critical role in mediating the positive effects of SPL-inhibition on muscle regeneration. This conclusion is supported by our findings that the muscles of THI-treated mdx mice exhibit enhanced STAT3 activation, whereas the increases in regenerating fiber numbers and SC recruitment after injury associated with THI treatment are attenuated by inhibition of STAT3-dependent signaling. Several reports have linked S1P signaling to the regulation of STAT3, including the recent study establishing that paracrine S1P signaling events are responsible for constitutive STAT3 signaling in cancer 
. In addition, S1P and S1P-containing HDL activate STAT3 signaling in ventricular cardiomyocytes and in prostate cancer cells 
. While interleukin-6 and STAT3 are known to participate in muscle biology and SC functions 
, the only published report we are aware of that links STAT3 to MD was an exploratory survey of transcription factors in mdx 
. Whether other STAT3 activators could be useful in MD remains to be tested. It is interesting to note that the STAT3 activator LIF enhances muscle regeneration through the incorporation of transplanted donor myoblasts into injured and diseased muscle fibers 
Our results have implicated a critical role for S1PR2 signaling in mediating SC recruitment and muscle regeneration. This notion is supported by our finding of reduced numbers of regenerating fibers and endogenous SCs in response to muscle injury in mice pretreated with the S1PR2 antagonist JTE-013. This effect occurs at least in part at the level of the myoblast, since activated STAT3 is markedly reduced in myoblasts treated with S1PR2 siRNA or with the S1PR2 inhibitor JTE-013 but not with other receptor inhibitors. Importantly, STAT3 has been shown to repress cell cycle inhibitor proteins including p21 and p27, which has been proposed as one of the major mechanisms responsible for STAT3's ability to promote cell proliferation 
. We found that S1PR2 inhibition by JTE-013 in myoblasts reduces STAT3 activation and results in marked upregulation of p21 and p27. Our cumulative findings suggest that the activation of SCs by S1P may be mediated through a pathway involving activation of S1PR2 and STAT3, leading to the repression of cell cycle inhibitors and de-repression of the quiescent state.
Our results suggest that S1PR2 signaling activates STAT3 through a process that involves its reciprocal effects on small Rho GTPases. Specifically, we find that inhibition of S1PR2 in myoblasts correlates with Rac1 activation concomitant with STAT3 inhibition, whereas Rac1 inhibition or RhoA overexpression in myoblasts leads to activation of STAT3. The finding that S1PR2 activation leads to inhibition of Rac1 in myoblasts is consistent with the known functions of S1PR2 in other cell types 
. In contrast to the effect of S1PR2 on Rac1, RhoA has been shown to be activated by S1PR2 signaling 
. There is considerable evidence that wild type and mutant Rac1 and other Rho small GTPases can activate STAT3, especially within the context of cancer 
. However, our findings suggest that in myoblasts inhibition of Rac1 and activation of RhoA by S1PR2 contribute to activation of STAT3.
STAT3 plays a role in rodent and human SC proliferation, migration and differentiation 
. Therefore, we consider it likely that S1P signaling plays a critical role in multiple SC functions through its impact on STAT3 signaling. However, in our investigation of primary SCs from WT and sphingolipid mutant mice, it was not possible to segregate proliferation from differentiation effects, since a change in proliferation rate would reduce the number of SCs available to undergo fusion, nuclear accretion, and myotube formation. Therefore, additional studies will be required to specifically delineate the involvement of S1P/S1PR2/STAT3 signaling in SC differentiation and migration.
In summary, we have found that S1P signaling changes dynamically in response to muscle injury, plays a role in SC activation and muscle regeneration through an S1PR2/STAT3-dependent signaling pathway, and that SPL can be targeted to enhance muscle regeneration in a model of MD. Many of the known functions of S1P, including its role in angiogenesis, nitric oxide metabolism, innate and adaptive immunity, calcium homeostasis and cytokine and growth factor signaling, are important components of the physiological response to muscle injury. It will be important to determine how S1P-dependent signaling and SPL inhibitory strategies impact the many facets of muscle regeneration. Considering the recently described role of S1P as a histone deacetylase inhibitor, it is also possible that S1P influences epigenetic reprogramming in regenerating muscle 
. The ubiquitous role of S1P signaling in physiology raises the concern that targeting SPL may be too toxic for practical use. However, humanized SPL mice exhibiting 10–20% of WT SPL activity levels live a normal lifespan, are healthy, reproductive and exhibit lymphopenia as their only phenotype 
. Human clinical trials using small molecule inhibitors of SPL appear promising for the treatment of autoimmune diseases 
. These findings suggest that brief, intermittent or incomplete pharmacological SPL suppression is a feasible therapeutic strategy for enhancing SC functions in MD and other diseases of skeletal muscle. Future studies employing other murine models and large animal models of MD should help to clarify this issue.