Investigation of Sulf regulation on the noncanonical Wnt signaling pathway

描述（由申请人提供）：肌肉再生是一个复杂的过程，涉及多种因素，这些因素在空间和时间上决定了称为卫星细胞（SC）的骨骼肌干细胞的活化、增殖、迁移和分化以及新生成肌细胞（分化的SC）的融合。因此，了解参与肌肉再生每个阶段的调节因素将为设计更好的肌肉相关疾病（如肌肉萎缩症和萎缩症）治疗方法开辟新的途径。我们以前的研究表明，硫酸乙酰肝素-6-O-内硫酸酯酶（Sulfs）的丢失导致肌肉再生延迟和SC群体的短暂增加，这种表型可能是由Sulf介导的SC分化过程中FGF 2信号转导的抑制引起的。硫是否在骨骼肌再生过程中发挥额外的作用尚不清楚。初步研究表明，硫抑制肌肉再生和卫星细胞培养过程中的非经典Wnt信号通路。由于非经典的Wnt信号被证明是通过驱动对称的细胞分裂来促进SC扩增，我们假设Sulfs调节非经典的Wnt信号以影响肌肉再生过程中的SC群体。我们的研究结果有望提供基质依赖性机制，调节骨骼肌再生和退行性疾病过程中的肌生成和SC保存。这些发现可能为硫酸乙酰肝素及其衍生物在干细胞工程和肌肉退行性疾病治疗中的应用提供依据。为了研究这样的假设，我们将利用体外细胞模型（单肌纤维）和通过损伤和通过肌营养不良再生的动物模型的组合，所述动物模型分别在卫星细胞Pax 7Sulf-DN（Pax 7-Cre：ER/Rosa 26;eYFP/Sulf 1/2flox）和Pax 7Sulf-DN;mdx中具有特异性失活的Sulfs。我们还将利用表达显性阴性Sulf 2的慢病毒来急性感染Sulfs。

DESCRIPTION (provided by applicant): Muscle regeneration is an intricate process involving multiple factors that spatially and temporally dictate the activation, proliferation, migration, and differentiation of skeletal muscle stem cells known as satellite cells (SC) and the fusion of nascent myoblasts (differentiated SC). Understanding the regulatory factors participating in each individual stage of muscle regeneration would therefore open new venues for designing better treatments for muscle related diseases such as muscular dystrophy and atrophy. Our previous study has shown that loss of heparan sulfate-6-O-endosulfatases (Sulfs) leads to a delay in muscle regeneration and a transient increase in SC population, and such phenotypes may be caused by Sulf-mediated repression of FGF2 signaling during SC differentiation. Whether Sulfs play additional roles during skeletal muscle regeneration is unknown. Here preliminary studies show that Sulfs repress the noncanonical Wnt signaling pathway during muscle regeneration and in satellite cell cultures. Since noncanonical Wnt signaling is shown to promote SC expansion by driving the symmetric cell division, we hypothesize that Sulfs regulate the noncanonical Wnt signaling to affect SC population during muscle regeneration. Results of our studies are expected to provide matrix-dependent mechanisms that regulate myogenesis and SC preservation during skeletal muscle regeneration and degenerative diseases. These findings may provide basis for future application of heparan sulfate (HS) and HS-derivatives in stem cell engineering and treatment of muscular degenerative diseases. To investigate such hypothesis, we will utilize a combination of in vitro cell model (single myofibers) and animal models of regeneration by injury and by muscular dystrophy in mice having Sulfs specifically inactivated in the satellite cells Pax7Sulf-DN (Pax7-Cre:ER/Rosa26;eYFP/Sulf1/2flox) and Pax7Sulf-DN;mdx, respectively. We will also utilize lentivirus expressing the dominant negative Sulf2 to acutely inactivate Sulfs.