Investigation of Sulf regulation on the noncanonical Wnt signaling pathway

描述(申请人提供)：肌肉再生是一个复杂的过程，涉及多种因素，在空间和时间上决定着被称为卫星细胞(SC)的骨骼肌干细胞的激活、增殖、迁移和分化，以及新生成肌细胞(已分化SC)的融合。因此，了解参与肌肉再生每个单独阶段的调控因素将为设计更好的肌肉营养不良和萎缩等肌肉相关疾病的治疗方法开辟新的途径。我们先前的研究表明，硫酸乙酰肝素-6-O-内切酶(Sulf)的缺失会导致肌肉再生的延迟和SC数量的一过性增加，这种表型可能是由于Sulf在SC分化过程中介导的FGF2信号的抑制所致。目前尚不清楚Sulf在骨骼肌再生过程中是否发挥了额外的作用。在这里，初步研究表明，在肌肉再生和卫星细胞培养中，Sulf抑制非典型的Wnt信号通路。由于非典范的Wnt信号被证明通过驱动对称的细胞分裂来促进SC的扩张，我们假设Sulf调控非典范的Wnt信号来影响肌肉再生过程中的SC群体。我们的研究结果有望提供在骨骼肌再生和退行性疾病期间调节肌肉生成和SC保存的基质依赖机制。这些发现可能为未来硫酸肝素(HS)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。为了研究这一假说，我们将利用体外细胞模型(单肌纤维)和动物模型的组合，分别在卫星细胞Pax7Sulf-Dn(Pax7-CRE：ER/rosa26；EYFP/Sulf1/2flx)和Pax7Sulf-Dn；MDX中特异性失活Sulf的小鼠损伤和肌肉营养不良再生。我们还将利用表达显性负Sulf2的慢病毒来迅速灭活Sulf。

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.