Role of proteoglycan sulfation during muscle regeneration in dystrophic animals

描述(申请人提供)：骨骼肌的发育和修复需要肌肉干细胞(MUSC)。在这些肌肉生长和再生的动态时期，MUSC必须选择性地解释一系列信号分子来自我更新。这种能力在Duchenne肌营养不良症(DMD)中逐渐受损，Duchenne肌营养不良症是一种毁灭性的肌肉萎缩疾病，目前还没有治愈的方法。DMD的特征是微环境信号的改变，对MUSC介导的再生潜力产生负面影响。为了更好地了解MUSC如何与其细胞外环境进行通信，我探索了几种膜结合的信号转导介体。硫酸乙酰肝素蛋白多糖(HSPG)，尤其是合成酶，在MUSC中高表达，并能调节配体的生物利用度和受体的形成。HSPG上的不同硫化模式通过翻译后修饰增加了行为的复杂性，导致了关键信号通路的不同活动。因为成人的再生概括了肌肉发生的几个方面，所以我的初步研究集中在胎儿MUSC中的HSPG硫酸盐化，这是成人MUSC的发育前体。我已经证明了HSPG 6-O-硫化的特异性调节因子Sulf1在胎儿MUSC中表达下调。我提供的证据表明，胎儿MUSC在体外对肌源性承诺具有抵抗力，能够在体内快速扩张，并在移植后长期自我更新。此外，未融合的胎儿骨髓间充质干细胞对强大的有丝分裂原FGF2优先敏感，而对Wnt/β-Catenin介导的肌源性分化反应较差。事实上，Sulf1以前已经被证明同时促进Wnt/β-catenin和抑制FGF2信号转导。在肌肉萎缩的情况下，Sulf1和HSPG的表达是失调的。因此，本项目的目标是研究HSPG 6-O-硫酸盐在营养不良的MUSC自我更新和再生潜能调节中的作用。为了实现这一点，我建议(目标1a)验证HSPG硫酸酯酶和成纤维细胞生长因子以及Wnt/β-catenin信号成分在整个肌肉发生和营养不良动物中的表达水平。为了确定骨髓间充质干细胞对成纤维细胞生长因子和Wnt/β-连环蛋白通路激活的反应性(目标1b)，我将测量下游信号通路效应物的表达和磷酸化水平。抑制HSPG 6-O-硫化将确定其在成纤维细胞生长因子受体复合体形成和WNT3a配体生物利用度中的作用，从而确定HSPG调节适当途径激活的机制。(目标1c)。我将确定Sulf1抑制对营养不良、肌纤维相关的MUSC自我更新的影响(目标2a)。最后，我将通过抑制(Aim 2b)移植、供体MUSC和(Aim 2c)原生营养不良MUSC中Sulf1的表达，来评估HSPG 6-O-磺化在DMD临床前模型中的功能作用。通过这些研究，我将研究HSPG6-O-硫化的分子机制，它能够调节营养不良动物成纤维细胞生长因子和WNT/β-连环蛋白信号和骨髓间充质干细胞的再生能力。这项工作将可能确定Sulf1作为促进MUSC自我更新和改善DMD的新的治疗靶点。

DESCRIPTION (provided by applicant): Muscle stem cells (MuSC) are required for the development and repair of skeletal muscle. During these dynamic periods of muscle growth and regeneration, MuSC must selectively interpret a host of signaling molecules to self-renew. This ability is progressively impaired in Duchenne muscular dystrophy (DMD), a devastating muscle wasting disease for which there is no cure. DMD is characterized by altered microenvironmental signaling that negatively influences MuSC-mediated regenerative potential. To better understand how MuSC communicate with their extracellular environment, I explored several membrane-bound mediators of signal transduction. Heparan sulfate proteoglycans (HSPG), in particular syndecans, are highly expressed in MuSC and can regulate ligand bioavailability and receptor formation. Heterogeneous sulfation patterns on HSPG via post-translational modifications increase behavioral complexity, leading to a differential activity of critical signalng pathways. Because regeneration in the adult recapitulates several aspects of myogenesis, I focused my preliminary studies on HSPG sulfation in fetal MuSC, the developmental precursors of adult MuSC. I have demonstrated that Sulf1, a specific regulator of HSPG 6-O-sulfation, is downregulated in fetal MuSC. I provide evidence that fetal MuSC are resistant to myogenic commitment in vitro and capable of rapid in vivo expansion and long-term self-renewal following transplantation. Furthermore, uncommitted fetal MuSC are preferentially sensitive to the potent mitogen FGF2 while less responsive to Wnt/β-catenin-mediated myogenic differentiation. Indeed, Sulf1 has been previously shown to simultaneously promote Wnt/β-catenin and inhibit FGF2 signaling. Sulf1 and HSPG expression are dysregulated in muscle wasting conditions. Therefore, the goal of this project is to examine the role of HSPG 6-O-sulfation on the regulation of dystrophic MuSC self-renewal and regenerative potential. To accomplish this, I propose (Aim 1a) to validate HSPG sulfatase and FGF and Wnt/β-catenin signaling component expression levels throughout myogenesis and in dystrophic animals. To determine MuSC responsiveness to FGF and Wnt/β-catenin pathway activation (Aim 1b), I will measure the expression and phosphorylation levels of downstream signaling pathway effectors. Inhibition of HSPG 6-O-sulfation will ascertain its role in FGF receptor complex formation and Wnt3a ligand bioavailability, defining the mechanisms underlying HSPG regulation of proper pathway activation. (Aim 1c). I will determine the impact of Sulf1 inhibition on dystrophic, myofiber-associated MuSC self-renewal (Aim 2a). Finally, I will evaluate the functional role of HSPG 6-O-sulfation in a preclinical model of DMD by inhibiting Sulf1 expression in (Aim 2b) transplanted, donor MuSC and (Aim 2c) native, dystrophic MuSC. Through these studies I will investigate a molecular mechanism, HSPG 6-O-sulfation, able to regulate FGF and Wnt/β-catenin signaling and MuSC regenerative potential in dystrophic animals. This work will potentially identify Sulf1 as a novel therapeutic target for the enhancement of MuSC self-renewal and the amelioration of DMD.