Role of proteoglycan sulfation during muscle regeneration in dystrophic animals

蛋白多糖硫酸化在营养不良动物肌肉再生过程中的作用

• 批准号: 8650140
• 负责人: Matthew Tierney
• 金额: $ 2.67万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8650140
• 关键词: Adult; Animals; Behavior; Behavioral; Binding; Biological Assay; Biological Availability; Cells; Communication; Complex; Cues; Development; Disease Progression; Donor person; Duchenne muscular dystrophy; Environment; Event; Exhibits; FGF2 gene; Fetal Tissues; Fibroblast Growth Factor; Fibroblast Growth Factor Receptors; Fluorescence-Activated Cell Sorting; Gene Expression; Goals; Growth; Growth Factor; Health; Heparan Sulfate Proteoglycan; Impairment; In Vitro; Intervention; Ligands; Measures; Mediating; Mediator of activation protein; Membrane; Mitogens; Molecular; Monitor; Mus; Muscle; Muscle satellite cell; Natural regeneration; Pathway interactions; Patients; Pattern; Phosphorylation; Play; Post-Translational Protein Processing; Pre-Clinical Model; Proteoglycan; Quality of life; Regulation; Resistance; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Skeletal Muscle; Staging; Stem cells; Sulfatases; Testing; Therapeutic Effect; Transplantation; Wasting Syndrome; Western Blotting; Work; bioluminescence imaging; cell behavior; extracellular; fetal; functional disability; human disease; improved; in vivo; innovation; insight; mouse model; muscle regeneration; myogenesis; neutralizing antibody; new therapeutic target; pre-clinical; prospective; receptor; regenerative; repaired; response; self-renewal; sulfation; syndecan; therapy design; tool; wasting

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.

描述（由申请人提供）：肌肉干细胞（MuSC）是骨骼肌发育和修复所必需的。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进行自我更新。这种能力在杜氏肌营养不良症（DMD）中逐渐受损，这是一种无法治愈的毁灭性肌肉萎缩疾病。DMD的特点是微环境信号改变，对musc介导的再生潜能产生负面影响。为了更好地了解MuSC如何与其细胞外环境进行交流，我探索了几种信号转导的膜结合介质。硫酸肝素蛋白聚糖（HSPG），特别是syndecans，在MuSC中高表达，可以调节配体的生物利用度和受体的形成。通过翻译后修饰在HSPG上的异质硫酸化模式增加了行为复杂性，导致关键信号通路的差异活性。因为成人的再生概括了肌肉发生的几个方面，我把我的初步研究集中在HSPG硫酸盐在胎儿MuSC中的作用上，这是成人MuSC的发育前体。我已经证明，在胎儿MuSC中，HSPG 6- o -硫酸化的特定调节因子Sulf1被下调。我提供的证据表明，胎儿MuSC在体外对肌源性承诺有抵抗力，并且能够在体内快速扩张和移植后长期自我更新。此外，未确定的胎儿MuSC对有效的有丝分裂原FGF2优先敏感，而对Wnt/β-连环蛋白介导的肌源性分化反应较弱。事实上，先前已经证明Sulf1可以同时促进Wnt/β-catenin并抑制FGF2信号传导。Sulf1和HSPG在肌肉萎缩状态下表达失调。因此，本项目的目的是研究HSPG 6- o -硫酸化对营养不良的MuSC自我更新和再生潜能的调节作用。为了实现这一目标，我建议（Aim 1a）验证HSPG硫酸酯酶、FGF和Wnt/β-连环蛋白信号成分在肌发生和营养不良动物中的表达水平。为了确定MuSC对FGF和Wnt/β-catenin通路激活（Aim 1b）的响应性，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 -硫酸化将确定其在FGF受体复合物形成和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1 c)。我将确定硫酸抑制对营养不良、肌纤维相关的MuSC自我更新的影响（Aim 2a）。最后，我将评估HSPG 6- o -磺化在DMD临床前模型中的功能作用，通过抑制（Aim 2b）移植的、供体的MuSC和（Aim 2c）原生的、营养不良的MuSC中Sulf1的表达。通过这些研究，我将研究一种分子机制，HSPG 6- o -硫酸化，能够调节营养不良动物的FGF和Wnt/β-catenin信号和MuSC再生潜力。这项工作将潜在地确定f1作为一种新的治疗靶点，以增强MuSC自我更新和改善DMD。