Mechanism of Wnt5a signaling in skeletal development and diseases

Wnt5a信号在骨骼发育和疾病中的机制

• 批准号: 10683310
• 负责人: Rosa A. Serra
• 金额: $ 62.41万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10683310
• 关键词: Acute Respiratory Distress Syndrome; Amino Acids; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Biophysics; Cell Maintenance; Complex; Cysteine; Cysteine-Rich Domain; Data; Defect; Development; Digit structure; Dimerization; Disease; Distal; Dose; Dwarfism; Engineering; Etiology; Family; Family member; Foundations; Frizzled Domain; Future; Genetic; Genetic study; Homeostasis; Human; Knowledge; Ligand Binding; Ligands; Limb Bud; Limb Development; Limb structure; Malignant Neoplasms; Mammals; Measures; Mediating; Modeling; Molecular; Morphogenesis; Mus; Mutation; N-terminal; Natural regeneration; Nature; Osteogenesis; Output; Pathogenesis; Pathogenicity; Pathway interactions; Pattern; Process; Property; Protein Isoforms; Proteins; Regenerative Medicine; Robinow syndrome; Role; Series; Signal Transduction; Signaling Molecule; Skeletal Development; Skeletal system; Skeleton; Structure; Syndrome; Testing; Therapeutic Intervention; Thumb structure; Tissues; Transgenes; Transgenic Mice; Variant; WNT5A gene; WNT5A protein; Xenopus; autosome; congenital skeletal disorder; convergent extension; design; disulfide bond; gain of function; insight; long bone; morphogens; mouse genetics; mutant; planar cell polarity; predictive modeling; receptor; skeletal; skeletal disorder; skeletal dysplasia; skeletal regeneration; stem cells; structural biology

The skeletal system provides crucial structural and functional support to the body. The long-term objective of this project is to uncover the pathogenesis of congenital skeletal disorders and develop strategies for skeletal regeneration by elucidating the key signaling mechanisms governing skeletal development. Early mouse genetic studies have uncovered a critical role of Wnt5a in limb skeletal development. Human mutations in WNT5A have also been implicated in Autosomal Dominant Robinow syndrome (ADRS), a disorder that manifests dwarfism and widespread skeletal dysplasia. Studies over the past 20 years have revealed that Wnt5a is a non-canonical Wnt ligand that signals through receptor Frizzled (Fz) and co-receptors Ror1/2 to activate the planar cell polarity (PCP) pathway for limb morphogenesis during skeletal formation. The objective of the current proposal is to characterize several fundamental properties of Wnt5a as a potent morphogen, such as its signaling potency and range, the structural foundation that governs these properties, and how human ADRS mutations alter its structure to modify WNT5A signaling. The objective will be achieved by specifically testing 1) how two Wnt5a isoforms that differ by 18aa residues at the N-terminus may function differently in PCP signaling to impact limb development; 2) how the Wnt5a isoforms and ADRS mutations may alter the mode through which Wnt5a clusters together Fz and Ror to trigger PCP signaling; and 3) how the ADRS mutations alter Wnt5a’s signaling potency and range during early limb development and endochondral bone formation. Building on our preliminary studies that mouse transgenes encoding different Wnt5a isoforms display remarkably different functional range in the limb, and that the two isoforms display different potency in activating PCP in the Xenopus model, we will first determine whether their function range is solely determined by the activity level difference, or additional factors such as dispersal ability during limb development. Secondly, we will use a set of biochemical and biophysical assays to test a structural biology based model, in which different N-terminal residues in Wnt5a isoforms and ADRS variants may alter the mode through which Wnt5a brings together Fz and Ror to form distinct type of ligand/receptor complexes with different activity levels. Thirdly, we will use a set of quantitative functional and molecular readout to determine how the ADRS variants may cause elevated PCP signaling activity in Xenopus, and how these variants impact Wnt5a- mediated skeletal development and endochondral bone formation in the mouse. These studies will elucidate mechanistically how the N-terminal region may regulate Wnt5a’s signaling activity in PCP, and how alterations in the N-terminus leads to skeletal defects in ADRS in humans.

骨骼系统为身体提供重要的结构和功能支持。的长期目标是 该项目旨在揭示先天性骨骼疾病的发病机制，并制定骨骼疾病的治疗策略。 通过阐明控制骨骼发育的关键信号机制来实现再生。早起的老鼠 遗传学研究揭示了Wnt5a在肢体骨骼发育中的关键作用。人类基因突变 Wnt5A还与常染色体显性遗传性Robinow综合征(ADRS)有关，这种疾病 表现为侏儒症和广泛性骨骼发育不良。过去20年的研究表明， Wnt5A是一种非正则的Wnt配体，通过受体FrizzledFz和辅受体Ror1/2传递信号 在骨骼形成过程中，激活四肢形态发生的平面细胞极性(PCP)途径。目标是 本提案的目的是表征WNT5a作为一种有效的致形剂的几个基本性质， 例如它的信号效力和范围，管理这些属性的结构基础，以及如何 人类ADRs突变会改变其结构，从而改变Wnt5A信号。目标将通过以下方式实现 具体测试1)N端18aa残基不同的两个WNT5a亚型如何发挥作用 影响肢体发育的PCP信号的不同；2)WNT5a亚型和ADRS突变如何可能 改变WNT5a将FZ和ROR聚集在一起以触发PCP信令的模式；以及3)如何 ADRs突变改变Wnt5a在肢体早期发育和软骨内的信号效力和范围 骨形成。根据我们的初步研究，编码不同Wnt5a亚型的小鼠转基因 在肢体中表现出明显不同的功能范围，这两种异构体在 在非洲爪哇模型中激活PCP时，我们将首先确定它们的功能范围是否单独确定 活动水平的差异，或肢体发育过程中的其他因素，如分散能力。 其次，我们将使用一套生化和生物物理测试来测试一个基于结构生物学的模型，在 WNT5a异构体和ADRs变异体中哪些不同的N末端残基可能改变 WNT5A将FZ和ROR结合在一起，形成具有不同活性的不同类型的配体/受体复合体 级别。第三，我们将使用一组定量的功能和分子读数来确定ADRs是如何 变异可能导致非洲爪哇PCP信号活性升高，以及这些变异如何影响Wnt5a- 介导小鼠骨骼发育和软骨内骨形成。这些研究将阐明 N-末端区域如何调节PCP中Wnt5a的信号活性，以及如何改变 在人类的ADR中，N端的突变导致骨骼缺陷。