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.

骨骼系统为人体提供了至关重要的结构和功能支持。长期目标的 该项目是要揭示先天性骨骼疾病的发病机理，并制定骨骼的策略 通过阐明管理骨骼发育的关键信号传导机制来再生。早期鼠标 遗传研究发现了Wnt5a在肢体骨骼发育中的关键作用。人类突变 Wnt5a也与常染色体显性robinow综合征（ADR）有关，一种疾病 表现出矮人和宽度骨骼发育不良。在过去的20年中，研究表明 Wnt5a是一种非典型的Wnt配体，它通过受体毛躁（FZ）和共受体ROR1/2 TO信号 激活骨骼形成过程中肢体形态发生的平面细胞极性（PCP）途径。目标 当前的建议是将Wnt5a的几种基本特性表征为有效的形态学，即 例如其信号效力和范围，控制这些特性的结构基础以及如何 人类ADR突变改变了其结构以修改Wnt5a信号传导。目标将通过 特别测试1）在N末端在18AA残差不同的两个Wnt5a同工型可能起作用 PCP信号的不同，以影响肢体发育； 2）WNT5A同工型和ADR突变如何可能 更改WNT5A将FZ和ROR聚集在一起以触发PCP信号传导的模式； 3）如何 ADRS突变改变了Wnt5a在早期肢体发育和内侧方针期间的信号效力和范围 骨形成。在我们的初步研究的基础上，小鼠转基因编码不同的Wnt5a同工型 在肢体中显示出明显不同的功能范围，并且两个同工型在 在Xenopus模型中激活PCP，我们将首先确定其功能范围是否仅确定 通过活动水平差异或其他因素，例如肢体发育过程中的分散能力。 其次，我们将使用一组生化和生物物理测定法来测试基于结构生物学的模型， 在Wnt5a同工型和ADRS变体中，不同的N末端保留在 Wnt5a将FZ和ROR汇集在一起​​，形成具有不同活性的不同类型的配体/受体复合物 水平。第三，我们将使用一组定量功能和分子读数来确定ADR 变体可能会导致爪蟾的PCP信号传导活性升高，以及这些变体如何影响Wnt5a- 小鼠中介导的骨骼发育和骨软骨骨形成。这些研究将阐明 机械地，N末端区域如何调节WNT5A在PCP中的信号转导活动，以及如何改变 在N末端会导致人类ADR的骨骼缺陷。