Phospho-ERK1/2 in beta-catenin-dependent cranial lineage restriction

β-连环蛋白依赖性颅谱系限制中的磷酸化 ERK1/2

• 批准号: 9794645
• 负责人: Beatriz Adrianna Ibarra
• 金额: $ 3.22万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2021-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9794645
• 关键词: Adult; Biological Assay; Bone Development; Bone Growth; Bone Regeneration; Calvaria; Cartilage; Cells; Cephalic; Chemicals; Chondrogenesis; Communities; Complex; Congenital Abnormality; Craniofacial Abnormalities; Data; Defect; Dermal; Development; Disease; Embryo; Embryonic Development; Etiology; Genetic; Genetic screening method; In Vitro; Infection; Knock-out; Lead; Limb Bud; Link; Live Birth; MAPK3 gene; MEK inhibition; MEKs; Malignant Neoplasms; Mediating; Mesenchyme; Methods; Morbidity - disease rate; Mus; Newborn Infant; Operative Surgical Procedures; Osteogenesis; Pathway interactions; Phosphorylation; Physiologic calcification; Preventive treatment; Process; Repression; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Testing; Tissues; United States; WNT Signaling Pathway; Wild Type Mouse; beta catenin; bone; bone cell; bone loss; cell type; craniofacial; craniofacial development; craniofacial disorder; cranium; improved; in vivo; insight; long bone; loss of function; mouse model; new therapeutic target; overexpression; partial recovery; small molecule inhibitor; therapeutic target; vector

Project Summary/Abstract Approximately 10,000 live births have defects in the craniofacial region in the United States annually. Severe craniofacial defects can result in lethality while less severe defects lead to morbidity and most surgical treatments only result in partial recovery. Identifying signaling factors that regulate cell fate decisions in craniofacial development is necessary for understanding the etiology of birth defects and identification of therapeutic targets. The Wnt/β-catenin signaling pathway functions in cell fate selection during development and cancer. Our lab has demonstrated that loss of Wnt/-catenin signaling in the cranial mesenchyme results in a cell fate switch from skull bone to cartilage. How Wnt signaling promotes bone and dermal fate while inhibiting alternative cell fates in the cranial mesenchyme remains unclear. Previous studies have demonstrated that Erk1/2 activation is required for endochondral bone formation and growth as well as repression of cartilage differentiation during embryonic development. My preliminary data suggests that phosphorylation of Erk1/2 is diminished in the absence of -catenin in cranial mesenchyme at E12.5. I hypothesize that Wnt/β-catenin signaling represses cartilage and promotes skull bone formation through activation of Erk1/2 signaling in mice. To test this hypothesis I propose the following aims. 1) Determine if loss of phospho-Erk1/2 promotes cranial mesenchyme differentiation to cartilage. Here I propose to utilize two methods to inhibit Erk signaling in vivo, including genetic loss of Erk1/2 using En1Cre and chemical inhibition of Erk1/2 phosphorylation and assay for ectopic cartilage formation in the cranial mesenchyme at E12.5. Additionally I will overexpress Erk1/2 activation using a CA-Mek lentiviral infection in En1Cre;-catfl/del calvarial mesenchyme in vitro. 2) Test whether phosphorylation of Erk1/2 is dependent on -catenin in the cranial mesenchyme. First I will demonstrate that loss of -catenin in cranial mesenchyme results in diminished Erk1/2 phosphorylation and determine whether this effect is due to Wnt signaling. Additionally, genetic interaction of -catenin and Erk1/2 will be determined by crossing En1Cre;- catfl/del and Erk1-/-; Erk2fl/fl mice to obtain triple heterozygous loss-of-function embryos. Demonstrating a functional link between these two signaling pathways in regulating skull bone cell fate decisions in vivo will improve our understanding of normal skull bone development and associated birth defects, and more broadly, provide new avenues for bone repair in adult tissues. Successful completion of this proposal will provide insights into how Wnt/-catenin signaling regulates cell fate decisions during cranial bone development and can be applied towards cell fate decisions in multiple cell types. Identifying new targets of Wnt signaling in bone development and cell fate could provide options for new therapeutic targets or preventative treatments of craniofacial disorders and would be applicable to a larger community that studies Wnt signaling and cell fate in other disease contexts, including cancer.

项目概要/摘要 在美国，每年约有 10,000 名活产婴儿存在颅面部缺陷。严重 颅面缺陷可能导致致命，而不太严重的缺陷则导致发病，大多数手术治疗 仅导致部分恢复。识别调节颅面部细胞命运决定的信号因子 发展对于了解出生缺陷的病因和确定治疗靶点是必要的。 Wnt/β-catenin 信号通路在发育和癌症过程中的细胞命运选择中发挥作用。我们实验室有 证明颅内间充质中 Wnt/-catenin 信号传导的丧失会导致细胞命运从 头骨到软骨。 Wnt 信号如何促进骨骼和真皮命运，同时抑制其他细胞命运 在颅骨间质中的作用仍不清楚。先前的研究表明，Erk1/2 的激活是 软骨内骨形成和生长以及抑制软骨分化所需的 胚胎发育。我的初步数据表明 Erk1/2 的磷酸化在 E12.5 的颅间质中缺乏 -连环蛋白。我假设 Wnt/β-连环蛋白信号传导抑制 通过激活小鼠体内的 Erk1/2 信号传导，促进软骨形成并促进颅骨形成。为了检验这个假设 我提出以下目标。 1) 确定磷酸-Erk1/2 的缺失是否会促进颅间充质 分化为软骨。在这里我建议利用两种方法来抑制体内的Erk信号，包括遗传方法 使用 En1Cre 丢失 Erk1/2 并化学抑制 Erk1/2 磷酸化和异位软骨测定 在 E12.5 处颅骨间质中形成。此外，我将使用 CA-Mek 过度表达 Erk1/2 激活 En1Cre;-catfl/del 颅骨间充质体外慢病毒感染。 2）测试Erk1/2是否磷酸化 依赖于颅内间充质中的 -连环蛋白。首先，我将证明颅脑中 -连环蛋白的丢失 间充质导致 Erk1/2 磷酸化减弱，并确定这种效应是否由 Wnt 引起 发信号。此外，-连环蛋白和 Erk1/2 的遗传相互作用将通过 En1Cre 杂交来确定；- catfl/del 和 Erk1-/-； Erk2fl/fl 小鼠获得三重杂合的功能丧失胚胎。展示功能性 这两种信号通路在调节体内颅骨细胞命运决定方面的联系将改善我们的研究 了解正常颅骨发育和相关出生缺陷，更广泛地说，提供新的 成人组织中骨修复的途径。该提案的成功完成将提供有关如何 Wnt/-连环蛋白信号调节颅骨发育过程中的细胞命运决定并可应用 多种细胞类型的细胞命运决定。鉴定骨骼发育中 Wnt 信号传导的新靶点 细胞命运可以为颅面疾病的新治疗靶点或预防性治疗提供选择 并将适用于研究其他疾病背景下的 Wnt 信号传导和细胞命运的更大社区， 包括癌症。