Regulation of skeletal development and homeostasis by IFT protein

IFT 蛋白对骨骼发育和稳态的调节

• 批准号: 9292979
• 负责人: SHUYING YANG
• 金额: $ 38.63万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9292979
• 关键词: Adult; Affect; BMP2 gene; Binding; Bone Development; Bone Marrow Cells; C-terminal; Calcium; Carrier Proteins; Cells; Cilia; Cilium Microtubule; Clinical; Databases; Disease; Economics; Emotional; Erinaceidae; Family; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Proteins; Gene Expression; Genes; Growth; Health; Homeostasis; Human; In Vitro; Individual; Jeune syndrome; Knockout Mice; Mechanics; Mediating; Medicine; Methods; Molecular; Mus; Mutant Strains Mice; Mutation; Organelles; Osteoblasts; Osteogenesis; Patients; Phenotype; Play; Polydactyly; Process; Proteins; RNA Interference; Regulation; Retroviridae; Role; Signal Transduction; Signaling Protein; Skeletal Development; Staging; Subfamily lentivirinae; Surface; Testing; Tooth structure; WD Repeat; base; bone; bone mass; bone morphogenetic protein 2; craniofacial; craniofacial development; fluid flow; gain of function; in vivo; insight; liquid chromatography mass spectrometry; loss of function; meetings; mouse model; novel; osteoblast differentiation; overexpression; protein protein interaction; response; rib bone structure; shear stress; skeletal; smoothened signaling pathway; transcription factor

DESCRIPTION (provided by applicant): Skeletal and craniofacial diseases affect millions of patients each year in the U.S. Identifying the function and mechanism of the new genes in craniofacial and skeletal development and homeostasis is critical for treatment of these diseases. Intraflagellar transport (IFT) protein is essential for cilia formation and crucial for processing of many signaling components such as Hedgehog and Gli signaling. It is known that Cilia-related proteins regulate craniofacial and bone development and mechanically regulate bone homeostasis in adults. However, how IFT protein regulates OB differentiation and function during those processes remains largely unknown. IFT80 is a newly-identified IFT protein. Reduced expression of IFT80 in humans causes Jeune asphyxiating thoracic dystrophy and short rib polydactyly type III with very severe bone abnormalities. However, the mechanism by which IFT80 functions in skeletal development and homeostasis is unknown. Our in vitro studies suggest that IFT80 plays an important role in osteogenesis. By deleting IFT80 in osteoblast (OB) specific lineage using Osterix-cre, we found that IFT80flox/flox/Osx-cre mice showed apparent growth retardation with severe tooth and craniofacial bone abnormalities and significant decrease in bone mass. Deletion of IFT80 impaired cilia formation and OB differentiation accompanied by down-regulated Hh-Gli signaling activities. Most importantly, deletion of IFT80 significantly inhibited Runx2 and BMP2 gene expression during OB differentiation. Overexpression of Gli2 or these two genes could rescue defective OB differentiation in IFT80 deleted cells. Thus, we hypothesize that IFT80 is required for cilia formation and OB differentiation and that loss of IFT80 results in the inability of pre-OBs and mature OBs to maintain their stage of differentiation, resulting in cells responding abnormally to mechanotransduction and altering skeletal development and homeostasis. We will test the hypothesis through three Specific Aims. In Aim 1. We will reveal the in vivo function of IFT80 at various stages of OB differentiation by analyzing bone phenotypes of IFT80flox/flox/Osx-cre and inducible IFT80flox/flox/col1a1-cre and determining whether deletion of IFT80 affects mechanical loading caused bone formation. We will further define whether mutation of different IFT gene causes different bone phenotype by comparing IFT80flox/flox/Osx-cre and IFT20flox/flox/Osx-cre mice. In Aim 2. We will elucidate in vitro the role and mechanism by which IFT80 regulates of OB differentiation at different stages by performing the studies with IFT80del cells and lentivirus mediated RNAi and retrovirus mediated overexpression with or without fluid flow shear stress stimulation. We will further investigate the mechanism by which IFT80 regulates transcription factors and critical signaling proteins in OB differentiation and mechanosensary function, with particular emphasis on its interplay with Hh, Gli, BMP2 and Runx2 signaling and IFT80 downstream genes. In Aim 3. We will dissect the molecular mechanism of IFT80 interactions that confers cilia formation and OB differentiation and function by characterizing IFT80 structural domains, interacting proteins and their functions.

描述（由申请人提供）：在美国，骨骼和颅面疾病每年影响数百万患者。确定新基因在颅面和骨骼发育及稳态中的功能和机制对于治疗这些疾病至关重要。鞭毛内转运 (IFT) 蛋白对于纤毛形成至关重要，并且对于 Hedgehog 和 Gli 信号等许多信号成分的处理至关重要。众所周知，纤毛相关蛋白调节颅面和骨骼发育，并机械地调节成人的骨骼稳态。然而，IFT 蛋白如何在这些过程中调节 OB 分化和功能仍然很大程度上未知。 IFT80是一种新鉴定的IFT蛋白。 IFT80 在人类中的表达减少会导致青年窒息性胸廓营养不良和 III 型短肋多指畸形，并伴有非常严重的骨异常。然而，IFT80 在骨骼发育和体内平衡中发挥作用的机制尚不清楚。我们的体外研究表明 IFT80 在成骨中发挥重要作用。通过使用 Osterix-cre 删除成骨细胞 (OB) 特异性谱系中的 IFT80，我们发现 IFT80flox/flox/Osx-cre 小鼠表现出明显的生长迟缓，伴有严重的牙齿和颅面骨异常，骨量显着下降。 IFT80 的缺失会损害纤毛形成和 OB 分化，并伴有 Hh-Gli 信号传导活性下调。最重要的是，IFT80 的缺失显着抑制了 OB 分化过程中 Runx2 和 BMP2 基因的表达。 Gli2 或这两个基因的过度表达可以挽救 IFT80 缺失细胞中有缺陷的 OB 分化。因此，我们假设 IFT80 是纤毛形成和 OB 分化所必需的，而 IFT80 的缺失会导致前 OB 和成熟 OB 无法维持其分化阶段，从而导致细胞对力转导反应异常，并改变骨骼发育和体内平衡。我们将通过三个具体目标来检验该假设。在目标1中，我们将通过分析IFT80flox/flox/Osx-cre和诱导型IFT80flox/flox/col1a1-cre的骨表型，并确定IFT80的缺失是否影响引起骨形成的机械负荷，揭示IFT80在OB分化的各个阶段的体内功能。我们将通过比较IFT80flox/flox/Osx-cre和IFT20flox/flox/Osx-cre小鼠，进一步明确不同IFT基因突变是否导致不同的骨表型。在目标2中，我们将通过对IFT80del细胞和慢病毒介导的RNAi和逆转录病毒介导的过表达（有或没有流体流动剪切应力刺激）进行研究，在体外阐明IFT80在不同阶段调节OB分化的作用和机制。我们将进一步研究 IFT80 在 OB 分化和机械感觉功能中调节转录因子和关键信号蛋白的机制，特别强调其与 Hh、Gli、BMP2 和 Runx2 信号以及 IFT80 下游基因的相互作用。在目标 3 中，我们将通过表征 IFT80 结构域、相互作用的蛋白质及其功能来剖析 IFT80 相互作用的分子机制，该相互作用赋予纤毛形成和 OB 分化和功能。