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，每年有数百万的患者受到骨骼和颅面疾病的影响。确定新基因在颅面和骨骼发育和体内平衡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）蛋白是纤毛形成所必需的，对许多信号成分如Hedgehog和Gli信号的加工至关重要。已知纤毛相关蛋白调节颅面和骨发育，并机械地调节成人的骨稳态。然而，IFT蛋白在这些过程中如何调节OB分化和功能仍然是未知的。IFT 80是一种新发现的IFT蛋白。在人类中，IFFT 80的表达减少导致青少年窒息性胸营养不良和III型短肋多指（趾）畸形，伴有非常严重的骨异常。然而，IFT 80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，IFFT 80在骨生成中起着重要作用。通过使用Osterix-cre删除成骨细胞（OB）特异性谱系中的IFFT 80，我们发现IFFT 80 flox/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以及骨量显著减少。删除IFFT 80损害纤毛形成和OB分化伴随着下调Hh-Gli信号传导活动。最重要的是，在OB分化过程中，IFFT 80的缺失显著抑制Runx 2和BMP 2基因的表达。Gli 2或这两个基因的过表达可以挽救在IFFT 80缺失的细胞中有缺陷的OB分化。因此，我们假设，ift 80是纤毛形成和OB分化所需的，ift 80的缺失导致前OB和成熟OB无法维持其分化阶段，导致细胞对机械转导的异常反应，并改变骨骼发育和稳态。我们将通过三个具体目标来检验这个假设。在目标1中。我们将揭示在OB分化的各个阶段的IFFT 80的体内功能，通过分析骨表型的IFFT 80 flox/flox/Osx-cre和诱导型的IFFT 80 flox/flox/col 1a 1-cre，并确定是否删除的IFFT 80影响机械负荷引起的骨形成。我们将通过比较IFT 80 flox/flox/Osx-cre和IFT 20 flox/flox/Osx-cre小鼠进一步确定不同IFT基因突变是否导致不同的骨表型。在目标2中。我们将在体外阐明的作用和机制，通过在不同阶段的成骨细胞的调控，通过进行研究与IFFT 80 del细胞和慢病毒介导的RNAi和逆转录病毒介导的过表达与或没有流体流切应力刺激。我们将进一步研究IFFT 80调节OB分化和机械感觉功能中的转录因子和关键信号蛋白的机制，特别强调其与Hh，Gli，BMP 2和Runx 2信号传导以及IFFT 80下游基因的相互作用。目标3.我们将通过表征IFFT 80结构域、相互作用蛋白及其功能来剖析赋予纤毛形成和OB分化和功能的IFFT 80相互作用的分子机制。