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(申请人提供)：在美国，骨骼和颅面部疾病每年影响数百万患者。识别新基因在颅面和骨骼发育和动态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，也是许多信号成分如Hedgehog和Gli信号处理的关键。已知纤毛相关蛋白调节成人的头面部和骨骼发育，并机械地调节骨骼的动态平衡。然而，IFT蛋白如何在这些过程中调节OB的分化和功能在很大程度上仍不清楚。IFT80是一种新发现的IFT蛋白。在人类中，IFT80的表达减少会导致Jeune窒息的胸部营养不良和III型短肋骨多指畸形，并伴有非常严重的骨骼异常。然而，IFT80在骨骼发育和动态平衡中的作用机制尚不清楚。我们的体外研究表明，IFT80在成骨过程中起着重要作用。通过使用Osterix-cre技术删除成骨细胞(OB)特异性谱系中的IFT80，我们发现IFT80/FLOX/OSX-cre小鼠表现出明显的生长迟缓，并伴有严重的牙齿和颅面骨异常，骨量显著减少。IFT80缺失阻碍纤毛形成和OB分化，并伴随HH-Gli信号活性下调。最重要的是，IFT80的缺失显著抑制了OB分化过程中Runx2和BMP2基因的表达。过表达Gli2或这两个基因可以挽救IFT80缺失细胞的OB分化缺陷。因此，我们假设IFT80是纤毛形成和OB分化所必需的，而IFT80的缺失导致前OB和成熟OB无法维持其分化阶段，从而导致细胞对机械转导的异常反应，改变骨骼发育和动态平衡。我们将通过三个具体目标来检验这一假设。目的1.通过分析IFT80/FLOX/OSX-cre和诱导型IFT80/FLOX/col1a1-cre的骨表型，确定IFT80的缺失是否影响机械负荷引起的骨形成，揭示IFT80在成骨细胞分化不同阶段的体内作用。我们将通过比较IFT80FLOX/FLOX/OSX-cre和IFT20FLOX/FLOX/OSX-cre小鼠来进一步确定不同IFT基因突变是否会导致不同的骨表型。目的2.通过对IFT80del细胞、慢病毒介导的RNAi和逆转录病毒介导的过度表达在有或没有流体切应力刺激下的研究，在体外阐明IFT80在不同阶段调控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，特别是它与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的结构域、相互作用的蛋白质及其功能来剖析IFT80相互作用导致纤毛形成、OB分化和功能的分子机制。