The role of IFT80 in bone formation

描述(申请人提供)：鞭毛内转运(IFT)蛋白是以微管为基础的运输机械，对所有纤毛和鞭毛的组装和维护至关重要。最近的发现不仅揭示了纤毛和鞭毛在运动、感觉接收和信号传递中的各种作用，而且还揭示了IFT在控制基因调控和表达、细胞增殖和分化以及动物发育和行为方面的功能。IFT蛋白的突变会导致各种生物体中纤毛和鞭毛的丢失或严重减少，从而导致许多人类疾病，其特征是肾脏、视网膜和骨骼的各种病理变化。有条件地消融IFT88会扰乱刺猬信号，出现多指和软骨内骨形成缺陷。Kif3a是Kinesin II的一个亚单位，破坏显示出肢体和颅骨的异常。最近，在软骨外胚层发育不良Ellis-van Creveld综合征(EVC)中发现了一种新的突变蛋白定位于纤毛底部，该基因在小鼠身上的破坏导致了各种骨骼和颅面的异常 以及牙齿和指甲的变化。靶向缺失IFT/纤毛的小鼠有广泛的 各种骨表型，提供了对单个IFT蛋白在骨形成中的作用的有趣见解。最近，Beales等人发现，IFT复合体B的新成分IFT80在人类体内的部分缺失会导致人类疾病，如Jeune窒息胸廓营养不良(JATD)和III型短肋骨多指(SRP)。这两种疾病都有严重的骨骼异常，包括长骨缩短和胸腔狭窄。然而，目前尚不清楚IFT80突变是如何导致骨骼异常的。我们最近的研究表明，IFT80在成骨细胞分化过程中高表达。沉默IFT80不仅损害纤毛的形成，而且通过下调成骨细胞标志基因Runx2和Osteocalin以及HH相关基因shh、ihh和Gli2的表达而显著抑制成骨细胞的分化。这些都是重要的结果；然而，仍然没有体内证据表明在成骨细胞分化和骨形成过程中普遍需要IFT80信号。最近，我们在成骨细胞特异性谱系中建立了IFT80条件性基因敲除模型，发现IFT80突变小鼠表现出明显的生长迟缓和严重的骨异常。根据这些结果，我们推测IFT80通过调节成骨细胞基因的表达、分化和HH/Gli途径，在脊椎动物的骨形成和正常的骨功能中发挥重要作用。在这项建议中，我们将通过产生IFT80条件基因敲除等位基因来检验这一假说，以研究IFT80在骨形成中的作用，并研究该基因缺失对成骨细胞基因表达、分化和增殖的影响。本工作的长期目标是了解IFT/纤毛蛋白在骨发育和骨疾病中的作用机制和相互作用。 公共卫生相关性：项目叙述IFT80在骨骼形成和维护中的作用和机制尚不清楚。利用条件遗传策略研究IFT80在体内骨形成、成骨细胞基因表达、分化和增殖中的作用，将有助于我们更好地理解骨形成和人类IFT相关骨病的发病机制，并将这些知识应用于开发这些疾病的新诊断和治疗替代方案。

DESCRIPTION (provided by applicant): Intraflagellar transport (IFT) proteins are microtubule based transport machinery, which are essential for the assembly and maintenance of all cilia and flagella. Recent findings have not only revealed the various roles of cilia and flagella in motility, sensory reception, and signaling, but also demonstrated the function of IFT in the control of gene regulation and expression, cell proliferation and differentiation, and animal development and behavior. The mutation of IFT proteins causes the loss or severe reduction of cilia and flagella in various organisms, which can lead to numerous human diseases characterized by various combinations of pathological changes of kidney, retina, and skeleton. Conditional ablation of IFT88 disrupts hedgehog signaling, with polydactyly and defects of endochondral bone formation. Disruption of Kif3a, a subunit of kinesin II, showed limb and cranial skeletal abnormalities. Recently, a novel protein mutated in chondroectodermal dysplasia Ellis-van Creveld syndrome (EVC) was found to be localized to the base of the cilia, and disruption of this gene in mice results in a variety of skeletal and craniofacial abnormalities as well as alterations in the teeth and nails. Mice with targeted deletion of IFT/cilia have a wide variety of bone phenotypes that provide interesting insights into the function of individual IFT protein in bone formation. Most recently, Beales et al group found that the partial loss of IFT80 in human, a novel component of the IFT complex B, causes human diseases such as Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly (SRP) type III. Both diseases have severe bone abnormalities including shortening of the long bones and constriction of the thoracic cage. However, it is unclear how IFT80 mutation leads to skeletal abnormalities. Our recent studies have shown that IFT80 is highly expressed during osteoblast differentiation. Silencing IFT80 not only impairs cilia formation, but also significantly inhibits osteoblast differentiation through down-regulating the expression of osteoblast marker genes- Runx2 and osteocalcin, and hedgehog signaling (Hh) related genes -shh, Ihh and Gli2. These are important results; nonetheless, there is still no in vivo evidence for a general requirement for IFT80 signaling in osteoblast differentiation and bone formation. Most recently, we have generated IFT80 conditional knockout model in osteoblast specific lineage and found that IFT80 mutant mice showed apparent growth retardation with severe bone abnormalities. Based on these results, we hypothesize that IFT80 plays an essential role in vertebrate bone formation and normal bone function through regulating osteoblast gene expression, differentiation and Hh/Gli pathway. In this proposal, we will test the hypothesis by generating an IFT80 conditional knockout allele to study the role of IFT80 in bone formation and investigating the effect of deletion of this gene on osteoblast gene expression, differentiation and proliferation. The long term objective of this work is to understand the functionmechanism and interactions of IFT/cilia proteins in bone development and bone diseases. PUBLIC HEALTH RELEVANCE: Project Narrative The role and mechanism of IFT80 in bone formation and maintenance is unknown. Studying the role of IFT80 in bone formation, osteoblast gene expression, differentiation and proliferation in vivo by using conditional genetic strategies will help us to better understand bone formation and the mechanisms responsible for human IFT-related bone diseases and to apply this knowledge towards the development of new diagnostic and therapeutic alternatives for these diseases.