Deciphering the role of CFTR in osteoblasts

背景：囊性纤维化跨膜传导调节因子(CFTR)是一种定位于上皮细胞顶膜上的蛋白质，它通过将氯离子转运出细胞而促进其分泌功能。尽管cftr作为离子通道的生理作用是毋庸置疑的，但最近的证据表明，cftr在非上皮性细胞，如成骨细胞中的表达，增加了它除了调节通道活性之外，可能还有其他作用的可能性。事实上，CFTR在成骨细胞中的点状分布，与在上皮细胞中主要观察到的顶端定位形成对比，强烈暗示了不同的细胞功能。此外，在不同类型细胞中的研究表明，CFTR通过其PDZ结合基序与其他蛋白质进行物理相互作用，并且这种相互作用组可能调节参与细胞增殖和分化的下游信号通路。尽管有这些观察，CFTR在成骨细胞中的功能一直没有得到彻底的研究，而且仍然不清楚。因此，我们的长期目标是研究CFTR在成骨细胞中的功能。*假设：根据我们的初步数据，CFTR的表达随着成骨细胞的分化而增加，我们假设CFTR通过与细胞内伙伴的物理相互作用和下游信号通路的调节，抑制成骨细胞的细胞周期进程，促进成骨细胞的分化。*实验方法和具体目的：利用处于不同分化阶段的小鼠成骨细胞，结合分子生物学、蛋白质生物化学、荧光显微镜和蛋白质组学的实验方法，我们将追求以下具体目标：*目的1.研究成骨细胞中β-catenin是否与cftr相互作用；*目的2.确定cftr调控的信号通路，参与成骨细胞的增殖和分化；*目的3.通过cftr相互作用组图寻找新的cftr在成骨细胞中相互作用的伙伴。*影响：总的来说，拟议的实验将通过建立CFTR通过与其他蛋白质的相互作用和下游信号通路的调节来调节细胞分化，从而促进我们对一个基本的细胞过程，即成骨细胞分化的理解。我们相信，这个项目将为CFTR在控制骨骼稳态方面的新功能提供相当大的帮助。**

Background: The cystic fibrosis transmembrane conductance regulator (CFTR) is a protein localised on the apical membrane of epithelial cells where it contributes to their secretory functions by transporting chloride out of the cells. While the physiological role of CFTR as an ion channel is indisputable, recent evidence showing CFTR expression in non-epithelial cells, such as osteoblasts, raises the possibility that it, in addition to channel activity regulation, might have additional roles, apart from its channel activity. Indeed, the punctate intracellular distribution of CFTR in osteoblasts, contrasting with the predominantly apical localization observed in epithelia, is strongly suggestive of distinct cellular functions. Furthermore, work performed in various cell types has revealed that CFTR interacts physically with other proteins through its PDZ-binding motif and that such interactome may regulate downstream signalling pathways involved in cell proliferation and differentiation. Despite these observations, the function of CFTR in osteoblasts has never been thoroughly investigated and remains unclear. Our long-term objective is thus to investigate the function of CFTR in osteoblasts. ******Hypothesis: Guided by our preliminary data showing that CFTR expression increases with osteoblast differentiation, we postulate that CFTR, through its physical interaction with intracellular partners and regulation of downstream signalling pathways, inhibits osteoblast cell-cycle progression and facilitates osteoblast differentiation. ******Experimental approach and Specific Aims: Using murine osteoblasts at various stages of differentiation and experimental approaches combining molecular biology, protein biochemistry, fluorescent microscopy and proteomics, we will pursue the following specific aims:***Aim 1. To investigate whether β-catenin interacts with CFTR in osteoblasts;***Aim 2. To identify CFTR-regulated signalling pathways involved in osteoblast proliferation and differentiation;***Aim 3. To identify new interacting partners of CFTR in osteoblasts through the mapping of the CFTR interactome. ******Impact: Collectively, the proposed experiments will advance our understanding of a fundamental cellular process, namely osteoblast differentiation, by establishing that the CFTR, through interactions with other proteins and the regulation of downstream signalling pathways, regulates cell differentiation. We believe this project will shed considerable light on a new function for the CFTR in the control of bone homeostasis.**