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，它通过将氯离子运输出细胞来促进上皮细胞的分泌功能。虽然CFTR作为离子通道的生理作用是无可争议的，但最近有证据表明，CFTR在非上皮细胞（如成骨细胞）中表达，这提出了一种可能性，即除了通道活性调节外，CFTR可能还有其他作用。事实上，成骨细胞中CFTR的点状细胞内分布，与上皮细胞中观察到的主要顶端定位形成鲜明对比，强烈暗示了不同的细胞功能。此外，在各种细胞类型中进行的研究表明，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。尽管有这些观察结果，CFTR在成骨细胞中的功能从未被彻底研究过，并且仍然不清楚。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表达随着成骨细胞分化而增加，我们假设CFTR通过与细胞内伴侣的物理相互作用和调节下游信号通路，抑制成骨细胞周期进程，促进成骨细胞分化。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，采用分子生物学、蛋白质生物化学、荧光显微镜和蛋白质组学相结合的实验方法，实现以下具体目的：探讨-catenin在成骨细胞中是否与CFTR相互作用；目标2。研究cftr调控的成骨细胞增殖和分化信号通路；目标3。通过CFTR相互作用组的定位，确定成骨细胞中CFTR的新相互作用伙伴。影响：总的来说，拟议的实验将通过确定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，从而促进我们对基本细胞过程（即成骨细胞分化）的理解。我们相信该项目将为CFTR在控制骨稳态中的新功能提供相当大的启示。

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 -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.