Mechanisms of Cystic Fibrosis-induced Osteoporosis

描述(申请人提供)：这项建议的目的是了解CFTR参与骨形成的细胞和分子机制，以及该蛋白的破坏(如囊性纤维化中自然发生的)如何损害骨形成从而产生骨量减少和骨质疏松。因此，这项建议属于NIAMS 2006-2009年长期计划所建议的研究领域，即“确定骨骼疾病病理基础的分子和细胞机制”(3C-3)。鉴于CFTR对碳酸氢盐的多效性，它在ATP分泌中的作用，它调节细胞内钙水平的能力，以及这些过程在骨骼内伸展中的重要性，我们设计了实验来研究CFTR是否在成骨细胞中调节这些过程以及由此对骨形成标志物的影响。在SA1中，我们将检测CFTR抑制(通过药理学和基因组方法)对成骨前体细胞、成骨前细胞和成熟成骨细胞的增殖(通过BrdU掺入)和分化(通过qPCR和Western免疫印迹检测骨形成标志，检测基质矿化)的影响。接下来，我们将研究CFTR抑制细胞的增殖和分化的改变是否是成骨细胞酸碱平衡改变的结果。在SA2中，我们将研究CFTR在调节细胞内和细胞周围pH以及由此导致的增殖和基质矿化变化中的作用。在这项研究中，我们将使用pH敏感的BCECF来检验在存在和不存在CFTR抑制剂的情况下，CFTR在从酸刺激中恢复的贡献。接下来，我们将研究CFTR在调节细胞外pH中的作用，这是已知的矿物质形成的关键。在SA3中，我们将研究CFTR是否是负荷诱导的ATP释放(荧光素-荧光素酶检测)和细胞内钙瞬变(使用比率荧光素Fura-2)所必需的。在SA4中，我们将通过体外骨吸收试验以及与破骨细胞生成标志物的相关性来检测CFTR对破骨细胞成熟和激活的影响。相关性：医学的进步大大延长了囊性纤维症先天患者的平均寿命。然而，随着CF人口开始老龄化，我们已经意识到CF的另一个症状是骨骼变弱，增加骨折的风险。这些实验表明CFTR是如何阻止骨骼的正常形成的。希望通过了解CFTR如何参与骨形成，我们将提供洞察力，帮助我们和其他人开发新的治疗策略，最终为肌肉骨骼病理提供洞察力，包括囊性纤维化和年龄相关性骨质疏松。

DESCRIPTION (provided by applicant): The objective of this proposal is to understand the cellular and molecular mechanisms whereby CFTR is involved in bone formation and how disruption of this protein, as occurs naturally in Cystic Fibrosis, impairs bone formation to produce osteopenia and osteoporosis. As such, this proposal falls into the areas of research recommended by NIAMS Long Range Plan 2006-2009, to "identify the molecular and cellular mechanisms underlying the pathology of bone diseases" (3C-3). Provided the pleiotropic capability of CFTR to conduct bicarbonate, its role in ATP secretion, and its ability to modulate intracellular calcium levels, and the importance of these processes in skeletal homesotasis, we have designed experiments to address whether CFTR mediates these process in osteoblastic cells and the resultant effect on markers of bone formation. In SA1, we will examine the effect of CFTR inhibition (by both pharmacologic and genomic approaches) on the proliferation (measured by BrdU incorporation) and differentiation (qPCR and Western immunoblotting of markers of bone formation, detection of matrix mineralization) of osteoprogenitors, preosteoblasts,and mature osteoblasts. We will next examine whether alterations in proliferation and differentiation in CFTR-inhibited cells is the result of alterations in acid-base balance in osteoblastic cells. In SA2, we will examine the role of CFTR on the regulation of both intracellular and pericellular pH and the resultant changes in proliferation and matrix mineralization. In the studies, we will use the pH-sensitive BCECF to examine the contribution of CFTR on recovery from acid challenge in the presence and absence of CFTR inhibitors. We will next examine the role of CFTR on the regulation of extracellular pH, which is known to be crucial for mineral formation. In SA3, we will examine whether CFTR is required for load-induced ATP release (luciferin-luciferase detection) and transients in intracellular calcium (using the ratiometric fluorophore Fura-2). In SA4, we will examine the effect of CFTR on the maturation and activation of osteoclasts using in vitro bone resorption assays and correlation with markers of osteoclastogenesis. Relevance: Advances in medicine has greatly prolonged the average lifespan of people born with cystic fibrosis. Yet, as the CF population begins to age, we have become aware that another symptom of CF is weakened bones, increases the risk of fractures. These experiments would suggest how CFTR prevents proper formation of bone. It is hoped that, by understanding how CFTR is involved in bone formation, we would provide insight that would help us and others to develop novel therapeutic strategies to ultimately provide insight into musculoskeletal pathologies, including Cystic Fibrosis- and age-related osteoporosis.