Mechanisms of Cystic Fibrosis-induced Osteoporosis

囊性纤维化诱发骨质疏松的机制

• 批准号: 7409373
• 负责人: DAMIAN C GENETOS
• 金额: $ 5.13万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-10-01 至 2011-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7409373
• 关键词: Acid-Base Equilibrium; Acids; Address; Adrenal Cortex Hormones; Adult; Affect; Age; Age-Related Bone Loss; Area; Bicarbonates; Biological Assay; Bone Diseases; Bone Resorption; Bromodeoxyuridine; Calcium; Caucasians; Caucasoid Race; Cell membrane; Cell physiology; Cells; Chloride Channels; Chloride Ion; Chlorides; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Detection; Development; Digestive System Disorders; Disease; Disruption; Exocrine Glands; Fracture; Functional disorder; Fura-2; Genes; Genomics; Goals; Hereditary Disease; In Vitro; Ion Channel; Knockout Mice; Lead; Life Expectancy; Live Birth; Longevity; Luciferases; Lung; Lung diseases; Measures; Mediating; Medicine; Minerals; Molecular; Musculoskeletal; Mutate; Mutation; Non-Steroidal Anti-Inflammatory Agents; Numbers; Osteoblasts; Osteoclasts; Osteocytes; Osteogenesis; Osteoporosis; Pancreas; Pathology; Persons; Play; Population; Process; Proteins; Range; Recovery; Regulation; Reporting; Research; Resort; Rib Fractures; Risk; Role; Signaling Molecule; Skeletal system; Symptoms; System; Testing; Western Blotting; Work; apical membrane; bone; bone cell; bone quality; cell type; cystic fibrosis patients; design; extracellular; falls; fluid flow; fluorophore; gastrointestinal system; inhibitor/antagonist; insight; luciferin; mineralization; novel therapeutics; osteoclastogenesis; prevent; research study; spine bone structure

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.

描述（由申请人提供）：本提案的目的是了解CFTR参与骨形成的细胞和分子机制，以及该蛋白质的破坏（如囊性纤维化中自然发生的）如何损害骨形成，从而产生骨质减少和骨质疏松症。因此，该建议福尔斯属于NIAMS长期计划2006-2009推荐的研究领域，以“确定骨疾病病理学基础的分子和细胞机制”（3C-3）。提供了CFTR的多效性能力，进行碳酸氢盐，其在ATP分泌中的作用，其调节细胞内钙水平的能力，以及这些过程在骨骼homesotasis的重要性，我们设计了实验，以解决CFTR是否介导这些过程中的成骨细胞和骨形成的标志物的结果的影响。在SA 1中，我们将检查CFTR抑制（通过药理学和基因组方法）对骨祖细胞、前成骨细胞和成熟成骨细胞增殖（通过BrdU掺入测量）和分化（骨形成标志物的qPCR和Western免疫印迹，基质矿化检测）的影响。接下来我们将研究CFTR抑制细胞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SA 2中，我们将研究CFTR对细胞内和细胞周围pH调节的作用以及由此产生的增殖和基质矿化的变化。在研究中，我们将使用pH敏感的BCECF来检查在存在和不存在CFTR抑制剂的情况下CFTR对从酸挑战中恢复的贡献。接下来，我们将研究CFTR在调节细胞外pH值方面的作用，这对于矿物质形成至关重要。在SA 3中，我们将检查CFTR是否需要负载诱导的ATP释放（荧光素酶检测）和细胞内钙瞬变（使用比率荧光团Fura-2）。在SA 4中，我们将使用体外骨吸收测定和与破骨细胞生成标志物的相关性来检查CFTR对破骨细胞成熟和活化的影响。相关性：医学的进步大大延长了囊性纤维化患者的平均寿命。然而，随着CF人群开始老龄化，我们已经意识到CF的另一个症状是骨质疏松，增加了骨折的风险。这些实验将表明CFTR如何阻止骨的正常形成。希望通过了解CFTR如何参与骨形成，我们将提供见解，帮助我们和其他人开发新的治疗策略，最终提供对肌肉骨骼病理学的见解，包括囊性纤维化和年龄相关的骨质疏松症。