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，以及该蛋白的破坏如何在囊性纤维化中自然发生，损害骨形成从而产生骨质减少和骨质疏松症。因此，本提案属于NIAMS 2006-2009年长期计划推荐的研究领域，即“确定骨病病理背后的分子和细胞机制”（3C-3）。考虑到CFTR传导碳酸氢盐的多效性、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原位定位中的重要性，我们设计了实验来研究CFTR是否在成骨细胞中介导这些过程，以及由此对骨形成标志物的影响。在SA1中，我们将研究CFTR抑制（通过药理学和基因组学方法）对成骨细胞、成骨前细胞和成熟成骨细胞的增殖（通过BrdU结合测量）和分化（qPCR和骨形成标记物的Western免疫印迹检测，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SA2中，我们将研究CFTR对细胞内和细胞周围pH的调节作用，以及由此引起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CFTR在存在和不存在CFTR抑制剂的情况下对酸攻击恢复的贡献。接下来，我们将研究CFTR在细胞外pH值调节中的作用，这对于矿物质的形成至关重要。在SA3中，我们将检查CFTR是否需要用于负载诱导的ATP释放（荧光素-荧光素酶检测）和细胞内钙的瞬态（使用比例荧光团Fura-2）。在SA4中，我们将通过体外骨吸收试验以及与破骨细胞发生标志物的相关性来研究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先天性囊性纤维化患者的平均寿命。然而，随着CF人群开始衰老，我们已经意识到CF的另一个症状是骨骼变弱，增加了骨折的风险。这些实验将揭示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，这将有助于我们和其他人开发新的治疗策略，最终提供洞察肌肉骨骼病理，包括囊性纤维化和年龄相关的骨质疏松症。