Role of Calcium-Activated Chloride Channels in Airway Disease Phenotypes

钙激活氯离子通道在气道疾病表型中的作用

• 批准号: 7188465
• 负责人: Anand Champak Patel
• 金额: $ 11.93万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-12 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7188465
• 关键词: Air; Allergens; Antisense Oligonucleotides; Asthma; Biological Assay; Calcium; Cell Line; Cells; Chloride Channels; Chloride Ion; Chlorides; Chronic; Chronic Obstructive Airway Disease; Condition; Dependovirus; Development; Disease; Epithelial Cells; Exhibits; Extrinsic asthma; Family; Family member; Gene Expression; Gene Family; Genes; Goblet Cells; Human; In Vitro; Inflammatory; Interleukin-13; Interleukin-4; Interleukin-9; Knockout Mice; Liquid substance; Lung; Maintenance; Metaplasia; Molecular; Monitor; Mucins; Mucous body substance; Mus; Mutagenesis; Pathway interactions; Pattern; Play; Protein Family; Research Personnel; Reverse Transcriptase Polymerase Chain Reaction; Role; Sequence Homology; Testing; Time; Tissues; Viral Bronchiolitis; Virus Diseases; Work; abstracting; adeno-associated viral vector; airway hyperresponsiveness; airway remodeling; base; cytokine; design; disease phenotype; gene transfer vector; in vivo; inhibitor/antagonist; member; mouse model; novel therapeutics; programs; research study; selective expression; therapeutic target; trait

DESCRIPTION (provided by applicant): Airway hyperresponsiveness and mucous cell metaplasia are essential features of inflammatory airway diseases (including asthma and COPD), but the cellular and molecular pathways leading to these disease traits still need to be better defined. Previous work by others suggested that a goblet cell-specific member of the calcium activated chloride channel family of proteins (mClca3 in mouse and hCLCAl in human) is necessary and sufficient for allergen-induced airway hyperresponsiveness and goblet cell metaplasia in mice and is overexpresssed in allergic asthma in humans. However, we found that a new /nC/ca3-null mouse still fully develops these asthma traits after viral infection or allergen challenge. These and other preliminary results suggest functional redundancy in the CLCA gene family. Indeed, we find that the CLCA locus consists of 6 distinct genes in the mouse and 4 in the human, and each exhibits a high degree of intra- and inter-species sequence homology but a distinct pattern of expression in tissues. We therefore propose that select members of the CLCA family of proteins play tissue-specific but convergent roles in the development and maintenance of airway hyperresponsiveness and goblet cell metaplasia. Thus, we propose to: I. Determine the patterns of gene expression for mClca family members in the setting of experimental airway disease driven by viral infection or allergen challenge, using real-time quantitative RT-PCR assays to precisely monitor the levels of mC/ca family gene expression in wild-type and mClca3-null mice. Studies will be performed in vivo using our mouse models of viral bronchiolitis and allergen challenge and in vitro using primary-culture mouse airway epithelial cells grown at air-liquid interface and stimulated with cytokines (IL-4, IL-9, and IL-13) to induce goblet cell metaplasia. II. Determine the effects of selectively expressing mClca family members on experimental airway disease phenotypes (i.e. goblet cell metaplasia; and airway hyperresponsiveness) using adeno-associated virus (AAV) gene transfer vectors. In vivo studies will again be compared to the actions of mClca expression done in vitro using primary-culture mouse and/or human airway epithelial cells. III. Determine the effects of selectively blocking expression of mClca family members on experimental airway disease phenotypes driven by viral infection or allergen challenge, using AAV vectors to deliver antisense oligonucleotides that selectively inhibit mClca gene expression. This will be compared to effects of targeted mutagenesis (i.e., our mC/ca3-null mouse) and of treatment with taniflumate (an inhibitor of calcium-dependent chloride flux). In vivo studies will again be compared to the actions of these inhibitors in vitro using primary-culture mouse and/or human airway epithelial cells. These proposed experiments should serve to define CLCA function in experimental asthma and thereby provide for new therapeutic targets in humans with airway disease. (End of Abstract)

描述（由申请人提供）： 气道高反应性和粘液细胞化生是炎症性气道疾病（包括哮喘和COPD）的基本特征，但导致这些疾病特征的细胞和分子途径仍需要更好地定义。其他人先前的工作表明，钙激活氯离子通道蛋白家族的杯状细胞特异性成员（小鼠中的mClca 3和人中的hCLCAl）对于小鼠中过敏原诱导的气道高反应性和杯状细胞化生是必要的和足够的，并且在人的过敏性哮喘中过表达。然而，我们发现一种新的/nC/ca 3-null小鼠在病毒感染或过敏原攻击后仍然完全发展这些哮喘特征。这些和其他初步结果表明，在CLCA基因家族的功能冗余。事实上，我们发现，CLCA基因座由6个不同的基因在小鼠和4个在人类中，每个表现出高度的物种内和物种间的序列同源性，但在组织中的表达模式不同。因此，我们提出CLCA蛋白家族的选定成员在气道高反应性和杯状细胞化生的发展和维持中发挥组织特异性但会聚的作用。因此，我们建议： I.在病毒感染或过敏原激发导致的实验性气道疾病背景下，使用实时定量RT-PCR测定确定mClca家族成员的基因表达模式，以精确监测野生型和mClca 3缺失小鼠中mC/ca家族基因表达水平。将使用我们的病毒性细支气管炎和过敏原激发的小鼠模型在体内进行研究，并使用在气-液界面生长并用细胞因子（IL-4、IL-9和IL-13）刺激以诱导杯状细胞化生的原代培养小鼠气道上皮细胞在体外进行研究。 二.使用腺相关病毒（AAV）基因转移载体确定选择性表达mClca家族成员对实验性气道疾病表型（即杯状细胞化生和气道高反应性）的影响。将再次将体内研究与使用原代培养小鼠和/或人气道上皮细胞在体外进行的mClca表达的作用进行比较。 三. 使用AAV载体递送选择性抑制mClca基因表达的反义寡核苷酸，确定选择性阻断mClca家族成员的表达对由病毒感染或过敏原激发驱动的实验性气道疾病表型的影响。这将与靶向诱变（即，我们的mC/ca 3-无效小鼠）和用他氟磺酸酯（钙依赖性氯离子通量的抑制剂）治疗。体内研究将再次与这些抑制剂在体外使用原代培养小鼠和/或人气道上皮细胞的作用进行比较。 这些拟议的实验应有助于确定CLCA在实验性哮喘中的功能，从而为人类气道疾病提供新的治疗靶点。 (End摘要）