Leukotriene C4 and STIM/Orai channels in airway smooth muscle remodeling

白三烯 C4 和 STIM/Orai 通道在气道平滑肌重塑中的作用

• 批准号: 9069952
• 负责人: Mohamed Trebak
• 金额: $ 39.83万
• 依托单位: PENNSYLVANIA STATE UNIV HERSHEY MED CTR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-30 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9069952
• 关键词: Address; Agonist; Arachidonic Acids; Asthma; Calcium; Cell membrane; Cells; Chimera organism; Chronic; Clinical; Coupled; Data; Diglycerides; Disease; Electrophysiology (science); Endoplasmic Reticulum; Functional disorder; Goals; Growth Factor Gene; Health; Homologous Gene; Hyperplasia; Hypertrophy; Image; Immune; Inflammatory; Inositol; Knock-out; Knockout Mice; Leukotriene C4; Ligation; Mammals; Mediating; Molecular; Mus; Obstructive Lung Diseases; Organ; Ovalbumin; Phospholipase C; Phosphorylation; Physiology; Platelet-Derived Growth Factor; Production; Proteins; Publishing; Regulation; Research Proposals; Role; STIM1 gene; Sarcoplasmic Reticulum; Second Messenger Systems; Signal Pathway; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; Thrombin; Tissues; Up-Regulation; Vertebrates; airway obstruction; airway remodeling; asthmatic; cysteinyl leukotriene receptor; in vitro Model; knock-down; mouse model; muscle form; new therapeutic target; novel; paracrine; patch clamp; protein expression; pulmonary function; pulmonary function decline; receptor; respiratory smooth muscle; response; second messenger; sensor; targeted treatment; therapeutic target; voltage

DESCRIPTION (provided by applicant): Airway smooth muscle (ASM) remodeling in chronic asthma involves structural and functional changes from healthy into hyper-reactive and proliferative/hypertrophic ASM. ASM remodeling is an important determinant in airway obstruction and decline pulmonary function in asthma that compounds the well- established immune/inflammatory components. These ASM phenotypic changes are of great clinical importance yet remains poorly understood. A more recent concept is that ASM not only contributes to physical obstruction of airways, but acts as immune effector in asthma. Orai1 encodes canonical, ubiquitous and evolutionarily-conserved Ca2+ release-activated Ca2+ (CRAC) channels regulated by the endoplasmic reticulum (ER) Ca2+ sensor STIM1. We showed that CRAC mediate proliferative signals during pathological smooth muscle remodeling. However, Orai1-mediated CRAC is widely functional in most, if not all, tissues rendering its specific targeting for therapy challenging. Orai1 has two homologs, Orai2 and Orai3 which are exclusive to vertebrates and mammals, respectively. We propose that Orai3 is a key determinant in ASM remodeling during asthma and that Orai3 is a better therapeutic target than Orai1. Our exciting data identified novel native Orai3/Orai1 heteromultimeric channels in ASM that are regulated by STIM1 and gated by cytosolic leukotrieneC4. This "intracrine" function of leukotrieneC4 is independent of paracrine actions on plasma membrane receptors. We showed upregulation of STIM1, Orai1 and Orai3 proteins in ASM from asthmatic mice. Furthermore, we generated Orai3 knockout (O3-KO) mice which, unlike Orai1 knockouts, are viable and healthy. Therefore, we hypothesize that: i) increased leukotrieneC4-regulated Ca2+ (LRC) currents are regulated by STIM1 and mediated by Orai3/Orai1 hetero-multimers in ASM is an important determinant for ASM remodeling in asthma. We propose two specific aims to address this hypothesis: Aim1) determine the mechanisms of leukotrieneC4-dependent activation of Orai3/Orai1 in ASM. i) We will determine the domains of STIM1 and Orai3 required for Orai3/Orai1 channel activation by LTC4. Using STIM1 truncations, Orai3/Orai1 chimeras and "erase and replace" strategies coupled to multi-cell Ca2+ imaging, we will determine the exact domains of STIM1 and Orai3 required for STIM1/Orai3 interaction and LTC4 action on Orai3/Orai1 channels. ii) We will determine the signaling pathways controlled by LRC-generated Ca2+ signals downstream Akt activation. Aim 2) We will determine the role of STIM1 and Orai3 in ASM remodeling in a mouse model of asthma. i) We will determine the increase in LRC current activity and Akt signaling in ASM from ovalbumin (OVA)-induced asthmatic mice. ii) We will determine the ability of smooth muscle-specific STIM1 and Orai3 knockout mice to develop asthma and ASM remodeling upon OVA challenge. These studies will enhance our understanding of native Orai Ca2+ channels regulation in ASM and will validate Orai3 and Orai3-encoded channels as therapeutic targets for ASM remodeling in chronic asthma.

描述（由申请人提供）：慢性哮喘中的气道平滑肌（ASM）重塑涉及从健康到过度反应和增殖/肥厚的 ASM 的结构和功能变化。 ASM 重塑是哮喘气道阻塞和肺功能下降的重要决定因素，它复合了公认的免疫/炎症成分。这些 ASM 表型变化具有重要的临床意义，但仍知之甚少。最近的一个概念是，ASM 不仅导致气道的物理阻塞，而且在哮喘中充当免疫效应器。 Orai1 编码规范、普遍存在且进化保守的 Ca2+ 释放激活 Ca2+ (CRAC) 通道，受内质网 (ER) Ca2+ 传感器 STIM1 调节。我们发现 CRAC 在病理性平滑肌重塑过程中介导增殖信号。然而，Orai1 介导的 CRAC 在大多数（如果不是全部）组织中广泛发挥作用，这使得其特定的靶向治疗具有挑战性。 Orai1 有两个同源物，Orai2 和 Orai3，分别为脊椎动物和哺乳动物所独有。我们认为Orai3是哮喘期间ASM重塑的关键决定因素，并且Orai3是比Orai1更好的治疗靶点。我们令人兴奋的数据在 ASM 中发现了新型天然 Orai3/Orai1 异多聚体通道，这些通道受 STIM1 调节并受胞质白三烯 C4 门控。白三烯C4 的这种“分泌”功能独立于质膜受体的旁分泌作用。我们发现哮喘小鼠的 ASM 中 STIM1、Orai1 和 Orai3 蛋白上调。此外，我们还培育出了 Orai3 敲除 (O3-KO) 小鼠，与 Orai1 敲除不同，该小鼠具有活力且健康。因此，我们假设：i) ASM 中增加的白三烯 C4 调节的 Ca2+ (LRC) 电流受 STIM1 调节并由 Orai3/Orai1 异多聚体介导，是哮喘 ASM 重塑的重要决定因素。我们提出了两个具体目标来解决这一假设： 目标 1) 确定 ASM 中 Orai3/Orai1 依赖于白三烯 C4 的激活机制。 i) 我们将确定 LTC4 激活 Orai3/Orai1 通道所需的 STIM1 和 Orai3 的域。使用 STIM1 截短、Orai3/Orai1 嵌合体和“擦除和替换”策略与多细胞 Ca2+ 成像相结合，我们将确定 STIM1/Orai3 相互作用和 Orai3/Orai1 通道上的 LTC4 作用所需的 STIM1 和 Orai3 的确切结构域。 ii) 我们将确定 LRC 生成的 Ca2+ 信号下游 Akt 激活所控制的信号通路。目标 2) 我们将确定 STIM1 和 Orai3 在哮喘小鼠模型 ASM 重塑中的作用。 i) 我们将确定卵清蛋白 (OVA) 诱导的哮喘小鼠 ASM 中 LRC 电流活性和 Akt 信号传导的增加。 ii) 我们将确定平滑肌特异性 STIM1 和 Orai3 敲除小鼠在 OVA 攻击后发生哮喘和 ASM 重塑的能力。这些研究将增强我们对 ASM 中天然 Orai Ca2+ 通道调节的理解，并将验证 Orai3 和 Orai3 编码通道作为慢性哮喘 ASM 重塑的治疗靶点。