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

描述（由申请人提供）：慢性哮喘气道平滑肌（ASM）重构涉及从健康到高反应性和增生性ASM的结构和功能变化。ASM重塑是哮喘患者气道阻塞和肺功能下降的重要决定因素，它与已有的免疫/炎症成分相结合。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不仅有助于气道的物理阻塞，而且在哮喘中起免疫效应。Orai1编码规范的，普遍存在的和进化保守的Ca2+释放激活的Ca2+ (CRAC)通道，由内质网（ER） Ca2+传感器STIM1调节。我们发现CRAC介导病理性平滑肌重塑过程中的增殖信号。然而，orai1介导的CRAC在大多数（如果不是全部）组织中具有广泛的功能，这使得其特异性靶向治疗具有挑战性。Orai1有两个同源基因Orai2和Orai3，它们分别只存在于脊椎动物和哺乳动物中。我们认为Orai3是哮喘期间ASM重构的关键决定因素，并且Orai3是比Orai1更好的治疗靶点。我们激动人心的数据在ASM中发现了新的原生Orai3/Orai1异聚通道，这些通道由STIM1调节，并由细胞质白三烯ec4门控。白三烯ec4的“分泌内”功能独立于对质膜受体的旁分泌作用。我们发现哮喘小鼠ASM中STIM1、Orai1和Orai3蛋白上调。此外，我们产生了Orai3敲除（O3-KO）小鼠，与Orai1敲除不同，这些小鼠是健康的。因此，我们假设：i)增加的白三烯ec4调节的Ca2+ (LRC)电流由STIM1调节，并由ASM中的Orai3/Orai1异源多聚体介导，是哮喘ASM重构的重要决定因素。我们提出了两个具体的目标来解决这一假设：目的1)确定白三enec4依赖性的Orai3/Orai1在ASM中的激活机制。i)我们将确定LTC4激活Orai3/Orai1通道所需的STIM1和Orai3结构域。利用STIM1截断、Orai3/Orai1嵌合体和“erase and replace”策略，结合多细胞Ca2+成像，我们将确定STIM1/Orai3相互作用和LTC4作用于Orai3/Orai1通道所需的STIM1和Orai3的确切结构域。ii)我们将确定lrc产生的Ca2+信号在Akt激活下游控制的信号通路。目的2)我们将在哮喘小鼠模型中确定STIM1和Orai3在ASM重构中的作用。i)我们将检测卵清蛋白（OVA）诱导的哮喘小鼠ASM中LRC电流活性和Akt信号的增加。ii)我们将确定平滑肌特异性STIM1和Orai3基因敲除小鼠在OVA攻击后发生哮喘和ASM重构的能力。这些研究将增强我们对ASM中原生oraica2 +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。

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.