Neurotrophins in the Lung

描述（由申请人提供）：神经营养因子（nt）是气道生理学和病理生理学中一个令人兴奋的新研究主题：包括脑源性神经营养因子（BDNF）在内的生长因子，因其在神经系统中的多种作用而闻名。NTs及其受体现已在包括气道平滑肌（ASM）在内的不同肺组织中被发现，在哮喘、过敏甚至肺癌中均有表达改变。虽然NTs可能来源于多种来源，但我们发表的初步数据表明，ASM是NTs的靶点，而且NTs不仅有助于正常情况下ASM的收缩性，而且还有助于气道炎症（如TNFa诱导的气道炎症）的收缩性增加。拟议研究的长期目标是了解NTs在ASM生理和病理生理中的作用。总的假设是，NTs增强a)肌浆网（SR） Ca2+释放和Ca2+内流；b) Ca2+对ASM受力的敏感性。我们认为BDNF是影响ASM收缩性的关键NT。最后，气道炎症增强BDNF信号，导致[Ca2+]i和力的整体增强。在这项提议中，我们将使用人类ASM和卵清蛋白（OVA）小鼠模型来检验BDNF受体（高亲和力TrkB和低亲和力p75NTR）对ASM收缩性的相对作用。我们假设TrkB对[Ca2+]i调节更重要，而p75NTR调节力。利用生物化学、药理学、分子生物学、免疫细胞化学、荧光Ca2+成像、力测量技术和肺力学，我们将重点关注BDNF可能调节的具体机制：第二信使IP3（通过磷脂酶C PLC）和环ADP核糖（通过CD38） （Aim 1）；SR Ca2+释放（IP3受体与ryanodine受体（RyR）通道）（Aim 2）；Ca2+内流通过储存操作Ca2+进入（SOCE） （Aim 3）和力调节机制肌球蛋白轻链（MLC20）和rhoA/ rhoA激酶（Aim 4）。这些人类ASM的体外研究将被整合到OVA小鼠模型中，用于TrkB敲入小鼠的重点研究（其中TrkB功能被可逆抑制）。我们将探讨TNFa诱导的炎症增加组成性BDNF受体表达，改变特异性[Ca2+]i和力调节机制，从而启动ASM增强对BDNF和支气管收缩剂的反应。具体目的是：目的1：确定BDNF调节人类ASM第二信使信号的机制；目的2：确定BDNF在人ASM中调节SR Ca2+调节的机制；目的3：确定BDNF调节人类ASM中SOCE的机制；目的4：确定BDNF调节人ASM力调节的机制；目的5：在气道炎症和高反应性小鼠模型中确定BDNF在ASM收缩性中的作用。公共卫生相关性。越来越多的人认识到，在哮喘和慢性支气管炎等临床重要疾病中，气道平滑肌收缩性异常（由炎症加剧）会导致气道过度狭窄并伴随呼吸短促。在这方面，被称为神经营养因子的生长因子在气道收缩性调节中的潜在作用是一个令人兴奋的新兴研究领域。通过确定神经营养因子在有或无炎症的气道狭窄中的作用，本研究将为更好地了解气道疾病和开发新的治疗靶点奠定基础。

DESCRIPTION (provided by applicant): An exciting, new investigative theme in airway physiology and pathophysiology is neurotrophins (NTs): growth factors including brain-derived neurotrophic factor (BDNF) known for their diverse roles in the nervous system. NTs and their receptors have now been found in different lung components including airway smooth muscle (ASM), with altered expression observed in asthma, allergy, and even lung cancer. While NTs may be derived from several sources, our published and preliminary data suggest that ASM is a target of NTs, and that NTs contribute not only to ASM contractility under normal circumstances, but also to increased contractility with airway inflammation (such as that induced by TNFa). The long term goal of the proposed studies is to understand the role of NTs in ASM physiology and pathophysiology. The overall hypothesis is that NTs enhance a) sarcoplasmic reticulum (SR) Ca2+ release and Ca2+ influx; and b) Ca2+ sensitivity for force generation in ASM. We propose that BDNF is a key NT influencing ASM contractility. Finally, airway inflammation enhances BDNF signaling, leading to an overall enhancement of [Ca2+]i and force. In this proposal, we will use human ASM and the ovalbumin (OVA) mouse model to examine the relative role of the BDNF receptors (high affinity TrkB vs. low affinity p75NTR) vis-¿vis ASM contractility. We hypothesize that TrkB is more important for [Ca2+]i regulation, while p75NTR regulates force. Using biochemistry, pharmacology, molecular biology, immunocytochemistry, fluorescence Ca2+ imaging, force measurement techniques, and lung mechanics, we will focus on specific mechanisms that may be regulated by BDNF: the second messengers IP3 (via phospholipase C PLC) and cyclic ADP ribose (via CD38) (Aim 1); SR Ca2+ release (IP3 receptor vs. ryanodine receptor (RyR) channels) (Aim 2); Ca2+ influx via store-operated Ca2+ entry (SOCE) (Aim 3) and the force regulatory mechanisms myosin light chain (MLC20) and rhoA/rho-kinase (Aim 4). These in vitro studies in human ASM will be integrated into the OVA mouse model applied in focus studies to the TrkB knockin mouse (where TrkB functionality is reversibly inhibited). We will explore the idea that inflammation induced by TNFa increases constitutive BDNF receptor expression, and alters specific [Ca2+]i and force regulatory mechanisms, thus priming ASM for enhanced response to both BDNF and bronchoconstrictor. The Specific Aims are: Aim 1: To determine mechanisms by which BDNF modulates second messenger signaling in human ASM; Aim 2: To determine mechanisms by which BDNF modulates SR Ca2+ regulation in human ASM; Aim 3: To determine mechanisms by which BDNF modulates SOCE in human ASM; Aim 4: To determine mechanisms by which BDNF modulates force regulation in human ASM; Aim 5: To determine the role of BDNF in ASM contractility in a mouse model of airway inflammation and hyperresponsiveness. PUBLIC HEALTH RELEVANCE. There is increasing recognition that abnormalities in airway smooth muscle contractility (exacerbated by inflammation) contribute to exaggerated airway narrowing and accompanying shortness of breath in clinically important diseases such as asthma and chronic bronchitis. In this regard, the potential role of growth factors called neurotrophins in regulation of airway contractility is an exciting and emerging area of research. By establishing the role of neurotrophins in airway narrowing with or without inflammation, the proposed studies will the foundation for better understanding of airway diseases, and potential development of new therapeutic targets.