Neurotrophins in the Lung

描述(申请人提供)：在呼吸道生理学和病理生理学中，一个令人兴奋的新研究主题是神经营养因子(NTS)：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不同作用而闻名。NTS及其受体目前已在不同的肺成分中被发现，包括呼吸道平滑肌(ASM)，并在哮喘、过敏甚至肺癌中观察到表达的改变。虽然NTS可能有几个来源，但我们已发表的和初步的数据表明，ASM是NTS的靶点，NTS不仅在正常情况下有助于ASM的收缩，而且还有助于增加与呼吸道炎症(如由TNFa引起的炎症)的收缩。拟议研究的长期目标是了解NTS在ASM生理学和病理生理学中的作用。总的假设是NTS增强了肌浆网(SR)的钙释放和钙内流；以及b)肌浆网(ASM)对钙的敏感性。我们认为BDNF是影响ASM收缩能力的关键因素。最后，气道炎症增强了BDNF信号，导致[Ca~(2+)]i和FORCE的整体增强。在这项建议中，我们将使用人ASM和卵蛋白(OVA)小鼠模型来研究BDNF受体(高亲和力TrkB与低亲和力p75NTR)与ASM收缩性能之间的相对作用。我们假设TrkB对[Ca~(2+)]i的调节更为重要，而p75NTR则调节力。我们将利用生物化学、药理学、分子生物学、免疫细胞化学、荧光钙成像、测力技术和肺力学，重点研究BDNF可能调节的特定机制：第二信使IP3(通过磷脂酶C PLC)和环状ADP核糖(通过CD38)(AIM 1)；SR钙释放(IP3受体与RyR受体(RyR)通道)(AIM 2)；通过商店操作的Ca+内流(SOCE)(AIM 3)(AIM 3)以及力调节机制肌球蛋白轻链(MLC20)和RhoA/Rho-Kinase(AIM 4)。这些对人类ASM的体外研究将被整合到OVA小鼠模型中，应用于TrkB敲门小鼠的焦点研究(其中TrkB功能被可逆抑制)。我们将探讨TNFa诱导的炎症增加BDNF受体的表达，改变特异性的[Ca2+]i和FORCE调节机制，从而为ASM对BDNF和支气管收缩药的增强反应做好准备。具体目的是：目的1：确定BDNF调节人ASM第二信使信号的机制；目的2：确定BDNF调节人ASM中SR钙离子调节的机制；目的3：确定BDNF调节人ASM中SOCE的机制；目的4：确定BDNF调节人ASM中力量调节的机制；目的5：在小鼠呼吸道炎症和高反应模型中确定BDNF在ASM收缩中的作用。与公共卫生相关。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，气道平滑肌收缩功能的异常(因炎症而加重)会导致气道过度狭窄和伴随的呼吸短促。在这方面，被称为神经营养因子的生长因子在调节呼吸道收缩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伴有或不伴有炎症的气道狭窄中的作用，拟议的研究将为更好地了解呼吸道疾病和潜在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.