Targeting compartmentalized phosphodiesterases to regulate CFTR function

描述（由申请人提供）：通过本提案，我们将测试信号微域cAMP水平的调节，而不是在全球范围内，决定囊性纤维化跨膜传导调节剂（CFTR）在健康和疾病中的功能的总体假设。CFTR是camp刺激的阴离子通道，对包括气道和胃肠道在内的许多上皮细胞的离子和水稳态至关重要。由于囊性纤维化（CF）的CFTR基因突变或慢性阻塞性肺疾病（COPD）的烟雾暴露导致CFTR功能低下，导致上皮顶端膜中CFTR通道水平降低，从而导致异常的液体和电解质运输。PKA磷酸化可以刺激CFTR的功能。然而，在过去，通过腺苷酸环化酶激活剂和广谱PDE抑制剂的组合将全局细胞cAMP增加到超生理水平来刺激CF模型系统中cftr依赖的离子运输的结果和成功程度不一。在这项研究计划中，我们希望测试两种新的方法来克服这一限制。本提案的具体目标1将验证环核苷酸磷酸二酯酶（PDEs）的特定同工异构体（降解和灭活cAMP的酶）的假设，这些酶在物理上与涉及CFTR的信号复合物相连，并在cAMP信号的区隔微域中控制该通道的活性。我们希望验证这样一种观点，即与增加全局cAMP水平相比，局部PDE活性池的失活或其从CFTR信号复合物中位移提供了一种更安全、更有效的刺激CFTR功能的方法，因为后者会诱导显著的细胞反馈反应和不良副作用，最终限制其功效。人类基因组编码21个PDE基因，这些基因可能表达为100多种蛋白质变体。4型PDEs （PDE4s）抑制剂已被证明可以刺激永生化上皮细胞系的CFTR。我们现在获得的初步数据显示，PDE4亚型是原代肺上皮细胞中CFTR活性的主要调节因子，并且物理上与该通道相连，这表明这些pde是控制CFTR微域cAMP的pde。在本提案的Specific Aim 1中，我们将表征CFTR与单个PDE4亚型的相互作用，并测试这些PDE4的选择性失活对非CF和CF细胞和组织中CFTR功能的影响。在本提案的具体目标2中，我们将生成新的基于fret的生物传感器，可以测量CFTR附近的cAMP水平和PKA活性。这将使我们能够追踪决定CFTR功能的cAMP池，确定该隔室中的关键调节因子，并探索CF和非CF上皮之间cAMP信号事件的可能差异。

DESCRIPTION (provided by applicant): With this proposal, we will test the overall hypothesis that regulation of cAMP levels in microdomains of signaling, rather than on the global scale, determines the function of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) in health and disease. The CFTR is a cAMP-stimulated anion channel that is critical for ion- and water homeostasis across many epithelia including in the airways and the gastrointestinal tract. CFTR hypofunction, as a result of mutations in the CFTR gene in Cystic Fibrosis (CF), or smoke exposure in Chronic Obstructive Pulmonary Disease (COPD), results in reduced levels of this channel in the apical membrane of epithelia which causes abnormal fluid and electrolyte transport. It is well established that CFTR function is stimulated by PKA phosphorylation. However, stimulating CFTR-dependent ion transport in CF model systems by increasing global cellular cAMP to supra-physiological levels with a combination of adenylyl cyclase activators and broad-spectrum PDE inhibitors has been met with mixed results and successes in the past. With this research proposal, we wish to test two novel approaches to overcome this limitation. Specific Aim 1 of this proposal will test the hypothesis that specific isoforms of cyclic nucleotide phosphodiesterases (PDEs), the enzymes that degrade and inactivate cAMP, are physically tethered to signaling complexes involving the CFTR and control the activity of this channel in compartmentalized microdomains of cAMP signaling. We wish to test the idea that inactivation of this localized pool of PDE activity or its displacement from CFTR signaling complexes provides a safer and more effective approach to stimulate CFTR function compared to increasing global cAMP levels, as the latter approach induces significant cellular feedback responses and undesirable side effects that ultimately limit its efficacy. The human genome encodes for 21 PDE genes, which are likely expressed as more than 100 protein variants. Inhibitors of type 4 PDEs (PDE4s) have been shown to stimulate CFTR in immortalized epithelial cell lines. We have now generated preliminary data showing that PDE4 isoforms are the major regulators of CFTR activity in primary lung epithelial cells and are physically tethered to this channel suggesting that these are the PDEs that control cAMP in CFTR microdomains. In Specific Aim 1 of this proposal, we will characterize the interaction of CFTR with individual PDE4 isoforms and test the effect of selective inactivation of these PDE4s on CFTR function in non-CF and CF cells and tissues. In Specific Aim 2 of this proposal, we will generate novel FRET-based biosensors that can measure cAMP levels and PKA activity in the immediate vicinity of the CFTR. This will allow us to trace the pools of cAMP that determine CFTR function, identify the key regulators in this compartment, and explore possible differences in cAMP signaling events between CF and non-CF epithelia. PUBLIC HEALTH RELEVANCE: Abnormal expression and function of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is central to several diseases with high public health relevance including cystic fibrosis (CF). The identification of specific cyclic nucleotide phosphodiesterase 4 (PDE4) isoforms as regulators of CFTR activity will open new opportunities to target PDE4s as a therapeutic approach in CF. This proposal aims to validate the idea that targeting microdomains of cAMP signaling, rather than global cAMP levels, is an effective therapeutic approach, which will advance the development of more selective and, thus, safer drugs.