Global and local cGMP signals in growth control

信号分子cGMP控制许多细胞功能，从生长到收缩和离子运输。目前的工作模型假设NO刺激的鸟苷酸环化酶产生全局胞浆cGMP信号，而跨膜鸟苷酸环化酶，例如心钠素(ANP)或C型利钠肽(CNP)的受体，建立局部的膜相关cGMP微区。CGMP信号转导的生理结果取决于cGMP信号的时空分布以及全球和局部cGMP池的盛行程度。在本项目中，我们将通过对小鼠活细胞和组织中cGMP信号和细胞行为的实时相关性分析来验证这一假说，重点是cGMP与血管平滑肌细胞(VSMCs)和背根节(DRG)神经元重塑过程的相互作用。在之前的资助期间，我们已经培育了转基因cGMP传感器小鼠，在所有组织中或在特定细胞类型中选择性地表达基于荧光共振能量转移(FRET)的cGi500传感器。FRET显微镜显示，从cGMP传感器小鼠分离的活的原代细胞和组织中确实可以看到cGMP信号。通过一种定位技术，我们可以直接将单个培养的VSMC的ANP/CNP反应与它们各自的分化状态联系起来。有趣的是，在ANP和CNP诱导的cGMP信号方面，VSMC群体表现出明显的异质性，单个细胞的ANP/CNP偏好似乎与其生长表型有关。在另一个子项目中，我们已经在原代DRG神经元中建立了cGMP成像。我们能够在这些神经元的亚细胞隔间(如轴突和生长锥)触发局部cGMP信号，并将它们与Ca(2+)信号联系起来。基于这些结果，我们现在将扩展我们的研究，以了解更多关于VSMCs和DRG神经元中形成cGMP信号的机制，例如，特定的cGMP降解磷酸二酯酶的作用。我们还将分析我们的发现与动脉粥样硬化和再狭窄期间的血管重构以及胚胎发育过程中cGMP介导的轴突分支的相关性。小鼠的体外和体内实验将探讨两个主要问题：(1)cGMP信号如何影响VSMCs和DRG神经元的生长和存活；(2)在这些细胞类型中，细胞生长和表型如何影响cGMP信号？为了实现我们的目标，我们将与这个研究单位的其他小组密切互动，分享专业知识以及老鼠和细胞模型。在哺乳动物细胞或生物体中信号转导过程中cGMP的可视化将有助于环核苷酸的基础研究以及靶点识别和药物开发。

The signalling molecule cGMP controls many cellular functions ranging from growth to contractility and ion transport. The current working model postulates that NO-stimulated guanylyl cyclases generate global cytoplasmic cGMP signals, whereas transmembrane guanylyl cyclases, for instance, the receptors for atrial natriuretic peptide (ANP) or C-type natriuretic peptide (CNP), establish local membrane-associated cGMP microdomains. The physiological outcome of cGMP signal transduction depends on the spatiotemporal profile of the cGMP signal and the prevalence of global vs local cGMP pools. In the present project, we will test this hypothesis by correlative analysis of cGMP signals and cell behaviour in real-time in living cells and tissues of mice, with a focus on the interplay between cGMP and remodelling processes in vascular smooth muscle cells (VSMCs) and dorsal root ganglion (DRG) neurons. In the previous funding period, we have generated transgenic cGMP sensor mice expressing the fluorescence resonance energy transfer (FRET)-based cGi500 sensor either globally in all tissues or selectively in specific cell types. FRET microscopy showed that cGMP signals can indeed be visualized in living primary cells and tissues isolated from cGMP sensor mice. By using a mapping technique, we could directly correlate ANP/CNP responsiveness of individual cultured VSMCs to their respective differentiation state. Interestingly, VSMC populations show pronounced heterogeneity with respect to ANP- and CNP-induced cGMP signals, and the ANP/CNP preference of an individual cell appears to be associated with its growth phenotype. In another subproject, we have established cGMP imaging in primary DRG neurons. We were able to trigger local cGMP signals in subcellular compartments of these neurons, such as axons and growth cones, and to correlate them with Ca(2+) signals. Based on these results, we will now extend our studies to learn more about the mechanisms that shape cGMP signals in VSMCs and DRG neurons, for instance, the role of specific cGMP-degrading phosphodiesterases. We will also analyse the relevance of our findings for vascular remodelling during atherosclerosis and restenosis and for cGMP-mediated axon branching during embryogenesis. Two major questions that will be approached by in vitro and in vivo experiments in mice are (1) how do cGMP signals affect the growth and survival of VSMCs and DRG neurons and (2) how do cell growth and phenotype affect cGMP signalling in these cell types? To achieve our goals, we will closely interact with the other groups of this research unit to share expertise as well as mouse and cell models. The visualization of cGMP during signal transduction in a living mammalian cell or organism will be useful for both basic research on cyclic nucleotides as well as for target identification and drug development.