Mechanical characterization of cell signaling mechanisms.

本研究计划的两个目的旨在探索G蛋白偶联受体（GPCR）信号传导与血管细胞机械特性和功能之间的关系。 1)细胞膜张力与GPCR机械活化的关系 机械力通过细胞信号通路的激活转化为生化信息，在生理和病理生理过程中起着重要作用。细胞膜蛋白作为机械传感器包括各种离子通道，磷脂酶和整合素。最近，越来越多的实验证据表明，G-蛋白偶联受体（GPCR）可能涉及作为机械传感器，特别是血管紧张素II的AT 1受体，这已经受到了极大的关注。事实上，AT 1受体（AT 1-R）可以在没有激动剂的情况下被机械刺激激活，这是一种被认为与心血管功能有关的特殊功能特性。机械力激活GPCR的机制仍然知之甚少。目前有两种模式在讨论气相化学还原的效力。第一个模型涉及它与存在于细胞表面或细胞骨架内的大分子元素的相互作用，这些大分子元素有效地充当机械天线来感知外部机械扰动。第二个模型假设，GPCR被激活，由于细胞膜的横向张力的扰动，这会影响在GPCR/磷脂双层界面的压力分布。 膜侧向张力的这种变化引起GPCR的构象变化并改变其活化状态。在这个目标中，我们提出了原始的AFM实验来研究细胞膜张力在GPCR激活中的作用。 2)选择性血管紧张素信号转导对血管平滑肌细胞力学表型的影响。 血管平滑肌细胞（VSMC）的一个重要特性是心血管系统的功能、适应和维持所需的可塑性。在正常生理条件下，功能性VSMC的收缩表型通过编码收缩和细胞骨架蛋白、细胞内酶以及细胞表面配体和受体的基因的表达来调节。然而，作为对血管应激的响应，VSMC下调收缩蛋白的表达并激活细胞周期，这导致所谓的“合成”表型。VSMC表型转换是血管功能调节的标志。心肌素（Myocardin，Myocd）是近年来发现的一种调节血管平滑肌细胞收缩表型的关键因子。Myocd与MADS盒转录因子SRF相互作用，以上调编码肌动球蛋白收缩机制的许多蛋白质如肌球蛋白重链（MyHC）和SM-α-肌动蛋白的基因的转录。在这种情况下，Myocd反式激活结构域可以被Erk 1/2磷酸化，导致VSMC收缩标志物的减少。NFkB也被证明与Myocd相互作用以抑制其活性，这可以解释在炎症条件下在VSMC中观察到的表型转换。因此，预期AT 1-R的典型（AngII）和偏向（SII）配体对Erk 1/2和NFkB信号传导途径的扰动可以不同地影响VSMC的收缩表型。在这个目标中，使用VSMC模型，我们将研究Erk 1/2，NFkB信号，收缩标志物的调制，VSMC的机械特性（等长张力）以及单个细胞的收缩能力之间存在的联系。

The two aims of this research program are designed to explore the relationship between G-proteins coupled receptors (GPCR) signalling and mechanical properties and function in vascular cells. 1) RELATION BETWEEN CELL MEMBRANE TENSION AND GPCR MECHANICAL ACTIVATION. Transduction of mechanical force into biochemical information trough the activation of cell signaling pathways plays a central role in physiological and pathophysiological processes. Cell membrane proteins acting as mechanosensor includes a variety of ion channels, phospholipases and integrins. More recently, a growing body of experimental evidence suggested that G-proteins coupled receptors (GPCR) could be implicated as mechanosensor, in particular the AT1 receptor of angiotensin II, which has received a significant attention. Indeed, the AT1 receptor (AT1-R) can be activated by mechanical stimuli in absence of agonist, a peculiar functional property believed to be involved in cardiovascular function. The mechanisms responsible for GPCR activation by mechanical forces are still poorly understood. Two models are currently debated as to how GPCR transduce force. The first model involves its interaction with macromolecular elements present at the cell surface of within the cell cytoskeleton, which effectively act as a mechanical antenna to perceive the external mechanical perturbation. The second model supposes that GPCR are activated due to a perturbation of the cell membrane lateral tension, which affect the pressure profile at the GPCR/phospholipid bilayer interface. Such changes in membrane lateral tension provoke conformational changes of GPCR and shift their activation status. In this objective, we propose original AFM experiments to investigate the role of cell membrane tension in GPCR activation. 2) IMPACT OF SELECTIVE ANGIOTENSIN SIGNALLING ON VASCULAR SMOOTH MUSCLE CELLS MECHANICAL PHENOTYPE. An important property of the vascular smooth muscle cells (VSMC) is the plasticity required for the function, adaptation and maintenance of the cardiovascular system. In normal physiological conditions, the contractile phenotype of functional VSMC is regulated through the expression of genes encoding contractile and cytoskeletal proteins, intracellular enzymes, as well as cell surface ligands and receptors. However, in response to vascular stress, VSMCs down-regulate the expression of contractile proteins and activates the cell cycle, which leads to the so-called “synthetic” phenotype. VSMC phenotype switching is the hallmark of vascular function function regulation. Myocardin (Myocd) was recently demonstrated as a key player in the regulation of the contractile phenotype of VSMC. Myocd interacts with the MADS box transcription factor, SRF, to upregulate the transcription of genes encoding for a number of proteins of the actomyosin contractile machinery such as Myosin Heavy Chain (MyHC) and SM-a-actin. In this situation, the Myocd transactivation domain can be phosphorylated by Erk 1/2 leading to a decrease of the VSMC contraction marker. NFkB was also demonstrated to interact with Myocd to inhibit its activity, which may explain phenotypic switch observed in VSMC in inflammatory conditions. Therefore, it is expected that perturbations of the Erk 1/2 and NFkB signaling pathways by canonical (AngII) and biased (SII) ligands of AT1-R could differently affect the contractile phenotype of VSMC. In this objective, using VSMC models, we will study the link existing between Erk 1/2, NFkB signaling, the modulation of contractile markers, the mechanical properties of VSMC (isometric tension) as well as the contractile capability of individual cells.