Laminin mediated alveolar cell mechano-transduction

机械通气会加重动物原有的肺损伤，并导致急性呼吸窘迫综合征(ARDS)患者的死亡。机械拉伸可增加细胞氧化剂的生成，诱导促炎基因的表达，抑制上皮损伤愈合，诱导细胞凋亡。然而，肺泡上皮细胞感知周期性拉伸的机制(S)尚未阐明。初步实验表明，基底膜蛋白可能在机械信号转导中起重要作用。 在肺中的转导。特别是，培养的肺泡细胞分泌一种富含层粘连蛋白的基质，其中含有α3亚单位的层粘连蛋白组织成纤维状阵列。此外，拉伸诱导的大鼠肺泡细胞中丝裂原活化蛋白激酶(MAPK)的一过性增加可被针对α3层粘连蛋白亚单位球状结构域的抗体和干扰β1整合素功能的抗体所抑制。这些数据支持一种假说，即肺泡细胞中的机械信号是由层粘连蛋白/整合素复合体介导的。我们将在三个具体目标上检验这一假设。在目标1中，我们将确定肺泡上皮细胞与细胞外基质中的层粘连蛋白黏附的功能后果。具体地说，我们建议测定肺泡细胞在体外和体内表达的层粘连蛋白亚型。此外，我们还将评估整合素受体在肺泡附着中的作用。 在不同的层粘连蛋白亚型上的扩散和迁移。在目标2中，我们将确定层粘连蛋白/整合素相互作用是否对培养细胞中的机械信号转导负责。我们将研究层粘连蛋白/整合素相互作用的抑制剂在暴露于周期性拉伸的原代培养的肺泡上皮细胞中的作用，重点是MAP激酶的激活及其下游后果对Na+-K+-ATPase活性和表达的影响。在目标3中，我们将确定alpha3层粘连蛋白亚基是否在体内负责机械信号转导。我们 将利用Cre重组酶介导的切除，开发一种转基因的肺特异性α3层粘连蛋白的条件性敲除。将评估缺乏Alpha3层粘连蛋白的成年动物的肺功能。这些研究将为细胞外基质在调节肺细胞生理中的作用提供新的见解。

Mechanical ventilation worsens pre-existing lung injury in animals and contributes to mortality in patients with the acute respiratory distress syndrome (ARDS). Mechanical stretch of cultured ceils increases oxidant generation, induces the expression of pro-inflammatory genes, inhibits epithelial wound healing and induces apoptosis. The mechanism(s) by which alveolar epithelial cells sense cyclic stretch, however, have not been elucidated. Preliminary experiments suggest that basement membrane proteins may play an important role in mediating mechanosignal transduction in the lung. In particular, cultured alveolar ceils secrete a matrix rich in laminin containing an alpha3 subunit The alpha3 subunit-containing laminin organizes into a fibrillar arrays. Moreover, a transient increase in mitogen activated protein kinase (MAPK) in rat alveolar cells induced by stretch is inhibited by an antibody against the alpha3 laminin subunit globular domain and by antibodies that perturb the function of beta1 integrin. These data provide support for a hypothesis that mechanosignaling in alveolar cells is mediated by a laminin/integrin complex. We will test this hypotheis in three specific aims. In aim 1, we will determine the functional consequences of alveolar epithelial cell adhesion to laminins in the extracellular matrix. Specifically, we propose to determine laminin isoforms expressed by alveolar cells in vitro and in vivo. In addition, we will assess the role of integrin receptors in alveolar attachment, spreading and migration on different laminin isoforms. In aim 2, we will determine whether a laminin/integrin interaction is responsible for mechanosignal transduction in cultured cells. We will examine the effects of inhibitors of the laminin/integrin interaction in primary cultures of alveolar epithelial cells exposed to cyclic stretch focusing on MAP kinase activation and its down stream consequences on the activity and expression of the Na+-K+-ATPase. In aim 3 we will determine whether the alpha3 laminin subunit is responsible for mechanosignal transduction in vivo. We will develop a transgenic lung-specific conditional knockout of alpha3 laminin using Cre recombinase mediated excision. Lung function will be evaluated in adult animals lacking the alpha3 laminin. These studies will provide new insight into the role of extracellular matrix in regulating lung cell physiology.