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）患者的死亡率。机械拉伸培养的细胞增加氧化剂的产生，诱导促炎基因的表达，抑制上皮伤口愈合，诱导细胞凋亡。然而，肺泡上皮细胞感知循环拉伸的机制尚未阐明。初步实验表明，基底膜蛋白可能在介导机械信号中起重要作用

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.