Laminin mediated alveolar cell mechano-transduction

层粘连蛋白介导的肺泡细胞机械转导

• 批准号: 7435396
• 负责人: JONATHAN C. JONES
• 金额: $ 32.88万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7435396
• 关键词: ATP phosphohydrolase; Acute; Adenoviruses; Adult Respiratory Distress Syndrome; Alveolar; Alveolar Cell; Animals; Antibodies; Apoptosis; Basement membrane; Binding; Biological Assay; CD29 Antigen; Cell Adhesion; Cell physiology; Cell surface; Cell-Matrix Junction; Chemicals; Complex; Cultured Cells; Data; Edema; Electroporation; Epithelial; Epithelial Cells; Excision; Exposure to; Extracellular Matrix; Generations; Genes; Genetic Transcription; Helper Viruses; Hour; In Vitro; Inflammatory; Integrins; K ATPase; Knock-out; Laminin; Lung; Measures; Mechanical ventilation; Mechanics; Mediating; Membrane Proteins; Mitogen-Activated Protein Kinases; Mitogens; Molecular; Mus; Na(+)-K(+)-Exchanging ATPase; Newborn Respiratory Distress Syndrome; Oxidants; Patients; Plasmids; Play; Protein Isoforms; Rattus; Respiratory Failure; Respiratory physiology; Role; Stream; Stretching; Technology; Testing; Tidal Volume; Transgenic Organisms; Treatment Protocols; Wound Healing; in vivo; inhibiting antibody; inhibitor/antagonist; insight; laminin alpha 3; lung injury; mature animal; migration; mortality; promoter; receptor; recombinase; research study

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.

机械通气可导致动物预先存在的肺损伤，并导致急性呼吸窘迫综合征（ARDS）患者的死亡率。培养细胞的机械拉伸增加氧化剂产生，诱导促炎基因的表达，抑制上皮伤口愈合并诱导细胞凋亡。然而，肺泡上皮细胞感知周期性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 在肺部的传导。特别地，培养的肺泡细胞分泌富含含有α 3亚基的层粘连蛋白的基质。含有α 3亚基的层粘连蛋白组织成纤维状阵列。此外，牵张诱导的大鼠肺泡细胞中丝裂原活化蛋白激酶（MAPK）的瞬时增加被抗α 3层粘连蛋白亚基球状结构域的抗体和干扰β 1整联蛋白功能的抗体抑制。这些数据提供了支持的假设，肺泡细胞中的机械信号是介导的层粘连蛋白/整合素复合物。我们将在三个具体目标中检验这一假设。在目标1中，我们将确定肺泡上皮细胞粘附到细胞外基质中的层粘连蛋白的功能后果。具体来说，我们建议确定层粘连蛋白亚型表达肺泡细胞在体外和体内。此外，我们将评估整合素受体在肺泡附着中的作用， 在不同层粘连蛋白同种型上的扩散和迁移。在目标2中，我们将确定层粘连蛋白/整合素相互作用是否负责培养细胞中的机械信号转导。我们将研究层粘连蛋白/整合素相互作用的抑制剂在肺泡上皮细胞暴露于周期性拉伸的原代培养物中的作用，重点是MAP激酶激活及其下游后果对Na+-K+-ATP酶的活性和表达。在目标3中，我们将确定是否α 3层粘连蛋白亚基是负责机械信号转导在体内。我们 将开发使用Cre重组酶介导的切除的α 3层粘连蛋白的转基因肺特异性条件敲除。将在缺乏α 3层粘连蛋白的成年动物中评价肺功能。这些研究将为细胞外基质在调节肺细胞生理学中的作用提供新的见解。