Role of beta-1 containing integrins in JNK activation in lung mechanical stretch

描述（由申请人提供）：重症监护室经常使用机械通气来支持危重患者。研究表明，机械牵张通过肺泡组织的过度扩张和炎性细胞因子的增加导致呼吸机诱导的肺损伤（VILI）。VILI中涉及的细胞信号传导尚不完全清楚。有丝分裂原活化蛋白激酶途径，特别是cJun氨基末端激酶（JNK）参与VILI的发病机制。JNK的上游信号传导对机械拉伸的响应还没有很好的描述。在机械拉伸的其他模型中，整合素信号传导是机械转导和JNK磷酸化的关键。这通过在粘着斑复合物处的整联蛋白聚集和通过包括粘着斑激酶（FAK）在内的多种蛋白质的信号传导而发生。我的初步研究结果表明：1）JNK磷酸化和随后的早期生长反应基因（Egr 1）表达随着机械拉伸而增加; 2）拉伸诱导的Egr 1表达依赖于JNK; 3）JNK激活需要含有1亚基的整合素的信号传导。基于这些数据，该提议的具体假设是，牵张诱导的肺泡上皮细胞中的JNK磷酸化需要含有整合素的α 1的活化和随后的粘着斑复合物的形成。具体目标是：1）鉴定在肺上皮细胞中参与牵张诱导的JNK磷酸化和随后的基因表达的特异性整联蛋白。使用显性负性<$1构建体，我将证实含有<$1的整合素对于牵张诱导的JNK磷酸化是重要的。然后，通过使用针对不同整联蛋白α亚基的阻断抗体和/或RNA干扰，我将进一步描述负责JNK磷酸化的特定整联蛋白。2)确定周期性牵张诱导的整合素激活导致下游JNK磷酸化的机制。我将染色粘着斑复合物，拉伸和不拉伸，以及使用FAK siRNA检查粘着斑复合物的形成和FAK激活在拉伸诱导的JNK激活的作用。 公共卫生相关性：机械通气在重症监护室中经常使用，并且已知会增加肺损伤，这带来了高死亡率。进一步了解参与肺机械牵张的细胞通路有助于对呼吸机患者进行潜在的治疗和方法。

DESCRIPTION (provided by applicant): Mechanical ventilation is frequently used in the intensive care unit to support critically ill patients. Studies have shown that mechanical stretch leads to ventilator induced lung injury (VILI) through overdistension of alveolar tissue and augmentation of inflammatory cytokines. The cell signaling involved in VILI is incompletely understood. The mitogen activated protein kinase pathways, particularly the cJun amino terminal kinase (JNK) are implicated in the pathogenesis of VILI. The upstream signaling of JNK in response to mechanical stretch is not well described. In other models of mechanical stretch, integrin signaling is key to mechanotransduction and JNK phosphorylation. This occurs through integrin clustering at focal adhesion complexes and signaling through multiple proteins including focal adhesion kinase (FAK). Results of my initial studies show that: 1) JNK phosphorylation and subsequent early growth response gene (Egr1) expression is increased with mechanical stretch; 2) stretch-induced Egr1 expression is dependent on JNK; and 3) JNK activation requires signaling by integrins containing the ¿1 subunit. Based on these data, the specific hypothesis for this proposal is that stretch induced JNK phosphorylation in alveolar epithelial cells requires activation of a ¿1 containing integrin and subsequent focal adhesion complex formation. The specific aims are to: 1) Identify the specific integrin(s) involved in stretch-induced JNK phosphorylation and subsequent gene expression in lung epithelial cells. Using a dominant negative ¿1 construct, I will confirm that ¿1 containing integrins are important for stretch-induced JNK phosphorylation. Then, by using blocking antibodies and/or RNA interference directed towards different integrin a subunits, I will further delineate the specific integrin(s) responsible for JNK phosphorylation. 2) Identify the mechanism by which cyclical stretch-induced integrin activation causes downstream JNK phosphorylation. I will stain focal adhesion complexes with and without stretch as well as using FAK siRNA to examine the role of focal adhesion complex formation and FAK activation in stretch-induced JNK activation. PUBLIC HEALTH RELEVANCE: Mechanical ventilation is used frequently in the intensive care unit and is known to augment lung injury, which carries a high mortality. Further understanding of the cellular pathways involved in lung mechanical stretch could help with potential therapies and approaches to patients on ventilators.