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

描述(申请人提供)：重症监护病房经常使用机械通风来支持危重病人。研究表明，机械拉伸通过肺泡组织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涉及的细胞信号转导机制尚不完全清楚。丝裂原活化蛋白激酶通路，尤其是cJun氨基末端激酶(JNK)参与了VILI的发病机制。JNK的上游信号对机械拉伸的反应没有得到很好的描述。在机械拉伸的其他模型中，整合素信号是机械转导和JNK磷酸化的关键。这是通过整合素聚集在粘着斑处，并通过包括粘着斑激酶(FAK)在内的多种蛋白质来传递信号来实现的。我的初步研究结果表明：1)JNK的磷酸化和随后的早期生长反应基因(Egr1)的表达随着机械拉伸的增加而增加；2)拉伸诱导的Egr1的表达依赖于JNK；3)JNK的激活需要包含？1亚单位的整合素的信号传递。基于这些数据，这一建议的特定假设是，拉伸诱导的肺泡上皮细胞JNK磷酸化需要激活含有整合素的1，并随后形成焦点黏附复合体。本研究的目的是：1)确定牵张诱导肺上皮细胞JNK磷酸化及随后基因表达的特异性整合素(S)。使用显性负1结构，我将证实含有整合素的？1在拉伸诱导的JNK磷酸化中是重要的。然后，通过针对不同整合素a亚基的阻断抗体和/或RNA干扰，进一步确定负责JNK磷酸化的特异性整合素(S)。2)确定周期性拉伸诱导的整合素激活导致JNK下游磷酸化的机制。我将对有无拉伸的焦点黏附复合体进行染色，并使用FAK siRNA来检测焦点黏附复合体的形成和FAK激活在拉伸诱导的JNK激活中的作用。 公共卫生相关性：重症监护病房中经常使用机械通风，众所周知，它会加重肺损伤，从而导致高死亡率。进一步了解肺机械拉伸所涉及的细胞通路有助于为使用呼吸机的患者提供潜在的治疗方法和途径。

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.