The role of alveolar epithelial type I cells in bleomycin-induced lung injury: Characterization of fibrosis-relevant signal pathways

在这个项目的开始，我们研究了博莱霉素处理人远端肺上皮细胞系NCI-H441和小鼠肺精密切片培养后I型肺泡上皮细胞中与嘌呤能受体P2X7R相关的信号通路。我们证实了在博莱霉素作用后的早期阶段存在着P2X7R-PKC-β-CaMKII轴的证据，并且我们仍然在研究这一通路对于维持肺泡屏障功能的重要性。这个项目的另一个重要方面涉及到小窝蛋白-1参与了适当维持肺泡屏障功能。在这里，我们可以证明小窝蛋白-1与Src激酶Fyn相关。Fyn激酶在肺泡I型上皮细胞中有选择性表达。博莱霉素降低NCI-H441细胞单层跨皮电阻(TEER)，抑制Fyn可减轻在此条件下TEER的损失。此外，小窝蛋白-1基因敲除的小鼠肺中出现了Fyn激动酶的细胞分布变化。在该项目的扩展过程中，主要目的是研究新近批准的抗纤维化药物吡非尼酮对肺泡上皮细胞抗纤维化通路的影响。计划中的实验应该会澄清，E-钙粘素-连环蛋白复合体的重要结合伙伴p120连环蛋白是否被Fyn磷酸化，以及Fyn激酶是否是吡非尼酮的靶标。吡非尼酮诱导的小窝蛋白-1和E-钙粘蛋白表达的变化，正如本项目中已经证明的那样，强调了Fyn激酶和小窝蛋白-1参与了肺泡上皮细胞间通讯的调节。

At the beginning of this project, we studied signaling pathways associated with the purinergic receptor P2X7R in alveolar epithelial type I cells after bleomycin treatment of the human distal lung epithelial cell line, NCI-H441 and of mouse precision-cut lung slice cultures. We confirmed the evidence of the P2X7R-PKC-β-CaMKII axis during earliest stages after bleomycin exposure and we still investigate the importance of this pathway for the maintenance of the alveolar barrier function.Another important aspect of this project relates to the involvement of caveolin-1 in a proper maintaining alveolar barrier function. Here we could demonstrate that caveolin-1 is associated with the Src kinase Fyn. Fyn kinase was found to be selectively expressed in alveolar epithelial type I cells. Bleomycin reduces the transepithelial electrical resistance (TEER) in NCI-H441 cell monolayers and inhibition of Fyn attenuated the loss of TEER under these conditions. In addition, caveolin-1 knockout in mouse lungs presented with an altered cellular distribution of Fyn kinase.It is the main aim during the extension of this project to investigate the effect of pirfenidone, a recently approved anti-fibrosis agent, on anti-fibrotic pathways in alveolar epithelial cells. The planned experiments should clarify, whether p120 catenin, an important binding partner of the E-cadherin-catenin complex, is phosphorylated by Fyn and whether Fyn kinase is a target of pirfenidone. The pirfenidone-induced changes in caveolin-1 and E-cadherin expression, as already demonstrated in this project, emphasize the involvement of Fyn kinase and caveolin-1 in the regulation of intercellular communication in the alveolar epithelium.