Department of Pathology, College of Medicine, University of Vermont, Burlington, Vermont, USA.
PLoS One. 2013;8(1):e54391. doi: 10.1371/journal.pone.0054391. Epub 2013 Jan 18.
The respiratory epithelium is subject to continuous environmental stress and its responses to injury or infection are largely mediated by transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling cascades. Based on previous studies indicating involvement of ATP-dependent activation of the NADPH oxidase homolog DUOX1 in epithelial wound responses, the present studies were performed to elucidate the mechanisms by which DUOX1-derived H(2)O(2) participates in ATP-dependent redox signaling and EGFR transactivation. ATP-mediated EGFR transactivation in airway epithelial cells was found to involve purinergic P2Y(2) receptor stimulation, and both ligand-dependent mechanisms as well as ligand-independent EGFR activation by the non-receptor tyrosine kinase Src. Activation of Src was also essential for ATP-dependent activation of the sheddase ADAM17, which is responsible for liberation and activation of EGFR ligands. Activation of P2Y(2)R results in recruitment of Src and DUOX1 into a signaling complex, and transient siRNA silencing or stable shRNA transfection established a critical role for DUOX1 in ATP-dependent activation of Src, ADAM17, EGFR, and downstream wound responses. Using thiol-specific biotin labeling strategies, we determined that ATP-dependent EGFR transactivation was associated with DUOX1-dependent oxidation of cysteine residues within Src as well as ADAM17. In aggregate, our findings demonstrate that DUOX1 plays a central role in overall epithelial defense responses to infection or injury, by mediating oxidative activation of Src and ADAM17 in response to ATP-dependent P2Y(2)R activation as a proximal step in EGFR transactivation and downstream signaling.
呼吸道上皮细胞不断受到环境压力的影响，其对损伤或感染的反应主要通过表皮生长因子受体 (EGFR) 的转激活及其下游信号级联来介导。基于先前的研究表明，ATP 依赖性 NADPH 氧化酶同源物 DUOX1 的激活参与了上皮细胞伤口反应，本研究旨在阐明 DUOX1 衍生的 H2O2 参与 ATP 依赖性氧化还原信号和 EGFR 转激活的机制。研究发现，气道上皮细胞中 ATP 介导的 EGFR 转激活涉及嘌呤能 P2Y2 受体刺激，以及配体依赖性和非受体酪氨酸激酶Src 引起的配体非依赖性 EGFR 激活。Src 的激活对于 ATP 依赖性 ADAM17 的激活也是必需的，ADAM17 负责 EGFR 配体的释放和激活。P2Y2R 的激活导致 Src 和 DUOX1 募集到信号复合物中，瞬时 siRNA 沉默或稳定 shRNA 转染证实了 DUOX1 在 ATP 依赖性 Src、ADAM17、EGFR 和下游伤口反应激活中的关键作用。使用硫醇特异性生物素标记策略，我们确定 ATP 依赖性 EGFR 转激活与 DUOX1 依赖性 Src 以及 ADAM17 中的半胱氨酸残基氧化有关。总之，我们的研究结果表明，DUOX1 通过介导 Src 和 ADAM17 的氧化激活来发挥核心作用，从而响应 ATP 依赖性 P2Y2R 激活，作为 EGFR 转激活和下游信号的近端步骤，从而介导上皮细胞对感染或损伤的整体防御反应。
