Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520-8057, USA.
MS & Proteomics Resource at Yale University, WM Keck Foundation Biotechnology Resource Laboratory, Department of Molecular Biophysics and Biochemistry, New Haven, CT 06520-8057, USA.
Free Radic Biol Med. 2016 Dec;101:102-115. doi: 10.1016/j.freeradbiomed.2016.10.001. Epub 2016 Oct 4.
Cigarette smoking is the primary risk factor for COPD which is characterized by excessive inflammation and airflow obstruction of the lung. While inflammation is causally related to initiation and progression of COPD, the mitochondrial mechanisms that underlie the associated inflammatory responses are poorly understood. In this context, we have studied the role played by Mitogen activated protein (MAP) kinase kinase 3 (MKK3), a dual-specificity protein kinase, in cigarette smoke induced-inflammation and mitochondrial dysfunction. Serum pro-inflammatory cytokines were significantly elevated in WT but not in MKK3 mice exposed to Cigarette smoke (CS) for 2 months. To study the cellular mechanisms of inflammation, bone marrow derived macrophages (BMDMs), wild type (WT) and MKK3, were exposed to cigarette smoke extract (CSE) and inflammatory cytokine production and mitochondrial function assessed. The levels of IL-1β, IL-6, and TNFα were increased along with higher reactive oxygen species (ROS) and P-NFκB after CSE treatment in WT but not in MKK3 BMDMs. CSE treatment adversely affected basal mitochondrial respiration, ATP production, maximum respiratory capacity, and spare respiratory capacity in WT BMDMs only. Mitophagy, clearance of dysfunctional mitochondria, was up regulated in CS exposed WT mice lung tissue and CSE exposed WT BMDMs, respectively. The proteomic analysis of BMDMs by iTRAQ (isobaric tags for relative and absolute quantitation) showed up regulation of mitochondrial dysfunction associated proteins in WT and higher OXPHOS (Oxidative phosphorylation) and IL-10 signaling proteins in MKK3 BMDMs after CSE exposure, confirming the critical role of mitochondrial homeostasis. Interestingly, we found increased levels of p-MKK3 by immunohistochemistry in COPD patient lung tissues that could be responsible for insufficient mitophagy and disease progression. This study identifies MKK3 as a negative regulator of mitochondrial function and inflammatory responses to CS and suggests that MKK3 could be a therapeutic target.
吸烟是 COPD 的主要危险因素，其特征是肺部过度炎症和气流阻塞。虽然炎症与 COPD 的发生和进展有关，但导致相关炎症反应的线粒体机制尚不清楚。在这种情况下，我们研究了丝裂原激活蛋白激酶激酶 3（MKK3），一种双特异性蛋白激酶，在香烟烟雾引起的炎症和线粒体功能障碍中的作用。在接触香烟烟雾（CS）2 个月的 WT 但不是 MKK3 小鼠中，血清促炎细胞因子显著升高。为了研究炎症的细胞机制，将骨髓来源的巨噬细胞（BMDMs）、野生型（WT）和 MKK3 暴露于香烟烟雾提取物（CSE），并评估炎性细胞因子的产生和线粒体功能。在用 CSE 处理后，WT 但不是 MKK3 BMDMs 中 IL-1β、IL-6 和 TNFα 的水平增加，同时 ROS 和 P-NFκB 增加。CSE 处理仅在 WT BMDMs 中对基础线粒体呼吸、ATP 产生、最大呼吸能力和备用呼吸能力产生不利影响。在 CS 暴露的 WT 小鼠肺组织和 CSE 暴露的 WT BMDMs 中，分别上调了线粒体自噬，即清除功能失调的线粒体。通过 iTRAQ（相对和绝对定量的同重同位素标记）对 BMDMs 的蛋白质组分析表明，WT 中与线粒体功能障碍相关的蛋白质上调，而 MKK3 BMDMs 中 OXPHOS（氧化磷酸化）和 IL-10 信号蛋白上调，证实了线粒体动态平衡的关键作用。有趣的是，我们通过免疫组织化学发现 COPD 患者肺组织中 p-MKK3 水平升高，这可能导致自噬不足和疾病进展。这项研究确定了 MKK3 是 CS 诱导的线粒体功能和炎症反应的负调节剂，并表明 MKK3 可能是一个治疗靶点。
