Hewson Christopher A, Edbrooke Mark R, Johnston Sebastian L
Department of Respiratory Medicine, National Heart and Lung Institute, Imperial College London, Norfolk Place, London W2 1PG, UK.
J Mol Biol. 2004 Nov 26;344(3):683-95. doi: 10.1016/j.jmb.2004.09.059.
Asthma and chronic obstructive pulmonary disease are highly prevalent and economically important inflammatory airway diseases associated with mucus hypersecretion. Considerable additional morbidity and mortality are related to acute exacerbations, which are associated with further mucus hypersecretion. MUC5AC is a prominent airway mucin; however, the signalling pathways regulating MUC5AC hypersecretion are not fully characterised. We investigated the signalling pathway regulating phorbol 12-myristate 13-acetate (PMA)-induced MUC5AC gene and protein expression in human respiratory epithelial cells. Using NCI-H292 cells, we demonstrated that treatment with PMA increased production of total and MUC5AC-specific mucin proteins. This increase was dependent on de novo MUC5AC gene transcription. We identified a short, proximal region of the MUC5AC promoter essential for this activity containing three specificity protein (Sp) 1 transcription factor-binding sites and a single CACCC site. By chemical inhibition, site-directed promoter mutagenesis and electrophoretic mobility-shift assay (EMSA), we demonstrated that PMA induced proteins binding to all three Sp1 sites and that they were all required for full induction of MUC5AC promoter activity. We then demonstrated a Ras-Raf-MEK/ERK signalling pathway was exclusively activated upstream of Sp1 activating the promoter and confirmed the requirement for matrix metalloproteinase activation leading to a ligand-dependent activation of the epidermal growth factor receptor. Finally, we demonstrated that activation of the novel protein kinase C isoforms delta and theta; was required upstream of the metalloproteinase activation. We have characterised a signalling pathway regulating PMA induction of MUC5AC. Studies such as this identify key signalling intermediates as targets for pharmacological intervention to treat mucus hypersecretion.
哮喘和慢性阻塞性肺疾病是高度流行且在经济上具有重要意义的炎症性气道疾病，与黏液分泌过多有关。相当多的额外发病率和死亡率与急性加重相关，而急性加重又与进一步的黏液分泌过多有关。MUC5AC是一种主要的气道黏蛋白；然而，调节MUC5AC分泌过多的信号通路尚未完全明确。我们研究了调节佛波酯12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）诱导的人呼吸道上皮细胞中MUC5AC基因和蛋白表达的信号通路。使用NCI - H292细胞，我们证明用PMA处理可增加总黏蛋白和MUC5AC特异性黏蛋白的产生。这种增加依赖于MUC5AC基因的从头转录。我们确定了MUC5AC启动子的一个短的近端区域，该区域对于此活性至关重要，包含三个特异性蛋白（Sp）1转录因子结合位点和一个单一的CACCC位点。通过化学抑制、定点启动子诱变和电泳迁移率变动分析（EMSA），我们证明PMA诱导蛋白与所有三个Sp1位点结合，并且它们对于MUC5AC启动子活性的完全诱导都是必需的。然后我们证明Ras - Raf - MEK/ERK信号通路在激活启动子的Sp1上游被特异性激活，并证实了基质金属蛋白酶激活的必要性，这导致表皮生长因子受体的配体依赖性激活。最后，我们证明新型蛋白激酶C同工型δ和θ的激活在金属蛋白酶激活的上游是必需的。我们已经确定了调节PMA诱导MUC5AC的信号通路。这样的研究确定了关键信号中间体作为治疗黏液分泌过多的药物干预靶点。
