Tsunoda Kou, Tsujino Ichiro, Koshi Ryosuke, Sugano Naoyuki, Sato Shuichi, Asano Masatake
Division of Applied Oral Sciences, Nihon University Graduate School of Dentistry, Tokyo, Japan.
Division of Respiratory Medicine, Nihon University School of Medicine, Tokyo, Japan.
J Cell Biochem. 2016 Apr;117(4):1009-15. doi: 10.1002/jcb.25387. Epub 2015 Oct 6.
Cigarette smoking is one of the most important risk factors for the development of various diseases. Nicotine is the most extensively investigated component of cigarette smoke, and a comprehensive analysis of the genes induced by nicotine stimulation revealed that interleukin-8 (IL-8) was induced in oral squamous cell carcinoma cell (OSCC). Based on this background, the signaling mechanisms of nicotine-mediated IL-8 induction in OSCC was investigated. Augmented IL-8 secretion by Ca9-22 cells was blocked by the NF-κB inhibitor L-1-4'-tosylamino-phenylethyl-chloromethyl ketone (TPCK) and the nicotinic acetylcholine receptor (nAChR)-specific inhibitor α-bungarotoxin (αBtx). The downstream signaling pathway was further examined by pre-incubating the cells with inhibitors against mitogen-activated protein kinase (MEK), protein kinase C (PKC), and Ca(2+)/calmodulin-dependent kinase II (CaMK II). Only the CaMK II inhibitor was found to exert an inhibitory effect on nicotine-mediated IL-8 secretion. Pre-treatment of the Ca9-22 cells with the Ca(2+) chelator BAPTA-AM drastically inhibited IL-8 secretion. Although nicotine stimulation induced the phosphorylation of the NF-κB p65 subunit, pre-treatment with BAPTA-AM was found to inhibit this activity significantly. CaMK II-dependent p65 phosphorylation was confirmed by pre-incubation of the cells with CaMK II inhibitor. The results from this study indicate that the binding of nicotine to nAChR induces Ca(2+) influx, which results in the activation and phosphorylation of CaMK II and NF-κB p65, respectively. Nicotine-mediated IL-8 induction should be a trigger for the initiation of various diseases.
吸烟是多种疾病发生的最重要风险因素之一。尼古丁是香烟烟雾中研究最为广泛的成分，对尼古丁刺激诱导的基因进行综合分析后发现，口腔鳞状细胞癌细胞（OSCC）中可诱导白细胞介素-8（IL-8）产生。基于这一背景，对尼古丁介导的OSCC中IL-8诱导的信号传导机制进行了研究。Ca9-22细胞中IL-8分泌的增加被NF-κB抑制剂L-1-4'-甲苯磺酰氨基苯乙基氯甲基酮（TPCK）和烟碱型乙酰胆碱受体（nAChR）特异性抑制剂α-银环蛇毒素（αBtx）所阻断。通过用丝裂原活化蛋白激酶（MEK）、蛋白激酶C（PKC）和钙/钙调蛋白依赖性激酶II（CaMK II）的抑制剂对细胞进行预孵育，进一步研究了下游信号通路。结果发现，只有CaMK II抑制剂对尼古丁介导的IL-8分泌具有抑制作用。用Ca(2+)螯合剂BAPTA-AM对Ca9-22细胞进行预处理可显著抑制IL-8分泌。尽管尼古丁刺激可诱导NF-κB p65亚基的磷酸化，但发现用BAPTA-AM预处理可显著抑制该活性。通过用CaMK II抑制剂对细胞进行预孵育，证实了CaMK II依赖性p65磷酸化。本研究结果表明，尼古丁与nAChR的结合诱导Ca(2+)内流，分别导致CaMK II和NF-κB p65的激活和磷酸化。尼古丁介导的IL-8诱导应是各种疾病发生的触发因素。
