Said Anan H, Hu Shien, Abutaleb Ameer, Watkins Tonya, Cheng Kunrong, Chahdi Ahmed, Kuppusamy Panjamurthy, Saxena Neeraj, Xie Guofeng, Raufman Jean-Pierre
Division of Gastroenterology and Hepatology, Department of Medicine, University of Maryland School of Medicine, 22 South Greene Street, N3W62, Baltimore, MD 21201-1595, U.S.A.
Veterans Affairs Maryland Health Care System, Baltimore, MD 21201-1595, U.S.A.
Biochem J. 2017 Feb 20;474(5):647-665. doi: 10.1042/BCJ20160704.
M3 muscarinic receptor (M3R) expression is increased in colon cancer; M3R activation stimulates colon cancer cell invasion via cross-talk with epidermal growth factor receptors (EGFR), post-EGFR activation of mitogen-activated protein kinase (MAPK) extracellular signal-related kinase 1/2 (ERK1/2), and induction of matrix metalloproteinase-1 () expression. expression is strongly associated with tumor metastasis and adverse outcomes. Here, we asked whether other MAPKs regulate M3R agonist-induced expression. In addition to activating ERK1/2, we found that treating colon cancer cells with acetylcholine (ACh) stimulated robust time- and dose-dependent phosphorylation of p38 MAPK. Unlike ERK1/2 activation, ACh-induced p38 phosphorylation was EGFR-independent and blocked by inhibiting protein kinase C-α (PKC-α). Inhibiting activation of PKC-α, EGFR, ERK1/2, or p38-α/β alone attenuated, but did not abolish ACh-induced expression, a finding that predicted potentiating interactions between these pathways. Indeed, ACh-induced expression was abolished by incubating cells with either an EGFR or MEK/ERK1/2 inhibitor combined with a p38-α/β inhibitor. Activating PKC-α and EGFR directly with the combination of phorbol 12-myristate 13-acetate (PMA) and EGF potentiated gene and protein expression, and cell invasion. PMA- and ACh-induced expression were strongly diminished by inhibiting Src and abolished by concurrently inhibiting both p38-α/β and Src, indicating that Src mediates the cross-talk between PKC-α and EGFR signaling. Using siRNA knockdown, we identified p38-α as the relevant p38 isoform. Collectively, these studies uncover novel functional interactions between post-muscarinic receptor signaling pathways that augment expression and drive colon cancer cell invasion; targeting these potentiating interactions has therapeutic potential.
M3毒蕈碱受体（M3R）在结肠癌中表达增加；M3R激活通过与表皮生长因子受体（EGFR）相互作用、EGFR激活后丝裂原活化蛋白激酶（MAPK）细胞外信号调节激酶1/2（ERK1/2）的激活以及基质金属蛋白酶-1（）表达的诱导来刺激结肠癌细胞侵袭。 表达与肿瘤转移和不良预后密切相关。在此，我们探讨了其他MAPK是否调节M3R激动剂诱导的 表达。除了激活ERK1/2外，我们发现用乙酰胆碱（ACh）处理结肠癌细胞可刺激p38 MAPK发生强烈的时间和剂量依赖性磷酸化。与ERK1/2激活不同，ACh诱导的p38磷酸化不依赖于EGFR，并可被抑制蛋白激酶C-α（PKC-α）所阻断。单独抑制PKC-α、EGFR、ERK1/2或p38-α/β的激活可减弱但不能消除ACh诱导的 表达，这一发现预示了这些信号通路之间的增强相互作用。事实上，通过将细胞与EGFR或MEK/ERK1/2抑制剂与p38-α/β抑制剂联合孵育，可消除ACh诱导的 表达。用佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）和表皮生长因子（EGF）联合直接激活PKC-α和EGFR可增强 基因和蛋白表达以及细胞侵袭。抑制Src可强烈减弱PMA和ACh诱导的 表达，同时抑制p38-α/β和Src可消除该表达，表明Src介导PKC-α和EGFR信号之间的相互作用。通过小干扰RNA敲低，我们确定p38-α是相关的p38亚型。总体而言，这些研究揭示了毒蕈碱受体后信号通路之间新的功能相互作用，这些相互作用增强了 表达并驱动结肠癌细胞侵袭；针对这些增强相互作用具有治疗潜力。
