PDF(Pubmed)
Abstract：
BACKGROUND: We have previously discovered clusters of sequentially negative and positive modulators of acute inflammation during cytokine stimulation in epithelial cells and identified potential targets for therapy within these clusters. MAP3K8 is a druggable kinase that we found to be a hub of a principal interaction network. We describe here the results of MAP3K8 knockdown in the A549 lung cancer cell line, the BEAS-2B epithelial cell line and normal human bronchial epithelial (NHBE) cells following IL-1β stimulation. We analysed signalling transduction and global gene expression after IL-1β stimulation with and without MAP3K8 knockdown, quantifying levels of the inflammatory cytokines IL-6, IL-8 and RANTES levels by qPCRs and/or by ELISAs. We also examined potential small molecule inhibitors for MAP3K8 in the same models.
RESULTS: IL-1β significantly and consistently increased MAP3K8 expression after 2 h in A549, BEAS-2B and NHBE cells. Phosphorylation of MAP3K8 occurred at 20 min after IL-1β stimulation and MAP3K8 protein was degraded at 30 min. MAP3K8 knockdown significantly reduced IL-6, IL-8 levels after IL-1β stimulation and yielded a 10-fold enhancement of the anti-inflammatory effects of dexamethasone. Phosphorylation of ERK1/2 (P-ERK1/2) and phosphorylation of SAPK/JNK (P-SAPK/JNK) decreased at 30 min after IL-1β stimulation with MAP3K8 knockdown. The combination of dexamethasone and MAP3K8 knockdown resulted in greater inhibition of phosphorylated ERK1/2 and SAPK/JNK. Nineteen genes including MMP1, MMP3, MMP10, ITGB8, LAMC2 and PLAT (P corrected < 0.01 respectively) demonstrated a distinct altered temporal response to IL-1β following suppression of MAP3K8. However, putative MAP3K8 inhibitors including Tpl2-1, Tpl2-2 and GSK2222867A only showed inhibition of IL-6 and IL-8 production at a high dose.
CONCLUSIONS: These results confirm that MAP3K8 is a key mediator of the early inflammatory response and that it is a potential target in inflammatory diseases. However, current tool compounds do not effectively inhibit its effects.
RESULTS: IL-1β significantly and consistently increased MAP3K8 expression after 2 h in A549, BEAS-2B and NHBE cells. Phosphorylation of MAP3K8 occurred at 20 min after IL-1β stimulation and MAP3K8 protein was degraded at 30 min. MAP3K8 knockdown significantly reduced IL-6, IL-8 levels after IL-1β stimulation and yielded a 10-fold enhancement of the anti-inflammatory effects of dexamethasone. Phosphorylation of ERK1/2 (P-ERK1/2) and phosphorylation of SAPK/JNK (P-SAPK/JNK) decreased at 30 min after IL-1β stimulation with MAP3K8 knockdown. The combination of dexamethasone and MAP3K8 knockdown resulted in greater inhibition of phosphorylated ERK1/2 and SAPK/JNK. Nineteen genes including MMP1, MMP3, MMP10, ITGB8, LAMC2 and PLAT (P corrected < 0.01 respectively) demonstrated a distinct altered temporal response to IL-1β following suppression of MAP3K8. However, putative MAP3K8 inhibitors including Tpl2-1, Tpl2-2 and GSK2222867A only showed inhibition of IL-6 and IL-8 production at a high dose.
CONCLUSIONS: These results confirm that MAP3K8 is a key mediator of the early inflammatory response and that it is a potential target in inflammatory diseases. However, current tool compounds do not effectively inhibit its effects.
摘要：
背景：我们先前已经发现了上皮细胞中细胞因子刺激期间急性炎症的顺序阴性和阳性调节剂簇，并确定了这些簇中潜在的治疗靶标。MAP3K8是一种药物激酶，我们发现它是主要相互作用网络的枢纽。我们在这里描述了A549肺癌细胞系中MAP3K8敲低的结果，IL-1β刺激后的BEAS-2B上皮细胞系和正常人支气管上皮（NHBE）细胞。我们分析了在有和没有MAP3K8敲低的情况下IL-1β刺激后的信号转导和全局基因表达，通过qPCR和/或ELISA定量炎性细胞因子IL-6、IL-8和RANTES水平。我们还在相同模型中检查了MAP3K8的潜在小分子抑制剂。
结果：IL-1β在A549，BEAS-2B和NHBE细胞中2小时后显着且一致地增加了MAP3K8的表达。IL-1β刺激后20分钟发生MAP3K8的磷酸化，30分钟时MAP3K8蛋白降解。MAP3K8敲低在IL-1β刺激后显著降低IL-6、IL-8水平,并使地塞米松的抗炎作用增强10倍。IL-1β刺激MAP3K8敲除后30分钟，ERK1/2（P-ERK1/2）的磷酸化和SAPK/JNK（P-SAPK/JNK）的磷酸化降低。地塞米松和MAP3K8敲低的组合导致磷酸化ERK1/2和SAPK/JNK的更大抑制。包括MMP1，MMP3，MMP10，ITGB8，LAMC2和PLAT在内的19个基因（分别校正为P<0.01）显示出抑制MAP3K8后对IL-1β的明显改变的时间反应。然而,假定的MAP3K8抑制剂,包括Tpl2-1,Tpl2-2和GSK2222867A在高剂量时仅显示出IL-6和IL-8产生的抑制。
结论：这些结果证实MAP3K8是早期炎症反应的关键介质，并且是炎性疾病的潜在靶标。然而,目前的工具化合物不能有效地抑制其作用。
结果：IL-1β在A549，BEAS-2B和NHBE细胞中2小时后显着且一致地增加了MAP3K8的表达。IL-1β刺激后20分钟发生MAP3K8的磷酸化，30分钟时MAP3K8蛋白降解。MAP3K8敲低在IL-1β刺激后显著降低IL-6、IL-8水平,并使地塞米松的抗炎作用增强10倍。IL-1β刺激MAP3K8敲除后30分钟，ERK1/2（P-ERK1/2）的磷酸化和SAPK/JNK（P-SAPK/JNK）的磷酸化降低。地塞米松和MAP3K8敲低的组合导致磷酸化ERK1/2和SAPK/JNK的更大抑制。包括MMP1，MMP3，MMP10，ITGB8，LAMC2和PLAT在内的19个基因（分别校正为P<0.01）显示出抑制MAP3K8后对IL-1β的明显改变的时间反应。然而,假定的MAP3K8抑制剂,包括Tpl2-1,Tpl2-2和GSK2222867A在高剂量时仅显示出IL-6和IL-8产生的抑制。
结论：这些结果证实MAP3K8是早期炎症反应的关键介质，并且是炎性疾病的潜在靶标。然而,目前的工具化合物不能有效地抑制其作用。
