CIHR Group in Fetal Development and Health, Department of Physiology, University of Toronto, Toronto, Ontario, Canada.
Biol Reprod. 2010 Sep;83(3):481-7. doi: 10.1095/biolreprod.109.082578. Epub 2010 May 12.
The identification of proinflammatory signal transduction pathways may suggest new therapeutic targets. In this study, we examine which signaling pathways are involved in tumor necrosis factor (TNF)-induced matrix metalloproteinase 9 (MMP9) secretion in human chorionic trophoblast (CT) cells. Purified CT cells were cultured in the presence of antibodies or chemical inhibitors that specifically block/inhibit distinct TNF receptors and kinase pathways. TNF-induced proMMP9 production, as measured by zymography, was significantly blocked/inhibited by TNF receptor 1 (TNFRSF1A) antibody, NFKB activation inhibitor (NFKBAI), and MAPK1/3 (ERK) inhibitor (U0126) (P < 0.01), but not by TNF receptor 2 (TNFRSF1B) antibody, MAPK14 (p38 MAPK) inhibitor (SB203580), and MAPK8/9/10 (JNK) inhibitor (SP600125). By Western blot analysis, we found that TNF rapidly and significantly increased phosphorylation of IKBKB, MAPK1/3, and MAPK8/9/10 and that the phosphorylation of these kinases by TNF was reduced significantly by TNFRSF1A neutralizing antibody, but not by TNFRSF1B neutralizing antibody. Moreover, we found that TNF increased TNF receptor-associated factor (TRAF) 1 and decreased TRAF2 protein expression through TNFRSF1A, but not TNFRSF1B. The CT cells that had increased TRAF1 and decreased TRAF2 after an initial TNF treatment demonstrated a dramatic deficiency in phosphorylation of the above protein kinases following a secondary TNF treatment. Localization of RELA subunit by immunocytochemistry was shifted to the nuclei after TNF treatment compared to cytosol in untreated controls. We also found cross-talk between the phosphoinositide 3-kinase pathway and ERK pathway. In summary, we have demonstrated that TNF stimulates proMMP9 production in CT cells through TNFRSF1A-TRAFs-IKBKB-NFKB and ERK signaling pathways, but not through TNFRSF1B and JNK/p38-AP-1 pathways.
鉴定促炎信号转导途径可能提示新的治疗靶点。在这项研究中，我们研究了哪些信号通路参与了肿瘤坏死因子（TNF）诱导的人绒毛膜滋养层（CT）细胞基质金属蛋白酶 9（MMP9）的分泌。纯化的 CT 细胞在存在特异性阻断/抑制不同 TNF 受体和激酶途径的抗体或化学抑制剂的情况下进行培养。通过酶谱法测量，TNF 诱导的 proMMP9 产生被 TNF 受体 1（TNFRSF1A）抗体、NFKB 激活抑制剂（NFKBAI）和 MAPK1/3（ERK）抑制剂（U0126）显著阻断/抑制（P<0.01），但不受 TNF 受体 2（TNFRSF1B）抗体、MAPK14（p38 MAPK）抑制剂（SB203580）和 MAPK8/9/10（JNK）抑制剂（SP600125）的影响。通过 Western blot 分析，我们发现 TNF 迅速且显著增加了 IKBKB、MAPK1/3 和 MAPK8/9/10 的磷酸化，并且 TNFRSF1A 中和抗体显著降低了这些激酶的磷酸化，但 TNFRSF1B 中和抗体没有。此外，我们发现 TNF 通过 TNFRSF1A 增加了 TNF 受体相关因子（TRAF）1 并降低了 TRAF2 蛋白表达，但不是通过 TNFRSF1B。最初用 TNF 处理后增加了 TRAF1 并降低了 TRAF2 的 CT 细胞在用第二次 TNF 处理后，上述蛋白激酶的磷酸化明显缺乏。与未处理对照相比，TNF 处理后 RELA 亚基的免疫细胞化学定位从细胞质转移到细胞核。我们还发现了磷酸肌醇 3-激酶途径和 ERK 途径之间的串扰。总之，我们已经证明 TNF 通过 TNFRSF1A-TRAFs-IKBKB-NFKB 和 ERK 信号通路而不是通过 TNFRSF1B 和 JNK/p38-AP-1 通路刺激 CT 细胞中 proMMP9 的产生。
Zotero 插件