Holvoet Sébastien, Vincent Claude, Schmitt Daniel, Serres Mireille
Laboratoire Peau Humaine et Immunité, Unité INSERM 346, Pavillon R, Hôpital E. Herriot, 69437 Lyon Cedex 03, France.
Exp Cell Res. 2003 Oct 15;290(1):108-19. doi: 10.1016/s0014-4827(03)00293-3.
MMP-9 (92 kDa) is the major gelatinase able to degrade collagen IV, secreted by keratinocytes that are actively involved in wound-healing or tumorigenesis. Since the invasive phenotype of cancers is dependent on MMP-9 expression, it appeared of interest to precisely characterize which signal transduction pathways activated by TNF-alpha are involved in MMP-9 up-regulation induced by TNF-alpha. In HaCaT cells, activation of MMP-9 occurs at the transcriptional level. Inhibition of the MAPK pathway using specific inhibitors of the Ras, Raf, MEK1/2, and Erk1/2 cascade was correlated with a marked inhibition of MMP-9 activity, as determined by gene and protein expression. MAPK pathway activation via TNF-alpha was confirmed by marked AP-1 activation detected in EMSA. Under our experimental conditions, p38 MAPK and SAPK/JNK pathways were not activated. Gene and protein expression of other MMPs that regulate MMP-9, such as MMP-1 and MMP-13, were also up-regulated by TNF-alpha and inhibited by UO126, providing evidence that the MAPK pathway plays a fundamental role in the regulation of MMP-9 secretion by keratinocytes. As TNF-alpha is known to be a main activator of NF-kappaB pathway, the effects of campthothecin and caffeic acid were investigated, such as, TNF-alpha campthothecin up-regulated MMP-9 activity but caffeic acid only weakly inhibited MMP-9 activation induced by TNF-alpha. However, NF-kappaB is activated as shown from immunostaining data, a nuclear staining and higher Western blotting expression of p50 and p65 NF-kappaB subunits were detected after TNF-alpha treatment. A higher specific signal was also detected in EMSA for TNF-alpha-treated cells.
基质金属蛋白酶-9（92 kDa）是主要的明胶酶，能够降解IV型胶原蛋白，由积极参与伤口愈合或肿瘤发生的角质形成细胞分泌。由于癌症的侵袭表型依赖于基质金属蛋白酶-9的表达，因此精确表征肿瘤坏死因子-α激活的哪些信号转导途径参与肿瘤坏死因子-α诱导的基质金属蛋白酶-9上调似乎很有意义。在HaCaT细胞中，基质金属蛋白酶-9的激活发生在转录水平。使用Ras、Raf、MEK1/2和Erk1/2级联的特异性抑制剂抑制丝裂原活化蛋白激酶（MAPK）途径与基质金属蛋白酶-9活性的显著抑制相关，这通过基因和蛋白质表达来确定。通过在电泳迁移率变动分析（EMSA）中检测到的显著激活蛋白-1（AP-1）激活，证实了通过肿瘤坏死因子-α激活的MAPK途径。在我们的实验条件下，p38 MAPK和应激激活蛋白激酶/应激活化蛋白激酶（SAPK/JNK）途径未被激活。调节基质金属蛋白酶-9的其他基质金属蛋白酶，如基质金属蛋白酶-1和基质金属蛋白酶-13的基因和蛋白质表达也被肿瘤坏死因子-α上调，并被UO126抑制，这证明MAPK途径在角质形成细胞调节基质金属蛋白酶-9分泌中起重要作用。由于已知肿瘤坏死因子-α是核因子-κB（NF-κB）途径的主要激活剂，因此研究了喜树碱和咖啡酸的作用，例如，肿瘤坏死因子-α喜树碱上调了基质金属蛋白酶-9活性，但咖啡酸仅微弱抑制肿瘤坏死因子-α诱导的基质金属蛋白酶-9激活。然而，如免疫染色数据所示，NF-κB被激活，在肿瘤坏死因子-α处理后检测到p50和p65 NF-κB亚基的核染色和更高的蛋白质印迹表达。在EMSA中也检测到肿瘤坏死因子-α处理细胞的更高特异性信号。
