Webster Jeffrey C, Huber Reid M, Hanson Rebecca L, Collier Paul M, Haws Thomas F, Mills Juliane K, Burn Timothy C, Allegretto Elizabeth A
Department of Inflammatory Diseases Research, Bristol-Myers Squibb Pharma, Wilmington, Delaware 19880, USA.
Endocrinology. 2002 Oct;143(10):3866-74. doi: 10.1210/en.2002-220188.
Using microarray technology, we analyzed 12,000 genes for regulation by TNF-alpha and the synthetic glucocorticoid, dexamethasone, in the human lung epithelial cell line, A549. Only one gene was induced by both agents, the cellular inhibitor of apoptosis 2 (c-IAP2), which was induced 17-fold and 5-fold by TNF-alpha at 2 h and 24 h, respectively, and increased 14-fold and 9-fold by dexamethasone at 2 h and 24 h, respectively. The combination of the two agents together led to an additive increase (34-fold) at 2 h and a more than additive effect (36-fold) at 24 h. The human c-IAP2 promoter contains two nuclear factor (NF)-kappaB sites that have been shown to be required for transcriptional activation by TNF-alpha. To test whether glucocorticoids regulate the c-IAP2 gene at the level of the promoter, a reporter vector containing 947 bases upstream of the start site of transcription of the human c-IAP2 promoter was linked to luciferase [IAP(-947-+54)-LUC] and transfected into A549 cells. Dexamethasone and TNF-alpha each induced reporter activity, whereas the combination of the two agents led to greater induction of luciferase than either one alone. Truncation of the promoter region containing a putative glucocorticoid response element (GRE) at -515 [IAP(-395-+54)-LUC] or mutation of the GRE in the context of the natural promoter [IAP(-947-+54mutGRE)-LUC] resulted in a loss of dexamethasone-mediated induction of reporter activity. Although the functional NF-kappaB sites were retained in the truncated and mutant c-IAP2 promoter constructs, dexamethasone did not inhibit the TNF-alpha induction of luciferase activity, indicating that GR repression through the NF-kappaB sites did not occur. Regulation of the c-IAP2 gene is therefore unique, as GR and NF-kappaB signaling pathways are usually mutually antagonistic, not cooperative. Treatment of A549 cells with TNF-alpha and/or dexamethasone had no effect on cell death, but the two agents were able to inhibit interferon-gamma/anti-FAS antibody-mediated apoptosis. In human glioblastoma A172 cells, TNF-alpha and dexamethasone together elicited a greater than additive increase in c-IAP2 mRNA levels and also inhibited anti-FAS antibody-mediated A172 cell apoptosis. In contrast, in human CEM-C7 leukemic T cells, whereas TNF-alpha and dexamethasone treatment also led to an increase in c-IAP2 mRNA, the two agents were able to induce apoptosis on their own. However, TNF-alpha and dexamethasone were also able to blunt anti-FAS-induced apoptosis in the T cells. These data indicate that the induction of the antiapoptotic protein, c-IAP2, by glucocorticoids and TNF-alpha correlates with the ability of these agents to inhibit apoptosis in a variety of cell types.
我们运用微阵列技术，在人肺上皮细胞系A549中分析了12000个基因受肿瘤坏死因子-α（TNF-α）和合成糖皮质激素地塞米松调控的情况。两种药物仅共同诱导了一个基因，即细胞凋亡抑制因子2（c-IAP2），TNF-α在2小时和24小时分别使其诱导增加17倍和5倍，地塞米松在2小时和24小时分别使其增加14倍和9倍。两种药物联合使用在2小时导致相加性增加（34倍），在24小时产生超相加效应（36倍）。人c-IAP2启动子包含两个核因子（NF）-κB位点，已证明它们是TNF-α转录激活所必需的。为了测试糖皮质激素是否在启动子水平调控c-IAP2基因，将一个包含人c-IAP2启动子转录起始位点上游947个碱基的报告载体与荧光素酶连接[IAP(-947- +54)-LUC]，并转染到A549细胞中。地塞米松和TNF-α各自诱导报告基因活性，而两种药物联合使用比单独使用任何一种药物导致更强的荧光素酶诱导。在-515处截断包含假定糖皮质激素反应元件（GRE）的启动子区域[IAP(-395- +54)-LUC]或在天然启动子背景下使GRE突变[IAP(-947- +54mutGRE)-LUC]导致地塞米松介导的报告基因活性诱导丧失。尽管在截断和突变的c-IAP2启动子构建体中功能性NF-κB位点得以保留，但地塞米松并未抑制TNF-α诱导的荧光素酶活性，表明未发生通过NF-κB位点的糖皮质激素受体（GR）抑制作用。因此，c-IAP2基因的调控是独特的，因为GR和NF-κB信号通路通常相互拮抗而非协同。用TNF-α和/或地塞米松处理A549细胞对细胞死亡无影响，但这两种药物能够抑制干扰素-γ/抗FAS抗体介导的细胞凋亡。在人胶质母细胞瘤A172细胞中，TNF-α和地塞米松共同作用使c-IAP2 mRNA水平产生超相加性增加，并且也抑制抗FAS抗体介导的A172细胞凋亡。相反，在人CEM-C7白血病T细胞中，虽然TNF-α和地塞米松处理也导致c-IAP2 mRNA增加，但这两种药物自身能够诱导细胞凋亡。然而，TNF-α和地塞米松也能够减弱T细胞中抗FAS诱导的细胞凋亡。这些数据表明，糖皮质激素和TNF-α对抗凋亡蛋白c-IAP2的诱导与这些药物在多种细胞类型中抑制细胞凋亡的能力相关。
