title: Dexamethasone and tumor necrosis factor-alpha act together to induce the cellular inhibitor of apoptosis-2 gene and prevent apoptosis in a variety of cell types.
abstract: 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.
authors.affiliation: Department of Inflammatory Diseases Research, Bristol-Myers Squibb Pharma, Wilmington, Delaware 19880, USA. jwebster@ttpharma.com
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Webster
authors.forename: Reid M
authors.initials: RM
authors.lastname: Huber
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Hanson
authors.forename: Paul M
authors.initials: PM
authors.lastname: Collier
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Haws
authors.forename: Juliane K
authors.initials: JK
authors.lastname: Mills
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Burn
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Allegretto
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D003907:Dexamethasone / Q000494:pharmacology*; D004357:Drug Synergism; D005786:Gene Expression Regulation / Q000187:drug effects*; D005909:Glioblastoma / Q000378:metabolism / Q000473:pathology; D005938:Glucocorticoids / Q000494:pharmacology* / Q000502:physiology; D006801:Humans; D008168:Lung / Q000166:cytology / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D020218:Response Elements / Q000502:physiology; D013601:T-Lymphocytes / Q000502:physiology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D005938:Glucocorticoids; D011506:Proteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D003907:Dexamethasone