Abstract
The antiapoptotic function of NF-kappaB is believed to be mediated through the induction of antiapoptotic genes. Among the antiapoptotic genes, cellular inhibitor of apoptosis protein 2 (c-IAP2/HIAP-1/MIHC) is originally identified as a molecule recruited to the tumor necrosis factor (TNF) receptor complex, and its expression is preferentially up-regulated by TNF and other stimuli activating NF-kappaB. However, direct evidence of transcriptional regulation of NF-kappaB on the c-IAP2 gene is still missing. Here, we have cloned and characterized the promoter region required for NF-kappaB-dependent transcription of the c-IAP2 gene. Sequencing of a 3.5-kilobase fragment of the 5'-flanking region of the c-IAP2 gene has identified a TATA-like sequence and potential binding sites for nuclear factor of activated T cells, interferon regulatory factor 1, activator protein 1, glucocorticoid response element, and three putative NF-kappaB binding elements. Deletion and mutational analysis of the 5'-flanking region linked to the luciferase gene revealed that transcriptional activation by TNF or interleukin 1 is mediated cooperatively by two NF-kappaB binding sites. Electrophoretic mobility shift assays characterized that the two NF-kappaB sites can be recognized and bound by the NF-kappaB p50/p65 heterodimer. In addition, the transcription of c-IAP2 promoter was strongly up-regulated when CD40 or Epstein-Barr virus latent membrane protein 1 was overexpressed.
Highlights
Apoptotic cell death and the other that antagonizes the apoptotic signal by activating transcription factor NF-B [1]
Because it has been reported that c-IAP2 gene is induced by tumor necrosis factor (TNF) [10, 17], we initially tried to analyze whether the region upstream from the 5Ј-end of the longest cDNA filed (GenBankTM accession number AF070674) possesses the promoter activity conferring TNF inducibility by reporter gene transfection analysis
It is becoming evident that NF-B appears to exert its antiapoptotic function through the induction of antiapoptotic genes, which include c-IAP1, c-IAP2, XIAP, Bcl2, IEX-1L, and A20 (4 –10)
Summary
Apoptotic cell death and the other that antagonizes the apoptotic signal by activating transcription factor NF-B [1]. It has been demonstrated that the transcriptional activation of other antiapoptotic genes such as A20, Bcl-X, and Bfl/A1 by NF-B is through one or two NF-B binding sites resided in their promoter regions [5, 20, 21]. It remains to be elucidated, whether a similar transcription-regulating mechanism is involved in c-IAP2 gene expression. Electrophoretic mobility shift assays (EMSA) and site-directed mutagenesis analysis of the NF-B binding sites demonstrated that two NF-B elements are required for promoter activity and that they function cooperatively in mediating TNF-induced c-IAP2 promoter activation. We showed that the c-IAP2 promoter activity is strongly enhanced in cells transfected with expression plasmids for CD40 and Epstein-Barr virus (EBV) oncoprotein latent membrane protein 1 (LMP1)
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