Involvement of two NF-κB binding elements in tumor necrosis factor α, CD40, and Epstein-Barr virus latent membrane protein 1-mediated induction of the cellular inhibitor of apoptosis protein 2 gene

The antiapoptotic function of NF-kappa B 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/MHC) 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-kappa B. However, direct evidence of transcriptional regulation of NF-kappa B on the c-IAP2 gene is still missing. Here, we have cloned and characterized the promoter region required for NF-kappa B-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-kappa B 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-kappa B binding sites. Electrophoretic mobility shift assays characterized that the two NF-kappa B sites can be recognized and bound by the NF-kappa B 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.