INVOLVEMENT OF NF-κB p50/p65 HETERODIMER IN ACTIVATION OF THE HUMAN PRO-INTERLEUKIN-1β GENE AT TWO SUBREGIONS OF THE UPSTREAM ENHANCER ELEMENT

A region between–3134 and –2729 bp upstream from the transcription site of the human pro-interleukin 1β (proIL-1β) gene was identified as an LPS-responsive enhancer element. In this study, the influence of the sequences located between –3134 and –2987 on the transcriptional activity of the proIL-1β gene in LPS-stimulated Raw 264.7 cells was examined in detail. The results obtained by transient transfection offos-CAT constructs that contained serial 5′-deletion mutations showed that the region between –3134 and –3059 appears to be required for the induction of transcription by LPS. Gel shift assay studies with synthetic oligonucleotides corresponding to partial sequences of the latter region and nuclear extracts from stimulated cells revealed specific protein binding sites between –3110 and –3090 and between –3079 and –3059. These specific bindings were time and LPS dose dependent. The results of supershift analysis using specific antibodies against transcription factors suggested that both binding complexes contained the NF-κB components p50 and p65, and did not contain other NF-κB proteins (p52, c-Rel, Rel B), AP-1 proteins (c-Fos, C-Jun), CREB or C/EBPβ (NF-IL6). Mutation of either of the putative NF-κB-binding sites in the enhancer element decreased the LPS-stimulated transcriptional activity. These data indicated that two NF-κB-binding sites, which are located between –3134 and –3059, are critical for the activation of proIL-1β gene transcription.