CYCLIC-AMP SIGNALING PATHWAYS ARE IMPORTANT IN IL-1-BETA TRANSCRIPTIONAL REGULATION

An intact cAMP response element (CRE) in the upstream regulatory seque nce of IL-1 beta (-2755/-2762) has been shown to be essential for main taining full IL-1 beta inducibility following treatment with LPS, PMA, or TNF-alpha. In the present study, using the recombinant plasmid pIL -1(4.0 kb)-chloramphenicol acetyltransferase, containing 4.0 kb of the IL-1 beta upstream regulatory sequence, we have demonstrated that dib utyryl cAMP treatment alone is capable of induction. Due to the critic al nature of the CRE for the induction of IL-1 beta transcription, an effort was made to determine the importance of the cAMP signaling path way(s) by determining whether CRE binding protein (CREB) and other CRE B/activating transcription factor (ATF) family members that responded to cAMP were associated with the DNA-protein complex that forms at thi s site. Nuclear extracts prepared from LPS-treated THP-1 5A cells were fractionated by ammonium sulfate precipitation and heparin-Sepharose chromatography, and the resulting fractions were characterized in elec trophoretic gel mobility shift assays. These purification steps result ed in an approximately 100-fold enrichment of the proteins binding to the CRE site. Western blot analysis of isolated fractions, using CREB- and ATF-1-specific Ab showed an increased level of these proteins in the enriched fractions. Tryptic digest and DNase I protection studies showed the presence of CREE protein in the complex at the CRE site. Su pershift electrophoretic gel mobility shift assays and immunoprecipita tion analysis provided further evidence that both CREB and ATF-1 are p resent in the complex. In addition, an increase in CREB phosphorylatio n was observed when THP-1 5A cells were treated with dibutyryl cAMP, L PS, or both. These studies confirm the importance of a cAMP signaling pathway(s) in the regulation of IL-1 beta at the transcriptional level .