Characterization of GTP cyclohydrolase I gene expression in the human neuroblastoma SKN-BE(2)M17: enhanced transcription in response to cAMP is conferred by the proximal promoter

GTP cyclohydrolase 1 (GTPCH) gene expression was investigated in the human monoamine-containing neuroblastoma cell line SK-N-BE(2)M17. Northern blot a nalysis revealed a single GTPCH mRNA transcript that was confirmed by RNase protection assay to encode for Type 1 GTPCH; no alternatively spliced form s of GTPCH mRNA were detected with this assay. Incubation with 8Br-cAMP, bu t not nerve growth factor or leukemia inhibitory factor, produced a rapid i ncrease in GTPCH mRNA and protein levels; protein levels remained elevated during the entire treatment period while mRNA content declined rapidly betw een 10 and 24 h. Treatment with 8Br-cAMP did not significantly modify the s tability of GTPCH mRNA but did increase GTPCH transcription as determined b y transient transfection assays of a luciferase reporter construct containi ng 1171 bp of human GTPCH 5'-flanking sequence. Cis-acting elements require d for maximal basal and cAMP-dependent transcription were localized by dele tion analysis to the 146 bp proximal promoter. DNase I footprint analysis o f the proximal promoter using SK-N-BE(2)M17 nuclear extracts identified two protein binding domains: one an upstream Spl-like site and the other a com bined CRE-Sp1-CCAAT-box element. EMSA and supershift assays demonstrated th at the combined CRE-Spl-CCAAT-box element recruits ATF-2 and NF-Y but not S p1-4 or Egr-1-3. NF-Y binding was confirmed using pure recombinant human NF -Y protein. Transcription of the human GTPCH gene in human SK-N-BE(2)M17 ce lls is thus enhanced by cAMP acting through regulatory elements located in the proximal promoter and may involve the transcription factors NF-Y and AT F-2.