C/EBP alpha and beta mimic retinoic acid activation of IGFBP-5 in neuroblastoma cells by a mechanism independent from binding to their site.

Signal transduction mediated by insulin-like growth factors is implicated in the aggressive behavior of neuroblastoma (NB), a childhood tumor originating from the neural crest. IGFBP-5, a protein that binds IGFs with high affinity, is expressed in many NB cell lines exerting opposite effects, depending on its concentration. We found that IGFBP-5 expression increased during retinoic acid (RA)-mediated differentiation of NB cells. This was due to transcriptional activation as demonstrated by reporter assays carried out in basal and differentiating conditions. We defined the shortest region of the human IGFBP-5 promoter (from nucleotide -83 to +53) which is sensitive to RA. Mutation of a CCAAT enhancer binding protein (C/EBP) element inside this region increased transcription, suggesting a repressive role of this sequence. DNA Affinity Precipitation Assays (DAPA) and chromatin immunoprecipitation demonstrated that the binding of C/EBPalpha and beta to the C/EBP site decreased upon treatment with RA. C/EBPalpha and beta induced an increase in IGFBP-5 transcription in human and murine NB cells similar to that obtained upon RA treatment. Activation by C/EBP alpha and beta did not depend on their binding to the C/EBP site, since they still activated IGFBP-5 promoter carrying a mutation in the C/EBP site. Of interest, we found that both transcription factors were able to interact with the TATA box, but only C/EBPalpha interaction increased during RA-induced differentiation.