Analyses of the Inducible Cyclic Adenosine 3′,5′-Monophosphate Early Repressor (ICER) and cAMP Response Element Binding Protein (CREB) in HL60 Cells: New Insight Leukemogenesis?.

The cyclic AMP response-element binding protein (CREB) is a transcription factor that controls genes that regulate cell differentiation, proliferation and survival. CREB protein overexpression has previously been demonstrated in lymphoid and myeloid leukemia. In contrast, it is lower in healthy samples. To understand CREB role in hematopoiesis and leukemogenesis we focused on ICER (Inducible Cyclic Adenosine 3′,5′-monophosphate early repressor). We hypothesize that ICER, CREB endogenous antagonist, deserves a special consideration in CREB function and in the activation of gene expression. ICER directs its specific binding to cAMP response elements (CREs) functioning as a potent repressor of CREB binding and therefore of cAMP-induced transcription. It has been already demonstrated that ICER directly participates in cell fate in other systems. We have previously found that ICER mRNA was at low level at diagnosis of leukemia, whereas it increased in documented remission samples collected during follow up. We constructed an expression vector for ICER and induced its exogenous expression in HL60 cells. We then tested transcription and translation of a series of genes known to have a direct link with the members of the cAMP/CREB pathway by quantitative gene expression analysis and western blot. To determine wheter ICER protein affected identical cellular targets of CREB by repressing CRE containing promoters, we examined luciferase activity when directed by a promoter made up of 4XCREs sequences post ICER transient induction. By chromatin immunoprecipitation assay (ChIP) we looked at specific genes promoters binding, also in permanent ectopic ICER expression system. Results revealed that ICER protein was detected after 24 hours post transfection with sustained induction after 48 hours, whereas CREB mRNA and protein are down regulated. Density Array made up of 96 genes cited in CREB database (http://natural.salk.edu/CREB) for the high predictive value to contain CRE consensus sequence in their promoter revealed a wide genes expression alterations occurring over time post ICER exogenous expression, counteracting CREB transcriptional function. For some genes we confirmed that mRNA down regulation was representative of protein downregulation. We revealed that luciferase activity was strongly reduced by ICER transient induction. Moreover ChIP analyses revealed that CREB binds to the Bcl-2, ICER and CyA1 promoters in HL60 controlling their transcription. This binding was strongly reduced, in particular for Bcl-2, after ICER stable transfection in HL60 confirming its important role in gene expression reduction. Finally, we hypothesize that CREB over expression might up-regulate target genes, affecting cell proliferation and survival at diagnosis of leukaemia. Insufficient ICER expression might fail to counteract these events. These findings represent an important first step in the understanding of the physiological processes linked to the cAMP/CREB/ICER pathway. The future understanding of ICER role in blocking cAMP activation pathway and the finding of a pool of CREB target genes in HL60 may help understanding leukemogenesis.