Role of c-Myc in repression of FOXO3a-mediated regulation of p27(Kip1) expression in WEHI 231 B cells treated with anti-IgM

B Cell Receptor (BCR) engagement of murine WEHI 231 immature B lymphoma cells leads sequentially to a drop in Nuclear Factor kappa B (NF-kappaB) and c-Myc, and induction of p27 cyclin-dependent kinase inhibitor (CKI), promoting growth arrest and apoptosis. BCR engagement also induces a drop in phosphatidylinositol 3-kinase (PI3K)/Akt signaling, preceding the p27 increase. Since induction of p27Kip1 is due to an increase in gene transcription, the role of the Forkhead box O (FOXO) transcription factor family was investigated. FOXO factors have been shown to potently induce p27Kip1 promoter activity and to be negatively regulated by PI3K/Akt phosphorylation. BCR engagement or PI3K inhibition in WEHI 231 cells caused an increase in functional nuclear FOXO3a levels. Inhibition of PI3K/Akt signaling led to an increase in nuclear p27 levels, while decreasing the levels of nuclear c-Myc, which represses p27Kip1 promoter activity via binding to the +1 initiator (Inr) element in a complex with Myc Associated Factor X (Max). Further, the role of Sirtuins (SIRT1) deacetylases, known to block FOXO3a activity was also studied. BCR engagement caused a modest increase in SIRTI levels and inhibition of SIRT1 activity increased nuclear FOXO3a and p27 levels in WEHI 231 cells. Since c-Myc repressed p27 Kip1 promoter activation by FOXO3a, the roles of c-Myc and FOXO3a in regulation of p27Kip1 promoter activity were explored. A functional Forkhead binding site was identified at -110 bp in the p27Kip1 promoter. In electrophoretic mobility shift and oligonucleotide precipitation assays, c-Myc via its Myc Box 11 domain interacted with FOXO3a while bound to the -110 bp Forkhead site and FOXO3a removed c-Myc/Max complexes from the +1 Mr. In vivo association of c-Myc and FOXO3a was confirmed and the interaction was mapped to the N-terminal DNA binding domain of FOXO3a. Thus, these studies identify a novel association between c-Myc and Forkhead factors that regulate the p27Kip1 CKI gene. Lastly, these findings were extended to a second FOXO3a target, the pro-apoptotic Bcl-2 family member bim (Bcl-2 interacting mediator of cell death). Thus, c-Myc and FOXO3a protein interactions function to mediate control of expression of genes regulating both cell cycle and survival.