C/Ebpβ Inhibits Apoptosis in Hematopoietic Cells Via Interaction with NF-κb p50.

The CCAAT/enhancer binding protein (C/EBP) family of proteins bind DNA via their C-terminal basic region – leucine zipper (BR-LZ or bZIP) domains and activate transcription via N-terminal trans-activation domains. We previously demonstrated that C/EBPα inhibits apoptosis when the immature hematopoietic Ba/F3 cell line is withdrawn from IL-3 and that a C/EBPα BR mutant that does not bind NF-κB p50 but retains the ability to bind NF-κB p65 is inactive. We have now sought to extend these findings to C/EBPβ. We first created two point mutations in C/EBPβ, R240G and ΔK253. The C/EBPβ(R240G) is analogous to the C/EBPα(R300G) BR variant that does not bind NF-κB p50 or inhibit apoptosis, and C/EBPβ(ΔK253) is analogous to the C/EBPα(ΔK313) LZ variant that does not bind DNA but retains the ability to interact with NF-κB p50 and inhibit apoptosis. In co-ip experiments using transiently transfected 293T extracts, both C/EBPβ and C/EBPβ(ΔK253) interacted with NF-κB p50, whereas C/EBPβ(R240G) did not, as predicted. In contrast, all three isoforms bound NF-κB p65. And in gel shift assay, C/EBPβ bound a consensus C/EBP site, whereas neither C/EBPβ(R240G) nor C/EBPβ(ΔK253) bound DNA, again as expected. Ba/F3 cell lines were then generated stably expressing C/EBPβ-ER, C/EBP(R240G)-ER, or C/EBPβ(ΔK253)-ER proteins, via transduction with pBabePuro retroviral vectors and puromycin selection of pooled populations. Western blot analysis confirmed similar expression of each transgene. Ba/F3 lines were removed from IL-3 and cultured with or without estradiol, to activate the ER fusion proteins. Remarkably, both CEBPβ-ER and C/EBPβ(ΔK253)-ER prolonged survival of the cells during the first 48 hrs after IL-3 withdrawal, similar to the activity of C/EBPα-ER, whereas C/EBPβ(R240G)-ER was ineffective. Current efforts focus on identifying genetic targets of C/EBPβ:NF-κB p50 cooperation relevant to the observed inhibition of apoptosis, on assessing activities of C/EBPβ and it variants in normal hematopoietic cells isolated from adult marrow, and on determining the activities of the long and short (LAP and LIP) C/EBPβ translational isoforms. Overall, the finding that C/EBPβ inhibits apoptosis in cooperation with NF-κB p50 is significant due to the expression of both C/EBPβ and activated NF-κB in several human lymphoma subsets and in solid tumors such as breast cancer. Targeting this interaction with small molecules may provide a novel therapeutic approach for these malignancies.