CCAAT/enhancer-binding proteins alpha and beta negatively influence the capacity of tumor necrosis factor alpha to up-regulate the human cytomegalovirus IE1/2 enhancer/promoter by nuclear factor kappaB during monocyte differentiation

Recently we demonstrated that the ability of tumor necrosis factor alpha (TNFalpha) to stimulate the human cytomegalovirus (HCMV) IE1/2 enhancer/promoter activity in myeloid progenitor-like cells decreases when these cells differentiate into promonocytic cells. In addition, TNFalpha stimulation in the progenitor-like cell line HL-60 was shown to be mediated by nuclear factor kappaB (NF-kappaB) activation and its binding to the 18-base pair sequence motifs of the IE1/2 enhancer. We demonstrate here that the cell differentiation-dependent reduction of TNFalpha stimulation is not due to insufficient NF-kappaB activation but correlates with increased synthesis of the monocyte differentiation-associated factors CCAAT/enhancer-binding protein (C/EBP) alpha and beta. Overexpression of C/EBPalpha/beta in HL-60 cells, which normally produce only very small amounts of C/EBP, stimulated the basal activity of the promoter in the absence of NF-kappaB but suppressed the stimulatory effect of TNFalpha. A novel C/EBP-binding site was identified in the IE1/2 enhancer directly downstream of a NF-kappaB site. In order to understand the mechanisms of interaction, we used an IE1/2 promoter mutant that failed to bind C/EBP at this position and several constructs that contained exclusively NF-kappaB- and/or C/EBP-binding sites upstream of the minimal IE1/2 promoter. We could demonstrate that C/EBPalpha/beta interacts with NF-kappaB p65 and displays inhibitory activity even in the absence of direct DNA binding by forming p65-C/EBP-containing protein complexes bound to the NF-kappaB site. Moreover, C/EBP binding to the DNA adjacent to NF-kappaB supports the down-regulatory effect of C/EBPs possibly due to stabilization of a multimeric NF-kappaB-C/EBP complex. Our results show that cell differentiation factors may interfere with TNFalpha-induced human cytomegalovirus gene (re)activation.     

C/EBPalpha inactivation in FAK-overexpressed HL-60 cells impairs cell differentiation

We previously demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as oxidative stress, ionizing radiation and TNF-receptor-induced ligand (TRAIL) compared with vector-transfected (HL-60/Vect) cells. Here, we show that HL-60/FAK cells are highly resistant to all-trans retinoic acid (ATRA)-induced differentiation, whereas original HL-60 or HL-60/Vect cells are sensitive. Treatment with ATRA at 1 muM for 5 days markedly inhibited the proliferation and increased the expression of differentiation markers (CD38, CD11b) in HL-60/Vect cells, but showed no such effect in HL-60/FAK cells. Electrophoretic mobility shift assay (EMSA) using an oligonucleotide for the c/EBP consensus binding sequence showed that c/EBPalpha was activated in ATRA-treated HL-60/Vect cells but not in HL-60/FAK cells, indicating that c/EBPalpha activation by ATRA was impaired in HL-60/FAK cells. In addition, the association of retinoblastoma protein (pRb) and c/EBPalpha after treatment with ATRA was seen in HL-60/Vect cells but not in HL-60/FAK cells. Further, hyperphosphorylation of pRb was observed in HL-60/FAK cells. Finally, the introduction of FAK siRNA into HL-60/FAK cells resulted in the recovery of sensitivity to ATRA-induced differentiation, confirming that the inhibition of HL-60/FAK differentiation resulted from both the induction of pRb hyperphosphorylation and the inhibition of association of pRb and c/EBPalpha.