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.
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Departments of Virology, Humboldt University, Medical School Charité, D-10098 Berlin, Germany. susanna.proesch@charite.de
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D001483:Base Sequence; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism / Q000502:physiology*; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D017931:DNA Primers; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics*; D009000:Monocytes / Q000737:chemistry*; D016328:NF-kappa B / Q000502:physiology*; D011401:Promoter Regions, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000502:physiology; D015854:Up-Regulation / Q000502:physiology*; D014764:Viral Proteins
D022763:CCAAT-Enhancer-Binding Protein-alpha; D022782:CCAAT-Enhancer-Binding Protein-beta; D017931:DNA Primers; C088645:IE1 protein, cytomegalovirus; D017874:Immediate-Early Proteins; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D014764:Viral Proteins