Abstract
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.
Highlights
Megalovirus (HCMV) IE1/2 enhancer/promoter activity (TNF␣) has been identified as a powerful mediator of human cytomegalovirus (HCMV) in myeloid progenitor-like cells decreases when these stimulation and reactivation in human and murine monocyte/
We could demonstrate that CCAAT/enhancerbinding protein (C/EBP-binding site (EBP))␣/ interacts with nuclear factor B (NF-B) 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-B site
This TNF␣-induced signal transduction pathway was identified in U937 and THP-1 cells indicating that the observed differentiation-dependent changes in TNF␣-mediated regulation of the HCMV IE1/2 enhancer/promoter cannot be explained by decreased activation of NF-B p65/p50
Summary
Cell Culture, Transfection, and CAT Assays—The cell lines HL-60, U937, and THP-1 (ATCC CCL 240, CRL 1593 and TIB 202) were maintained in RPMI 1640 medium (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (both certified for low endotoxin). Transfection efficacy of different plasmids was determined by dot blot hybridization of DNA isolated from comparable cell extracts (identical protein concentration) with a 32P-labeled BamHI-DNA fragment of plasmid pRR55 containing the CAT gene as described previously [15]. The plasmid p4-18PCAT carries the minimal IE1/2 promoter between positions Ϫ52 and ϩ52 in combination with four in-tandem copies of the 18-bp repetitive sequence motif of the IE1/2 enhancer upstream of the CAT gene [40]. Both plasmids were kindly provided by T. Cambridge, UK, contain the coding sequences for NF-B p65 and p50, respectively, under control of the CMV promoter in the pcDNA3 vector
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