CCAAT/Enhancer-binding Proteins α and β Negatively Influence the Capacity of Tumor Necrosis Factor α to Up-regulate the Human Cytomegalovirus IE1/2 Enhancer/Promoter by Nuclear Factor κB during Monocyte Differentiation

Abstract

Recently we demonstrated that the ability of tumor necrosis factor ␣ (TNF␣) 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, TNF␣ stimulation in the progenitor-like cell line HL-60 was shown to be mediated by nuclear factor B (NF-B) 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 TNF␣ stimulation is not due to insufficient NF-B activation but correlates with increased synthesis of the monocyte differentiation-associated factors CCAAT/enhancerbinding protein (C/EBP) ␣ and ␤. Several clinical studies have shown that systemic inflammation is a key factor for human cytomegalovirus (HCMV) 1 (re)activation in both immunosuppressed and non-immunosuppressed patients (1-6). The proinflammatory cytokine tumor necrosis factor ␣ (TNF␣) has been identified as a powerful mediator of HCMV stimulation and reactivation in human and murine monocyte/ macrophage progenitor cells (7,8). These are a main target cell for HCMV latency (7, 9 -11). However, TNF␣ did not directly influence reactivation of the virus in peripheral blood mononuclear leukocytes induced by allogenic stimulation (12) 2 suggesting distinct regulatory events in progenitor and mature peripheral blood mononuclear leukocytes.The HCMV IE1/2 enhancer/promoter regulates ie1/2 gene expression and initiation of viral replication (13). Recently, we observed that TNF␣ strongly activates in vitro HCMV IE1/2 enhancer/promoter in the undifferentiated granulocyte/macrophage progenitor-like cell line HL-60 (5, 14, 15). The stimulatory effect of TNF␣ on the IE1/2 promoter decreased as myeloid progenitor cells differentiated into (pro-)monocytic cells. In U937 and THP-1 cells (representing the phenotype of promonocytes and mature monocytes, respectively), the stimulatory effect of TNF␣ was reduced by about 70% and was completely abrogated, respectively, compared with HL-60 cells (14). Differentiation of HL-60 cells also diminished the TNF␣ effect on the IE1/2 enhancer/promoter (14).The stimulatory effect of TNF␣ on the IE1/2 enhancer/promoter in undifferentiated HL-60 cells is mediated via binding to its p55 type I receptor and subsequent activation of NF-B heterodimer p65/p50 that binds in the nucleus to the 18-bp repetitive sequence motifs in the IE1/2 enhancer (5, 15). Interestingly, this TNF␣-induced signal transduction pathway was also 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.The C/EBP family of transcription factors together with AP-1

Transcriptional Inhibition of Interleukin-8 Expression in Tumor Necrosis Factor-tolerant Cells

“…C/EBP␤ is a transcriptional protein that is involved in negative and positive regulation of a variety of genes including IL-8 (23)(24)(25)28). It was also suggested earlier (35) that simultaneous binding of C/EBP proteins and NF-B to DNA may strengthen the inhibitory effect of C/EBP possibly by forming a more stable protein-protein-DNA complex. In addition, in coimmunoprecipitation studies we observed an increased association of C/EBP␤ protein with p65-containing NF-B complexes in the nucleus of tolerant cells, which was most intensive after TNF re-stimulation.…”

“…As an example for the latter, C/EBP family proteins have been shown to negatively influence NF-B-mediated activation of the angiotensinogen gene acute-phase response element (46) and p65-dependent cytomegalovirus IE1/2 enhancer/promoter activity (35) as well as expression of the NF-B target gene c-myc (47). In the case of the IL-8 promoter, in which the C/EBP binding site is adjacent to the NF-B site (23), similar to the IE1/2 enhancer/promoter (35), a subtle interaction between NF-B and C/EBP appears to determine whether the IL-8 promoter is activated or inhibited (28). As discussed above, in TNF-tolerant cells we find an increased binding of C/EBP␤ to its IL-8 promoter motif as well as association of this protein with p65.…”

“…4A). Previous studies demonstrate a functional and physical association between NF-B and C/EBP family proteins (27,28,35). Therefore, in the following we attempted to determine by coimmunoprecipitation studies whether C/EBP␤ associates with NF-B complexes in cells that were first pretreated with TNF and then re-stimulated with this cytokine as described above.…”

Experimental human cytomegalovirus latency in CD14+monocytes

“…Furthermore, IL-6 can activate nuclear factor for IL-6 (NF-IL-6) in monocytic cells (42,43); NF-IL-6 is a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors (44). The HCMV major immediate-early promoter contains a C/EBP binding site (45), and therefore, IL-6 might simultaneously initiate monocyte differentiation and help to induce expression of the IE1 and IE2 proteins.…”

Immunobiology of congenital cytomegalovirus infection of the central nervous system—the murine cytomegalovirus model

“…35,46,47 The block in viral replication occurs at the level of transcription from the MIEP 38 and may be, at least in part, explained by the presence of the transcription factor C/EBP and its dominant-negative isoform. 38,48 The dominant-negative isoform of C/EBP, which has retained its DNA binding domain but has lost its transcriptional activation domain, 49 binds to and inhibits transcription from the CMV MIEP. However, inhibition of MIEP-mediated transcription can be reversed by neuron membrane depolarization dependent on activation of the cyclic AMP response element binding protein.…”