CCAAT/enhancer-binding protein beta inhibits proliferation in monocytic cells by affecting the retinoblastoma protein/E2F/cyclin E pathway but is not directly required for macrophage morphology.

Monocytic differentiation is orchestrated by complex networks that are not fully understood. This study further elucidates the involvement of transcription factor CCAAT/enhancer-binding protein ß (C/EBPß). Initially, we demonstrated a marked increase in nuclear C/EBPß-liver-enriched activating protein* (LAP*)/liver-enriched activating protein (LAP) levels and LAP/liver-enriched inhibiting protein (LIP) ratios in phorbol 12-myristate 13-acetate (PMA)-treated differentiating THP-1 premonocytic cells accompanied by reduced proliferation. To directly study C/EBPß effects on monocytic cells, we generated novel THP-1-derived (low endogenous C/EBPß) cell lines stably overexpressing C/EBPß isoforms. Most importantly, cells predominantly overexpressing LAP* (C/EBPß-long), but not those overexpressing LIP (C/EBPß-short), exhibited a reduced proliferation, with no effect on morphology. PMA-induced inhibition of proliferation was attenuated in C/EBPß-short cells. In C/EBPß(WT) macrophage-like cells (high endogenous C/EBPß), we measured a reduced proliferation/cycling index compared with C/EBPß(KO). The typical macrophage morphology was only observed in C/EBPß(WT), whereas C/EBPß(KO) stayed round. C/EBPα did not compensate for C/EBPß effects on proliferation/morphology. Serum reduction, an independent approach known to inhibit proliferation, induced macrophage morphology in C/EBPß(KO) macrophage-like cells but not THP-1. In PMA-treated THP-1 and C/EBPß-long cells, a reduced phosphorylation of cell cycle repressor retinoblastoma was found. In addition, C/EBPß-long cells showed reduced c-Myc expression accompanied by increased CDK inhibitor p27 and reduced cyclin D1 levels. Finally, C/EBPß-long and C/EBPß(WT) cells exhibited low E2F1 and cyclin E levels, and C/EBPß overexpression was found to inhibit cyclin E1 promoter-dependent transcription. Our results suggest that C/EBPß reduces monocytic proliferation by affecting the retinoblastoma/E2F/cyclin E pathway and that it may contribute to, but is not directly required for, macrophage morphology. Inhibition of proliferation by C/EBPß may be important for coordinated monocytic differentiation.

ITD- and FL-induced FLT3 signal transduction leads to increased C/EBPbeta-LIP expression and LIP/LAP ratio by different signalling modules.

FLT3 receptor-associated signalling plays a role in proliferation and leukaemia. The transcription factor C/EBPbeta may be involved in malignancy with its alternative translation product C/EBPbeta-LIP. We investigated a potential connection between FLT3 signalling and the C/EBPbeta system in FLT3-internal tandem duplication (ITD)-positive leukaemia cells and FLT3-ITD- or FLT3-wild type (WT)-transfected 32D cells. In FLT3-ITD-positive cells or when ITD sequences were inserted into the FLT3-WT receptor, significant LIP levels, increased LIP/LAP ratios, and enhanced proliferation rates were detected, which were reduced by FLT3 inhibition. In FLT3-WT cells, incubation with FLT3 receptor ligand (FL) also elevated LIP, LIP/LAP, and proliferation, albeit to a lesser extent. CEBPB-directed siRNA decreased both LIP and proliferation rates in FLT3-ITD-positive and FL-stimulated FLT3-WT-positive cells. PI3K inhibition affected ITD-associated and FL-induced LIP levels. Rapamycin, an inhibitor of mTOR involved in CEBPB translation, completely blocked the increase in LIP in FL-stimulated FLT3-WT- but not FLT3-ITD-positive cells. In contrast, the ITD-associated LIP elevation was mediated by p(90)-ribosomal-S6-kinase. This is the first report showing a LIP increase in the presence of ITD or following FL exposure. Our data suggest fundamental differences in the signalling cascades activated via ITD mutations or following FL stimulation, indicating the need for adapted molecular therapy.

C/EBPbeta enhances NF-kappaB-associated signalling by reducing the level of IkappaB-alpha.

NF-kappaB and C/EBPbeta proteins are involved in the regulation of genes which play a role in inflammation, immunity and malignant processes. The present study focuses on the question of how these systems cross talk "upstream" of the promoter level and investigates the regulation of NF-kappaB-associated signalling by C/EBPbeta. In C/EBPbeta(ko) macrophage-like cells stimulated with TNF or LPS a reduced 3kappaB-dependent transcription was detected compared to the wild type. This was accompanied by elevated nuclear p65 and NF-kappaB activity in the presence of C/EBPbeta. In addition, overexpression of C/EBPbeta in HeLa cells increased the nuclear level of coexpressed p65. Remarkably, the constitutive level of IkappaB-alpha was significantly higher in C/EBPbeta(ko) cells; and this higher level was readjusted following stimulus-induced proteolysis. The IkappaB-alpha protein stability was comparable in both macrophage-like cell types with a somewhat higher stability in unstimulated C/EBPbeta(ko) cells. Following stimulation with TNF, higher IkappaB-alpha mRNA levels were induced in C/EBPbeta(ko) cells. The autoregulatory recovery of IkappaB-alpha protein following activation was completely blocked by transcriptional inhibition, regardless if C/EBPbeta was present. Finally, we showed that C/EBPbeta overexpression in HeLa cells blocked TNF-mediated inducibility of the IkappaB-alpha promoter. Taken together, our results indicate that regulation of the IkappaB-alpha level is one of the underlying mechanisms by which C/EBPbeta controls NF-kappaB-associated signalling. C/EBPbeta may belong to the group of proteins in the regulatory machinery which adjusts the IkappaB-alpha level in different cell types under various conditions with physiological and pathophysiological implications.

Modified vaccinia virus ankara triggers chemotaxis of monocytes and early respiratory immigration of leukocytes by induction of CCL2 expression.

Orthopoxviruses commonly enter into humans and animals via the respiratory tract. Herein, we show that immigration of leukocytes into the lung is triggered via intranasal infection of mice with modified vaccinia virus Ankara (MVA) and not with the vaccinia virus (VACV) Elstree, Wyeth, or Western Reserve (WR) strain. Immigrating cells were identified as monocytes, neutrophils, and CD4(+) lymphocytes by flow cytometry and could be detected 24 h and 48 h postinfection. Using an in vitro chemotaxis assay, we confirmed that infection with MVA induces the expression of a soluble chemotactic factor for monocytes, identified as CCL2 (monocyte chemotactic protein-1 [MCP-1]). In contrast to infection with several other VACV strains, MVA induced the expression of CCL2, CCL3, CCL4, and CXCL10 in the human monocytic cell line THP-1 as well as in primary human monocytes. Thus, MVA, and not the VACV Elstree, Wyeth, or WR strain, consistently triggered the expression of a panel of chemokines, including CCL2, in the murine lung, correlating considerably with the immigration of leukocytes. Using CCL2-deficient mice, we demonstrate that CCL2 plays a key role in MVA-triggered respiratory immigration of leukocytes. Moreover, UV irradiation of MVA prevented CCL2 expression in vitro and in vivo as well as respiratory immigration of leukocytes, demonstrating the requirement for an activated molecular viral life cycle. We propose that MVA-triggered chemokine expression causes early immigration of leukocytes to the site of infection, a feature that is important for rapid immunization and its safety and efficiency as a viral vector.