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 β (C/EBPβ). Initially, we demonstrated a marked increase in nuclear C/EBPβ-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/EBPβ effects on monocytic cells, we generated novel THP-1-derived (low endogenous C/EBPβ) cell lines stably overexpressing C/EBPβ isoforms. Most importantly, cells predominantly overexpressing LAP* (C/EBPβ-long), but not those overexpressing LIP (C/EBPβ-short), exhibited a reduced proliferation, with no effect on morphology. PMA-induced inhibition of proliferation was attenuated in C/EBPβ-short cells. In C/EBPβWT macrophage-like cells (high endogenous C/EBPβ), we measured a reduced proliferation/cycling index compared with C/EBPβKO. The typical macrophage morphology was only observed in C/EBPβWT, whereas C/EBPβKO stayed round. C/EBPα did not compensate for C/EBPβ effects on proliferation/morphology. Serum reduction, an independent approach known to inhibit proliferation, induced macrophage morphology in C/EBPβKO macrophage-like cells but not THP-1. In PMA-treated THP-1 and C/EBPβ-long cells, a reduced phosphorylation of cell cycle repressor retinoblastoma was found. In addition, C/EBPβ-long cells showed reduced c-Myc expression accompanied by increased CDK inhibitor p27 and reduced cyclin D1 levels. Finally, C/EBPβ-long and C/EBPβWT cells exhibited low E2F1 and cyclin E levels, and C/EBPβ overexpression was found to inhibit cyclin E1 promoter-dependent transcription. Our results suggest that C/EBPβ 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/EBPβ may be important for coordinated monocytic differentiation.

Summary FLT3 receptor‐associated signalling plays a role in proliferation and leukaemia. The transcription factor C/EBPβ may be involved in malignancy with its alternative translation product C/EBPβ‐LIP. We investigated a potential connection between FLT3 signalling and the C/EBPβ 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 p90‐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.