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