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

Abstract

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.

Identification of Two Forms of TNF Tolerance in Human Monocytes: Differential Inhibition of NF-κB/AP-1– and PP1-Associated Signaling

“…The following primers were applied: IL8 (59-TCCTGTTTCTGCAGCTCTGG-39, 59-GGCCACT-CTCAATCACTCTC-39), IL6 (59-ACAGCCACTCACCTCTTCAG-39, 59-GTGCCTCTTTGCTGCTTTCAC-39), IL1A (59-TGACTGCCCAAGATG-AAGAC-39, 59-CCAAGCACACCCAGTAGTC-39), CCL20 (59-GAAGGC-TGTGACATCAATGC-39, 59-GGGCTATGTCCAATTCCATTC-39), PTGS2 (59-GGGCCAGCTTTCACCAAC-39, 59-ATCTTTGACTGTGGGAGGATAC-39), and IkBa (59-CGAGCAGATGGTCAAGGAGC-39, 59-CAGCCAAGT-GGAGTGGAGTC-39). IL-8 mRNA expression was alternatively assessed using universal probe library probe 72 (Roche) and IL-8-specific primers (59-AGACAGCAGAGCACACAAGC-39, 59-CACAGTGAGATGGTTCCTTCC-39), according to the standard protocol (21). Target gene expression levels were normalized to hGAPDH (59-AGGTCGGAGTCAACGGAT-39, 59-TCCTGG-AAGATGGTGATG-39) or b 2 -microglobulin (59-TGTGCTCGCGCTACTC-TCTCTT-39, 59-CGGATGGATGAAACCCAGACA-39).…”

“…Target gene expression levels were normalized to hGAPDH (59-AGGTCGGAGTCAACGGAT-39, 59-TCCTGG-AAGATGGTGATG-39) or b 2 -microglobulin (59-TGTGCTCGCGCTACTC-TCTCTT-39, 59-CGGATGGATGAAACCCAGACA-39). Generation of external standard curves and normalization of cDNA amount were performed, as previously described (21). Relative expression values and fold changes were calculated, as published (22).…”

“…Next, membranes were incubated (1 h, room temperature) with HRP-conjugated secondary Ab (Alexis Biochemicals). Proteins were visualized, as previously described (21). Densitometric analysis was performed with TotalLab TL100 software.…”

Heterologous Viral Promoters Incorporated into the Human Cytomegalovirus Genome Are Silenced during Experimental Latency

“…09-301-149; Fisher) by capillary blotting. Single-stranded, 32 P-labeled probes were generated with a MAXIscript T7 kit (Ambion) using primers listed in Table 1. Hybridization conditions included prehybridization with Ultrahyb (catalog no.…”

Involvement of C/EBPβ in monocytic differentiation of acute myeloid leukemia cells induced by LW-218, a new synthesized flavonoid

“…The CCAAT/enhancerbinding protein (C/EBP) family of transcription factors plays an important role in controlling cell proliferation and differentiation. C/EBPα and C/EBPβ have been reported to be key regulators of granulocytic and monocytic differentiation, respectively [16,17]. After treatment of LW-218, the C/EBPα expression was barely changed in U937 cells (Fig.…”