Regulation of C/EBPβ isoforms by MAPK pathways in HL60 cells induced to differentiate by 1,25-dihydroxyvitamin D3

C/EBPβ is known to be important for monocytic differentiation and macrophage function. Here, we found that expression of all three C/EBPβ isoforms induced in HL60 cells by 1,25-dihydroxyvitamin D3 (1,25D) was upregulated in a sustained manner that correlates with the appearance of monocytic phenotype and with the G1 phase cell cycle arrest. In 1,25D-resistant HL60-40AF cells, isoforms β-1 and β-3 were expressed at levels comparable to 1,25D-sensitive HL60-G cells, but isoform β-2 was difficult to detect. Treatment of sensitive HL60 cells with 1,25D resulted in predominantly nuclear localization of C/EBP isoforms β-2 and β-3, while a large proportion of C/EBPβ-1 remained in the cytoplasm. Attenuation of the MEK-ERK MAPK pathway by the inhibitor PD98059 markedly reduced the expression, 1,25D-induced phosphorylation and nuclear localization of C/EBPβ-2 and C/EBPβ-3. Interestingly, only the lower molecular mass isoforms of C/EBPβ phosphorylated on Thr235 were found in the nuclei, while C/EBPβ-1 was constitutively phosphorylated and was detected principally in the cytoplasmic fraction. Although the role of C/EBPβ isoforms in 1,25D-induced differentiation is complex, our results taken together strongly suggest that the phosphorylation of C/EBPβ isoforms on Thr235 takes place mainly via the MEK-ERK pathway and that C/EBPβ-2 is the principal transcription factor in this cell system.