Transcriptional regulation by C/EBPα and -β in the expression of the gene for the MRP14 myeloid calcium binding protein

Abstract

Transcriptional regulation of the gene for the myeloid calcium binding protein, MRP14, was investigated in human monocytic leukemia cell lines. The MRP14 gene was not expressed in monoblastic ML-1 cells, promonocytic U-937 cells, or promyelocytic HL-60 cells. On the other hand, the gene was expressed in monocytic THP-1 cells and in the HL-60 cells treated with 1,25-dihydroxyvitamin D3 (VD3). The level of MRP14 in VD3-treated HL-60 cells was two-fold higher than that in THP-1 cells. Among several known transcription factor binding motifs, nuclear protein(s) of VD3-treated HL-60 cells and THP-1 cells bound to the CCAAT/enhancer binding protein (C/EBP)-binding motif that was located in the upstream region of the MRP14 gene (-81), as evidenced by the competitive gel mobility-shift assay. An antibody for C/EBPα super-shifted the nucleoprotein complex in THP-1 cells but not in the VD3-treated HL-60 cells, whereas an antibody for C/EBPβ blocked the formation of the complex with the nuclear factor of the HL-60 cells but not with that of THP-1 cells. An anti-C/EBPδ antibody had no effect on the complex in either cell. Thus, it was concluded that C/EBPα and -β were able to bind to the C/EBP motif, and that C/EBPα bound to the motif in THP-1 cells and C/EBPβ bound to that in the VD3-treated HL-60 cells. Furthermore, to examine the transcriptional activity of the C/EBP motif, we transfected several constructed luciferase reporter DNAs into HL-60 cells and THP-1 cells. The luciferase activity of the C/EBP motif in HL-60 cells was increased by VD3 treatment. The C/EBP motif in the MRP14 gene was confirmed to function as a regulatory region in VD3-treated HL-60 cells and THP-1 cells by the assay. Since C/EBPβ was also detected in VD3-untreated HL-60 cells by immunoblotting, VD3 activated C/EBPβ to bind to the motif, probably through post-translational modification.