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/EBP α supershifted the nucleoprotein complex in THP-1 cells but not in the VD3-treated HL-60 cells, whereas an antibody for C/EBP β 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/EBP δ antibody had no effect on the complex in either cell. Thus, it was concluded that C/EBP α and -β were able to bind to the C/EBP motif, and that C/EBP α bound to the motif in THP-1 cells and C/EBP β 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/EBP β was also detected in VD3-untreated HL-60 cells by immunoblotting, VD3 activated C/EBP β to bind to the motif, probably through post-translational modification.
