Kuruto-Niwa R, Nakamura M, Takeishi K, Nozawa R
Laboratory of Microbiology and Host Defenses, University of Shizuoka, Japan.
Cell Struct Funct. 1998 Jun;23(3):109-18. doi: 10.1247/csf.23.109.
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 alpha super-shifted the nucleoprotein complex in THP-1 cells but not in the VD3-treated HL-60 cells, whereas an antibody for C/EBP beta 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 delta antibody had no effect on the complex in either cell. Thus, it was concluded that C/EBP alpha and -beta were able to bind to the C/EBP motif, and that C/EBP alpha bound to the motif in THP-1 cells and C/EBP beta 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 beta was also detected in VD3-untreated HL-60 cells by immunoblotting, VD3 activated C/EBP beta to bind to the motif, probably through post-translational modification.
在人单核细胞白血病细胞系中研究了髓系钙结合蛋白MRP14基因的转录调控。MRP14基因在单核母细胞性ML-1细胞、前单核细胞性U-937细胞或早幼粒细胞性HL-60细胞中不表达。另一方面，该基因在单核细胞性THP-1细胞以及用1,25-二羟基维生素D3（VD3）处理的HL-60细胞中表达。经VD3处理的HL-60细胞中MRP14的水平比THP-1细胞中的高两倍。在几个已知的转录因子结合基序中，经VD3处理的HL-60细胞和THP-1细胞的核蛋白与位于MRP14基因上游区域（-81）的CCAAT/增强子结合蛋白（C/EBP）结合基序序结合，竞争性凝胶迁移率变动分析证明了这一点。针对C/EBPα的抗体使THP-1细胞中的核蛋白复合物发生超迁移，但未使经VD3处理的HL-60细胞中的核蛋白复合物发生超迁移，而针对C/EBPβ的抗体阻断了HL-60细胞的核因子形成复合物，但未阻断THP-1细胞的核因子形成复合物。抗C/EBPδ抗体对两种细胞中的复合物均无影响。因此，可以得出结论，C/EBPα和-β能够结合到C/EBP基序上，并且C/EBPα在THP-1细胞中结合到该基序上，C/EBPβ在经VD3处理的HL-60细胞中结合到该基序上。此外，为了检测C/EBP基序的转录活性，我们将几个构建的荧光素酶报告基因DNA转染到HL-60细胞和THP-1细胞中。VD3处理可增加HL-60细胞中C/EBP基序的荧光素酶活性。通过该分析证实，MRP14基因中的C/EBP基序在经VD3处理的HL-60细胞和THP-1细胞中起调控区域的作用。由于通过免疫印迹在未经VD3处理的HL-60细胞中也检测到了C/EBPβ，因此VD3可能通过翻译后修饰激活C/EBPβ使其结合到该基序上。
