Sp1 and NF-Y Synergistically Mediate the Effect of Vitamin D3 in the p27Kip1 Gene Promoter That Lacks Vitamin D Response Elements

Abstract Vitamin D3 promotes myeloid leukemic cell lines to differentiate terminally into monocytes/macrophages. It has been reported that overexpression of thecdk inhibitor p27Kip1 results in the differentiation of the myelomonocytic U937 cell line and that this gene is the target of vitamin D3. To identify the sequences required for the positive regulation of p27Kip1transcription by vitamin D3, a 3.6-kilobase 5′-flanking region of the human p27Kip1 gene was examined by transiently transfecting luciferase reporter constructs into U937 cells. The transcriptional activity of this construct was activated by vitamin D3. Deletion and mutational analysis revealed that both a GGGCGG sequence (−545/−539) and a CCAAT sequence (−525/−520) were necessary to induce p27Kip1 gene expression. Importantly, the region containing both of these elements conferred positive responsiveness to vitamin D3 to a heterologous promoter. Gel shift assays showed that Sp1 binds to the GGGCGG sequence and that NF-Y binds to the CCAAT sequence. Consistent with the roles of these transcription factors, treatment with vitamin D3stimulated the DNA binding activities of these factors to each element and induced the change of one NF-Y subunit. We conclude that vitamin D3 stimulates transcription of the p27Kip1 gene by a novel mechanism involving Sp1 and NF-Y, but not the vitamin D receptor, during the early stages of U937 cell differentiation.