Transcriptional activation of the Cdk inhibitor p21 by vitamin D3 leads to the induced differentiation of the myelomonocytic cell line U937.

The hormonal form of vitamin D, 1,25-dihydroxyvitamin D3, acting through its cognate nuclear receptor (vitamin D3 receptor, VDR) will induce myeloid leukemic cell lines to terminally differentiate into monocytes/macrophages. Because VDR acts by transcriptionally regulating responsive genes in a ligand-dependent manner, we sought target genes of the receptor that initiate, the differentiation process in response to ligand. We screened a cDNA library prepared from the myelomonocytic U937 cell line with probes generated from either 1,25-dihydroxyvitamin D3-treated or untreated cells. We report here that a candidate clone that hybridized differentially is the Cdk inhibitor p21WAF1, CIP1. Furthermore, we show that p21 is transcriptionally induced by 1,25-dihydroxyvitamin D3 in a VDR-dependent, but not p53-dependent, manner, and we identify a functional vitamin D response element in the p21 promoter. Transient overexpression of p21 and/or the related Cdk inhibitor p27 in U937 cells in the absence of 1,25-dihydroxyvitamin D3 results in the cell-surface expression of monocyte/macrophage-specific markers, suggesting that ligand-modulated transcriptional induction of the p21 gene facilitates the induced differentiation of this monoblastic cell line. We believe that this is the first report demonstrating that the ectopic overexpression of a Cdk inhibitor such as p21 or p27 directly leads to a terminal differentiation program.

A mitogen-responsive promoter region that is synergistically activated through multiple signalling pathways.

A regulatory region of the human transferrin receptor gene promoter was found to be required for increased expression in response to serum or growth factors. This region contains two elements that appear to cooperate for full responsiveness. We found that sodium orthovanadate treatment of cells significantly activated expression of promoter constructs containing these elements. 12-O-Tetradecanoylphorbol-13-acetate alone induced a twofold increase in expression but acted synergistically with vanadate to generate a highly elevated level of expression. Dibutyryl cyclic AMP alone had no effect on expression, but when added together with vanadate and 12-O-tetradecanoylphorbol-13-acetate, led to superinduction of the promoter construct. Induction of expression by these reagents was delayed several hours, and the kinetics were identical to those observed for serum induction.