Increased nuclear expression and transactivation of vitamin D receptor by the cardiotonic steroid bufalin in human myeloid leukemia cells

The active form of vitamin D3, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], is a potent ligand for the nuclear receptor vitamin D receptor (VDR) and induces myeloid leukemia cell differentiation. The cardiotonic steroid bufalin enhances vitamin D-induced differentiation of leukemia cells and VDR transactivation activity. In this study, we examined the combined effects of 1,25(OH)2D3 and bufalin on differentiation and VDR target gene expression in human leukemia cells. Bufalin in combination with 1,25(OH)2D3 enhanced the expression of VDR target genes, such as CYP24A1 and cathelicidin antimicrobial peptide, and effectively induced differentiation phenotypes. An inhibitor of the Erk mitogen-activated protein (MAP) kinase pathway partially inhibited bufalin induction of VDR target gene expression. 1,25(OH)2D3 treatment induced transient nuclear expression of VDR in HL60 cells. Interestingly, bufalin enhanced 1,25(OH)2D3-induced nuclear VDR expression. The MAP kinase pathway inhibitor increased nuclear VDR expression induced by 1,25(OH)2D3 and did not change that by 1,25(OH)2D3 plus bufalin. A proteasome inhibitor also enhanced 1,25(OH)2D3-induced CYP24A1 expression and nuclear VDR expression. Bufalin-induced nuclear VDR expression was associated with histone acetylation and VDR recruitment to the CYP24A1 promoter in HL60 cells. Thus, the Na+,K+-ATPase inhibitor bufalin modulates VDR function through several mechanisms, including Erk MAP kinase activation and increased nuclear VDR expression.