Demonstration of vitamin D receptor expression in a human megakaryoblastic leukemia cell line: Regulation of vitamin D receptor mRNA expression and responsiveness by forskolin

Abstract

We have shown earlier that 1,25-dihydroxyvitamin D 3[1,25(OH) 2D 3] induces cell growth suppression and cell differentiation of a human megakaryoblastic leukemia cell line, HIMeg. However, the molecular mechanism of 1,25(OH) 2 D 3 action is still unknown. Prompted by this, we have searched here for the presence of 1,25(OH) 2 D 3 receptor (VDR) expression in HIMeg cells by reverse transcription-polymerase chain reaction (RT-PCR). The amplified product showed an identical size to the product amplified from the control human VDR cDNA and hybridized specifically with the digoxigenin-labeled human VDR cDNA fragment. As expected, VDR mRNA is also expressed in HOS-8603, a human osteosarcoma cell line. These results represent the first reported evidence that VDR mRNA is expressed in megakaryoblastic cells. In addition, the regulation of VDR mRNA expression in HIMeg cells was studied by quantitative RT-PCR. It was found the VDR mRNA expression in HIMeg cells could be down-regulated rapidly by 1,25(OH) 2 D 3 (10 nM) in a time-dependent manner, reaching a maximal reduction to about 15% of control. However, VDR mRNA expression in HOS-8603 cells was not regulated by 1,25(OH) 2 D 3 at any time-point tested. Treatment of HIMeg cells with forskolin (1 μM), an activator of adenylate cyclase, caused an increase in VDR mRNA levels. Similarly, VDR mRNA expression in HOS-8603 cells was also up-regulated by forskolin. Consistent with the functionality of the VDR in other target cells, we found that the up-regulation of VDR expression in HIMeg cells by forskolin was accompanied by an increased responsiveness of HIMeg cells to 1,25(OH) 2 D 3, even though forskolin alone had no effects. Exposure to 1,25(OH) 2 D 3 in combination with forskolin resulted in a much more significant inhibition of cell proliferation than to 1,25(OH) 2 D 3 alone. Similarly, forskolin could also augment the differentiation induced by 1,25(OH) 2 D 3, reflected by a more evident morphological change and a higher percentage of development of cells with multilobular nuclei. These alterations were accompanied by a loss of clonogenic capacity and a decrease in the number of cells in the S phase. These data establish that HIMeg cells express functional VDR, which served to mediate actions of its ligand on the proliferation and differentiation of these cells.