Song L N
Department of Pathophysiology, Second Military Medical University, Shanghai, People's Republic of China.
J Steroid Biochem Mol Biol. 1996 Mar;57(5-6):265-74. doi: 10.1016/0960-0760(95)00266-9.
We have shown earlier that 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3] induces cell growth suppression and cell differentiation of a human megakaryoblastic leukemia cell line, HIMeg. However, the molecular mechanism of 1,25(OH)2 D3 action is still unknown. Prompted by this, we have searched here for the presence of 1,25(OH)2 D3 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 that [correction of the] VDR mRNA expression in HIMeg cells could be down-regulated rapidly by 1,25(OH)2 D3 (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 D3 at any time-point tested. Treatment of HIMeg cells with forskolin (1 microM), 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 D3 even though forskolin alone had no effects. Exposure to 1,25(OH)2 D3 in combination with forskolin resulted in a much more significant inhibition of cell proliferation than to 1,25(OH)2 D3 alone. Similarly, forskolin could also augment the differentiation induced by 1,25(OH)2 D3 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.
我们先前已表明，1,25 - 二羟基维生素D3 [1,25(OH)2 D3] 可诱导人巨核母细胞白血病细胞系HIMeg的细胞生长抑制和细胞分化。然而，1,25(OH)2 D3作用的分子机制仍不清楚。受此启发，我们在此通过逆转录 - 聚合酶链反应（RT - PCR）寻找HIMeg细胞中1,25(OH)2 D3受体（VDR）表达的情况。扩增产物显示出与从对照人VDR cDNA扩增的产物相同的大小，并与地高辛标记的人VDR cDNA片段特异性杂交。正如预期的那样，VDR mRNA也在人骨肉瘤细胞系HOS - 8603中表达。这些结果代表了首次报道的巨核母细胞中VDR mRNA表达的证据。此外，通过定量RT - PCR研究了HIMeg细胞中VDR mRNA表达的调节。发现1,25(OH)2 D3（10 nM）可在时间依赖性方式下迅速下调HIMeg细胞中的VDR mRNA表达，最大降低至对照的约15％。然而，在任何测试的时间点，1,25(OH)2 D3均未调节HOS - 8603细胞中的VDR mRNA表达。用腺苷酸环化酶激活剂福斯高林（1 microM）处理HIMeg细胞会导致VDR mRNA水平升高。同样，福斯高林也上调了HOS - 8603细胞中的VDR mRNA表达。与VDR在其他靶细胞中的功能一致，我们发现福斯高林上调HIMeg细胞中VDR表达的同时，HIMeg细胞对1,25(OH)2 D3的反应性增加，尽管单独的福斯高林没有作用。与单独使用1,25(OH)2 D3相比，1,25(OH)2 D3与福斯高林联合使用对细胞增殖的抑制作用更为显著。同样，福斯高林也可增强1,25(OH)2 D3诱导的分化，表现为更明显的形态变化和具有多叶核的细胞发育百分比更高。这些改变伴随着克隆形成能力的丧失和S期细胞数量减少。这些数据表明HIMeg细胞表达功能性VDR，其用于介导其配体对这些细胞增殖和分化的作用。
