Vitamin D receptor (VDR) is present in multiple blood cells, and the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is essential for the proper functioning of the immune system. The role of retinoic acid receptor α (RARα) in hematopoiesis is very important, as the fusion of RARα gene with PML gene initiates acute promyelocytic leukemia where differentiation of the myeloid lineage is blocked, followed by an uncontrolled proliferation of leukemic blasts. RARα takes part in regulation of VDR transcription, and unliganded RARα acts as a transcriptional repressor to VDR gene in acute myeloid leukemia (AML) cells. This is why we decided to examine the effects of the combination of 1,25D and all-trans-retinoic acid (ATRA) on VDR gene expression in normal human and murine blood cells at various steps of their development. We tested the expression of VDR and regulation of this gene in response to 1,25D or ATRA, as well as transcriptional activities of nuclear receptors VDR and RARs in human and murine blood cells. We discovered that regulation of VDR expression in humans is different from in mice. In human blood cells at early stages of their differentiation ATRA, but not 1,25D, upregulates the expression of VDR. In contrast, in murine blood cells 1,25D, but not ATRA, upregulates the expression of VDR. VDR and RAR receptors are present and transcriptionally active in blood cells of both species, especially at early steps of blood development.

BackgroundAcute myeloid leukemia (AML) cells can be induced to undergo terminal differentiation with subsequent loss of tumorigenicity using 1,25-dihydroxyvitamin D3 (1,25D) alone or in combination with hematopoietic cytokines. KG1 cells are resistant to 1,25D-induced cell differentiation. These cells have the aberrant signal transduction resulting from a constitutively active fusion protein FOP2-FGFR1, a constitutively active STAT1 and a high level of interferon (IFN) stimulated genes (ISGs).MethodsIn this paper we report that in KG1 cells with constitutively activated protein FOP2-FGFR1 delivery of plasmid DNA disrupted FOP2-FGFR1 fusion gene.ResultsAs a consequence, STAT1 signal transduction pathway became switched off, the expression of vitamin D receptor (VDR) gene was increased and sensitivity to 1,25D-induced differentiation was restored. The activation of ISGs in KG1 cells resulted in resistance to externally added IFNs, and also this effect was reversed in cells with disrupted FOP2-FGFR1 fusion gene.DiscussionIn this paper we have documented for the first time a link between constitutively active STAT1 signal transduction pathway, high level of ISGs and low expression of VDR gene.ConclusionsWe show in this paper that delivery of plasmid DNA to the cells may disrupt fusion gene FOP2-FGFR1 which occurs in a disease entity called 8p11 myeloproliferative syndrome. Inhibition of the FOP2-FGFR1 signal transduction pathway restored sensitivity of the cells to 1,25D-induced cell differentiation.

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1,25-dihydroxyvitamin D3 (1,25D3) is implicated in many cellular functions, including cell proliferation and differentiation, thus exerting potential antitumor effects. A major limitation for therapeutic use of 1,25D3 are potent calcemic activities. Therefore, synthetic analogs of 1,25D3 for use in anticancer therapy should retain cell differentiating potential, with calcemic activity being reduced. To obtain this goal, the analogs should effectively activate transcription of genes responsible for cell differentiation, leaving the genes responsible for calcium homeostasis less active. In order to better understand this phenomenon, we selected a series of structurally related 19-nor analogs of 1,25D (PRI-5100, PRI-5101, PRI-5105, and PRI-5106) and tested their activities in blood cells and in cells connected to calcium homeostasis. Affinities of analogs to recombinant vitamin D receptor (VDR) protein were not correlated to their pro-differentiating activities. Moreover, the pattern of transcriptional activities of the analogs was different in cell lines originating from various vitamin D-responsive tissues. We thus hypothesized that receptors which participate in transport of the analogs to the cells might contribute to the observed differences. In order to study this hypothesis, we produced renal cells with knock-out of the megalin gene. Our results indicate that megalin has a minor effect on semi-selective activities of vitamin D analogs.

Continuing our structure-activity studies on the vitamin D analogs with the altered intercyclic seco-B-ring fragment, we designed compounds possessing dienyne system conjugated with the benzene D ring. Analysis of the literature data and the docking experiments seemed to indicate that the target compounds could mimic the ligands with a good affinity to the vitamin D receptor (VDR). Multi-step synthesis of the C/D-ring building block of the tetralone structure was achieved and its enol triflate was coupled with the known A-ring fragments, possessing conjugated enyne moiety, using Sonogashira protocol. The structures of the final products were confirmed by NMR, UV and mass spectroscopy. Their binding affinities for the full-length human VDR were determined and it was established that compound substituted at C-2 with exomethylene group showed significant binding to the receptor. This analog was also able to induce monocytic differentiation of HL-60 cells.