The Journal of Steroid Biochemistry and Molecular Biology
Increased nuclear expression and transactivation of vitamin D receptor by the cardiotonic steroid bufalin in human myeloid leukemia cells
Introduction
The active form of vitamin D3, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], regulates calcium and bone homeostasis, immunity, and cellular growth and differentiation through binding to the vitamin D receptor (VDR) [1]. VDR is a member of the nuclear receptor superfamily of ligand-activated transcription factors that regulate many physiological processes including cell growth and differentiation, embryogenic development, and metabolic homeostasis [2]. Upon ligand binding, nuclear receptors undergo a conformational change that induces nuclear translocation and formation of an active transcription factor complex, resulting in transcription of specific target genes. Natural and synthetic VDR ligands, such as 1,25(OH)2D3 and its derivatives, inhibit the proliferation and/or induce the differentiation of various types of malignant cells, including myeloid leukemia, breast, and colon cancer cells [3]. The administration of 1,25(OH)2D3, or 1α-hydroxyvitamin D3, which is rapidly metabolized to 1,25(OH)2D3, prolongs survival in a mouse model of myeloid leukemia [4]. Although vitamin D3 and its synthetic derivatives are utilized in the treatment of bone and skin disorders, adverse effects, especially hypercalcemia, limit their use in the treatment of leukemia and cancer. The development of synthetic vitamin D analogs with low calcemic activity and identification of VDR ligands with selective action offer promising strategies in overcoming the adverse effects [5], [6]. Another potential solution is combined administration of 1,25(OH)2D3 with other drugs, such as differentiation inducers [7]. Investigation of VDR-regulated mechanisms of leukemia differentiation should be helpful in incorporating vitamin D3 into cancer therapy.
Bufalin is a major active component of the toad venom preparation Chan Su or Senso, which has been used as a cardiotonic and local anesthetic agent in China and Japan for centuries and has been shown to be a potent inducer of human leukemia cell differentiation [8], [9]. Like other cardiotonic steroids including ouabain and digoxin, bufalin inhibits the membrane Na+,K+-ATPase and activates various intracellular signaling pathways presumably by a Na+,K+-ATPase-dependent mechanism [10]. While high concentrations of bufalin induce apoptosis, bufalin at low concentrations dramatically enhances the differentiation of myeloid leukemia cells induced by tumor necrosis factor-α and 1,25(OH)2D3 [9]. We previously reported that bufalin enhances VDR transactivation in a cell transfection assay and activates VDR-mediated endogenous gene expression [11]. Since bufalin does not bind to VDR or change the binding affinity of 1,25(OH)2D3 for VDR, and the α subunit of Na+,K+-ATPase is the only established receptor for bufalin, a Na+,K+-ATPase-mediated mechanism is likely involved in the VDR-modulating effects of bufalin. In this study, we investigated the effects of bufalin on VDR-mediated differentiation of human leukemia cells and found that bufalin modulates VDR function by multiple mechanisms, including increased nuclear expression of VDR protein.
Section snippets
Compounds
Bufalin, ouabain, digitoxigenin, cinobufagin, and SP600125 were purchased from Sigma–Aldrich (St. Louis, MO), 1,25(OH)2D3 was from Wako (Osaka, Japan), and PD98059, SB203580 and MG-132 were from EMD Chemicals (San Diego, CA).
Cell culture, cell growth, and nitroblue tetrazolium-reducing activity
Human myeloid leukemia HL60, THP-1 and U937 cells (RIKEN Cell Bank, Tsukuba, Japan) were cultured in RPMI1640 medium containing 10% fetal bovine serum, 100 unit/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. Cells were counted
Differentiation and VDR target gene induction in HL60 cells
Bufalin stimulates the differentiation of human myeloid leukemia cells and enhances the effects of other inducers, including 1,25(OH)2D3, on leukemia differentiation [9], [17]. We examined the combined effects of 1,25(OH)2D3 and bufalin on the proliferation and differentiation of myeloid leukemia HL60 cells. Bufalin inhibited proliferation of HL60 cells at a concentration-dependent range of 7.5–12.5 nM, and 12.5 nM bufalin induced NBT-reducing activity, a marker for differentiation [12] (Fig. 1A
Discussion
We show here that bufalin stabilizes 1,25(OH)2D3-dependent nuclear expression of VDR in HL60 cells. Nuclear expression of VDR is regulated by several mechanisms, including import, export, synthesis and degradation. Studies using fluorescent protein chimeras of VDR demonstrated that VDR is located in nuclei in the absence of ligand and that addition of ligand increases nuclear localization [30]. 1,25(OH)2D3 induces a rapid increase in synthesis of VDR protein and its transport to the nucleus in
Acknowledgements
We thank Dr. Kiyoshi Kawakami of Jichi Medical University, and members of the Makishima lab for technical assistance and helpful comments, and Dr. Andrew I. Shulman for editing assistance. This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (Grant-in-Aid for Scientific Research on Priority Areas, 18077005 to M. Makishima) and from the Ministry of Health, Labor, and Welfare, Japan. Y. Cho is a Postdoctoral Fellow supported by
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