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. 2008 Jun;110(3-5):269-77.
doi: 10.1016/j.jsbmb.2007.11.007. Epub 2008 Apr 22.

1alpha,25-Dihydroxyvitamin D(3)-26,23-lactam analogues function as vitamin D receptor antagonists in human and rodent cells

Seiichi Ishizuka  1 , Noriyoshi Kurihara, Yuko Hiruma, Daishiro Miura, Jun-ichi Namekawa, Azusa Tamura, Yuko Kato-Nakamura, Yusuke Nakano, Kazuya Takenouchi, Yuichi Hashimoto, Kazuo Nagasawa, G David Roodman
Affiliations

1alpha,25-Dihydroxyvitamin D(3)-26,23-lactam analogues function as vitamin D receptor antagonists in human and rodent cells

Seiichi Ishizuka et al. J Steroid Biochem Mol Biol. 2008 Jun.

Abstract

(23S,25S)-N-Benzyl-1alpha,25-dihydroxyvitamin D(3)-26,23-lactam ((23S,25S)-N-benzyl-1alpha,25-(OH)(2)D(3)-26,23-lactam, (23S,25S)-DLAM-1P) antagonizes nuclear vitamin D receptor (VDR)-mediated differentiation of human promyelocytic leukemia (HL-60) cells [Y. Kato, Y. Nakano, H. Sano, A. Tanatani, H. Kobayashi, R. Shimazawa, H. Koshino, Y. Hashimoto, K. Nagasawa, Synthesis of 1alpha,25-dihydroxy vitamin D(3)-26,23-lactams (DLAMs), a novel series of 1alpha,25-dihydroxy vitamin D(3) antagonist, Bioorg. Med. Chem. Lett. 14 (2004) 2579-2583]. To enhance its VDR antagonistic actions, we synthesized multiple analogues of 1alpha,25-(OH)(2)D(3)-26,23-lactam. Among these analogues, (23S,25S)-N-phenetyl-1alpha,25-(OH)(2)D(3)-26,23-lactam, ((23S,25S)-DLAM-2P) had the strongest VDR binding affinity, which was 3 times higher than that of (23S,25S)-DLAM-1P. The 1alpha,25-(OH)(2)D(3)-26,23-lactam analogues never induced HL-60 cell differentiation even at 10(-6)M, but (23S,25S)-DLAM-1P and (23S,25S)-DLAM-2P significantly and dose-dependently inhibited HL-60 differentiation induced by 10(-8)M 1alpha,25-dihydroxyvitamin D(3) (1alpha,25-(OH)(2)D(3)). These compounds also inhibited human and mouse cultures of osteoclast formation by marrow cells treated with 1alpha,25-(OH)(2)D(3). Moreover, the 1alpha,25-(OH)(2)D(3)-26,23-lactam analogues minimally induced 25-hydroxyvitamin D(3)-24-hydroxylase gene expression in HL-60 cells and human and mouse osteoblastic cells, but 10(-6)M (23S,25S)-DLAM-1P or (23S,25S)-DLAM-2P significantly blocked 24-hydroxylase gene expression induced by 10(-8)M 1alpha,25-(OH)(2)D(3). (23S,25S)-DLAM-2P was 5-12 times more potent as a vitamin D antagonist than (23S,25S)-DLAM-1P in HL-60 cells, human and mouse bone marrow cultures. These results demonstrate that (23S,25S)-DLAM-1P and (23S,25S)-DLAM-2P antagonize HL-60 cell differentiation and osteoclast formation by human and mouse osteoclast precursors induced by 1alpha,25-(OH)(2)D(3) through blocking VDR-mediated gene transcription. In contrast, (23S)-25-deoxy-1alpha-hydroxyvitamin D(3)-26,23-lactone, which only blocks human VDR, these vitamin D antagonists can block VDR in human cells and rodent cells.

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Figures

Figure 1
Figure 1
Structures of 1α,25-(OH)2D3-26,23-lactone analogues and 1α,25-(OH)2D3-26,23-lactam analogues. The (23S,25R)-1α,25-(OH)2D3-26,23-lactone is a naturally occurring metabolite derived from 1α,25-(OH)2D3.
Figure 2
Figure 2
Effects of 4 diastereoisomers of N-benzyl-1α,25-(OH)2D3-26,23-lactam analogues on 1α,25-(OH)2D3-induced HL-60 cell differentiation as determined by NBT-reducing activity. (A) HL-60 cells were treated with 1α,25-(OH)2D3 alone or N-benzyl-1α,25-(OH)2D3-26,23-lactam alone for 96 hrs, and NBT-reducing activity was examined. (B) HL-60 cells were treated with N-benzyl-1α,25-(OH)2D3-26,23-lactam in the presence of 10−8M 1α,25-(OH)2D3 for 96 hrs, and NBT-reducing activity was examined. Rectangles and bars show mean ± S.D. of triplicate experiments, respectively. ap<0.001, compared with cells treated with media alone. *p<0.05, **p<0.01 and ***p<0.001, compared with cells treated with 10−8M 1α,25-(OH)2D3, respectively. Similar results were seen in two independent experiments.
Figure 3
Figure 3
Effects of 4 diastereoisomers of N-phenetyl-1α,25-(OH)2D3-26,23-lactam analogues on 1α,25-(OH)2D3-induced HL-60 cell differentiation as determined by NBT-reducing activity. (A) HL-60 cells were treated with 1α,25-(OH)2D3 alone or N-phenetyl-1α,25-(OH)2D3-26,23-lactam alone for 96 hrs, and NBT-reducing activity was examined. (B) HL-60 cells were treated with N-phenetyl-1α,25-(OH)2D3-26,23-lactam in the presence of 10−8M 1α,25-(OH)2D3 for 96 hrs, and NBT-reducing activity was examined. Rectangles and bars show mean ± S.D. of triplicate experiments, respectively. ap<0.001, compared with cells treated with media alone. *p<0.05, **p<0.01 and ***p<0.001, compared with cells treated with 10−8M 1α,25-(OH)2D3, respectively. Similar results were seen in two independent experiments.
Figure 4
Figure 4
Effects of N-benzyl-1α,25-(OH)2D3-26,23-lactam analogues and N-phenetyl-1α,25-(OH)2D3-26,23-lactam analogues on OCL formation by human MVNP-transduced CFU-GM cells (A) and mouse bone marrow cells (B) treated with 1α,25-(OH)2D3. The experimental procedures for OCL formation from human MVNP-transduced CFU-GM cells and mouse bone marrow cells were carried out as described in Materials and Methods. Multinucleated cells that cross-reacted with the 23C6 antibody in human MVNP-transduced CFU-GM cells and TRAP-positive in mouse bone marrow cells and had three or more nuclei were scored an OCL. Data are expressed as the mean ± S.D. of quadruplicate determinations. ap<0.001, compared with cells treated with media alone. *p<0.05, **p<0.01 and ***p<0.001, compared with cells treated with 10−9M 1α,25-(OH)2D3 or 10−8M 1α,25-(OH)2D3, respectively. Similar results were seen in two independent experiments.
Figure 5
Figure 5
Effects of N-benzyl-1α,25-(OH)2D3-26,23-lactam analogues and N-phenetyl-1α,25-(OH)2D3-26,23-lactam analogues on 25-OH-D3-24-hydroxylase gene expression induced by 1α,25-(OH)2D3 in HL-60 cells (A), human osteoblastic cell line (HOS cells) (B) and primary mouse osteoblastic cells (C). The experimental procedures for gene expression induced by vitamin D analogues were carried out as described in Materials and Methods. 1, vehicle; 2, 10−8M 1α,25-(OH)2D3; 3, 10−6M TEI-9647; 4, 10−6M (23S,25S)-DLAM-1P; 5, 10−6M (23S,25R)-DLAM-1P; 6, 10−6M (23S,25S)-DLAM-2P; 7, 10−6M (23S,25R)-DLAM-2P; 8, 10−6M 1α,25-(OH)2D3-26,23-lactone; 9, 10−8M 1α,25-(OH)2D3 + 10−6M TEI-9647; 10, 10−8M 1α,25-(OH)2D3 + 10−6M (23S,25S)-DLAM-1P; 11, 10−8M 1α,25-(OH)2D3 + 10−6M (23S,25R)-DLAM-1P; 12, 10−8M 1α,25-(OH)2D3 + 10−6M (23S,25S)-DLAM-2P; 13, 10−8M 1α,25-(OH)2D3 + 10−6M (23S,25R)-DLAM-2P; 14, 10−8M 1α,25-(OH)2D3 + 10−6M 1α,25-(OH)2D3-26,23-lactone. Similar results were seen in two independent experiments.
Figure 6
Figure 6
Effect of (23S,25S)-DLAM-2P on reporter gene activity induced by 1α,25-(OH)2D3 in MVNP-transduced NIH3T3 cells. The reporter plasmid containing 25-OH-D3-24-hydroxylase promoter and β-galactosidase vector were transfected into MVNP-transduced NIH3T3 cells. 10−9M 1α,25-(OH)2D3 and various concentration of (23S,25S)-DLAM-2P were added for 24 hours after the transfection of MVNP-transduced NIH3T3 cells. Results are expressed as the mean ± SEM. ap<0.001, compared with cells treated with media alone. *p< 0.01 and **p<0.001 compared with cells treated with 10−9M 1α,25-(OH)2D3. Similar results were seen in three independent experiments.
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