Analysis of the molecular mechanism for the antagonistic action of a novel 1alpha,25-dihydroxyvitamin D(3) analogue toward vitamin D receptor function.
We have recently reported that 23(S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) efficiently blocks the differentiation of HL-60 cells induced by 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) (Miura, D., Manabe, K., Ozono, K., Saito, M., Gao, Q., Norman, A. W., and Ishizuka, S. (1999) J. Biol. Chem. 274, 16392-16399). To clarify the molecular mechanisms of this antagonism, we examined whether TEI-9647 antagonizes the genomic effects of 1alpha,25(OH)(2)D(3). 10(-7) to 10(-9) M TEI-9647 inhibited the transactivation effect of 10(-8) M 1alpha,25(OH)(2)D(3) in a dose-dependent manner, while TEI-9647 alone did not activate the reporter activity driven by SV40 promoter containing two vitamin D response elements in Saos-2 cells. The antagonistic effect of TEI-9647 was also observed using the rat 24-hydroxylase gene promoter, but the effect was weaker in HeLa and COS-7 cells than in Saos-2 cells. TEI-9647 also exhibited antagonism in an assay system where the VDR fused to the GAL4 DNA-binding domain and the reporter plasmid containing the GAL4 binding site were used in Saos-2 cells, but did not in HeLa cells. TEI-9647 reduced the interaction between VDR and RXRalpha according to the results obtained from the mammalian two-hybrid system in Saos-2 cells, but did not in HeLa cells. The two-hybrid system also revealed that the interaction between VDR and SRC-1 was reduced by TEI-9647 in Saos-2 cells. These results demonstrate that the novel 1alpha,25(OH)(2)D(3) analogue, TEI-9647, is the first synthetic ligand for the VDR that efficiently antagonizes the action of 1alpha, 25(OH)(2)D(3), although the extent of its antagonism depends on cell type.
Ozono
K
K
Department of Environmental Medicine, Osaka Medical Center, 840 Murodo-cho, Izumi, Osaka 594-1101, Japan. j61642@center.osaka-u.ac.jp
Saito
M
M
Miura
D
D
Michigami
T
T
Nakajima
S
S
Ishizuka
S
S
D000818:Animals; D019556:COS Cells; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000494:pharmacology; D004268:DNA-Binding Proteins; D005656:Fungal Proteins / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D010006:Osteoblasts / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D018167:Receptors, Calcitriol / Q000187:drug effects / Q000502:physiology*; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D047488:Retinoid X Receptors; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000378:metabolism
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D008024:Ligands; D008164:Luminescent Proteins; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D011993:Recombinant Fusion Proteins; D047488:Retinoid X Receptors; D029701:Saccharomyces cerevisiae Proteins; C400545:TEI 9647; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D002117:Calcitriol