Selective interaction of vitamin D receptor with transcriptional coactivators by a vitamin D analog

Abstract

The nuclear vitamin D receptor (VDR) is a member of a nuclear receptor superfamily and acts as a ligand-dependent transcription factor. A family of cotranscriptional activators (SRC-1, TIF2, and AIB-1) interacts with and activates the transactivation function of nuclear receptors in a ligand-dependent way. We examined interaction of VDR with these coactivators that was induced by several vitamin D analogs, since they exert differential subsets of the biological action of vitamin D through unknown mechanisms. Unlike other vitamin D analogs tested, OCT (22-oxa-1alpha,25-dihydroxyvitamin D3) induced interaction of VDR with TIF2 but not with SRC-1 or AIB-1. Consistent with these interactions, only TIF2 was able to potentiate the transactivation function of VDR bound to OCT. Thus, the present findings suggest that the structure of VDR is altered in a vitamin D analog-specific way, resulting in selective interactions of VDR with coactivators. Such selective interaction of coactivators with VDR may specify the array of biological actions of a vitamin D analog like OCT, possibly through activating a particular set of target gene promoters.

FIG. 1

Differential interactions between VDR and coactivators induced by vitamin D analogs in the yeast two-hybrid system. pGBT9(GAL4-DBD)-VDR(DEF) fusion protein and one of the indicated coactivators (SRC-1, TIF2, or AIB-1) or interacting proteins (RIP140, SUG1, or VAF-1) were expressed in yeast containing the lacZ gene controlled by the GAL4 enhancer. After incubation for 16 h in the presence of the 1α,25(OH)₂D₃ (10⁻⁸ M), 24R,25(OH)₂D₃ (10⁻⁸ M), or vitamin D analogs (10⁻⁸ M), as indicated, interaction of VDR with the coactivator or interacting protein was assessed by measuring β-galactosidase activity. Results are means ± standard deviations of three independent experiments.

FIG. 2

Selective interaction of VDR with coactivators induced by OCT in the mammalian two-hybrid system. COS-1 cells were transiently transfected with a reporter plasmid (17M2-G-CAT) and pVP(VP16)-VDR(DEF) with or without pM(GAL4-DBD)-SRC-1, pM(GAL4-DBD)-TIF2, or pM(GAL4-DBD)-AIB-1 expression plasmid. Twelve hours after transfection, the transfected cells were treated with the indicated analogs at 10⁻⁷ to 10⁻¹⁰ M concentrations and harvested for CAT assays at 48 h posttransfection. Results are means ± standard deviations of three independent experiments.

FIG. 3

Vitamin D analog-induced interactions between GST-VDR and SRC-1/TIF2 family proteins in a GST pull-down assay. GST-VDR was expressed in E. coli and immobilized on glutathione-Sepharose beads. In vitro-translated SRC-1 and TIF2 labeled with [³⁵S]methionine were incubated with the beads in the absence of added ligand (−) or in the presence of 1α,25(OH)₂D₃, F₆-1α,25-(OH)₂D₃, ED-71, OCT, or 24R,25(OH)₂D₃ at a concentration of 10⁻⁸ M. The bound proteins were analyzed by SDS–7.5% PAGE and visualized by autoradiography. As a control, 1/10 of the amount of labeled SRC-1 (left panel) and TIF2 (right panel) included in the binding reactions are shown in the first lane. Representative GST pull-down assays and graphs corresponding to means ± standard deviations for triplicate independent experiments are shown. Note that any interaction of GST with these coactivators was not induced in the presence of ligands (data not shown).

FIG. 4

Selective interaction of the OCT-VDR-RXR heterodimer bound to VDREs with SRC-1/TIF2 family proteins. EMSA analysis was performed with [γ-³²P]ATP-labeled VDREs (DR3, and opn-VDRE). rVDR and mRXRα were expressed in transfected COS-1 cells, and the nuclear extracts therefrom were used for this assay. Bacterially expressed SRC-1 and TIF2 fused to GST were purified with glutathione-Sepharose beads, and SRC-1 and TIF2 were excised from the fusion protein. These proteins were incubated at room temperature with approximately 10 ng of labeled DR3 or opn-VDRE in the absence or presence of 10⁻⁷ M 1α,25(OH)₂D₃ (lanes 3, 6, 9, 13, 16, and 19) or OCT (lanes 4, 7, 10, 14, 17, and 20).

FIG. 5

VDR-mediated transactivation stimulated by OCT is potentiated by TIF2 but not by SRC-1. The effects of SRC-1 (upper panel) and TIF2 (lower panel) on the AF-2 of VDR bound to vitamin D analog were examined in COS-1 cells. The CAT assay was performed with a reporter plasmid (17M2-G-CAT) and pM(GAL4-DBD)-VDR(DEF), pM(GAL4-DBD)-VDR(L417S), or pM(GAL4-DBD)-VDR(E420Q) mutant expression vector with or without an expression plasmid of the SRC-1/TIF2 protein family, as described in the legend for Fig. 1. Values are expressed as means ± standard deviations of triplicate transfections.