Promoter-, cell-, and ligand-specific transactivation responses of the VDRB1 isoform

The vitamin D receptor (VDR) mediates the effects of 1,25(OH)(2)D(3), the active form of vitamin D. The human VDRB1 isoform differs from the originally described VDR by an N-terminal extension of 50 amino acids. Here we investigate cell-, promoter-, and ligand-specific transactivation by the VDRB1 isoform. Transactivation by these isoforms of the cytochrome P450 CYP24 promoter was compared in kidney (HEK293 and COS1), tumor-derived colon (Caco-2, LS174T, and HCT15), and mammary (HS578T and MCF7) cell lines. VDRB1 transactivation in response to 1,25(OH)(2)D(3) was greater in COS1 and HCT15 cells (145%), lower in HEK293 and Caco-2 cells (70-85%) and similar in other cell lines tested. By contrast, on the cytochrome P450 CYP3A4 promoter, 1,25(OH)(2)D(3)-induced VDRB1 transactivation was significantly lower than VDRA in Caco-2 (68%), but comparable to VDRA in HEK293 and COS1 cells. Ligand-dependence of VDRB1 differential transactivation was investigated using the secondary bile acid lithocholic acid (LCA). On the CYP24 promoter LCA-induced transactivation was similar for both isoforms in COS1, whereas in Caco-2 and HEK293 cells VDRB1 was less active. On the CYP3A4 promoter, LCA activation of VDRB1 was comparable to VDRA in all the cell lines tested. Mutational analysis indicated that both the 1,25(OH)(2)D(3) and LCA-regulated activities of both VDR isoforms required a functional ligand-dependent activation function (AF-2) domain. In gel shift assays VDR:DNA complex formation was stronger in the presence of 1,25(OH)(2)D(3) than with LCA. These results indicate that regulation of VDRB1 transactivation activity is dependent on cellular context, promoter, and the nature of the ligand.     

Culture serum-induced conversion from agonist to antagonist of a Vitamin D analog, TEI-9647

The nuclear receptor for Vitamin D (VDR) mediates many of the effects of Vitamin D in target tissues by regulating gene expression. The transactivation function of ligand-bound VDR in target tissues is thought to depend on the tissue-type and the cellular-environment, but the molecular basis for these differences has not been fully understood. In this study, during characterization of TEI-9647 as a synthetic ligand for the VDR, we found that depletion of serum from the culture medium converted TEI-9647 from an antagonist to an agonist of VDR-mediated transactivation, whereas it retained antagonistic activity in the presence of serum. Consistent with these results, using a mammalian two-hybrid system, we found that TEI-9647 recruited different coactivators to the VDR in the presence and absence of serum. These findings suggest that an unknown serum factor modulates the transactivation function of the VDR.     

Critical role of helix 12 of the vitamin D(3) receptor for the partial agonism of carboxylic ester antagonists.

The carboxy-terminal alpha-helix of a nuclear receptor ligand-binding domain (LBD), helix 12, contains a critical, ligand-modulated interface for the interaction with coactivator proteins. In this study, using the example of the vitamin D receptor (VDR) and the partial antagonist ZK159222, the role of helix 12 (residues 417-427) for both antagonistic and agonistic receptor actions was investigated. Amino acid residue G423 was demonstrated to be critical for partial agonism of ZK159222, but not for the activity of the natural VDR agonist, 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)). The amount of partial agonism of ZK159222 increased when helix 12 was truncated by the last four amino acid residues (Delta424-27) and augmented even more, when in addition helix 12 of VDR's dimerization partner, retinoid X receptor (RXR), was truncated. In contrast, the low agonism of a structural derivative of ZK159222, ZK168281, was not affected comparably, whereas other close structural relatives of ZK159222 even demonstrated the same agonistic activity as that of 1alpha,25(OH)(2)D(3). The amount of agonism of ZK159222 and ZK168281 at different variations of helix 12 correlated well with VDR's ability to complex with coactivator proteins and inversely correlated with the strength of the compound's antagonistic action on 1alpha,25(OH)(2)D(3) signalling. Molecular dynamics simulations of the LBD complexed with the two antagonists could explain their different action by demonstrating a more drastic displacement of helix 12 through ZK168281 than through ZK159222. Moreover, the modelling could indicate a kink of helix 12 at amino acid residue G423, which provides the last four amino acid residues of helix 12 with a modulatory role for the partial agonism of some VDR antagonists, such as ZK159222. In conclusion, partial agonism of a VDR antagonist is lower the more it disturbs helix 12 in taking the optimal position for coactivator interaction.     

Interferon gamma-induced EGF receptor transactivation depends on the level of expression of EGF receptor in epithelial cells

Recently, we demonstrated the transactivation of the epidermal growth factor receptor (EGFR) in response to interferon γ (IFNγ) in epidermoid carcinoma A431 cells. It was shown that IFNγ-induced EGFR transactivation is impossible in the some cancer epithelial cells. Here, we hypothesize that IFNγ-dependent EGFR transactivation in these cells correlates to the amount of EGFR on the surface of the cell. To test this suggestion, a line of stably transfected HEK293 cells (HEK293Δ99 cells) expressing a high level of mutant EGFR that lacked 99 C-terminal residues was obtained. Unlike the parent HEK293 cells, which lacked transactivation, HEK293Δ99 cells demonstrated EGFR transactivation in response to IFNγ. In HEK293Δ99 and A431 cells, the time courses of EGFR activation induced by IFNγ have the same pattern. In HEK293Δ99, as in A431 cells, IFNγ-induced EGFR transactivation requires EGFR kinase activity and occurs via an autophosphorylation mechanism. Taken together, these data provide direct evidence for the dependence of IFNγ-induced EGFR transactivation upon EGFR expression level in epithelial cells.     

Ligand-specific structural changes in the vitamin D receptor in solution

Vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily. When bound to a variety of vitamin D analogues, VDR manifests a wide diversity of physiological actions. The molecular mechanism by which different vitamin D analogues cause specific responses is not understood. The published crystallographic structures of the ligand binding domain of VDR (VDR-LBD) complexed with ligands that have differential biological activities have exhibited identical protein conformations. Here we report that rat VDR-LBD (rVDR-LBD) in solution exhibits differential chemical shifts when bound to three ligands that cause diverse responses: the natural hormone, 1,25-dihydroxyvitamin D(3) [1,25(OH)?D?], a potent agonist analogue, 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D? [2MD], and an antagonist, 2-methylene-(22E)-(24R)-25-carbobutoxy-26,27-cyclo-22-dehydro-1α,24-dihydroxy-19-norvitamin D? [OU-72]. Ligand-specific chemical shifts mapped not only to residues at or near the binding pocket but also to residues remote from the ligand binding site. The complexes of rVDR-LBD with native hormone and the potent agonist 2MD exhibited chemical shift differences in signals from helix-12, which is part of the AF2 transactivation domain that appears to play a role in the selective recruitment of coactivators. By contrast, formation of the complex of rVDR-LBD with the antagonist OU-72 led to disappearance of signals from residues in helices-11 and -12. We present evidence that disorder in this region of the receptor in the antagonist complex prevents the attachment of coactivators.     

Molecular mechanism of the vitamin D antagonistic actions of (23S)-25-dehydro-1alpha-hydroxyvitamin D3-26,23-lactone depends on the primary structure of the carboxyl-terminal region of the vitamin d receptor

We reported that (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) antagonizes vitamin D receptor (VDR)-mediated genomic actions of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] in human cells but is agonistic in rodent cells. Human and rat VDR ligand-binding domains are similar, but differences in the C-terminal region are important for ligand binding and transactivation and might determine the agonistic/antagonistic effects of TEI-9647. We tested TEI-9647 on 1alpha,25(OH)(2)D(3) transactivation using SaOS-2 cells (human osteosarcoma) or ROS 24/1 cells (rat osteosarcoma) cotransfected with human or rodent VDR and a reporter. In both cell lines, TEI-9647 was antagonistic with wild-type human (h)VDR, but agonistic with overexpressed wild-type rat (r)VDR. VDR chimeras substituting the hVDR C-terminal region (activation function 2 domain) with corresponding rVDR residues diminished antagonism and increased agonism of TEI-9647. However, substitution of 25 C-terminal rVDR residues with corresponding hVDR residues diminished agonism and increased antagonism of TEI-9647. hVDR mutants (C403S, C410N) demonstrated that Cys403 and/or 410 was necessary for TEI-9647 antagonism of 1alpha,25(OH)(2)D(3) transactivation. These results suggest that species specificity of VDR, especially in the C-terminal region, determines the agonistic/antagonistic effects of TEI-9647 that determine, in part, VDR interactions with coactivators and emphasize the critical interaction between TEI-9647 and the two C-terminal hVDR Cys residues to mediate the antagonistic effect of TEI-9647.     

A unique insertion/duplication in the VDR gene that truncates the VDR causing hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia

Hereditary vitamin D resistant rickets (HVDRR) is caused by mutations in the vitamin D receptor (VDR). Here we describe a patient with HVDRR who also exhibited some hypotrichosis of the scalp but otherwise had normal hair and skin. A 102 bp insertion/duplication was found in the VDR gene that introduced a premature stop (Y401X). The patient's fibroblasts expressed the truncated VDR, but were resistant to 1,25(OH)2D3. The truncated VDR weakly bound [3H]-1,25(OH)2D3 but was able to heterodimerize with RXR, bind to DNA and interact with the corepressor hairless (HR). However, the truncated VDR failed to bind coactivators and was transactivation defective. Since the patient did not have alopecia or papular lesions of the skin generally found in patients with premature stop mutations this suggests that this distally truncated VDR can still regulate the hair cycle and epidermal differentiation possibly through its interactions with RXR and HR to suppress gene transactivation.