The vitamin D receptor (VDR) is a transcription factor that transmits incoming 1,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) signaling via combined contact with coactivator proteins and specific DNA binding sites (VDREs), which ultimately results in activation of transcription. In contrast, the mechanisms of transcriptional repression via the VDR are less well understood. This study documents VDR-dependent transcriptional repression largely via histone deacetylase (HDAC) activity. Direct, ligand-sensitive protein-protein interaction of the VDR with the nuclear receptor corepressor (NCoR) and a novel corepressor, called Alien, was demonstrated to be comparable but independent of the VDR AF-2 trans-activation domain. Functional assays indicated that Alien, but not NCoR, displays selectivity for different VDRE structures for transferring these repressive effects into gene regulatory activities. Moreover, superrepression via Alien was found to be affected only in part by HDAC inhibitors such as trichostatin A. Finally, for a dissociation of VDR-Alien complexes in vitro and in vivo, higher ligand concentrations were needed than for a dissociation of VDR-NCoR complexes. This suggests that Alien and NCoR are using different interfaces for interaction with the VDR and different pathways for mediating superrepression, which in turn characterizes Alien as a representative of a new class of corepressors. Taken together, association of the VDR with corepressor proteins provides a further level of transcriptional regulation, which is emerging as a complex network of protein-protein interaction-mediated control.

1 -mediated gene transcription is the regulation of calcium homeostasis and bone mineralization (1), but the nuclear hormone also plays a role in controlling cellular growth, differentiation, and apoptosis (2). Various analogues of 1␣,25-(OH) 2 D 3 , which mainly contain modifications of the side chain, have been developed with the goal to improve the biological profile of the natural hormone for a potential therapeutic application (3). The genomic effects of 1␣,25-(OH) 2 D 3 and its analogues are principally mediated through the 1␣,25-(OH) 2 D 3 receptor (VDR) (4), which is a member of the nuclear receptor transcription factor superfamily (5). VDR binds as a heterodimer with the retinoid X receptor (RXR) (6) to specific sequences in promoter regions of 1␣,25-(OH) 2 D 3 target genes, referred to as 1␣,25-(OH) 2 D 3 response elements (VDREs) (7). Simple VDREs consist of two hexameric nuclear receptor binding sites, which are commonly arranged as a direct repeat with 3 spacing nucleotides (DR3-type VDREs) (4). The VDR consists of several functional domains, which includes the DNA-binding domain (DBD) and the ligandbinding domain (LBD). The LBD of the VDR is formed by 12 ␣-helical structures, of which the last one, helix 12, contains a short transactivation function 2 (AF-2) domain. A critical step in 1␣,25-(OH) 2 D 3 signaling is the specific ligand-triggered induction of a conformational change within the LBD of the VDR. This conformational change induces the dissociation of corepressors, such as NCoR (8) or Alien (9), and facilitates the interaction with coactivator proteins with members of the p160 family, such as SRC-1/ERAP160/NCoA1 (10, 11), TIF2/Grip-1/NCoA2 (12, 13), and RAC3/AIB1/ACTR/ pCIP (14 -16). This VDR-coactivator interaction then further facilitates recruitment of other factors to form a larger complex that modulates chromatin structure and initiates transcription (17). This also involves the recently described DRIP/ ARC cofactor complexes (18,19), which appear to contact the VDR and other nuclear receptors preceding their interaction with p160 family of coactivators (20).For some members of the nuclear hormone receptor superfamily, such as the estrogen receptor (ER) and the progesterone receptor, antagonists in addition to agonists have been known for some time (21). Recently, 25-carboxylic ester and 26,23-lactone 1␣,25-(OH) 2 D 3 analogues have been described as the first types of VDR antagonists (22, 23). The molecular mechanisms of action of ER antagonists have been explained by incorrect positioning and blocking of the AF-2 domain (24, 25). In contrast, the molecular mechanisms of the antagonistic action of the 26,23-lactone analogue have been explained by reduced VDR-RXR heterodimer complex formation (26).In this study, a 25-carboxylic ester analogue of 1␣,25-(OH) 2 D 3 , ZK159222, was characterized as a novel type of 1␣,25-(OH) 2 D 3 antagonist. ZK159222 was not able to promote a ligand-dependent interaction of the VDR with coactivator proteins of the p160 family neither in solution nor in a...

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A novel, more potent VDR antagonist, ZK168281, was identified, which stabilizes VDR-RXR heterodimers in living cells in a different conformation than agonists. In addition, the VDR/RXR ratio was found as the major discriminating factor for understanding cell-specific effects of VDR antagonists.

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