CDP/cut is an osteoblastic coactivator of the vitamin D receptor (VDR).

Vitamin D plays an important role in regulating bone and calcium metabolism. The actions of vitamin D are mediated through the nuclear vitamin D receptor (VDR), and gene disruption of the VDR in mice causes skeletal disorders. However, the precise role of the VDR in each stage of osteoblastogenesis is not well understood. To address this issue, we used a biochemical approach to identify an osteoblast-specific coregulator of the VDR. Using a GST-fused VDR ligand-binding domain as bait, proteins associated with liganded VDR were purified from nuclear extracts of HOS osteoblastic cells and compared with those of HeLa cells. Among the interactants identified by mass fingerprinting, CCAAT displacement protein (CDP) was found as a novel ligand-dependent VDR interactant in HOS cells, together with other previously reported DRIP/TRAP complex components. Further biochemical analysis showed that complex formation between the VDR and CDP was distinct from the previously known DRIP/TRAP complex and the p160 family coactivator complexes. Transient expression of CDP potentiated VDR-mediated transcriptional activation in HOS cells. Furthermore, modulation of CDP expression levels in osteoblastic SaM-1 cells affected vitamin D-dependent osteoblast differentiation before the maturation (mineralization) stage. These findings suggest that CDP is a novel differentiation stage-specific coactivator of the VDR in osteoblasts.

Mechanisms of transcriptional repression by 1,25(OH)2 vitamin D.

PURPOSE OF REVIEW: Vitamin D has diverse biological actions, and consequently the mechanisms behind how it regulates gene transcription are diverse. Unlike its well described positive effects on gene transcription, little is known about how vitamin D induces transcriptional repression. RECENT FINDINGS: Vitamin D-induced transcriptional repression of several negative vitamin D receptor target genes has been studied on a molecular level. A new class of negative vitamin D response elements, which are E-box-type motifs, bind the bHLH-type transcriptional activator (VDIR) together with a histone acetyltransferase coactivator. The vitamin D receptor, activated by vitamin D, does not directly bind to the negative vitamin D response elements, but instead associates with VDIR. This leads to the dissociation of the histone acetyltransferase coactivator and recruitment of a histone deacetylase corepressor to transrepress transcription of the target gene promoter. SUMMARY: Histone inactivation induced by histone deacetylase co-repressors appears to facilitate vitamin D-induced transcriptional repression via the vitamin D receptor. Following vitamin D binding, structural alteration of the DNA-unbound vitamin D receptor triggers transcriptional repression. Given this, the mechanisms behind vitamin D-induced transcriptional repression are probably more complex than those of vitamin D-induced transactivation.

1Alpha,25(OH)2D3-induced transrepression by vitamin D receptor through E-box-type elements in the human parathyroid hormone gene promoter.

Although transactivation by the liganded vitamin D receptor (VDR) is well described at the molecular level, the precise molecular mechanism of negative regulation by the liganded VDR remains to be elucidated. We have previously reported a novel class of negative vitamin D response element (nVDRE) called 1alphanVDRE in the human 25(OH)D31alpha-hydroxylase [1alpha(OH)ase] gene by 1alpha,25(OH)2D3-bound VDR. This element was composed of two E-box-type motifs that bound to VDIR for transactivation, which was attenuated by liganded VDR. Here, we explore the possible functions of VDIR and E-box motifs in the human (h) PTH and hPTHrP gene promoters. Functional mapping of the hPTH and hPTHrP promoters identified E-box-type elements acting as nVDREs in both the hPTH promoter (hPTHnVDRE; -87 to -60 bp) and in the hPTHrP promoter (hPTHrPnVDRE; -850 to -600 bp; -463 to -104 bp) in a mouse renal tubule cell line. The hPTHnVDRE alone was enough to direct ligand-induced transrepression mediated through VDR/retinoid X receptor and VDIR. Direct DNA binding of hPTHnVDRE to VDIR, but not VDR/retinoid X receptor, was observed and ligand-induced transrepression was coupled with recruitment of VDR and histone deacetylase 2 (HDAC2) to the hPTH promoter. These results suggest that negative regulation of the hPTH gene by liganded VDR is mediated by VDIR directly binding to the E-box-type nVDRE at the promoter, together with recruitment of an HDAC corepressor for ligand-induced transrepression.

Multiple co-activator complexes support ligand-induced transactivation function of VDR.

Vitamin D receptor (VDR) mediates a wide variety of vitamin D actions through transcriptional controls of target genes as a ligand-dependent transcription factor. The transactivation by VDR is known to associate with two co-activator complexes, DRIP/TRAP and p160/CBP, through physical interaction with DRIP205 and p160 members (TIF2) components, respectively. However, functional difference between the two co-activator complexes for VDR co-activation remains unclear. In the present study, to address this issue, a series of point mutants in VDR helix 12 were generated to test the functional association. Alanine replacement of VDR valine 418 resulted in loss of DRIP205 interaction, but it was still transcriptionally potent with ability to interact with TIF2. Surprisingly, the V421A mutant was only partially impaired in transactivation without co-activator interaction, implying presence of a putative co-activator/complex. Thus, these findings suggest that ligand-induced transcriptional controls by VDR require a number of known and unknown co-regulator complexes, that may support the tissue-specific function of VDR.

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.

Nuclear receptor mediated gene regulation through chromatin remodeling and histone modifications.

Nuclear steroid/thyroid vitamin A/D receptor genes form a gene superfamily and encode DNA-binding transcription factors that control the transcription of target genes in a ligand-dependent manner. It has become clear that chromatin remodeling and the modification of histones, the main components of chromatin, play crucial roles in gene transcription, and many distinct classes of NR-interacting co-regulators have been identified that perform significant roles in gene transcription. Since NR dysfunction can lead to the onset or progression of endocrine disease, elucidation of the mechanisms of gene regulation mediated by NRs, as well as the identification and characterization of co-regulator complexes (especially chromatin remodeling and histone-modifying complexes), is essential not only for better understanding of NR ligand function, but also for pathophysiological studies and the development of therapeutic interventions in humans.

Identification and characterization of noncalcemic, tissue-selective, nonsecosteroidal vitamin D receptor modulators.

Vitamin D receptor (VDR) ligands are therapeutic agents for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. VDR ligands also show immense potential as therapeutic agents for autoimmune diseases and cancers of skin, prostate, colon, and breast as well as leukemia. However, the major side effect of VDR ligands that limits their expanded use and clinical development is hypercalcemia that develops as a result of the action of these compounds mainly on intestine. In order to discover VDR ligands with less hypercalcemia liability, we sought to identify tissue-selective VDR modulators (VDRMs) that act as agonists in some cell types and lack activity in others. Here, we describe LY2108491 and LY2109866 as nonsecosteroidal VDRMs that function as potent agonists in keratinocytes, osteoblasts, and peripheral blood mononuclear cells but show poor activity in intestinal cells. Finally, these nonsecosteroidal VDRMs were less calcemic in vivo, and LY2108491 exhibited more than 270-fold improved therapeutic index over the naturally occurring VDR ligand 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in an in vivo preclinical surrogate model of psoriasis.

Synthesis of 2,2-dimethyl-1,25-dihydroxyvitamin D3: A-ring structural motif that modulates interactions of vitamin D receptor with transcriptional coactivators.

A concise synthesis of all four possible A-ring stereoisomers of 2,2-dimethyl-1,25-dihydroxyvitamin D3 and characterization of their distinct transcriptional features, which appear to have been inherited from the corresponding 2alpha-methyl derivatives, is reported.