The vitamin D receptor (VDR) binds the vitamin D-responsive element (VDRE) as a heterodimer with an unidentified receptor auxiliary factor (RAF) present in mammalian cell nuclear extracts. VDR also interacts with the retinoid X receptors (RXRs), implying that RAF may be related to the RXRs. Here we demonstrate that highly purified HeLa cell RAF contained RXR beta immunoreactivity and that both activities copurified and precisely coeluted in high-resolution hydroxylapatite chromatography. Furthermore, an RXR beta-specific antibody disrupted VDR-RAF-VDRE complexes in mobility shift assays. These data strongly indicate that HeLa RAF is highly related to or is identical to RXR beta. Consequently, the effect of the 9-cis retinoic acid ligand for RXRs was examined in 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-activated gene expression systems. Increasing concentrations of 9-cis retinoic acid (1 nM to 1 microM) markedly reduced 1,25(OH)2D3-dependent accumulation of osteocalcin mRNA in osteoblast-like ROS 17/2.8 cells. All-trans retinoic acid also interfered with vitamin D responsiveness, but it was consistently less potent than the 9-cis isomer. Transient transfection studies revealed that attenuation by 9-cis retinoic acid was at the transcriptional level and was mediated through interactions at the osteocalcin VDRE. Furthermore, overexpression of both RXR beta and RXR alpha augmented 1,25(OH)2D3 responsiveness in transient expression studies. Direct analysis of VDRE binding in mobility shift assays demonstrated that heteromeric interactions between VDR and RXR were enhanced by 1,25(OH)2D3 and were not affected appreciably by 9-cis retinoic acid, except that inhibition was observed at high retinoid concentrations. These data suggest a regulatory mechanism for osteocalcin gene expression that involves 1,25(OH)2D3-induced heterodimerization of VDR and unliganded RXR. 9-cis retinoic acid may attenuate 1,25(OH)2D3 responsiveness by diverting RXRs away from VDR-mediated transcription and towards other RXR-dependent transcriptional pathways.

Alopecia is a feature of vitamin D receptor (VDR) mutations in humans and in VDR null mice. This alopecia results from an inability to initiate the anagen phase of the hair cycle after follicle morphogenesis is complete. Thus, once the initial hair is shed it does not regrow. VDR expression in the epidermal component of the hair follicle, the keratinocyte, is critical for maintenance of the hair cycle. To determine which functional domains of the VDR are required for hair cycling, mutant VDR transgenes were targeted to the keratinocytes of VDR null mice. Keratinocyte-specific expression of a VDR transgene with a mutation in the hormone-binding domain that abolishes ligand binding restores normal hair cycling in VDR null mice, whereas a VDR transgene with a mutation in the activation function 2 domain that impairs nuclear receptor coactivator recruitment results in a partial rescue. Mutations in the nuclear receptor corepressor Hairless are also associated with alopecia in humans and mice. Hairless binds the VDR, resulting in transcriptional repression. Neither VDR mutation affects Hairless interactions or its ability to repress transcription. These studies demonstrate that the effects of the VDR on the hair follicle are ligand independent and point to novel molecular and cellular actions of this nuclear receptor.

Nuclear coactivator-62 kDa/Ski-interacting protein (NCoA62/SKIP) is a putative vitamin D receptor (VDR) and nuclear receptor coactivator protein that is unrelated to other VDR coactivators such as those in the steroid receptor coactivator (SRC) family. The mechanism through which NCoA62/SKIP functions in VDRactivated transcription is unknown. In the present study, we identified a nuclear localization sequence in the COOH terminus of NCoA62/SKIP and showed that NCoA62/SKIP was targeted to nuclear matrix subdomains. Chromatin immunoprecipitation studies revealed that endogenous NCoA62/SKIP associated in a 1,25-dihydroxyvitamin D 3 -dependent manner with VDR target genes in ROS17/2.8 osteosarcoma cells. A cyclic pattern of promoter occupancy by VDR, SRC-1, and NCoA62/SKIP was observed, with NCoA62/SKIP entering these promoter complexes after SRC-1. These studies provide strong support for the proposed role of NCoA62/SKIP as a VDR transcriptional coactivator, and they indicate that key mechanistic differences probably exist between NCoA62/SKIP and SRC coactivators. To explore potential mechanisms, NCoA62/SKIP-interacting proteins were purified from HeLa cell nuclear extracts and identified by mass spectrometry. The identified proteins represent components of the spliceosome as well as other nuclear matrix-associated proteins. Here, we show that a dominant negative inhibitor of NCoA62/SKIP (dnNCoA62/SKIP) interfered with appropriate splicing of transcripts derived from 1,25-dihydroxyvitamin D 3 -induced expression of a growth hormone minigene cassette. Taken together, these data show that NCoA62/SKIP has properties that are consistent with those of nuclear receptor coactivators and with RNA spliceosome components, thus suggesting a potential role for NCoA62/SKIP in coupling VDR-mediated transcription to RNA splicing. The vitamin D receptor (VDR)1 is a nuclear receptor (NR) family member that mediates the biological actions of 1,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ), the active hormone of the vitamin D endocrine system. VDR forms a heterodimer with retinoid X receptor (RXR) and binds to specific vitamin Dresponsive elements (VDREs) in the promoter region of target genes to regulate transcription by RNA polymerase II (1, 2). Transcriptional activation through 1,25-(OH) 2 D 3 and VDR is enhanced by nuclear receptor coactivator proteins such as steroid receptor coactivators (SRCs) (3-5) and proteins of the vitamin D receptor-interacting protein (DRIP) complex (6, 7). The SRCs and DRIPs utilize leucine-rich LXXLL motifs (8, 9) to interact in a 1,25-(OH) 2 D 3 -dependent manner with a complementary hydrophobic cleft on the surface of the VDR ligand binding domain. This hydrophobic surface is composed of helices H3, H4, H5, and H12, and it is the ligand-dependent folding of H12 that creates the interaction cleft for SRC binding to NRs (10 -15). The H12 helix of the NRs contains the critical ligand-dependent activation function-2 domain that is essential for ligand activated transcription (16, 17) and ligand-dependent S...

The vitamin D receptor (VDR) heterodimerizes with retinoid X receptors (RXR) on many vitamin D-responsive promoter elements, suggesting that this complex is the active factor in vitamin D-mediated transcription. However, the mechanism of transcriptional regulation following VDR-RXR binding to DNA is not well characterized. Using a yeast two-hybrid protein interaction assay, we demonstrate that VDR forms specific protein: protein contacts with the basal transcription factor TFIIB. Deletion analysis indicated that the carboxyl-terminal ligand binding domain of VDR interacted with a 43-residue amino-terminal domain in TFIIB. The interaction with TFIIB showed selectivity for the ligand binding domain of VDR as similar regions of RXR alpha or of retinoic acid receptor alpha did not couple with TFIIB. Binding assays with purified proteins showed a direct interaction between VDR and TFIIB in vitro. These data suggest a mechanism for VDR-dependent transcription in which protein contacts between VDR and TFIIB may impart regulatory information to the transcription preinitiation complex.

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