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 D 3 [1,25-(OH) 2 D 3 ] in an in vivo preclinical surrogate model of psoriasis.

The vitamin D receptor (VDR) belongs to the thyroid hormone/retinoid receptor subfamily of nuclear receptors and functions as a heterodimer with retinoid X receptor (RXR). The RXR-VDR heterodimer, in contrast to other members of the class II nuclear receptor subfamily, is nonpermissive where RXR does not bind its cognate ligand, and therefore its role in VDR-mediated transactivation by liganded RXR-VDR has not been fully characterized. Here, we show a unique facet of the intermolecular RXR-VDR interaction, in which RXR actively participates in vitamin D3-dependent gene transcription. Using helix 3 and helix 12 mutants of VDR and RXR, we provide functional evidence that liganded VDR allosterically modifies RXR from an apo (unliganded)- to a holo (liganded)-receptor conformation, in the absence of RXR ligand. As a result of the proposed allosteric modification of RXR by liganded VDR, the heterodimerized RXR shows the "phantom ligand effect" and thus acquires the capability to recruit coactivators steroid receptor coactivator 1, transcriptional intermediary factor 2, and amplified in breast cancer-1. Finally, using a biochemical approach with purified proteins, we show that RXR augments the 1,25-dihydroxyvitamin D3-dependent recruitment of transcriptional intermediary factor 2 in the context of RXR-VDR heterodimer. These results confirm and extend the previous observations suggesting that RXR is a significant contributor to VDR-mediated gene expression and provide a mechanism by which RXR acts as a major contributor to vitamin D3-dependent transcription.

We provide evidence of a cross-talk between nuclear receptor and Ser/Thr protein phosphatases and show that vitamin D receptor (VDR) interacts with the catalytic subunit of protein phosphatases, PP1c and PP2Ac, and induces their enzymatic activity in a ligand-dependent manner. PP1c specifically interacts with VDR but not retinoic acid receptor ␣ and retinoid X receptor ␣ in yeast. Although VDR-PP1c and VDR-PP2Ac interaction is ligand-independent in vivo, 1␣,25-dihydroxy-vitamin D 3 induces VDR-associated phosphatase activity. Further, VDR modulation of PP1c/PP2Ac activity results in a rapid and specific dephosphorylation and inactivation of their substrate, p70 S6 kinase (p70 S6k ). Finally, we demonstrate that the endogenous VDR, PP1c or PP2Ac, and p70S6k are present in a ternary complex in vivo, and the interaction of p70S6k with the VDR-PP complex is modulated by the phosphorylation state of the kinase. Since p70S6k is essential for G 1 -S transition, our results provide a molecular basis of 1␣,25-dihydroxyvitamin D 3 -induced G 1 block in colon cancer cells. Vitamin D receptor (VDR),1 a sequence-specific ligand-dependent transcription factor belonging to the family of nuclear receptors, mediates biological actions of 1␣,25-dihydroxy-vitamin D 3 (1,25(OH) 2 D 3 ). VDR heterodimerizes with retinoid X receptor (RXR), and at the molecular level VDR-RXR heterodimers induce gene expression via interaction with vitamin D response elements present in the promoter regions of responsive genes (1). This mode of action is known as the "genomic action" of VDR. However 1,25-(OH) 2 D 3 also induces gene expression by a mechanism distinct from its classical mode of action. For example, 1,25-(OH) 2 D 3 -induced expression of monocytic differentiation markers CD14 and CD11b in THP-1 cells requires phosphatidylinositol 3-kinase (PI 3-kinase) via liganddependent interaction of VDR with the regulatory (p85) subunit of PI 3-kinase (2). Similarly estrogen receptor interacts with the p85 regulatory subunit of the PI 3-kinase where estrogen receptor-PI 3-kinase interaction leads to the activation of protein kinase B/AKT and endothelial nitric-oxide synthase (3). It thus appears that cross-talk between nuclear receptors and other signal transduction pathways can lead to either induction of gene expression in a nuclear receptor-responsive element-independent manner or to extranuclear/non-genomic induction of enzymatic activities that are physiologically important, for example, in explaining the vasoprotective effects of estrogen (3). Further, nuclear receptor ligands (dexamethasone, triiodothyronine, and retinoic acid) also induce a rapid dephosphorylation of c-Jun N-terminal kinase independently of their transcriptional activation (4). Therefore, nuclear receptors appear to have a functional role both outside and inside the nucleus. Ser/Thr phosphatases are implicated in the regulation of a wide variety of cellular functions, namely metabolism, transcription, translation, development, cell growth, and differentiation (5). There are ...

Ligand activation of retinoic acid receptors (RARs) involves coordinated changes in their interaction with coregulatory molecules. Binding of the agonist all-transretinoic acid to the RAR results in increased interaction with coactivator molecules as well as a decreased interaction with corepressor molecules. Thus, an all-transretinoic acid antagonist might function either by preventing agonist induction of such events or, additionally, by actively increasing repression via corepressor recruitment. We demonstrate that the repression of the transcriptional activity of a constitutively active RAR␥-VP-16 chimeric receptor by the inverse agonist AGN193109 requires a functional Co-R box and that binding of this ligand to RAR␥ leads to an increased interaction with the corepressor N-CoR both in glutathione S-transferase pull-down and yeast two-hybrid analyses. Detection of nuclear receptor corepressor (N-CoR) association with RAR␥ was greatly facilitated by inclusion of a RARE oligonucleotide in coimmunoprecipitation analyses, a result of an increase in association of the ternary complex consisting of RAR, RXR, and DNA. Similarly, this DNA-dependent increase in heterodimer formation likewise resulted in an increase in agonistmediated recruitment efficiency of the coactivator SRC-1. Under conditions which favor ternary complex formation, a RAR neutral antagonist is distinguished from an inverse agonist with respect to corepressor recruitment as is a RAR partial agonist distinguished from an agonist with respect to coactivator recruitment. These results indicate that it is possible to design RAR ligands with distinct recruitment capabilities for coregulators, both coactivators as well as corepressors. In addition, using this recruitment assay, we show that SRC-1 and the related coactivator molecule ACTR associate with the ternary complex via utilization of different helical motifs within their conserved receptor interaction domains.

The syntheses and full retinoid receptor characterization of a novel series of retinoic acid receptor alpha (RAR alpha) antagonists, 1-5, are described. These compounds bind with high affinity to RAR alpha but were completely inactive in gene transactivation. They were also potent and effective antagonists of retinoic acid (RA) induced gene transcription at RAR alpha. Compounds 1-5 exhibited varying degrees of selectivity for RAR alpha relative to RAR beta/gamma, with compound 5 being the most selective in both binding and functional antagonism assays. These compounds will be invaluable tools in delineating the physiological roles of RAR alpha in development and in the adult animal and may themselves be useful therapeutic agents in human diseases associated with RAR alpha.