Elizabeth A. Allegretto

Novel nonsecosteroidal vitamin D mimics exert VDR-modulating activities with less calcium mobilization than 1,25-dihydroxyvitamin D3

BACKGROUND The secosteroid 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) acts through the vitamin D receptor (VDR) to elicit many activities that make it a promising drug candidate for the treatment of a number of diseases, including cancer and psoriasis. Clinical use of 1,25(OH)2D3 has been limited by hypercalcemia elicited by pharmacologically effective doses. We hypothesized that structurally distinct, nonsecosteroidal mimics of 1,25(OH)2D3 might have different activity profiles from vitamin D analogs, and set out to discover such compounds by screening small-molecule libraries. RESULTS A bis-phenyl derivative was found to activate VDR in a transactivation screening assay. Additional related compounds were synthesized that mimicked various activities of 1,25(OH)2D3, including growth inhibition of cancer cells and keratinocytes, as well as induction of leukemic cell differentiation. In contrast to 1, 25(OH)2D3, these synthetic compounds did not demonstrate appreciable binding to serum vitamin D binding protein, a property that is correlated with fewer calcium effects in vivo. Two mimics tested in mice showed greater induction of a VDR target gene with less elevation of serum calcium than 1,25(OH)2D3. CONCLUSIONS These novel VDR modulators may have potential as therapeutics for cancer, leukemia and psoriasis with less calcium mobilization side effects than are associated with secosteroidal 1,25(OH)2D3 analogs.

Retinoid X Receptor (RXR) Ligands Activate the Human 25-Hydroxyvitamin D3-24-hydroxylase Promoter via RXR Heterodimer Binding to Two Vitamin D-responsive Elements and Elicit Additive Effects with 1,25-Dihydroxyvitamin D3

We have previously shown that RNA levels of kidney 25-hydroxyvitamin D3-24-hydroxylase (24(OH)ase), a key metabolic enzyme for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is up-regulated by retinoids in mice within hours. Deletion analysis of ∼5500 base pairs of the human 24(OH)ase promoter showed that the sequence between −316 and −142 contained the information necessary and sufficient for retinoid-induced activation of the promoter. This region contains two previously defined vitamin D-responsive elements (VDREs) at −294 to −274 and −174 to −151. Mutation of either VDRE diminished responsiveness of the −316 to −22 promoter sequence to retinoids or 1,25(OH)2D3, while mutation of both VDREs essentially abolished the activity of the ligands via the promoter. Heterologous promoter vectors driven by the VDREs were responsive to a retinoid X receptor (RXR)-selective ligand (LG100268), a retinoic acid receptor (RAR)-selective ligand (TTNPB), or 1,25(OH)2D3, while combinations of LG100268 with either TTNPB or 1,25(OH)2D3 resulted in additive increases in activity. Band shift analyses showed that vitamin D receptor, RAR, or RXR alone did not bind to the VDREs; however, the combination of either vitamin D receptor or RAR with RXR led to retardation of each of the labeled probes. Treatment of nontransfected CV-1 cells with retinoids or 1,25(OH)2D3resulted in induction of 24(OH)ase RNA, and ligand combinations led to increased RNA levels. These data imply that either or both of the heterodimer partners can be occupied with ligand to induce this enzyme, with dual receptor occupation leading to increased activation.

The Vitamin D Response Element-binding Protein A NOVEL DOMINANT-NEGATIVE REGULATOR OF VITAMIN D-DIRECTED TRANSACTIVATION

Vitamin D resistance in certain primate genera is associated with the constitutive overexpression of a non-vitamin D receptor (VDR)-related, vitamin D response element-binding protein (VDRE-BP) and squelching of vitamin d-directed transactivation. We used DNA affinity chromatography to purify proteins associated with non-VDR-VDRE binding activity from vitamind-resistant New World primate cells. In electrophoretic mobility shift assays, these proteins bound specifically to either single-strand or double-strand oligonucleotides harboring the VDRE. Amino acid sequencing of tryptic peptides from a 34-kDa (VDRE-BP1) and 38-kDa species (VDRE-BP-2) possessed sequence homology with human heterogeneous nuclear ribonucleoprotein (hnRNP) A1 and hnRNPA2, respectively. cDNAs bearing the open reading frame for both VDRE-BPs were cloned and used to transfect wild-type, hormone-responsive primate cells. Transient and stable overexpression of the VDRE-BP2 cDNA, but not the VDRE-BP1 cDNA, in wild-type cells with a VDRE-luciferase reporter resulted in significant reduction in reporter activity. These data suggest that the hnRNPA2-related VDRE-BP2 is a dominant-negative regulator of vitamin D action.

Retinoid X Receptor Acts as a Hormone Receptor in Vivo to Induce a Key Metabolic Enzyme for 1,25-Dihydroxyvitamin D3

We demonstrate here that RNA levels of 25-hydroxyvitamin D3-24-hydroxylase (24-(OH)ase), a key catabolic enzyme for 1,25-dihydroxyvitamin D3, are increased by a highly selective retinoid X receptor (RXR) ligand, LG100268, in mice within hours. Correspondingly, upon LG100268 treatment, kidney 24-(OH)ase enzymatic activity increases 5-10-fold. The endogenous retinoid hormones, all-trans-retinoic acid and 9-cis-retinoic acid, and the synthetic retinoic acid receptor-selective compound, TTNPB, also stimulate 24-(OH)ase. Additionally, we show that LG100268 stimulates transcription of a luciferase reporter plasmid driven by 24-(OH)ase promoter sequences in the presence of RXR in CV-1 cell cotransactivation assays. This first demonstration of a gene that is regulated in the intact animal through an RXR-mediated pathway confirms earlier hypotheses that RXR is a bona fide hormone receptor. Regulation of a key gene in the vitamin D signaling pathway by a retinoid transducer may provide a molecular basis for some of the documented biological effects of vitamin A on bone and vitamin D metabolism.

[23]Intracellular receptor characterization and ligand screening by transactivation and hormone-binding assays

Publisher Summary The assessment of receptor hormone-binding activity remains today an essential tool for the characterization of both the receptors with known ligands and the putative orphan receptors. The chapter discusses the utilization of transactivation and hormone-binding assays for the characterization of known receptors and presents a brief overview of their application in the drug screening of these known receptors. Specifically, the six retinoid receptors, including the three known retinoic acid receptors (RARs) and the three retinoid X receptors (RXRs), and their known retinoid isomer ligands, all- trans -retinoic acid (tRA) and 9- cis -retinoic acid (9cRA), are presented as an example system in the chapter. These receptors have been characterized by both hormone-binding assays and transactivation assays, the latter performed in both mammalian cells and yeast. Both hormone-binding and transactivation assays are indispensable tools for the characterization of receptors, for screening potential drug candidate ligands for a known receptor, and for screening compounds that are potential orphan receptor ligands. Because the receptors exhibit modular functional domain structure, the hormone-binding domain (HBD) of a known receptor can be replaced with the HBD of an orphan receptor (within the context of the intact known receptor) to generate a chimeric receptor. The chimeric receptor transcribes a reporter enzyme plasmid containing the hormone response element (HRE) of the known receptor in response to a ligand that binds to the HBD of the orphan receptor.