Paul D. Thompson

The nuclear vitamin D receptor: Biological and molecular regulatory properties revealed

IN THE DECADE SINCE THE VITAMIN D RECEPTOR (VDR) was cloned and recognized as a member of the superfamily of nuclear receptors that regulate gene expression in a ligand-dependent manner, the central role of VDR in the biology of vitamin D action has been illuminated and is being defined at the molecular level. Following renal production as the hormonal metabolite of vitamin D, 1a,25-dihydroxyvitamin D3 (1,25(OH)2D3) functions as the ligand for VDR, with the hormone–receptor complex inducing calcemic and phosphatemic effects that result in normal bone mineralization and remodeling. VDR not only mediates the action of 1,25(OH)2D3 in calcium/phosphate translocating tissues, primarily intestine, but also elicits a myriad of apparent bioactivities in other major cell systems in the organism, including immune, neural, epithelial, and endocrine. The scope of this review will be limited to highlighting the actions of 1,25(OH)2D3 mediated by nuclear VDR and discussing new developments in the structure/function analysis of the receptor, including the phenotype of VDR knockout mice and the biochemical classification of patients with point mutations in the receptor. These new advances, along with other recent research, will be interpreted to update our understanding of the molecular role of VDR, ranging from characterization of its natural gene and clinically significant polymorphisms, through its DNA contact sites and protein partners, to novel ligand analogs that hold the promise of influencing VDR conformation in a therapeutically beneficial fashion. VDR BIOLOGY

Molecular Nature of the Vitamin D Receptor and its Role in Regulation of Gene Expression

Clinical and molecular genetic data from the last decade have provided unequivocal evidence for the obligatory role of the nuclear vitamin D receptor (VDR) in mediating the actions of vitamin D. As illustrated in Fig. 1, following its renal production as the hormonal metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) functions as the ligand for VDR, with the hormone-receptor complex inducing calcemic and phosphatemic effects that result in normal bone mineralization and remodeling [1]. Familial target tissue insensitivity to 1,25(OH)2D3, known as hereditary hypocalcemic vitamin D-resistant rickets (HVDRR), is an autosomal recessive disorder with a phenotype of severe bowing of the lower extremities, short stature and often alopecia [2]. The cause of this syndrome is usually a defect in the gene encoding human (h)VDR [1], although potential exceptions have been described [3]. The fact that the phenotype of HVDRR patients, excluding alopecia, mimics classic nutritional as well as renal rickets, indicates that 1,25(OH)2D3-liganded VDR not only executes the bone mineral homeostatic actions of vitamin D, but suggests that VDR also participates in the normal hair growth cycle in skin (Fig. 1, upper left). Beyond calcium/phosphate translocating tissues, like intestine, kidney, and bone, there are a myriad of apparent vitamin D bioactivities in nontraditional targets, including cells of the immune, neural, and endocrine systems (Fig. 1, lower left). Notable examples include maintenance of insulin secretion by 1,25(OH)2D3, the exploitation of the prodifferentiation/antiproliferative actions of 1,25(OH)2D3 in the treatment of psoriasis, and a neuromodulatory effect of the vitamin D hormone in the central nervous system [1]. One key target for the nonclassical actions of 1,25(OH)2D3 is the immune system [4], where 1,25(OH)2D3 functions as a general suppressor, especially of T-helper cells (subset type 1), suggesting that analogs of vitamin D might be useful therapeutic agents in procedures such as organ transplants, or in the treatment of autoimmune disorders [5]. Although of potential therapeutic signi®cance, many of the nonclassical effects of 1,25(OH)2D3-VDR may be biologically redundant with other systemic modulators, as suggested by studies of the VDR knockout mice [6,7] which reveal apparently normal heterozygotes but homozygotes that display a phenotype very similar to HVDRR, including alopecia. Despite lack of VDR throughout early development, VDR null mice are born phenotypically normal, exhibiting symptoms of rickets/ osteomalacia and secondary hyperparathyroidism primarily after weaning [6]. In addition, Li et al. [8] showed that the prevention of many, but not all, of the phenotypic effects of VDR knockout can be achieved by a ``rescue diet, consisting of high levels of lactose, calcium, and phosphate. By arti®cially maintaining blood calcium and phosphate levels in this manner, parathyroid hormone (PTH) was normalized and bone mineralization was greatly improved in the VDR knockout animals, to a degree that the histology of the growth plate was indistinguishable from that of normal littermates. Similar results have been reported in HVDRR patients after frequent therapy with overnight intravenous calcium infusions [9]. These observations suggest that the vitamin D endocrine system is uniquely required

The nuclear vitamin D receptor controls the expression of genes encoding factors which feed the "Fountain of Youth" to mediate healthful aging.

The nuclear vitamin D receptor (VDR) binds 1,25-dihydroxyvitamin D3 (1,25D), its high affinity renal endocrine ligand, to signal intestinal calcium and phosphate absorption plus bone remodeling, generating a mineralized skeleton free of rickets/osteomalacia with a reduced risk of osteoporotic fractures. 1,25D/VDR signaling regulates the expression of TRPV6, BGP, SPP1, LRP5, RANKL and OPG, while achieving feedback control of mineral ions to prevent age-related ectopic calcification by governing CYP24A1, PTH, FGF23, PHEX, and klotho transcription. Vitamin D also elicits numerous intracrine actions when circulating 25-hydroxyvitamin D3, the metabolite reflecting vitamin D status, is converted to 1,25D locally by extrarenal CYP27B1, and binds VDR to promote immunoregulation, antimicrobial defense, xenobiotic detoxification, anti-inflammatory/anticancer actions and cardiovascular benefits. VDR also affects Wnt signaling through direct interaction with beta-catenin, ligand-dependently blunting beta-catenin mediated transcription in colon cancer cells to attenuate growth, while potentiating beta-catenin signaling via VDR ligand-independent mechanisms in osteoblasts and keratinocytes to function osteogenically and as a pro-hair cycling receptor, respectively. Finally, VDR also drives the mammalian hair cycle in conjunction with the hairless corepressor by repressing SOSTDC1, S100A8/S100A9, and PTHrP. Hair provides a shield against UV-induced skin damage and cancer in terrestrial mammals, illuminating another function of VDR that facilitates healthful aging.

Liganded VDR induces CYP3A4 in small intestinal and colon cancer cells via DR3 and ER6 vitamin D responsive elements

The nuclear vitamin D receptor (VDR) mediates the effects of 1,25-dihydroxyvitamin D(3) (1,25D(3)) to alter intestinal gene transcription and promote calcium absorption. Because 1,25D(3) also exerts anti-cancer effects, we examined the efficacy of 1,25D(3) to induce cytochrome P450 (CYP) enzymes. Exposure of human colorectal adenocarcinoma cells (HT-29) to 10(-8)M 1,25D(3) resulted in >/=3-fold induction of CYP3A4 mRNA and protein as assessed by RT-PCR and Western blotting, respectively. Six vitamin D responsive element (VDRE)-like sequences in the promoter region of the CYP3A4 gene were then individually tested for their ability to enhance transcription. A canonical DR3-type element in the distal region of the promoter (-7719-GGGTCAgcaAGTTCA-7733), and a proximal, non-classical everted repeat with a spacer of 6 bp (ER6; -169-TGAACTcaaaggAGGTCA-152) were identified as functional VDREs in this CYP gene. These data suggest that 1,25D(3)-dependent, VDR-mediated induction of CYP3A4 may constitute a chemoprotective mechanism for detoxification of enteric xenobiotics and carcinogens.

New understanding of the molecular mechanism of receptor-mediated genomic actions of the vitamin D hormone.

The nuclear vitamin D receptor (VDR) binds the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]hormone with high affinity and elicits its actions to regulate gene expression in target cells by binding to vitamin D-responsive elements (VDREs). VDREs in positively controlled genes such as osteocalcin, osteopontin, beta 3-integrin, and vitamin D-24-OHase are direct hexanucleotide repeats with a spacer of three nucleotides. The VDR associates with these VDREs with the greatest affinity as a heterodimer with one of the family of retinoid X receptors (RXRs). VDR consists of an N-terminal zinc finger domain that determines DNA binding, a hinge segment and a C-terminal hormone binding domain which also contains two conserved regions that engage in heterodimerization with an RXR on the VDRE. The role of the 1,25(OH)2D3 ligand in transcriptional activation by the VDR-RXR heterodimer is to alter the conformation of the hormone-binding domain of VDR to facilitate strong dimerization with RXR, which results in ligand-enhanced association with the VDRE. Thus RXR is recruited into a heterocomplex by liganded VDR. The natural ligand for the RXR coreceptor, 9-cis retinoic acid, suppresses both VDR-RXR binding to the VDRE and 1,25(OH)2D3-stimulated transcription, indicating that 9-cis retinoic acid diverts RXR away from being the silent partner of VDR to instead form RXR homodimers. Recent data reveal that after binding RXR, a subsequent target for VDR in the vitamin D signal transduction cascade is basal transcription factor IIB (TFIIB).(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular and functional comparison of 1,25‐dihydroxyvitamin D3 and the novel vitamin D receptor ligand, lithocholic acid, in activating transcription of cytochrome P450 3A4

The vitamin D receptor (VDR) binds to and mediates the effects of the 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) hormone to alter gene transcription. A newly recognized VDR ligand is the carcinogenic bile acid, lithocholic acid (LCA). We demonstrate that, in HT‐29 colon cancer cells, both LCA and 1,25(OH)2D3 induce expression of cytochrome P450 3A4 (CYP3A4), an enzyme involved in cellular detoxification. We also show that LCA‐VDR stimulates transcription of gene reporter constructs containing DR3 and ER6 vitamin D responsive elements (VDREs) from the human CYP3A4 gene. Utilizing gel mobility shift, pulldown, and mammalian two‐hybrid assays, we observe that: (i) 1,25(OH)2D3 enhances retinoid X receptor (RXR) heterodimerization with VDR more effectively than LCA, (ii) the 1,25(OH)2D3‐liganded VDR‐RXR heterodimer recruits full‐length SRC‐1 coactivator, whereas this interaction is minimal with LCA unless LXXLL‐containing fragments of SRC‐1 are employed, and (iii) both 1,25(OH)2D3 and LCA enhance the binding of VDR to DRIP205/mediator, but unlike 1,25(OH)2D3‐VDR, LCA‐VDR does not interact detectably with NCoA‐62 or TRIP1/SUG1, suggesting a different pattern of LCA‐VDR comodulator association. Finally, residues in the human VDR (hVDR) ligand binding domain (LBD) were altered to create mutants unresponsive to 1,25(OH)2D3‐ and/or LCA‐stimulated transactivation, identifying S237 and S225/S278 as critical for 1,25(OH)2D3 and LCA action, respectively. Therefore, these two VDR ligands contact distinct residues in the binding pocket, perhaps generating unique receptor conformations that determine the degree of RXR and comodulator binding. We propose that VDR is a bifunctional regulator, with the 1,25(OH)2D3‐liganded conformation facilitating high affinity endocrine actions, and the LCA‐liganded configuration mediating local, lower affinity cellular detoxification by upregulation of CYP3A4 in the colon. J. Cell. Biochem. 94: 917–943, 2005.

Isolation of baculovirus-expressed human vitamin D receptor: DNA responsive element interactions and phosphorylation of the purified receptor

Two controversial aspects in the mechanism of human vitamin D receptor (hVDR) action are the possible significance of VDR homodimers and the functional role of receptor phosphorylation. To address these issues, milligram quantities of baculovirus‐expressed hVDR were purified to 97% homogeneity, and then tested for binding to the rat osteocalcin vitamin D responsive element (VDRE) via electrophoretic mobility shift and half‐site competition assays in the presence or absence of a CV‐1 nuclear extract containing retinoid X receptor (RXR). Methylation interference analysis revealed that both the hVDR homodimer and the VDR‐RXR heterodimer display similar patterns of VDRE G‐base protection. However, in competition studies, the relative dissociation of the homodimeric hVDR complex from the VDRE was extremely rapid (t1/2u2009<u200930 s) compared to the dissociation of the heteromeric complex (t1/2u2009>u20095 min), thus illustrating the relative instability and low affinity of homodimeric VDR binding to DNA. These results indicate that VDR‐RXR heterodimers are the preferred VDRE binding species. Further, two dimensional gel electrophoresis of hVDR demonstrated several isoelectric forms of the receptor, suggesting that it is subject to multiple phosphorylation events. In vitro kinase assays confirmed that purified hVDR is an efficient substrate for protein kinases A and Cβ, as well as casein kinase II. In vivo studies of the expressed receptor in intact cells, namely baculovirus vector infected Sf9 insect cells and transfected mammalian COS‐7 cells, demonstrated that hVDR was phosphorylated in a hormone‐enhanced fashion. Functional consequences of hVDR phosphorylation were suggested by the observations that: (i) potato acid phosphatase (PAP)‐treated hVDR no longer interacted with the VDRE as either a homodimer or a heteromeric complex with RXR, and (ii) treatment of transfected COS‐7 cells with a phosphatase inhibitor (okadaic acid) along with 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) resulted in a synergistic enhancement of both hVDR phosphorylation and transactivation of a VDRE‐linked reporter gene, compared to the effect of treatment with either agent alone. These studies point to a significant role for phosphorylation of VDR in regulating high‐affinity VDR‐RXR interactions with VDREs, and also in modulating 1,25(OH)2D3‐elicited transcriptional activation in target cells. J. Cell. Biochem. 85: 435–457, 2002.

Vitamin D receptor displays DNA binding and transactivation as a heterodimer with the retinoid X receptor, but not with the thyroid hormone receptor†

The vitamin D receptor (VDR) is a transcription factor believed to function as a heterodimer with the retinoid X receptor (RXR). However, it was reported [Schräder et al., 1994] that, on putative vitamin D response elements (VDREs) within the rat 9k and mouse 28k calcium binding protein genes (rCaBP 9k and mCaBP 28k), VDR and thyroid hormone receptor (TR) form heterodimers that transactivate in response to both 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) and triiodothyronine (T3). We, therefore, examined associations of these receptors on the putative rCaBP 9k and mCaBP 28k VDREs, as well as on established VDREs from the rat osteocalcin (rOC) and mouse osteopontin (mOP) genes, plus the thyroid hormone response element (TRE) from the rat myosin heavy chain (rMHC) gene. In gel mobility shift assays, we found no evidence for VDR‐TR heterodimer interaction with any tested element. Further, employing these hormone response elements linked to reporter genes in transfected cells, VDR and TR mediated responses to their cognate ligands only from the rOC/mOP and rMHC elements, respectively, while the CaBP elements were unresponsive to any combination of ligand(s). Utilizing the rOC and mOP VDREs, two distinct repressive actions of TR on VDR‐mediated signaling were demonstrated: a T3‐independent action, presumably via direct TR‐RXR competition for DNA binding, and a T3‐dependent repression, likely by diversion of limiting RXR from VDR‐RXR toward the formation of TR‐RXR heterodimers. The relative importance of these two mechanisms differed in a response element‐specific manner. These results may provide a partial explanation for the observed association between hyperthyroidism and bone demineralization/osteoporosis. J. Cell. Biochem. 75:462–480, 1999.