Targeted ablation of the vitamin D receptor: An animal model of vitamin D-dependent rickets type II with alopecia

Vitamin D, the major steroid hormone that controls mineral ion homeostasis, exerts its actions through the vitamin D receptor (VDR). The VDR is expressed in many tissues, including several tissues not thought to play a role in mineral metabolism. Studies in kindreds with VDR mutations (vitamin D-dependent rickets type II, VDDR II) have demonstrated hypocalcemia, hyperparathyroidism, rickets, and osteomalacia. Alopecia, which is not a feature of vitamin D deficiency, is seen in some kindreds. We have generated a mouse model of VDDR II by targeted ablation of the second zinc finger of the VDR DNA-binding domain. Despite known expression of the VDR in fetal life, homozygous mice are phenotypically normal at birth and demonstrate normal survival at least until 6 months. They become hypocalcemic at 21 days of age, at which time their parathyroid hormone (PTH) levels begin to rise. Hyperparathyroidism is accompanied by an increase in the size of the parathyroid gland as well as an increase in PTH mRNA levels. Rickets and osteomalacia are seen by day 35; however, as early as day 15, there is an expansion in the zone of hypertrophic chondrocytes in the growth plate. In contrast to animals made vitamin D deficient by dietary means, and like some patients with VDDR II, these mice develop progressive alopecia from the age of 4 weeks.

Cloning and characterization of the mouse vitamin D receptor promoter

The gene encoding the mouse vitamin D receptor has been cloned. A new exon 1 has been found that changes the numbering established for the human VDR gene. Exons 2 and 3 in the human VDR gene (coding for the zinc fingers 1 and 2, respectively) are named exons 3 and 4 in the mouse vitamin D receptor. The 1.5-kb 5′-flanking region of the new exon 1 was analyzed and revealed the presence of putative cis-acting elements. Despite the absence of a TATA box, this 5′-flanking region contains several characteristics of a GC-rich promoter including four Sp1 sites present in tandem and two CCAAT boxes. Interestingly, the Sp1 site that is the most proximal to the new exon 1 overlaps a perfect site for Krox-20/24. Krox-20 is a transcription factor involved in brain development, and also in bone remodeling. In luciferase reporter gene expression assays, we showed that sequences from this 5′-flanking region elicit high transactivation activity. Furthermore, in the NIH 3T3 cell line, a 3- to 5-fold increase in response to forskolin treatment (an activator of adenylate cyclase and in turn of protein kinase A pathway) was observed.

Three-dimensional modeling of and ligand docking to vitamin D receptor ligand binding domain

The ligand binding domain of the human vitamin D receptor (VDR) was modeled based on the crystal structure of the retinoic acid receptor. The ligand binding pocket of our VDR model is spacious at the helix 11 site and confined at the β-turn site. The ligand 1α,25-dihydroxyvitamin D3 was assumed to be anchored in the ligand binding pocket with its side chain heading to helix 11 (site 2) and the A-ring toward the β-turn (site 1). Three residues forming hydrogen bonds with the functionally important 1α- and 25-hydroxyl groups of 1α,25-dihydroxyvitamin D3 were identified and confirmed by mutational analysis: the 1α-hydroxyl group is forming pincer-type hydrogen bonds with S237 and R274 and the 25-hydroxyl group is interacting with H397. Docking potential for various ligands to the VDR model was examined, and the results are in good agreement with our previous three-dimensional structure-function theory.

Multiple promoters direct the tissue-specific expression of novel N-terminal variant human vitamin D receptor gene transcripts

The effects of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] are mediated by the vitamin D receptor (VDR), a member of the nuclear receptor superfamily of transcriptional regulators. We have identified upstream exons of the human (h) VDR gene that are incorporated into variant transcripts, two of which encode N-terminal variant receptor proteins. Expression of the hVDR gene, which spans more than 60 kb and consists of at least 14 exons, is directed by two distinct promoters. A tissue-specific distal promoter generates unique transcripts in tissues involved in calcium regulation by 1,25-(OH)2D3 and can direct the expression of a luciferase reporter gene in a cell line-specific manner. These major N-terminal differences in hVDR transcripts, potentially resulting in structural differences in the expressed receptor, may contribute to cellular responsiveness to 1,25-(OH)2D3 through tissue differences in the regulation of VDR expression.

Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands

The crystal structures of the ligand-binding domain (LBD) of the vitamin D receptor complexed to 1α,25(OH)2D3 and the 20-epi analogs, MC1288 and KH1060, show that the protein conformation is identical, conferring a general character to the observation first made for retinoic acid receptor (RAR) that, for a given LBD, the agonist conformation is unique, the ligands adapting to the binding pocket. In all complexes, the A- to D-ring moieties of the ligands adopt the same conformation and form identical contacts with the protein. Differences are observed only for the 17β-aliphatic chains that adapt their conformation to anchor the 25-hydroxyl group to His-305 and His-397. The inverted geometry of the C20 methyl group induces different paths of the aliphatic chains. The ligands exhibit a low-energy conformation for MC1288 and a more strained conformation for the two others. KH1060 compensates this energy cost by additional contacts. Based on the present data, the explanation of the superagonist effect is to be found in higher stability and longer half-life of the active complex, thereby excluding different conformations of the ligand binding domain.

Dendritic cell modulation by 1α,25 dihydroxyvitamin D3 and its analogs: A vitamin D receptor-dependent pathway that promotes a persistent state of immaturity in vitro and in vivo

Dendritic cells (DCs) play a central role in regulating immune activation and responses to self. DC maturation is central to the outcome of antigen presentation to T cells. Maturation of DCs is inhibited by physiological levels of 1α,25 dihydroxyvitamin D3 [1α,25(OH)2D3] and a related analog, 1α,25(OH)2-16-ene-23-yne-26,27-hexafluoro-19-nor-vitamin D3 (D3 analog). Conditioning of bone marrow cultures with 10−10 M D3 analog resulted in accumulation of immature DCs with reduced IL-12 secretion and without induction of transforming growth factor β1. These DCs retained an immature phenotype after withdrawal of D3 analog and exhibited blunted responses to maturing stimuli (CD40 ligation, macrophage products, or lipopolysaccharide). Resistance to maturation depended on the presence of the 1α,25(OH)2D3 receptor (VDR). In an in vivo model of DC-mediated antigen-specific sensitization, D3 analog-conditioned DCs failed to sensitize and, instead, promoted prolonged survival of subsequent skin grafts expressing the same antigen. To investigate the physiologic significance of 1α,25(OH)2D3/VDR-mediated modulation of DC maturity we analyzed DC populations from mice lacking VDR. Compared with wild-type animals, VDR-deficient mice had hypertrophy of subcutaneous lymph nodes and an increase in mature DCs in lymph nodes but not spleen. We conclude that 1α,25(OH)2D3/VDR mediates physiologically relevant inhibition of DC maturity that is resistant to maturational stimuli and modulates antigen-specific immune responses in vivo.

Conformational change and enhanced stabilization of the vitamin D receptor by the 1,25-dihydroxyvitamin D3 analog KH1060.

The 1,25-dihydroxyvitamin D3 [1,25-(OH)2vitamin D3] analog KH1060 exerts very potent effects on cell proliferation and cell differentiation via the vitamin D receptor (VDR). However, the activities of KH1060 are not associated with an increased affinity for the VDR. We now show that increased stabilization of the VDR-KH1060 complex could be an explanation for its high potencies. VDR half-life studies performed with cycloheximide-translational blocked rat osteoblast-like ROS 17/2.8 cells demonstrated that, in the absence of ligand, VDR levels rapidly decreased. After 2 hr, less than 10% of the initial VDR level could be measured. In the presence of 1,25-(OH)2vitamin D3, the VDR half-life was 15 hr. After 24 hr. less than 20% of the initial VDR content was detectable, whereas, at this time-point, when the cells were incubated with KH1060 80% of the VDR was still present. Differences in 1,25-(OH)2vitamin D3- and KH1060-induced conformational changes of the VDR could underlie the increased VDR stability. As assessed by limited proteolytic digestion analysis, both 1,25-(OH)2vitamin D3 and KH1060 caused a specific conformational change of the VDR. Compared with 1,25-(OH)2vitamin D3, KH1060 induced a conformational change that led to a far more dramatic protection of the VDR against proteolytic degradation. In conclusion, the altered VDR stability and the possibly underlying change in VDR conformation caused by KH1060 could be an explanation for its enhanced bioactivity.

Human vitamin D receptor phosphorylation by casein kinase II at Ser-208 potentiates transcriptional activation.

The potential functional significance of human 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] receptor (hVDR) phosphorylation at Ser-208 was evaluated by cotransfecting COS-7 kidney cells with hVDR constructs and the catalytic subunit of human casein kinase 11 (CK-11). Under these conditions, hVDR is intensely phosphorylated in a reaction that depends on both CK-II and the presence of Ser-208. The resulting hyperphosphorylated receptor is unaltered in its kinetics for binding the 1,25(OH)2D3 ligand, its partitioning into the nucleus, and its ability to associate with a vitamin D responsive element. Replacement of Ser-208 with glycine or alanine indicates that phosphorylation of hVDR at Ser-208 is not obligatory for 1,25(OH)2D3 action, but coexpression of wild-type hVDR and CK-11 elicits a dose-dependent enhancement of 1,25(OH)2D3-stimulated transcription of a vitamin D responsive element reporter construct. This enhancement by CK-II is abolished by mutating Ser-208 to glycine or alanine and does not occur with glucocorticoid receptor-mediated transcription. Therefore, phosphorylation of hVDR by CK-11 at Ser-208 specifically modulates its transcriptional capacity, suggesting that this covalent modification alters the conformation of VDR to potentiate its interaction with the machinery for DNA transcription.

A possible role of vitamin D receptors in regulating vitamin D activation in the kidney.

The vitamin D endocrine system is regulated reciprocally by renal 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylases. Previously, we reported that renal proximal convoluted tubules, the major site of 1 alpha, 25-dihydroxyvitamin D3 production, have vitamin D receptors. In the presence of vitamin D receptors, renal proximal convoluted tubules cannot maintain the state of enhanced production of 1 alpha, 25-dihydroxyvitamin D3. To clarify this discrepancy, we proposed a working hypothesis for the reciprocal control of renal 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylase activities. In rat models of enhanced renal production of 1 alpha, 25-dihydroxyvitamin D3, expression of vitamin D receptors and 25-hydroxyvitamin D3 24-hydroxylase mRNAs was strikingly suppressed in renal proximal convoluted tubules but not in the cortical collecting ducts. In vitamin D-deficient rats with up-regulated renal 25-hydroxyvitamin D3 1 alpha-hydroxylase activity, expression of vitamin D receptor mRNA in renal proximal convoluted tubules was also down-regulated, indicating that the down-regulation of vitamin D receptor mRNA is not the result of the enhanced production of 1 alpha, 25-dihydroxyvitamin D3. In Japanese quail models with up-regulated renal 25-hydroxyvitamin D3 1 alpha-hydroxylase activity by sex steroids, expression of vitamin D receptor mRNA was also down-regulated in the kidney but not in the duodenum. These results suggest that the down-regulation of vitamin D receptors plays a critical role in production of 1 alpha, 25-dihydroxyvitamin D3 in renal proximal convoluted tubules.

Targeted ablation of the 25-hydroxyvitamin D 1α-hydroxylase enzyme: Evidence for skeletal, reproductive, and immune dysfunction

The active form of vitamin D, 1α,25-dihydroxyvitamin D [1α,25(OH)2D], is synthesized from its precursor 25 hydroxyvitamin D [25(OH)D] via the catalytic action of the 25(OH)D-1α-hydroxylase [1α(OH)ase] enzyme. Many roles in cell growth and differentiation have been attributed to 1,25(OH)2D, including a central role in calcium homeostasis and skeletal metabolism. To investigate the in vivo functions of 1,25(OH)2D and the molecular basis of its actions, we developed a mouse model deficient in 1α(OH)ase by targeted ablation of the hormone-binding and heme-binding domains of the 1α(OH)ase gene. After weaning, mice developed hypocalcemia, secondary hyperparathyroidism, retarded growth, and the skeletal abnormalities characteristic of rickets. These abnormalities are similar to those described in humans with the genetic disorder vitamin D dependent rickets type I [VDDR-I; also known as pseudovitamin D-deficiency rickets (PDDR)]. Altered non-collagenous matrix protein expression and reduced numbers of osteoclasts were also observed in bone. Female mutant mice were infertile and exhibited uterine hypoplasia and absent corpora lutea. Furthermore, histologically enlarged lymph nodes in the vicinity of the thyroid gland and a reduction in CD4- and CD8-positive peripheral T lymphocytes were observed. Alopecia, reported in vitamin D receptor (VDR)-deficient mice and in humans with VDDR-II, was not seen. The findings establish a critical role for the 1α(OH)ase enzyme in mineral and skeletal homeostasis as well as in female reproduction and also point to an important role in regulating immune function.

Evolutionary importance for the membrane enhancement of the production of vitamin D3 in the skin of poikilothermic animals.

The photoproduction of vitamin D in the skin was essential for the evolutionary development of terrestrial vertebrates. During exposure to sunlight, previtamin D3 formed in the skin is isomerized to vitamin D3 (calciol) by a temperature-dependent process. Since early land vertebrates were poikilothermic, the relatively slow conversion of previtamin D3 to vitamin D3 at ambient temperature put them at serious risk for developing vitamin D deficiency, thus leading to a poorly mineralized skeleton that could have ultimately halted further evolutionary development of vertebrates on land. We evaluated the rate of isomerization of previtamin D3 to vitamin D3 in the skin of iguanas and found the isomerization rate was enhanced by 1100% and 1700% at 25 degrees C and 5 degrees C, respectively. It is likely that the membrane entrapment of previtamin D3 in its s-cis,s-cis conformation is responsible for the markedly enhanced conversion of previtamin D3 to vitamin D3. The membrane-enhanced production of vitamin D3 ensures the critical supply of vitamin D3 to poikilothermic animals such as iguanas.

Cloning and expression of rat 25-hydroxyvitamin D3-1α-hydroxylase cDNA

A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D3-1α-hydroxylase (P4501α), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(+). When P4501α cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D3-1α-hydroxylase activity. The sequence analysis showed that P4501α was of 2,469 bp long and contained an ORF encoding 501 amino acids. The deduced amino acid sequence showed a 53% similarity and 44% identity to the vitamin D3-25-hydroxylase (CYP27), whereas it has 42.6% similarity and 34% identity with the 25-hydroxyvitamin D3-24-hydroxylase (CYP24). Thus, it composes a new subfamily of the CYP27 family. Further, it is more closely related to the CYP27 than to the CYP24. The expression of P4501α mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1α,25-dihydroxyvitamin D3 (rats fed a low Ca diet), P4501α mRNA was greatly increased in the renal proximal convoluted tubules.