Deletion of Deoxyribonucleic Acid Binding Domain of the Vitamin D Receptor Abrogates Genomic and Nongenomic Functions of Vitamin D

Abstract

The vitamin D hormone 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], the biologically active form of vitamin D, is essential for an intact mineral metabolism. Using gene targeting, we sought to generate vitamin D receptor (VDR) null mutant mice carrying the reporter gene lacZ driven by the endogenous VDR promoter. Here we show that our gene-targeted mutant mice express a VDR with an intact hormone binding domain, but lacking the first zinc finger necessary for DNA binding. Expression of the lacZ reporter gene was widely distributed during embryogenesis and postnatally. Strong lacZ expression was found in bones, cartilage, intestine, kidney, skin, brain, heart, and parathyroid glands. Homozygous mice are a phenocopy of mice totally lacking the VDR protein and showed growth retardation, rickets, secondary hyperparathyroidism, and alopecia. Feeding of a diet high in calcium, phosphorus, and lactose normalized blood calcium and serum PTH levels, but revealed a profound renal calcium leak in normocalcemic homozygous mutants. When mice were treated with pharmacological doses of vitamin D metabolites, responses in skin, bone, intestine, parathyroid glands, and kidney were absent in homozygous mice, indicating that the mutant receptor is nonfunctioning and that vitamin D signaling pathways other than those mediated through the classical nuclear receptor are of minor physiological importance. Furthermore, rapid, nongenomic responses to 1,25-(OH)(2)D(3) in osteoblasts were abrogated in homozygous mice, supporting the conclusion that the classical VDR mediates the nongenomic actions of 1,25-(OH)(2)D(3).

Inactivation of the 25-Hydroxyvitamin D 1α-Hydroxylase and Vitamin D Receptor Demonstrates Independent and Interdependent Effects of Calcium and Vitamin D on Skeletal and Mineral Homeostasis

“…This constellation of abnormalities is observed in humans with VDR mutations in the inherited disorder vitamin D-dependent rickets type II (also called hereditary vitamin D-resistant rickets) (23). Rescue of this phenotype has been successfully accomplished with a high calcium, high phosphorus, high lactose diet administered for at least 1 month after weaning (22,24). Consequently, it has been postulated that the major action of the VDR in skeletal growth, maturation, and remodeling is its role in intestinal calcium absorption (25).…”

Rapid modulation of osteoblast ion channel responses by 1α,25(OH) 2 -vitamin D 3 requires the presence of a functional vitamin D nuclear receptor

“…Osteoblasts, which are secretory cells, produce a variety of bone matrix proteins and actively participate in the mineralization process under the influence of hormones involved in mineral metabolism, such as 1,25D. Recently, several strains of functional VDR knockout (KO) mice have been created by means of deletion of selected portions of the VDR gene (10)(11)(12)(13). They constitute a valuable tool for the study of a vast range of physiological conditions that arise from an impaired 1,25D physiology.…”

The Rapid Effects of 1,25-Dihydroxyvitamin D3 Require the Vitamin D Receptor and Influence 24-Hydroxylase Activity

“…Control cells responded within seconds to 1,25-(OH) 2 D 3 by increasing intracellular calcium concentration. The profile of the calcium response, the dose-response curve (maximal effect at 100 pM), and the inhibition of the response by the two L-type calcium channel blockers (verapamil and nifedipine) and the PLC inhibitor (U-73122) were identical to those found in other cell types such as osteoblasts, enterocytes, and parathyroid cells (7,20,21,36). Interestingly, fibroblasts bearing the K45E mutation showed no calcium response to 1,25-(OH) 2 D 3 , whereas fibroblasts bearing the W286R mutation showed a calcium response similar to controls as regards time course profile, the active range of 1,25-(OH) 2 D 3 concentrations, and the inhibitory effects of calcium channel and PLC inhibitors.…”

“…Cells from patients 2 and 3, with a W286R mutation in the ligandbinding domain, were selected as models with markedly destabilized ligand binding capacity, as shown by previous biological studies (26) and as suggested by x-ray crystal structure analysis of the mutated VDR. 2 In the three cases, the remaining portion of the VDR gene was normal, unlike in the previously reported osteoblast models, which bore large deletions of the VDR gene (21,22).…”

“…A first set of data supports the hypothesis that VDR itself mediates 1,25-(OH) 2 D 3 rapid effects. Among those findings are the observations that functional VDR expression and rapid response to 1,25-(OH) 2 D 3 in enterocytes have a simultaneous onset during rat post-natal development (20) and that the generation of transgenic mice lacking the first zinc finger of the VDR blocks rapid and longterm responses of osteoblasts to 1,25-(OH) 2 D 3 (21). But other data suggest that the presence of a functional nuclear VDR is not compulsory, as these findings showed persistent rapid effects of 1,25-(OH) 2 D 3 on protein kinase C (PKC) activation and Ca 2ϩ increase in osteoblasts of transgenic mice expressing a non-functional VDR lacking the second zinc finger and most of the ligand-binding domain (22).…”