In vivo function of VDR in gene expression-VDR knock-out mice

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Abstract

Vitamin D exerts many biological actions through nuclear vitamin D receptor (VDR)-mediated gene expression. The transactivation function of VDR is activated by binding 1α,25-dihydroxyvitamin D3[1α,25(OH)2D3], an active form of vitamin D. Conversion from 25(OH)D3 is finely regulated in kidney by 25(OH)D3 1α-hydroxylase[25(OH)D 1α-hydroxylase], keeping serum levels of 1α,25(OH)2D3 constant. Deficiency of vitamin D and mutations in the genes like VDR (type II genetic rickets) are known to cause rickets like lowered serum calcium, alopecia and impaired bone formation. However, the molecular basis of vitamin D–VDR system in the vitamin D action in intact animals remained to be established. In addition, the 1α-hydroxylase gene from any species had not yet been cloned, irrespective of its biological significance and putative link to the type I genetic rickets. We generated VDR-deficient mice (VDR KO mice). VDR KO mice grew up normally until weaning, but after weaning they developed abnormality like the type II rickets patients. These results demonstrated indispensability of vitamin D–VDR system in mineral and bone metabolism only in post-weaning life. Using a newly developed cloning system, we cloned the cDNA encoding a novel P450 enzyme, mouse and human 1α-hydroxylase. The study in VDR KO mice demonstrated the function of liganded VDR in the negative feed-back regulation of 1α,25(OH)2D3 production. Finally, from the analysis of type I rickets patients, we found missense genetic mutations in 1α-hydroxylase, leading to the conclusion that this gene is responsible for the type I rickets.

Introduction

The most biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3[1α,25(OH)2D3] has roles in a variety of biological actions such as calcium homeostasis, cell proliferation and cell differentiation to many target tissues [1], [2]. Most of such biological actions of 1α,25(OH)2D3 are thought to be exerted through gene expression mediated by VDR [3]. VDR is a member of the nuclear hormone receptor superfamily and acts as a ligand-inducible transcription factor [4], [5]. 1α,25(OH)2D3 is a most potent form of vitamin D and acts as a specific ligand for VDR.

1α,25(OH)2D3 is biosynthesized from cholesterol, and at the final steps two hydroxylations (hepatic 25-hydroxylation and renal 1α-hydroxylation) occur for its metabolic activation into a hormonal form [6], [7]. Renal hydroxylation of 25(OH)D3 is crucial for the biosynthesis, and is conducted by 25(OH)D3 1α-hydroxylase[25(OH)D 1α-hydroxylase] in the proximal tubule of the kidney [8]. It has been shown that the activity of 25(OH)D3 1α-hydroxylase is inhibited by its end product, 1α,25(OH)2D3, and activated by calciotropic peptide hormones such as calcitonin and PTH [6], [7], though the molecular mechanism underlying these regulations remained unclear.

To investigate the roles of 1α,25(OH)2D3–VDR in intact animals, we generated the mice lacking VDR by targeted gene disruption, and analyzed the phenotype of the mice.

Section snippets

Vitamin D receptor inactivation by homologous gene targeting in mice

We generated mice deficient of VDR by gene targeting in order to investigate the function of VDR in vivo [8]. A targeting plasmid was constructed to disrupt the VDR gene by inserting a neor gene into the exon 2 that encodes the first Zn finger motif in the DNA binding domain essential for the biological functions of VDR. Two lines of mice heterozygous for the mutation showed no obvious defects and were interbred to generate homologous gene targeted mice (VDR−/−).

Moreover, no bone malformation

Appearance of rickets with growth retardation only after weaning in VDR KO mice

Unexpectedly, the VDR-null mutant mice did not differ from the heterozygous or wild-type littermates in growth rate (Fig. 1a) or behavior, and seemed functionally normal after birth until weaning. However, after weaning (about 3 weeks), the VDR-null mutant mice unexpectedly showed marked growth retardation, and the body weight of the null mutant mice at 10 weeks was about 50% of those of the heterozygous and wild-type mice (Fig. 1a). After weaning the VDR-null mutant mice developed rickets, and

Impaired bone formation in VDR KO mice

Severe malformation induced by the inactivation of VDR only after weaning was detected in bone. Radiographic analysis of the VDR-null mutant mice at 7 weeks revealed growth retardation with loss of bone density. In gross appearance and on X-ray analysis of tibia and fibula, typical features of advanced rickets were observed including widening of epiphyseal growth plates, thinning of the cortex, fraying, cupping and widening of the metaphysis. In addition, orderly columns of hypertrophic

Cloning of mouse 25(OH)D 1α-hydroxylase cDNA from VDR KO mice by a newly developed cloning system, and identification as the responsible gene for the vitamin D dependent rickets type I

From the elevated levels of serum 1α,25(OH)2D in the VDR KO mice, it appeared that the VDR KO mice have the increased activity of 25(OH)D 1α-hydroxylase. Though the enzymatic activity of 25(OH)D 1α-hydroxylase is known over 25 years, the cDNA of this enzyme had not yet been cloned. From the VDR KO mice, we were able to, for the first time, clone the cDNA encoding mouse 25(OH)D 1α-hydroxylase by a newly developed expression cloning method [17]. Furthermore, using this cloned mouse cDNA, we

VDR is essential for the negative regulation of the 25(OH)D 1α-hydroxylase gene by vitamin D

As 1α,25(OH)2D3 plays a primary role in calcium homeostasis, the renal activity is positively regulated by calcitropic hormones, responding to serum calcium levels. 1α,25(OH)2D3 has been well characterized as a negative regulator for the renal activity of 25(OH)D 1α-hydroxylase [19], [20]. A study using VDR knock-out mice showed that one of the negative regulators, 1α,25(OH)2D3, acts at the transcriptional level, and this negative regulation requires the liganded-VDR [17], since no negative

Molecular basis for hereditary rickets (type I and II)

We have demonstrated here that the 1α,25(OH)2D3–VDR system is essential for growth, bone formation, and hair development only after weaning. The phenotype of the VDR(−/−) mice, but not heterozygous(+/−) mice, was similar to a human recessive genetic disease, vitamin D dependent rickets type II VDDR II [21]. However, in patients suffering from this disease, no null mutants for VDR have yet been reported [21] raising a possibility that the null mutation causes early lethality and/or infertility.

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