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Hypervitaminosis vitamin D(3) has been recently implicated in premature aging through the regulation of 1alpha hydroxylase expression by klotho and fibroblast growth factor-23 (Fgf-23). Here we examined whether the lack of hormonal function of vitamin D(3) in mice is linked to aging phenomena. For this, we used vitamin D(3) receptor (VDR) "Tokyo" knockout (KO) mice (fed with a special rescue diet) and analyzed their growth, skin and cerebellar morphology, as well as overall motor performance. We also studied the expression of aging-related genes, such as Fgf-23, nuclear factor kappaB (NF-kappaB), p53, insulin like growth factor 1 (IGF1) and IGF1 receptor (IGF1R), in liver, as well as klotho in liver, kidney and prostate tissues. Overall, VDR KO mice showed several aging related phenotypes, including poorer survival, early alopecia, thickened skin, enlarged sebaceous glands and development of epidermal cysts. There was no difference either in the structure of cerebellum or in the number of Purkinje cells. Unlike the wildtype controls, VDR KO mice lose their ability to swim after 6 months of age. Expression of all the genes was lower in old VDR KO mice, but only NF-kappaB, Fgf-23, p53 and IGF1R were significantly lower. Since the phenotype of aged VDR knockout mice is similar to mouse models with hypervitaminosis D(3), our study suggests that VDR genetic ablation promotes premature aging in mice, and that vitamin D(3) homeostasis regulates physiological aging.

Vitamin D is a seco-steroid hormone with multiple actions in the brain, mediated through the nuclear vitamin D receptor (VDR). We have recently shown that mutant mice lacking functional VDR demonstrate altered emotional behavior and specific motor deficits. Here we further examine phenotype of these mice, testing their novelty responses, as well as cognitive and sensory (olfactory and gustatory) functions in the novel food, two-trial Y-maze and tastant consumption tests. In addition, we study depression-like behavior in these mice, using anhedonia-based sucrose preference test. Overall, VDR mutant mice showed neophobic response in several different tests, but displayed unimpaired olfactory and gustatory functions, spatial memory and baseline hedonic responses. Collectively, these data confirm that mutation of VDR in mice leads to altering emotional/anxiety states, but does not play a major role in depression, as well as in the regulation of some sensory and cognitive processes. These results support the role of the vitamin D/VDR neuroendocrine system in the regulation of behavior, and may have clinical relevance, enabling a better focus on psychiatric and behavioral disorders associated with dysfunctions in this neuroendocrine system.

Epidemiological studies suggest that serum calcidiol (25(OH)-Vitamin D3) seems to be associated with several cancers including prostate cancer. We have made several experimental studies in order to clarify the mechanism(s) involved in the association. Calcidiol has been regarded as an inactive prohormone for calcitriol, which possesses the highest biological activity of the Vitamin D metabolites, when it is evaluated on the basis of bioactivity/nmol. However, we found recently that at the physiological concentration calcidiol (100-200 nM) is an active hormone, whereas calcitriol (1alpha,25(OH)2-Vitamin D3) (100 pM) is inactive in human primary prostate stromal cells. Calcidiol is able to inhibit cell growth and to induce or inhibit several genes including 1alpha-hydroxylase and 24-hydroxylase genes. This suggests that calcidiol might be an independent endocrine system involved in the control of cell differentiation and proliferation, whereas calcitriol might be mainly involved in the regulation of calcium and phosphorous balance. Several mechanisms may mediate the action of Vitamin D in the prostate. This is a review of some recent studies on the role of (1) Vitamin D metabolism, (2) growth factors and (3) fatty acid metabolism.

25-Hydroxyvitamin D(3) 1alpha-hydroxylase encoded by CYP27B1 converts 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3), a vitamin D receptor ligand. 25-Hydroxyvitamin D(3) has been regarded as a prohormone. Using Cyp27b1 knockout cells and a 1alpha-hydroxylase-specific inhibitor we provide in four cellular systems, primary mouse kidney, skin, prostate cells and human MCF-7 breast cancer cells, evidence that 25-hydroxyvitamin D(3) has direct gene regulatory properties. The high expression of megalin, involved in 25-hydroxyvitamin D(3) internalisation, in Cyp27b1(-/-) cells explains their higher sensitivity to 25-hydroxyvitamin D(3). 25-Hydroxyvitamin D(3) action depends on the vitamin D receptor signalling supported by the unresponsiveness of the vitamin D receptor knockout cells. Molecular dynamics simulations show the identical binding mode for both 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) with the larger volume of the ligand-binding pocket for 25-hydroxyvitamin D(3). Furthermore, we demonstrate direct anti-proliferative effects of 25-hydroxyvitamin D(3) in human LNCaP prostate cancer cells. The synergistic effect of 25-hydroxyvitamin D(3) with 1alpha,25-dihydroxyvitamin D(3) in Cyp27b1(-/-) cells further demonstrates the agonistic action of 25-hydroxyvitamin D(3) and suggests that a synergism between 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) might be physiologically important. In conclusion, 25-hydroxyvitamin D(3) is an agonistic vitamin D receptor ligand with gene regulatory and anti-proliferative properties.

The vitamin D endocrine system is essential for calcium and bone homeostasis. Vitamin D deficits are associated with muscle weakness and osteoporosis, whereas vitamin D supplementation may improve muscle function, body sway and frequency of falls, growth and mineral homeostasis of bones. The loss of muscle strength and mass, as well as deficits in bone formation, lead to poor balance. Poor balance is one of the main causes of falls, and may lead to dangerous injuries. Here we examine balance functions in vitamin D receptor deficient (VDR-/-) mice, an animal model of vitamin D-dependent rickets type II, and in 1alpha-hydroxylase deficient (1alpha-OHase-/-) mice, an animal model of pseudovitamin D-deficiency rickets. Recently developed methods (tilting box, rotating tube test), swim test, and modified accelerating rotarod protocol were used to examine whether the absence of functional VDR, or the lack of a key vitamin D-activating enzyme, could lead to mouse vestibular dysfunctions. Overall, VDR-/- mice, but not 1alpha-OHase-/- mice, showed shorter latency to fall from the rotarod, smaller fall angle in the tilting box test, and aberrant poor swimming. These data suggest that VDR deficiency in mice is associated with decreased balance function, and may be relevant to poorer balance/posture control in humans with low levels of vitamin D.

Vitamin D insufficiency has been reported to be associated with increased blood cholesterol concentrations. Here we used two strains of VDR knock-out (VDR-KO) mice to study whether a lack of vitamin D action has any effect on cholesterol metabolism. In 129S1 mice, both in male and female VDR-KO mice serum total cholesterol levels were significantly higher than those in wild type (WT) mice (20.7% (P=0.05) and 22.2% (P=0.03), respectively). In addition, the serum high-density lipoprotein-bound cholesterol (HDL-C) level was 22% (P=0.03), respectively higher in male VDR-KO mice than in WT mice. The mRNA expression levels of five cholesterol metabolism related genes in livers of 129S1 mice were studied using quantitative real-time PCR (QRT-PCR): ATP-binding cassette transporter A1 (ABCA1), regulatory element binding protein (SREBP2), apolipoprotein A-I (ApoAI), low-density lipoprotein receptor (LDLR) and liver X receptor beta (LXRbeta). In the mutant male mice, the mRNA level of ApoAI and LXRbeta were 49.2% (P=0.005) and 38.8% (P=0.034) higher than in the WT mice. These changes were not observed in mutant female mice, but the female mutant mice showed 52.5% (P=0.006) decrease of SREBP2 mRNA expression compared to WT mice. Because the mutant mice were fed with a special rescue diet, we wanted to test whether the increased cholesterol levels in mutant mice were due to the diet. Both the WT and mutant NMRI mice were given the same diet for 3 weeks before the blood sampling. No difference in cholesterol or in HDL-C between WT and mutant mice was found. The results suggest that the food, gender and genetic background have an effect on the cholesterol metabolism. Although VDR seems to regulate some of the genes involved in cholesterol metabolism, its role in the regulation of serum cholesterol seems to be minimal.

1Alpha,25(OH)2D3, the hormonal form of vitamin D, is a neuroactive seco-steroid hormone with multiple functions in the brain. Most of these effects are mediated through the nuclear vitamin D receptor (VDR), widely distributed in the central nervous system. Our earlier studies showed that mutant mice lacking functional VDR have specific behavioural abnormalities, including anxiety and aberrant maternal behaviour, which may be hormonally regulated. Here we describe impaired nest building behaviour in VDR mutant mice. Since prolactin plays a key role in the regulation of nest building in both sexes, we also examine whether VDR mutant mice have altered prolactin levels. Overall, serum prolactin levels were increased in VDR mutant mice, accompanied by marked impairments in their nest building activity. In contrast, there were no differences in prolactin mRNA expression levels between wildtype control mice and VDR mutant mice. Collectively, these data suggest that partial genetic ablation of VDR affects prolactin system in mice, and that altered serum prolactin levels in VDR mutants may underlie some of their behavioural abnormalities, such as impaired nest building.