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Abstract: Recent studies using genetically modified mice, such as FGF23−/− and Klotho−/− mice that exhibit altered mineral homeostasis due to a high vitamin D activity showed features of premature aging that include retarded growth, osteoporosis, atherosclerosis, ectopic calcification, immunological deficiency, skin and general organ atrophy, hypogonadism and short lifespan. The phenotype reversed by normalizing vitamin D and/or mineral homeostasis. Thus, hypervitaminosis D due to an increased 1α-hydroxylase activity seems to be a cause of the premature aging. In several studies, we have described that a complete or partial lack of vitamin D action (VDR−/− mice and CYP27B1−/−) show almost similar phenotype as FGF23−/− or Klotho−/− mice. VDR mutant mice have growth retardation, osteoporosis, kyphosis, skin thickening and wrinkling, alopecia, ectopic calcification, progressive loss of hearing and balance as well as short lifespan. CYP27B1−/− mice do not show alopecia nor balance deficit, which might be apoVDR-dependent or calcidiol-dependent. The features are typical to premature aging. The phenotype is resistant to a normalization of the mineral homeostasis by a rescue diet containing high calcium and phosphate. Taken together, aging shows a U-shaped dependency on hormonal forms of vitamin D suggesting that there is an optimal concentration of vitamin D in delaying aging phenomena. Our recent study shows that calcidiol is an active hormone. Since serum calcidiol but not calcitriol is fluctuating in physiological situations, calcidiol might determine the biological output of vitamin D action. Due to its high serum concentration and better uptake of calcidiol-DBP by the target cells through the cubilin–megalin system, calcidiol seems to be an important circulating hormone. Therefore, serum calcidiol might be associated with an increased risk of aging-related chronic diseases more directly than calcitriol. Aging and cancer seem to be tightly associated phenomena. Accumulation of damage on DNA and telomeres cause both aging and cancer, moreover the signalling pathways seem to converge on tumour suppressor protein, p53, which seems to be regulated by vitamin D. Also, the insulin-like growth factor signalling pathway (IGF-1, IGFBPs, IGFR) and fibroblast growth factor-23 (FGF-23) regulate growth, aging and cancer. Vitamin D can regulate these signalling pathways, too. Also NF-κB and telomerase reverse transcriptase (TERT) might be molecular mechanisms mediating vitamin D action in aging and cancer. Calcidiol serum concentrations show a U-shaped risk of prostate cancer suggesting an optimal serum concentration of 40–60nmol/L for the lowest cancer risk. Therefore, it is necessary to study several common aging-associated diseases such as osteoporosis, hypertension and diabetes known to be vitamin D-dependent before any recommendations of an optimal serum concentration of calcidiol are given. [Copyright &y& Elsevier]

Abstract: Vitamin D seems to be involved in the control of prostate cancer cell growth. 17β-Hydroxysteroid dehydrogenases type 2, type 4 and type 5 are enzymes which regulate intracellular concentration of active sex steroid hormones, which in turn, regulate the development, growth, and function of the prostate and play a role in the development and progression of prostate cancer. Using quantitative real-time PCR we find that calcitriol up-regulates HSD17B type 2, type 4 and type 5 in human prostate cancer LNCaP and PC3 cells but not in stromal cells. LXR agonist, TO-901317, suppresses the expression of HSD17B2 mRNA and inhibits calcitriol induced HSD17B2 expression. TO-901317 up-regulates the expression of HSD17B5 but not that of HSD17B4. 5α-Dihydrotestosterone up-regulates the expression of HSD17B2 and HSD17B4 but it significantly inhibits HSD17B5 expression by 70%. Calcitriol has no effect on DHT mediated expression of the three genes. The regulation of HSD17B2, HSD17B4 and HSD17B5 by ligands of LXR and VDR as well as AR in prostate cancer cells suggests a complex interaction of these signaling systems in the prostate. [Copyright &y& Elsevier]

Abstract: 25-Hydroxyvitamin D3-24-hydroxylase (24-hydroxylase, CYP24) is an important inactivating enzyme controlling the concentrations of both active metabolites 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3. In this paper, we demonstrate that 25-hydroxyvitamin D3 at 500nM significantly increases the expression of 24-hydroxylase mRNA and the increase is significantly decreased by 5α-dihydrotestosterone (DHT) at concentrations of 1–100nM in androgen-sensitive prostate cancer cells LNCaP. 25-Hydroxyvitamin D3 at 500nM and 1α,25-dihydroxyvitamin D3 at 10nM inhibit LNCaP cell growth, and the growth inhibition is enhanced by 1nM DHT. Neither 25-hydroxyvitamin D3 nor 1α,25-dihydroxyvitamin D3 at physiological concentrations has growth effect. However, in the presence of 1nM DHT, both 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 at physiological concentrations are clearly antiproliferative. These data demonstrate that DHT enhances the antiproliferative activity of Vitamin D3 hormones by inhibiting their inactivating enzyme. Most previous studies on Vitamin D3 action in cell cultures have used pharmacological concentrations of 1α,25-dihydroxyvitamin D3, the present results demonstrate, for the first time, that both 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 at physiological concentrations are active in the presence of physiological concentration of androgen. The combined use of androgen and Vitamin D3 metabolites could be a promising treatment for prostate cancer. [Copyright &y& Elsevier]

Abstract: Estrogens and androgens are proposed to play a role in the pathogenesis of prostate cancer. The effective metabolites, estradiol and 5α-dihydrotestosterone are produced from testosterone by aromatase and 5α-reductase, respectively. Metabolites of vitamin D have shown to inhibit the growth of prostate cancer cells. The aim of the present study was to verify whether 25-hydroxyvitamin D3 (25OHD3), 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3], dexamethasone, and progesterone regulate the expression of aromatase and 5α-reductase in human prostate cancer cells. LNCaP and PC3 cells were treated with 25OHD3, 1α,25-(OH)2D3, dexamethasone, or progesterone. Aromatase and 5α-reductase mRNA was quantified by real-time RT-PCR and aromatase enzyme activity was measured by the [3H] water assay. Aromatase enzyme activity in LNCaP and PC3 cells was increased by both 10nM dexamethasone, 1–100nM 1α,25-(OH)2D3 and 100nM–10μM progesterone. The induction was enhanced when hormones were used synergistically. Real-time RT-PCR analysis showed no regulation of the expression of aromatase mRNA by any steroids tested in either LNCaP or PC3 cells. The expression of 5α-reductase type I mRNA was not regulated by 1α,25-(OH)2D3 and no expression of 5α-reductase type II was detected in LNCaP. [Copyright &y& Elsevier]

Abstract: A20 is a TNF-inducible primary response gene and its product, a zinc finger protein, has antiapoptotic function in several cancer cells. We studied A20 gene expression in the Vitamin D- and TNF-sensitive LNCaP cell line and in the Vitamin D- and TNF-resistant PC-3 cell line. The results of the quantitative real-time RT-PCR analyses demonstrated that the basal level of A20 mRNA production in PC-3 cells was considerably higher than in LNCaP cells that is associated with the resistance of PC-3 cells. TNF induced A20 gene expression in both cell lines, but with different effect. A20 mRNA expression was down-regulated by 10nM calcitriol within 3–9h after treatment and up-regulated by androgen reaching maximal values by 6h after stimulation in LNCaP cells. We conclude that A20 may be involved in the regulation of cell proliferation by TNF, Vitamin D, and androgen in prostate cancer. [Copyright &y& Elsevier]

Abstract: 25-Hydroxyvitamin D3 1α-hydroxylase encoded by CYP27B1 converts 25-hydroxyvitamin D3 into 1α,25-dihydroxyvitamin D3, a vitamin D receptor ligand. 25-Hydroxyvitamin D3 has been regarded as a prohormone. Using Cyp27b1 knockout cells and a 1α-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 D3 has direct gene regulatory properties. The high expression of megalin, involved in 25-hydroxyvitamin D3 internalisation, in Cyp27b1 −/− cells explains their higher sensitivity to 25-hydroxyvitamin D3. 25-Hydroxyvitamin D3 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 D3 and 1α,25-dihydroxyvitamin D3 with the larger volume of the ligand-binding pocket for 25-hydroxyvitamin D3. Furthermore, we demonstrate direct anti-proliferative effects of 25-hydroxyvitamin D3 in human LNCaP prostate cancer cells. The synergistic effect of 25-hydroxyvitamin D3 with 1α,25-dihydroxyvitamin D3 in Cyp27b1 −/− cells further demonstrates the agonistic action of 25-hydroxyvitamin D3 and suggests that a synergism between 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 might be physiologically important. In conclusion, 25-hydroxyvitamin D3 is an agonistic vitamin D receptor ligand with gene regulatory and anti-proliferative properties. [Copyright &y& Elsevier]

Abstract: Hypervitaminosis vitamin D3 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 D3 in mice is linked to aging phenomena. For this, we used vitamin D3 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 D3, our study suggests that VDR genetic ablation promotes premature aging in mice, and that vitamin D3 homeostasis regulates physiological aging. [Copyright &y& Elsevier]

Abstract: 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 1α-hydroxylase deficient (1α-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 1α-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. [Copyright &y& Elsevier]

Abstract: 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 (LXRβ). In the mutant male mice, the mRNA level of ApoAI and LXRβ 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 1α,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. [Copyright &y& Elsevier]

Abstract: Calcitriol, a hormonal form of Vitamin D, regulates growth of normal and cancer cells of various origins by modulation of peptide growth factors signaling. Platelet-Derived Growth Factor (PDGF) signaling pathway is involved in prostate cancer progression. We studied the expression of PDGF receptors in human prostate primary stromal cells and cancer epithelial cell lines and growth response to PDGF-BB isoform. We found that the expression of PDGF receptors and PDGF-BB-mediated cell growth are regulated by calcitriol in prostate cells. Quantitative RT-PCR analysis revealed a lower level of mRNA for PDGF receptors in LNCaP and PC-3 cells than in primary stromal cells. Western blotting showed a high amount of PDGFRα and β proteins in primary stromal cells that could not be detected in LNCaP, which may explain the resistance of LNCaP cells to growth-promoting effect of PDGF-BB. Addition of Epidermal Growth Factor (EGF) to the culture medium induces the expression of PDGFRβ and restores responsiveness of LNCaP to PDGF-BB to some extent. Calcitriol down-regulates PDGFRβ expression and negatively regulates PDGF-mediated cell growth. Calcitriol does not affect PDGFRα and PDGF-B mRNA expression. We suggest that inhibition of PDGFRβ expression by calcitriol might reduce responsiveness of prostate cells to mitogenic action of PDGF-BB. [Copyright &y& Elsevier]

1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) and its derivatives are a potential treatment of human prostate cancer. The antiproliferative action of 1α,25(OH)2D3 is mainly exerted through nuclear vitamin D receptor (VDR)-mediated control of target gene transcription. To explore the target genes which are regulated by 1α,25(OH)2D3 in human prostate cancer LNCaP cells, cDNA microarray was performed by using a chip that contains 3000 gene probes. The results showed that 24 genes were regulated by 1α,25(OH)2D3. Five of them encode proteins which belong to metabolic enzymes and fatty acid biosynthesis. Fatty acid synthase (FAS) was found to be down-regulated by 1α,25(OH)2D3, and the regulation was confirmed by real-time quantitative RT-PCR analysis. Inhibition of FAS expression by 1α,25(OH)2D3 in LNCaP cells was more than 50% at 6 h. Inhibitory effect of 1α,25(OH)2D3 on FAS expression was completely blocked in the presence of protein synthesis inhibitor cycloheximide, indicating that the down-regulation of FAS gene expression by 1α,25(OH)2D3 was indirect in LNCaP cells. An inhibition of FAS activity by cerulenin resulted in a strong inhibition of LNCaP cell proliferation. The inhibition of FAS expression and cell proliferation by 1α,25(OH)2D3 seemed to be androgen-dependent, since antiandrogen, casodex and DCC-treatment of serum blocked the vitamin D action. The findings suggest that FAS is involved in the antiproliferative effect of 1α,25(OH)2D3 in presence of androgens on prostate cancer LNCaP cells. [Copyright &y& Elsevier]

Progesterone action in target tissues is mediated through two progesterone receptor (PR) isoforms, PR-A and PR-B, which display different regulatory functions in target cells. Relative expression ratio of these isoforms varies depending on cell and tissue types. Here, we studied the regulation of PR isoform expression by estradiol (E2), insulin, IGF-1 and cAMP in different breast cancer cell lines. Although, E2 induced PR expression in all cell lines studied, the expression ratio of PR-A/PR-B induced by E2 was dependent on the cell line. The differential regulation of the isoforms was also seen at the mRNA level suggesting that the PR-A and PR-B promoters are differentially regulated by E2 in different breast cancer cells. Insulin, IGF-1 or cAMP previously reported to induce PR expression however failed to alter the PR expression in our study. This is the first report describing that in different breast cancer cell lines the expression of PR-A and PR-B is regulated by E2 in a distinct way. [Copyright &y& Elsevier]