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Background To evaluate the individual and combined effects of enterolactone, vitamin D, free testosterone, Chlamydia trachomatis and HPV-18 on the risk of prostate cancer in a large population-based biochemical material that combined three Nordic serum sample banks. Material and methods A joint cohort of 209 000 healthy men was followed using cancer registry linkages. From this cohort altogether 699 incident cases of prostate cancer were identified. Four controls were selected by incidence density sampling and matching for country, age and date of the blood sampling. Complete data for all investigated exposures was available for 483 eligible cases and 1055 eligible controls. Multivariate regression analyses were performed to investigate the solitary and combined effects. Results The solitary effects were small. Significantly increased risk [rate ratio 1.6 (95% CI 1.0-2.5)] was found in those seronegative for C. trachomatis infection. The joint effect in risk levels of enterolactone and vitamin D was antagonistic [observed rate ratio (RR) 1.4 (1.0-2.1), expected RR 2.0 (1.0-4.1)] as well as that of HPV-18 and C. trachomatis [observed RR 1.9 (0.8-4.5), expected RR 9.9 (1.1-87.0)]. Conclusion A large follow-up study combining data from several previously investigated exposures to investigate joint effects found no evidence that exposure to two risk factors would increase the risk of prostate cancer from that expected on basis of exposure to one risk factor. If anything, the results were consistent with antagonistic interactions.

1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3) had earlier been regarded as the only active hormone. The newly identified actions of 25-hydroxyvitamin D3 (25(OH)D3) and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) broadened the vitamin D3 endocrine system, however, the current data are fragmented and a systematic understanding is lacking. Here we performed the first systematic study of global gene expression to clarify their similarities and differences. Three metabolites at physiologically comparable levels were utilized to treat human and mouse fibroblasts prior to DNA microarray analyses. Human primary prostate stromal P29SN cells (hP29SN), which convert 25(OH)D3 into 1α,25(OH)2D3 by 1α-hydroxylase (encoded by the gene CYP27B1), displayed regulation of 164, 171, and 175 genes by treatment with 1α,25(OH)2D3, 25(OH)D3, and 24R,25(OH)2D3, respectively. Mouse primary Cyp27b1 knockout fibroblasts (mCyp27b1 (-/-)), which lack 1α-hydroxylation, displayed regulation of 619, 469, and 66 genes using the same respective treatments. The number of shared genes regulated by two metabolites is much lower in hP29SN than in mCyp27b1 (-/-). By using DAVID Functional Annotation Bioinformatics Microarray Analysis tools and Ingenuity Pathways Analysis, we identified the agonistic regulation of calcium homeostasis and bone remodeling between 1α,25(OH)2D3 and 25(OH)D3 and unique non-classical actions of each metabolite in physiological and pathological processes, including cell cycle, keratinocyte differentiation, amyotrophic lateral sclerosis signaling, gene transcription, immunomodulation, epigenetics, cell differentiation, and membrane protein expression. In conclusion, there are three distinct vitamin D3 hormones with clearly different biological activities. This study presents a new conceptual insight into the vitamin D3 endocrine system, which may guide the strategic use of vitamin D3 in disease prevention and treatment.

We found previously that long-chain fatty-acid-CoA ligase 3 (FACL3), a critical enzyme for activation of long-chain fatty acids, was upregulated by 1α, 25(OH)(2)D(3) at an mRNA and enzyme activity levels in prostate cancer cells. Our further study indicated that the FACL3 mediated 1α,25(OH)(2)D(3) inhibition of fatty acid synthase (FAS), which is associated with many cancers, including prostate cancer. In the current study, we investigated an FACL3 protein expression and its regulation by 1α, 25(OH)(2)D(3) and its synthetic analogs EB1089 and CB1093 in prostate cancer cells. The results showed that the expression of an FACL3 protein was upregulated by 1α, 25(OH)(2)D(3), EB1089 and CB1093 in LNCaP cells, consistent with their upregulation of an FACL3 mRNA expression. In addition, the FACL3 expression was found to be markedly low at both mRNA and protein levels in more transformed prostate cancer PC-3 and DU145 cells compared with less transformed LNCaP cells. The data suggest that decreased FACL3 expression might be associated with a more malignant phenotype of prostate cancer., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Vitamin D is becoming increasingly recognized as a nontraditional drug target for different brain pathologies. Although widely known for their role in calcium metabolism, vitamin D and its receptor have been linked to several brain disorders, including cognitive decline, epilepsy, affective disorders, and schizophrenia. Here we discuss mounting evidence, and parallel recent clinical and animal behavioral, genetic and pharmacological data to emphasize the emerging role of the neurosteroid vitamin D system in brain function.

The objective of the study was to compare Bacillus subtilis spore film dosimeters with a Robertson Berger UV meter (RB meter) and diary records for assessing personal UV-B doses during a 13-day heliotherapy (HT) for atopic dermatitis (AD). In addition, the relationship between the personal UV-B dose and change in serum 25-hydroxyvitamin D (25(OH)D) was studied. Altogether 21 adult patients with AD completed the study arranged in the Canary Islands, either in January or March 2005. The spore film dosimeters were used throughout the day during the HT. Serum 25(OH)D was analyzed using radioimmunoassay. The mean personal UV-B dose measured with the dosimeters was 75 SED in January and 131 SED in March. The respective results gained from the RB meter combined with diary records were 63 SED and 119 SED showing a close correlation with the dosimeter results. Serum 25(OH)D concentration increased by 9.7nmol L(-1) in January and by 26.0 7nmol L(-1) in March. The increase in serum 25(OH)D correlated with the UV-B dose received. The patients complied well to use the dosimeters. We conclude spore films to be a feasible and reliable personal UV dosimeter in vivo in field conditions., (© 2010 The Authors. Journal Compilation. The American Society of Photobiology.)

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.

Background: Several laboratory and epidemiological studies have inversely linked endogenous vitamin D and the risk of breast cancer. The acquisition of vitamin D over time on the relative risk (RR) of the disease development is not known. In a longitudinal study, we evaluated the association between vitamin D levels at pregnancy over time with the risk of breast cancer, and pregnancy-associated breast cancer., Method: The risk for subsequent development of breast cancer associated with serum 25-hydroxyvitamin (25-OHD) levels was assessed for consecutive (1st and 2nd pregnancy) samples of 100 cases, with mean lag times (micro(t)) of 7.4 and 4.6 years between sampling and the diagnosis, and matched (parity, age, year, season) controls. Pregnancy-associated breast cancer (PABC, 111 case-control pairs, micro(t)=1 year) risk was also studied. Odds ratios (ORs) with 95% confidence intervals (CI) were calculated using the lowest quintile as the reference., Results: Serum 25-OHD level was not associated with an increased risk neither at the 1st nor at the 2nd pregnancy samples (OR=1.4, 95%CI 0.6-3.4; OR 1.4, 95%CI 0.7-2.8, respectively), but was associated with an increased risk of PABC (OR=2.7, 95%CI 1.04-6.7)., Conclusion: Generally, vitamin D may not be related to breast cancer risk but the increased PABC risk fits the association of vitamin D with the most aggressive cancers, and warrants caution with vitamin D supplementation during pregnancy., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Introduction: Ecological and experimental studies suggest that vitamin D may be associated with a reduced risk of ovarian cancer. In this study, we sought to determine the risk of developing ovarian cancer according to serum 25-hydroxyvitamin D (25-OHD) concentrations assessed on average 5 years before the diagnosis., Methods: We conducted a population-based longitudinal case-control study nested within the Finnish Maternity Cohort (FMC) which contains serum samples of virtually all pregnant women in Finland since 1983. Among them, 201 ovarian cancers diagnosed within 10 years of serum sampling were randomly selected as cases for this study. For each case, we selected two controls matched for age, parity and sampling season (+/-4 weeks) and one control matched for age and parity but for the opposite sampling season (6 months+/-4 weeks)., Results: The relative risks (estimated as odds ratio, OR) for ovarian cancer comparing the lowest quintile to the highest quintile of serum 25-OHD concentration were 1.8 (95% CI 0.9-3.5) among controls matched for the same season, and 1.1 (95% CI 0.6-2.2) among controls matched for the opposite season. The OR among women with insufficient (<75 nmol/L) serum 25-OHD concentration was 2.7 (95% CI 1.0-7.9, lower limit, 0.95) compared to that among those with sufficient (75 nmol/L) serum 25-OHD concentration. No differences in the point estimates were observed between serous or mucinous histological subtypes of ovarian cancer., Conclusion: Overall, we did not observe a significant association between serum 25-OHD concentrations and the risk of ovarian cancer. However, we found evidence suggestive of an increased risk among women with low to insufficient serum 25-OHD concentrations., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Knowledge of the stability of serum samples stored in large biobanks is pivotal for reliable assessment of hormone-dependent disease risks. We studied the effects of sample storage time and season of serum sampling on the stability of 25-hydroxy vitamin D (25-OHD) and androstenedione in a stratified random sample of 402 women, using paired sera from the Finnish Maternity Cohort. Serum samples selected were donated between 6 and 24 yr ago. The storage time did not affect serum 25-OHD and androstenedione levels. However, there was a significant mean difference in the 25-OHD levels of sera withdrawn during winter (first sample) vs. during summer (second sample; -18.4 nmol/l, P

We propose a new hypothesis pointing of a functional hypoxia in vasa vasorum, which might explain the initiation and the early development of the atherosclerosis in the deep layer of intima. Since vasa vasorum are end arteries, they easily develop hypoxia and/or ischemia in the cells of intima or media of arterial wall. The most vulnerable sites for hypoxia are the arterial bifurcations due to the anatomical and physiological reasons. They are also known to be the most common sites of atherosclerosis. The known risk factors for atherosclerosis, high blood pressure and nicotine, reduce the blood flow in the end branches of the vasa vasorum. The local ischemia will affect the endothelial cell structure and causes a local inflammation, which, in turn, makes it permeable to large particles such as microbes and LDL-lipoproteins and other fatty acids, which are phagocytozed by macrophages transforming them into foam cells. The present hypothesis explains most problems of the previous theories and offers a logical sequence of the events involved in the development of atherosclerosis.

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.

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.

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 1alpha-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-kappaB 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 40-60 nmol/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.

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.

Deficiency of the prohormone calcidiol (25OH vitamin D(3)) seems to be associated with several aging-related chronic diseases including cancer. Our results suggest that calcidiol is mainly responsible for differentiation homeostasis, whereas calcitriol might be more involved in calcium homeostasis. Therefore, an imbalance of calcidiol rather than calcitriol is a risk factor for cancer and chronic diseases. Calcidiol insufficiency, as well as insufficient solar exposure, is associated with increased risk of several solid cancers. Both a vitamin D(3) deficiency and a high concentration of calcidiol may increase cancer risk. Similarly, aging phenomena show a U-shaped association with vitamin D bioactivity. Therefore, the chronic diseases and cancers related to aging might be prevented by an optimal concentration of serum calcidiol, which remains to be determined.

Vitamin D receptor (VDR) and liver X receptor (LXR) are nuclear receptors, which regulate gene transcription upon binding of their specific ligands. VDR seems to play a role in the regulation of prostate cancer cell proliferation. ATP-binding cassette transporter A1 (ABCA1) is known to be a target gene of LXR and it has been reported to be inhibited by androgen and to be involved in the regulation of LNCaP proliferation. We find that calcitriol (1 alpha,25(OH)(2)D(3)) inhibits both basal and a LXR agonist, TO-901317, induced ABCA1 mRNA expression but has no effect on the mRNA expression of ATP-binding cassette transporter G1 (ABCG1), LXR alpha nor LXR beta. TO-901317 increases both basal and calcitriol induced 25-hydroxyvitamin D(3)-24-hydroxylase (CYP24) mRNA expression and it slightly but significantly inhibits VDR mRNA expression. The inhibition of ABCA1 by calcitriol appears to be androgen-independent. Cell growth assay shows that when each of calcitriol and 5 alpha-dihydrotestosterone (DHT) was co-treated with ABCA1 blocker, glybenclamide, cell-growth is significantly decreased compared to their own treatments respectively. Our study suggests a possible interaction between calcitriol and TO-901317 in LNCaP cells. Alike DHT, the inhibition of ABCA1 by calcitriol may be involved in its regulation of LNCaP growth.

Animal behavioral models are crucial for neurobiological research, allowing for the thorough investigation of brain pathogenesis to be performed. In both animals and humans, anxiety has long been linked to vestibular disorders. However, although there are many tests of anxiety and vestibular deficits, there are few protocols that address the interplay between these two domains. The Suok test and its light-dark modification presented here appear to be suitable for testing this pathogenetic link in laboratory rodents. This protocol adds a new dimension to previously used tests by assessing animal anxiety and balancing simultaneously, resulting in efficient, high-throughput screens for testing psychotropic drugs, phenotyping genetically modified animals, and modeling clusters of human disorders related to stress/anxiety and balancing.

Background: Both hypo- and hypervitaminosis D can cause sensorineural hearing loss, and aural symptoms due to vitamin D insufficiency are especially common during gravidity. Hormonal forms of vitamin D regulate transcription by binding with the high-affinity vitamin D receptor (VDR)., Objective: To assess the effects of impaired vitamin D action in VDR knockout (KO) mice on hearing, cochlear morphology, and cochlear gene expression., Materials and Methods: Eighteen young male and female mice (10 VDR KO and 8 wild type, WT, < or =6 months old), 33 adult male and female mice (16 VDR KO and 17 WT, between 7 and 14 months old), and 11 aged male and female mice (5 VDR KO and 6 WT, > or =15 months old) on 129S1 genetic background were studied. Auditory thresholds were evaluated by auditory brain stem response. Morphological changes were analyzed using plastic embedding and light microscopy. The expression of key genes (known to play a role in the regulation of cochlear function), and caspase 3 activity, were assessed using immunofluorescent confocal microscopy., Results: There was a statistically significant difference between the young and the adult groups, and between the adult and aged groups of WT mice. There was also a statistically significant difference between the adult and aged groups in VDR KO mice, and between the young WT group and the young VDR KO group. Spiral ganglion cell loss was observed in the basal turn of adult VDR KO mice, a phenomenon infrequently found in WT mice. Expression of connexin 26, KCNJ10, and transient receptor potential channel vanilloid subfamily 4/6 was not affected by VDR KO-mediated hearing loss. Caspase 3 activation was detected in the spiral ganglion cell and its satellite cells, stria vascularis, spiral ligament fibrocytes, and the organ of Corti in both genotypes. However, the percentage of positive cells and the staining intensity were lower in the VDR KO (compared to the WT) mice., Conclusion: These data suggest that sensorineural hearing loss progressively developed at an earlier age in VDR KO mice. While the fundamental gene expressions in the cochlea were not influenced by VDR mutation, it resulted in decrease of caspase 3 activation, which may be one of the factors underlying accelerating age-related hearing loss observed in VDR KO mice., ((c) 2008 S. Karger AG, Basel)

Vitamin D seems to be involved in the control of prostate cancer cell growth. 17beta-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. 5alpha-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.

Background: The effects of vitamin D in regulating bone mineralization are well documented. The action of vitamin D as a key link between Toll-like receptor activation and antibacterial responses in innate immunity has recently been shown. The data suggest that differences in the ability of human populations to produce vitamin D may contribute to susceptibility to microbial infection., Objective: We aimed to explore whether an association exists between vitamin D insufficiency and acute respiratory tract infection in young Finnish men., Design: Young Finnish men (n = 800) serving on a military base in Finland were enrolled for this study. Their serum 25-hydroxyvitamin [25(OH)D] concentrations were measured in July 2002. They were followed for 6 mo, and the number of days of absence from duty due to respiratory infection were counted., Results: The mean (+/- SD) serum 25(OH)D concentrations were 80.2 +/- 29.3 nmol/L (n = 756). Subjects with serum 25(OH)D concentrations < 40 nmol/L (n = 24) had significantly (P = 0.004) more days of absence from duty due to respiratory infection (median: 4; quartile 1-quartile 3: 2-6) than did control subjects (2; 0-4; n = 628; incidence rate ratio 1.63; 95% CI: 1.15, 2.24). We found a significant (P = 0.004) association between serum 25(OH)D concentrations and the amount of physical exercise before induction into military service. We also found significantly (P < 0.001) lower serum 25(OH)D concentrations in subjects who smoked (72.8 +/- 26.6 nmol/L; n = 192) than in control subjects (82.9 +/- 29.7 nmol/L; n = 537)., Conclusion: Clinical trials of vitamin D supplementation are needed to investigate whether it enhances immunity to microbial infections.

Background: Skin cancers are known to be associated with sun exposure, whereas sunlight through the production of vitamin D may protect against some cancers. The aim of this study was to assess whether patients with skin cancer have an altered risk of developing other cancers., Methods: The study cohort consisted of 416,134 cases of skin cancer and 3,776,501 cases of non-skin cancer as a first cancer extracted from 13 cancer registries. 10,886 melanoma and 35,620 non-melanoma skin cancer cases had second cancers. The observed numbers (O) of 46 types of second primary cancer after skin melanoma, basal cell carcinoma or non-basal cell carcinoma, and of skin cancers following non-skin cancers were compared to the expected numbers (E) derived from the age, sex and calendar period specific cancer incidence rates in each of the cancer registries (O/E=SIR, standardised incidence ratios). Rates from cancer registries classified to sunny countries (Australia, Singapore and Spain) and less sunny countries (Canada, Denmark, Finland, Iceland, Norway, Scotland, Slovenia and Sweden) were compared to each other., Results: SIR of all second solid primary cancers (except skin and lip) after skin melanoma were significantly lower for the sunny countries (SIR(S)=1.03; 95% CI 0.99-1.08) than in the less sunny countries (SIR(L)=1.14; 95%CI 1.11-1.17). The difference was more obvious after non-melanoma skin cancers: after basal cell carcinoma SIR(S)/SIR(L)=0.65 (95%CI=0.58-0.72); after non-basal cell carcinoma SIR(S)/SIR(L)=0.58 (95%CI=0.50-0.67). In sunny countries, the risk of second primary cancer after non-melanoma skin cancers was lower for most of the cancers except for lip, mouth and non-Hodgkin lymphoma., Conclusions: Vitamin D production in the skin seems to decrease the risk of several solid cancers (especially stomach, colorectal, liver and gallbladder, pancreas, lung, female breast, prostate, bladder and kidney cancers). The apparently protective effect of sun exposure against second primary cancer is more pronounced after non-melanoma skin cancers than melanoma, which is consistent with earlier reports that non-melanoma skin cancers reflect cumulative sun exposure, whereas melanoma is more related to sunburn.

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.

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.

F1 and F2 mouse hybrids derived from different parental strains are becoming a useful tool in behavioral research, underlining the importance of their in-depth behavioral phenotyping. 129S1/SvImJ (S1), C57BL/6 (B6), NMRI (N) and BALB/c (BC) mice are commonly used in behavioral neuroscience, demonstrating marked behavioral differences. Here, we assess behavioral phenotypes of male mice of S1 and several hybrid strains (S1B6, S1N, S1BC) in a battery of behavioral tests, including the open field, novel odor exposure, novelty-induced grooming, horizontal rod (Suok) and the elevated plus maze tests. In addition, we assessed aggression and social barbering in these strains. Overall, the substantial differences observed here between these strains allow us to determine the influence of different genetic backgrounds on mouse behaviors, and more fully understand how different strain-specific behaviors overlap in the F1 progeny. Our results imply complex interplay between parental genotypes in anxiety, activity, grooming, aggression and barbering of their F1 progeny, further confirming the utility of F1 hybrids in behavioral neurogenetics.

Background: Factors related to the metabolic syndrome and low levels of vitamin D have been implicated as risk factors for prostate cancer. Insofar, no studies have assessed their joint effects on prostate cancer risk., Methods: We studied (a) the associations of vitamin D with the metabolic syndrome factors body mass index, systolic and diastolic blood pressure, and high-density lipoprotein cholesterol (HDL-C) and (b) the prostate cancer risk associated with these factors and especially their joint effects with vitamin D on risk of prostate cancer. We did a longitudinal nested case-control study on 132 prostate cancer cases and 456 matched controls from a cohort of 18,939 Finnish middle-aged men from the Helsinki Heart Study. The odds ratios (OR) of prostate cancer were assessed via conditional logistic regression analysis., Results: Apart from HDL-C, there was no linear association between the metabolic syndrome factors and vitamin D levels. In univariate analysis, men in the highest quartiles of body mass index (>28 kg/m(2)) and systolic blood pressure (>150 mmHg) showed a modest increase in risks of prostate cancer, with ORs of 1.37 (P = 0.16) and 1.53 (P = 0.05) when compared with the three lower quartiles, but low HDL-C entailed no prostate cancer risk. However, with all three factors present, the OR was 3.36 (P = 0.02), and jointly with low vitamin D (

Grooming is a commonplace, robust behavior in rodent species. It has been shown to be highly sensitive to a number of experimental factors, making it an ideal target for manipulation. The complex patterning of grooming in rodents, which usually proceeds in a cephalo-caudal direction and involves several distinct stages, can be dissected into its constituent parts and microstructures. Several grooming patterning analysis methods are described in the protocol that allow for an assessment of this behavior based on measurements of grooming activity and its sequencing. Additionally, grooming can be evaluated in reference to the regional distribution and syntax in which it occurs. Owing to the ever-increasing number of rodent models that have strong grooming phenotypes, this high-throughput in-depth analysis is becoming crucial for biomedical research.

Purpose of Review: Vitamin D is a seco-steroid hormone with multiple functions in the nervous system. We discuss clinical and experimental evidence of the role of vitamin D in normal and pathological brain functions, and analyze the relative importance of vitamin D-modulated brain mechanisms at different stages of life. We also outline perspectives for the use of vitamin D in clinical nutrition to prevent or treat various brain disorders., Recent Findings: Numerous brain dysfunctions are linked to vitamin D deficits and/or dysfunctions of its receptors. In both animals and humans, vitamin D serves as an important endogenous and/or exogenous regulator of neuroprotection, antiepileptic and anticalcification effects, neuro-immunomodulation, interplay with neurotransmitters and hormones, modulation of behaviors, brain ageing, and some other, less-explored, brain processes., Summary: Vitamin D emerges as an important neurosteroid hormone in the brain, with a strong potential for age-specific applications in clinical nutrition.

Unlabelled: Low vitamin D level may predict rickets, osteomalacia, or osteoporosis. We examined serum 25(OH)D concentration as a predisposing factor for bone stress fracture in 756 military recruits. The average serum 25(OH)D concentration was significantly lower in the group with fracture, suggesting a relationship between vitamin D and fatigue bone stress fracture., Introduction: Low vitamin D level may predict rickets, osteomalacia, or osteoporosis. Fatigue bone stress fracture is one of the most frequently seen types of overuse injuries in athletes and military recruits. An association was recently shown between vitamin D and BMC. A correlation has also been found between low femoral BMD and stress fractures. We measured serum 25(OH)D concentration in a population sample of military recruits to determine if vitamin D is a predisposing factor for fatigue bone stress fracture., Materials and Methods: We prospectively followed 800 randomly selected, healthy Finnish military recruits with a mean age of 19 years for developing stress fractures in homogenous circumstances. Blood for serum 25(OH)D concentration was drawn at entry into military service, and the weight, height, body mass index (BMI), muscle strength, and 12-minute running were measured for all subjects. Serum 25(OH)D concentrations were measured with enzyme immunoassay. At end of the 90-day follow-up, 756 subjects completed the study. Subjects without fracture constituted controls., Results: Twenty-two recruits with stress fracture were identified (2.9%), the incidence being 11.6 (95% CI: 6.8-16.5) per 100 person-years. In the final multivariate analysis, the significant risk factor for stress fracture in conscripts was a below median serum 25(OH)D level (75.8 nM), OR being 3.6 (95% CI: 1.2-11.1). No significant associations between BMI (p = 0.255), age (p = 0.216), or smoking (p = 0.851) and bone stress fracture were found in this study population., Conclusions: A lower level of serum 25(OH)D concentration may be a generally predisposing element for bone stress fractures. Considering the obvious need of additional vitamin D in prevention of stress fractures, the effects of vitamin D fortification of foods and supplementation will be subjects of interest for future research.

Vitamin D3 plays an important role in the control of cell proliferation and differentiation. Cholesterol 25-hydroxylase (CH25H) is an enzyme converting cholesterol into 25-hydroxycholesterol. Vitamin D3 as well as 25-hydroxycholesterol has been shown to inhibit cell growth and induce cell apoptosis. Here we show that 10 nM 1alpha,25(OH)2D3 and 500 nM 25OHD3 upregulate CH25H mRNA expression in human primary prostate stromal cells (P29SN). Protein synthesis inhibitor cycloheximide does not block 1alpha,25(OH)2D3 mediated upregulation of CH25H mRNA. Transcription inhibitor actinomycin D blocks basal level as well as 1alpha,25(OH)2D3 induced CH25H mRNA expression. 1alpha,25(OH)2D3 has no effect on CH25H mRNA stability. 25-Hydroxycholesterol significantly decreased the P29SN cell number. A CH25H enzyme inhibitor, desmosterol, increases basal cell number but has no significant effect on vitamin D3 treated cells. Our data suggest that ch25h could be a vitamin D3 target gene and may partly mediate anti-proliferative action of vitamin D3 in human primary prostate stromal cells.

25-Hydroxyvitamin D3-24-hydroxylase (24-hydroxylase, CYP24) is an important inactivating enzyme controlling the concentrations of both active metabolites 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3. In this paper, we demonstrate that 25-hydroxyvitamin D3 at 500 nM significantly increases the expression of 24-hydroxylase mRNA and the increase is significantly decreased by 5alpha-dihydrotestosterone (DHT) at concentrations of 1-100 nM in androgen-sensitive prostate cancer cells LNCaP. 25-Hydroxyvitamin D3 at 500 nM and 1alpha,25-dihydroxyvitamin D3 at 10 nM inhibit LNCaP cell growth, and the growth inhibition is enhanced by 1 nM DHT. Neither 25-hydroxyvitamin D3 nor 1alpha,25-dihydroxyvitamin D3 at physiological concentrations has growth effect. However, in the presence of 1 nM DHT, both 25-hydroxyvitamin D3 and 1alpha,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 1alpha,25-dihydroxyvitamin D3, the present results demonstrate, for the first time, that both 25-hydroxyvitamin D3 and 1alpha,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.

Vitamin D is a steroid hormone with many important functions in the brain, mediated through the nuclear Vitamin D receptor (VDR). Mounting clinical data link VDR mutations to various psychiatric phenotypes. We have reported previously that mutant mice lacking functional VDR ("Tokyo" VDR mutant mice) display several behavioural anomalies, including high anxiety and aberrant grooming. Given the important role of Vitamin D and VDR in brain development and functioning, we hypothesized that several other important behavioural domains may be affected by disruption of the VDR gene in mice. Here we report that VDR mutants display unaffected depressive-like behaviour, but show abnormal social behaviours, reduced social barbering and aggressiveness, impaired nest building and aberrant maternal (pup neglect, cannibalism) behaviours. Taken together, these findings confirm the important role postulated for the VDR in the regulation of behaviour, and suggest the mice lacking functional VDR may be a useful tool to model different brain disorders.

We investigated behavioural activity and temporal distribution (patterning) of mouse exploration in different open field (OF) arenas. Mice of 129S1 (S1) strain were subjected in parallel to three different OF arenas (Experiment 1), two different OF arenas in two trials (Experiment 2) or two trials of the same OF test (Experiment 3). Overall, mice demonstrated a high degree of similarity in the temporal profile of novelty-induced horizontal and vertical exploration (regardless of the size, colour and shape of the OF), which remained stable in subsequent OF exposures. In Experiments 4 and 5, we tested F1 hybrid mice (BALB/c-S1; NMRI-S1), and Vitamin D receptor knockout mice (generated on S1 genetic background), again showing strikingly similar temporal patterns of their OF exploration, despite marked behavioural strain differences in anxiety and activity. These results suggest that mice are characterised by stability of temporal organization of their exploration in different OF novelty situations.

Vitamin D is a neuroactive steroid hormone with multiple functions in the brain. Numerous clinical and experimental data link various Vitamin D-related dysfunctions to epilepsy. Here, we study the role of Vitamin D receptors (VDRs) in experimental epilepsy in mice. To examine this problem, we assessed the seizure profiles in VDR knockout mice following a systemic injection of pentylenetetrazole (70 mg/kg). Overall, compared to the wild-type (WT) 129S1 mice (n=10 in each group), the VDR knockout group significantly demonstrated shorter latencies to the onset, higher Racine scores and increased mortality rates. Our findings suggest that VDRs modulate seizure susceptibility in mice, and that the Vitamin D/VDR endocrine system may be involved in the pathogenesis of epilepsy.

25-Hydroxyvitamin D(3)-24-hydroxylase (24-hydroxylase) is an important inactivating enzyme and its expression is induced by 25-hydroxyvitamin D3 (25OHD3) and 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) through action of heterodimers of vitamin D receptor (VDR) and retinoid X receptor (RXR). RXRs also act as heterodimer partners for retinoic acid receptors (RARs), mediating the action of all-trans-retinoic acid (ATRA). Prostate stroma plays a crucial role in prostate cancer development and benign prostatic hyperplasia. We demonstrate here that ATRA markedly reduced the expression of 24-hydroxylase mRNA induced by 25OHD3 and 1alpha,25-(OH)2D3 in human prostatic stromal cells P29SN and P32S but not in epithelial cells PrEC or cancer cells LNCaP. By using transfection and RAR-selective ligands, we found that the inhibitory effect of ATRA on 24-hydroxylase expression in stromal cells was mediated by RARalpha but not by RARbeta. Moreover, the ATRA-induced expression of RARbeta was also mediated by RARalpha. The combined treatment of 1alpha,25-(OH)2D3 and RARalpha agonist Am80 at 10 nM exhibited strong growth-inhibitory effect whereas either alone had no effect. Our data suggest that ATRA suppresses 24-hydroxylase expression through RARalpha-dependent signaling pathway and can enhance vitamin D action in suppression of cell growth.

Exploration of the long elevated alley (the rat Suok test) enables behavioural characterisation of anxiety, activity and neurological phenotypes in rats. Here we show that this new test is sensitive to different types of anxiety in rats, including drugs (pentylenetetrazole)-induced, light-induced and socially induced (encounter with an unfamiliar male) anxiety, as assessed by reduced Suok test horizontal, vertical, directed exploration and stops. High anxiety also leads to higher motor incoordination (as assessed by the number of falls and hind-paw slips), suggesting that this test may be used for combined profiling of anxiety, motor-vestibular anomalies and anxiety-induced motor incoordination in rats. This new behavioural paradigm may be widely used in neurobehavioural stress research, including modelling of stress-evoked states and pharmacological screening of psychotropic drugs.

Here, we study the role of a neurosteroid hormone Vitamin D in epilepsy. To examine this problem, we used 1,25-dihydroxyvitamin D, an active form of Vitamin D, injected subcutaneously to NMRI mice (33 microg/20 microl) 40 min prior to seizures induced by systemic injection of pentylenenetrazole (PTZ, 70 mg/kg). Overall, compared to the vehicle-treated control animals (n=11 in each group), the Vitamin D-treated mice demonstrated reduced severity of PTZ-induced seizures (longer latency, shorter duration and lower mortality). In a separate experiment, we assessed the time-course of antiepileptic effects of 1,25-dihydroxyvitamin D. For this, we injected this compound (33 microg/20 microl) to NMRIx129S1 mice (n=11) 40 min, 3, 6, 12 and 24 h prior to seizures, showing that antiepileptic effects were short-term, almost disappearing 3h after administration. Our findings show that Vitamin D plays a direct anticonvulsant role in the brain and suggest that the Vitamin D endocrine system may represent a new target for the development of anticonvulsant drugs.

1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3 or calcitriol) is an active hormone that regulates cellular proliferation and induces apoptosis in cancer cells. Here we report on a new calcitriol target gene in prostate cancer cells, tumour necrosis factor alpha (TNF-alpha). Calcitriol and its analogue CB1093 up-regulate TNF-alpha mRNA expression in LNCaP and PC-3 cells. The stimulation is dose-dependent in both of these cell lines, demonstrated by the quantitative real-time polymerase chain reaction. Calcitriol and CB1093 act synergistically with human recombinant TNF-alpha in activation of TNF-alpha mRNA expression in LNCaP but not in PC-3 cells. Transcriptional activation of TNF-alpha gene by calcitriol or CB1093 does not lead to TNF-alpha protein secretion, however calcitriol and CB1093 enhance TPA-stimulated TNF-alpha production in LNCaP cells. We did not observe any significant effect of calcitriol on regulation of TNFR1 at the level of gene expression. Nor does calcitriol affect transcriptional regulation of cytokine (IL-1, IL-6) and cytokine receptor genes in LNCaP and PC-3 prostate cancer cell lines. Calcitriol and its analogue CB1093 at 10 nM concentration induce programmed cell death in LNCaP cells. Combined addition of human recombinant TNF-alpha with calcitriol or CB1093 cause enhanced effect in induction of apoptosis. We conclude that under physiological conditions vitamin D activates only the transcription of TNF-alpha gene, for TNF-alpha protein synthesis additional cofactors are required. Therefore a cooperation of vitamin D and TNF-alpha may play an important role in the control of cell growth in prostate cancer.

Background: A cross-talk between 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)] and 5alpha-dihydrotestosterone (DHT) in the growth inhibition has been demonstrated, but the mechanism is unknown., Methods: The expression of 25-hydroxyvitamin D(3) 24-hydroxylase (24-hydroxylase) was measured using a real-time quantitative RT-PCR assay and the catabolism of 1alpha,25-(OH)(2)D(3) was measured using a radioreceptor assay., Results: Real-time RT-PCR showed that DHT at 1-100 nM significantly inhibited 1alpha,25-(OH)(2)D(3)-induced expression of 24-hydroxylase in LNCaP cells. Furthermore, the catabolism of 1alpha,25-(OH)(2)D(3) was decreased by 10 nM DHT. An androgen receptor (AR) antagonist, Casodex antagonized the DHT effect, whereas an AR agonist (due to the mutant AR in LNCaP cells) hydroxyflutamide did not., Conclusions: We demonstrated, for the first time, that DHT reduces the ability of 1alpha,25-(OH)(2)D(3) to induce 24-hydroxylase expression. Our results not only support the earlier finding of a cross-talk between androgen and vitamin D in human prostate cancer cells but also provide a possible mechanism how androgen and vitamin D signaling pathways may interact., ((c) 2004 Wiley-Liss, Inc.)

129S1/SvImJ (129S1), NMRI and BALB/c mice are widely used in behavioural research, demonstrating marked strain differences in their behavioural phenotypes. Grooming is a complex and essential ritual in the rodent behavioural repertoire with a general cephalocaudal progression (forepaws-nose-face-body-legs-tail and genitals). Various stressors as well as genetic manipulations have been reported to alter mouse grooming and its patterning, underlying the importance of analysis of grooming behaviours. Although strain differences between these mice have been assessed in many studies, no comparative analyses of their grooming have been performed. Here we show strain differences in spontaneous (novelty-induced) grooming between 129S1, NMRI and BALB/c mice. Overall, 129S1 mice demonstrated lower grooming activity and impaired microstructure (more interrupted bouts and incorrect transitions contrary to the cephalocaudal rule), accompanied by lower vertical exploration. In contrast, BALB/c and NMRI mice showed high vertical activity and unimpaired grooming microstructure, also exhibiting different grooming levels (BALB/c>NMRI). Our study suggests that contrasting grooming phenotypes in these mice may not be due to the strain differences in their sensory abilities, general activity levels, brain anatomy or aggressiveness, but rather reflect a complex interplay between anxiety, motor and displacement activity in these strains (hypoactive anxious phenotype in 129S1 mice, active anxious phenotype in BALB/c and non-anxious high displacement phenotype in NMRI mice). We suggest that ethological analysis of mouse grooming, such as that reported here, may be a useful tool in neurobehavioural research.

Stress has long been known to affect grooming in rodent species, altering both its activity measures and behavioural microstructure. Since stress disturbs a general pattern of self-grooming uninterrupted cephalocaudal progression, the grooming analysis algorithm (Kalueff and Tuohimaa, Brain Res. Protocols, 2004; 15: 151-8) was previously designed for mice to enable the detection of stress by measuring alterations in grooming microstructure in different test situations. Since mice and rats are known to differ in their behaviours, including grooming, the aim of the current study was to test our approach in rats and evaluate the utility of this method for differentiation between high- and low-stress situations. For this, we have developed the rat grooming analysis algorithm (based on ethological analysis of incorrect transitions contrary to the cephalocaudal rule, interrupted grooming activity and the assessment of the regional distribution of grooming) and applied this algorithm to the light-exposed (high stress) and dark-exposed (low stress) groups of rats. Here, we show that the percentage of 'incorrect' transitions between different grooming patterns, the percentage of interrupted grooming bouts and altered regional distribution of grooming (less caudal grooming, more rostral grooming) may be used as behavioural markers of stress in rats. Our results suggest that this method can be a useful tool in neurobehavioural stress research including modelling stress-evoked states, psychopharmacological or behavioural neurogenetics research in rats.

In the present study, we suggest that long elevated horizontal rod (Suok test, ST) and its light-dark modification (LDST) may be used for behavioral characterization in mice, including simultaneous assessment of their anxiety, activity, and neurological phenotypes. To establish the ST and the LDST as murine models of anxiety, we used several different mouse strains which differ markedly in their anxiety and activity (C57BL/6, 129S1/SvImJ, NMRI, and BALB/c). Here we show that our tests are able to ethologically discriminate between high and low anxiety mouse strains, as assessed by horizontal and directed exploration, stops, and defecation boli. The spatial distribution of the LDST behaviors is also sensitive to these strain-specific anxiety phenotypes, showing clear avoidance of the brightly lit part of the test in stressed (rat exposed) vs. control NMRI mice. In addition, we validated the ST in 129S1/SvImJ and BALB/c mice by assessing the behavioral consequences of acute stress such as rat exposure. Finally, we showed that our test is able to detect high anxiety and poorer motor coordination in 129S1/SvImJ (vs. C57BL/6) mice. The results of our study show that the ST emerges as an experimental tool to analyze anxiety, motor-vestibular anomalies, as well as anxiety-induced motor impairments in mice. Overall, we suggest that the ST can be a useful protocol in neurobehavioral stress research including modeling stress-evoked states, pharmacological screening of potential anti-stress drugs, or behavioral phenotyping of genetically modified animals.

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.

Estrogens and androgens are proposed to play a role in the pathogenesis of prostate cancer. The effective metabolites, estradiol and 5alpha-dihydrotestosterone are produced from testosterone by aromatase and 5alpha-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 D(3) (25OHD(3)), 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)], dexamethasone, and progesterone regulate the expression of aromatase and 5alpha-reductase in human prostate cancer cells. LNCaP and PC3 cells were treated with 25OHD(3), 1alpha,25-(OH)(2)D(3), dexamethasone, or progesterone. Aromatase and 5alpha-reductase mRNA was quantified by real-time RT-PCR and aromatase enzyme activity was measured by the [(3)H] water assay. Aromatase enzyme activity in LNCaP and PC3 cells was increased by both 10nM dexamethasone, 1-100 nM 1alpha,25-(OH)(2)D(3) and 100 nM-10 microM 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 5alpha-reductase type I mRNA was not regulated by 1alpha,25-(OH)(2)D(3) and no expression of 5alpha-reductase type II was detected in LNCaP.

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 PDGFRalpha and beta 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 PDGFRbeta and restores responsiveness of LNCaP to PDGF-BB to some extent. Calcitriol down-regulates PDGFRbeta expression and negatively regulates PDGF-mediated cell growth. Calcitriol does not affect PDGFRalpha and PDGF-B mRNA expression. We suggest that inhibition of PDGFRbeta expression by calcitriol might reduce responsiveness of prostate cells to mitogenic action of PDGF-BB.

Grooming is an important part of rodent behavioural repertoire, representing a complex hierarchically ordered cephalo-caudal sequence of patterns sensitive to stress and various drugs. Gamma-aminobutyric acid (GABA) is involved in the regulation of both anxiety and grooming behaviours. This study investigated the predictive validity of grooming behavioural microstructure as a marker of anxiety, by examining the effects of two GABAergic reference compounds, anxiolytic diazepam (0.1 and 0.5 mg/kg i.p.) and anxiogenic pentylenetetrazole (5 and 10 mg/kg i.p.) on mouse grooming. Our data suggest that the percentage of pattern transitions not fitting to the cephalo-caudal progression, and the percentage of interrupted grooming bouts are more reliable behavioural markers of stress bidirectionally sensitive to GABAergic anxiogenic and anxiolytic drugs, compared to the frequency and duration scores. Our study also confirms that detailed ethological analyses of grooming microstructure can be a useful tool in behavioural pharmacology of anxiety.

Vitamin D is a steroid hormone with several important functions in the nervous system. Numerous human and animal data link alterations in the vitamin D system to various behavioral disorders. Grooming is an important element of rodent behavior with a general pattern of cephalocaudal progression (paw licking - nose/face wash - body wash - tail/genitals wash). Here we studied whether genetic ablation of vitamin D nuclear receptors (VDR) in mice may be associated with altered behavioral sequencing of grooming. Overall, VDR null mutant mice showed abnormal grooming, including a higher percentage of "incorrect" transitions and longer duration of "incorrect" grooming (contrary to the cephalocaudal progression); a higher percentage of interrupted grooming bouts; and the atypical regional distribution of grooming (more leg grooming, less body and tail/genitals grooming), compared to their wild-type controls. Grooming of heterozygous mice was similar to the wild-type group, indicating that abnormal grooming patterning is inherited as a recessive. In contrast, behavioral sequencing of another complex behavior (mating with a female) was unaltered in all three genotypes, suggesting grooming-specific abnormal sequencing in these mutant mice. Our results suggest that a neurosteroid vitamin D and VDR may play an important role in controlling sequencing of grooming in mice, and further confirm the important role of the vitamin D system and VDR in the regulation of behavior.

Calcitriol (1alpha,25-dihydroxycholecalciferol) seems to play an important role in the complex control of prostate cell growth. It inhibits proliferation and induces differentiation and apoptosis in prostate cancer cells. However, the molecular mechanisms of the antiproliferative activity of calcitriol are not completely understood. The expression of prostate-derived factor (PDF), a member of the transforming growth factor-beta (TGF-beta) superfamily, has been shown to be associated with proapoptotic and antimitotic activities. We show that calcitriol induces PDF expression in LNCaP human prostate cancer cells in a concentration- and time-dependent manner. In LNCaP cells, the suppression of cell growth by calcitriol is accompanied by stimulation of PDF mRNA and protein synthesis. Human recombinant PDF inhibits LNCaP cell growth. We do not detect any effect of PDF-specific antibody on the basal growth of LNCaP cells, but this antibody partially reverses the suppression of LNCaP cell growth by calcitriol, suggesting that the effect of calcitriol on cell growth is at least partially mediated by PDF. In PC-3 cells, which are less responsive to the growth-inhibitory effect of calcitriol, it has no effect on PDF expression. We do not detect an effect of recombinant PDF on SMAD phosphorylation in LNCaP cells, but ERK1/2 kinases are transiently phosphorylated in response to PDF, which suggests that in LNCaP cells PDF may exert its action through pathway alternative to the classical TGF-beta signaling pathway. The present study describes the regulation of PDF, the proapoptotic protein of the TGF-beta superfamily, by calcitriol in androgen-responsive prostate cancer cells. This is a new link between calcitriol and growth factors of TGF-beta superfamily in the control of prostate cell growth., ((c) 2004 Wiley-Liss, Inc.)

Since C57 and 129 mice are the commonly used background strains, a better knowledge of all their behavioural characteristics is important in neuroscience research. Grooming is a complex and essential ritual in the rodent behavioural repertoire, normally proceeding in a cephalocaudal progression (paws-nose-face-body-legs-tail and genitals). Various stressors as well as genetic manipulations have been reported to alter mouse grooming and its patterning, underlying the importance of analysis of grooming behaviours in detail. Although strain differences between C57BL/6 and 129S1/SvImJ substrains have been assessed in many studies, no ethological analyses of their grooming have been performed. Here we show strain differences between these mice in spontaneous (novelty-induced) and artificial (water-induced) grooming. Overall, 129S1/SvImJ mice demonstrated less grooming activity, more interrupted and incomplete bouts, and more incorrect transitions (contrary to the cephalocaudal rule) between patterns, accompanied by lower vertical activity and higher defecation/urination in both tests. These results are consistent with general hypoactive anxious phenotype in 129S1/SvImJ mice and suggest that ethological analysis of mouse grooming may be used in neurobehavioural stress research, including behavioural phenotyping of both mutant and background mice.

FAS and FACL3 are enzymes of fatty acid metabolism. In our previous studies, we found that FAS and FACL3 genes were vitamin D3-regulated and involved in the antiproliferative effect of 1alpha,25(OH)2D3 in the human prostate cancer LNCaP cells. Here, we elucidated the mechanism behind the downregulation of FAS expression by vitamin D3. Triacsin C, an inhibitor of FACL3 activity, completely abolished the downregulation of FAS expression by vitamin D3, whereas an inhibitor of FAS activity, cerulenin, had no significant effect on the upregulation of FACL3 expression by vitamin D3 in LNCaP cells. In human prostate cancer PC3 cells, in which FACL3 expression is not regulated by vitamin D3, no regulation of FAS expression was seen. This suggests that the downregulation of FAS expression by vitamin D3 is mediated by vitamin D3 upregulation of FACL3 expression. Myristic acid, one of the substrates preferential for FACL3, enhanced the repression of FAS expression by vitamin D3. The action of myristic acid was abrogated by inhibition of FACL3 activity, suggesting that the enhancement in the downregulation of FAS expression by vitamin D3 is due to the formation of myristoyl-CoA. The data suggest that vitamin D3-repression of FAS mRNA expression is the consequence of feedback inhibition of FAS expression by long chain fatty acyl-CoAs, which are formed by FACL3 during its upregulation by vitamin D3 in human prostate cancer LNCaP cells.

Since rodent self-grooming behaviours are elicited by both comfort and stressful conditions, traditional measures such as duration, latency of onset and the number of bouts may be not suitable to dissociate between these opposite conditions. The aim of the current study was to improve and optimize ethological measurement of self-grooming in neurobehavioural stress research enabling differentiation between stress and no-stress situations. This protocol assists in the correct interpretation of animal grooming behaviours and detection of stress by measuring alterations in grooming microstructure in different test situations. While a general pattern of self-grooming uninterrupted cephalocaudal progression is normally observed in no-stress (comfort) conditions in mice and other rodents, the percentage of "incorrect" transitions between different stages and the percentage of interrupted grooming bouts may be used as behavioural marker of stress. The protocol can be a useful tool in neurobehavioural stress research including modelling stress-evoked states, pharmacological screening of potential antistress drugs or behavioural phenotyping of genetically modified animals.