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Abstract: 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)2D3 at an mRNA and enzyme activity levels in prostate cancer cells. Our further study indicated that the FACL3 mediated 1α,25(OH)2D3 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)2D3 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)2D3, 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. [ABSTRACT FROM AUTHOR]

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: 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. [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: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Summary: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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 (33μg/20μl) 40min prior to seizures induced by systemic injection of pentylenenetrazole (PTZ, 70mg/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 (33μg/20μl) to NMRIx129S1 mice (n =11) 40min, 3, 6, 12 and 24h 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. [Copyright &y& Elsevier]

Abstract: 1α,25–dihydroxyvitamin D3 (1α,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 α (TNF–α). Calcitriol and its analogue CB1093 up–regulate TNF–α 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–α in activation of TNF–α mRNA expression in LNCaP but not in PC–3 cells. Transcriptional activation of TNF–α gene by calcitriol or CB1093 does not lead to TNF–α protein secretion, however calcitriol and CB1093 enhance TPA–stimulated TNF–α 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–α 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–α gene, for TNF–α protein synthesis additional cofactors are required. Therefore a cooperation of vitamin D and TNF–α may play an important role in the control of cell growth in 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: 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 (μt ) 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, μt =1year) 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 &y& Elsevier]

Abstract: 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 (<75nmol/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 (⩾75nmol/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 &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: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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 (70mg/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. [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]

Vitamin D is a neuroactive seco-steroid and its importance to the nervous system is receiving increasing recognition. Since numerous data link vitamin D dysfunctions to various neurological and behavioural disorders, we studied whether genetic ablation of vitamin D receptors (VDR) may be associated with motor impairments in mice subjected to several behavioural tests. The data obtained in the vertical screen and swim tests show that VDR genetic ablation produces severe motor impairment (shorter screen retention and poor swimming) in mutant mice compared to wild-type and heterozygous control animals. These impairments appear to be unrelated to visual, vestibular and activity/emotionality parameters of mice, and are likely associated with disturbed calcium homeostasis. This study confirms the important role of the vitamin D system in motor functions and suggests that animal genetic models targeting the vitamin D/VDR system may be a useful tool to study vitamin D-related motor/behavioural disorders. [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]

The distribution of androgen receptor (AR) in human skin was studied by an immunohistochemical method using a polyclonal antibody against the human AR. Skin samples of preputial skin and male and female nongenital skin were examined. The possible correlation of AR location to acne was studied in skin biopsies from skin areas affected or unaffected by acne. In preputial skin, AR was expressed in epidermal cells as well as in fibroblasts, smooth muscle cells, and endothelial cells of blood vessels in the dermal area. AR was found located also in the flat fibroblast-like cells of Pacinian corpuscles. In nongenital skin, AR was also expressed in the basal cells and glandular cells of sebaceous glands, in the outer root sheath of hair follicles, and in eccrine sweat glands. The presence of AR in different cell types in the skin reflects the numerous direct effects androgens may have on this target tissue. The distribution of AR was similar in male and female skin. [ABSTRACT FROM AUTHOR]