Vitamin D3 inhibits proinflammatory cytokines and nitric oxide production by the EOC13 microglial cell line

In recent years, a neuroimmunomodulatory role for 1,25-dihydroxyvitamine D(3) [1,25(OH)(2)D(3)] has emerged. Microglial cells present a potential target for the effects of this hormone in the brain. This study focuses on the effect of 1,25(OH)(2)D(3) on the expression and production of inflammatory cytokines and nitric oxide (NO) by the EOC13 microglial cell line. The presence of the vitamin D3 receptor in microglia was demonstrated by RT-PCR. 1,25(OH)(2)D(3) inhibited the production of tumor necrosis factor-alpha, interleukin-6, and NO by stimulated microglia in a concentration-related fashion. The production of transforming growth factor-beta1 (TGF-beta1), an anti-inflammatory cytokine, was not modified in the presence of 1,25(OH)(2)D(3), indicating that the effects of 1,25(OH)(2)D(3) may not involve TGF-beta1 regulation. These results show that 1,25(OH)(2)D(3) has direct anti-inflammatory properties on microglia. It further supports the hypothesis that 1,25(OH)(2)D(3) could be involved in the maintenance of the brain homeostasis and may have a therapeutic potential in inflammatory pathologies of the central nervous system.     

Vitamin D receptor stable transfection restores the susceptibility to 1,25-dihydroxyvitamin D3cytotoxicity in a rat glioma resistant clone

Recently, 1,25-dihydroxyvitamin D₃ (1,25-D₃) and less hypercalcemic analogs were shown to exert a delayed cytotoxic effect on rat C6 glioma cells. 1,25-D₃ induces in these cells a programmed cell death, accompanied by the induction of c-myc, p53 and gadd 45 genes. The involvement of the intracellular vitamin D receptor (VDR) remained to be determined. In this lethal process, we have investigated its role in a subclone of C6 cells, which was isolated on the basis of its resistance to 1,25-D3, and in which VDR expression was not detected either at the mRNA or protein levels. The stable transfection of a rat VDR cDNA into this clone restored its susceptibility to the cytotoxic effects of 1,25-D₃. This phenomenon was accompanied by a dramatic upregulation of c-myc mRNA expression, as already described in a C6-sensitive clone. These results provide the first evidence that VDR expression, if not sufficient, is necessary to mediate 1,25-D₃ cytotoxic effect in C6 glioma cells. Since VDR mRNA expression has been already reported in human brain tumors, our data imply that the identification of VDR expression could become a prerequisite in any strategy of glioma treatment with vitamin D analogs. J. Neurosci. Res. 52:210–219, 1998. © 1998 Wiley-Liss, Inc.     

1,25-dihydroxyvitamin D3 reverses experimental autoimmune encephalomyelitis by inhibiting chemokine synthesis and monocyte trafficking

Multiple sclerosis (MS) is a complex neurodegenerative disease whose pathogenesis involves genetic and environmental risk factors leading to an aberrant, neuroantigen-specific, CD4+ T cell-mediated autoimmune response. In support of the hypothesis that vitamin D3 may reduce MS risk and severity, we found that vitamin D3 and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) inhibited induction of experimental autoimmune encephalomyelitis (EAE), an MS model. To investigate how 1,25-(OH)2D3 could carry out anti-inflammatory functions, we administered 1,25-(OH)2D3 or a placebo to mice with EAE, and subsequently analyzed clinical disease, chemokines, inducible nitric oxide synthase (iNOS), and recruitment of dye-labeled monocytes. The 1,25-(OH)2D3 treatment significantly reduced clinical EAE severity within 3 days. Sharp declines in chemokines, inducible iNOS, and CD11b+ monocyte recruitment into the central nervous system (CNS) preceded this clinical disease abatement in the 1,25-(OH)2D3-treated animals. The 1,25-(OH)2D3 did not directly and rapidly inhibit chemokine synthesis in vivo or in vitro. Rather, the 1,25-(OH)2D3 rapidly stimulated activated CD4+ T cell apoptosis in the CNS and spleen. Collectively, these results support a model wherein inflammation stimulates a natural anti-inflammatory feedback loop. The activated inflammatory cells produce 1,25-(OH)2D3, and this hormone subsequently enhances the apoptotic death of inflammatory CD4+ T cells, removing the driving force for continued inflammation. In this way, the sunlight-derived hormone could reduce the risk of chronic CNS inflammation and autoimmune-mediated neurodegenerative disease.     

Effects of vitamin D in the nervous system: Special focus on interaction with steroid hormone signalling and a possible role in the treatment of brain cancer

The active vitamin D hormone, 1,25‐dihydroxyvitamin D₃, exerts many physiological actions in the body, including effects on the nervous system. Studies of steroidogenesis in cells of the nervous system and elsewhere not only indicate that 1,25‐dihydroxyvitamin D₃ affects steroidogenic pathways but also suggest varying responses in different cell types. For example, 1,25‐dihydroxyvitamin D₃ stimulates the expression of aromatase in human glioma but not in human neuroblastoma cells or rat astrocytes. However, in astrocytes, 1,25‐dihydroxyvitamin D₃ suppresses 3β‐hydroxysteroid dehydrogenase and steroid 17‐hydroxylase/lyase. Other studies indicate cross‐talk between vitamin D signalling and signalling of oestrogens, progesterone or glucocorticoids. Reported data indicate synergistic effects of combinations of 1,25‐dihydroxyvitamin D₃ and other steroid hormones on neuroinflammation, neurite outgrowth and neuroprotection. Also, dysregulation of steroid pathways affecting brain cells is found in vitamin D deficiency. Thus, several studies suggest that active vitamin D may affect steroid hormone synthesis and/or signalling in the nervous system, although the potential mechanisms for these responses remain unclear. 1,25‐Dihydroxyvitamin D₃ suppresses proliferation in several cell types and is therefore of interest in cancer treatment. Also, epidemiological studies associate vitamin D levels with cancer risk or outcomes. Reported data on tumours of the nervous system are mainly on glioma, a common type of brain cancer. Expression of the vitamin D receptor in glioma tumours is associated with improved survival. Several studies show that 1,25‐dihydroxyvitamin D₃ and vitamin D analogues (synthetic vitamin D‐like compounds) suppress proliferation and migration in human vitamin D receptor‐expressing glioma cell lines. Studies on mechanisms for actions of 1,25‐dihydroxyvitamin D₃ or its analogues indicate regulation of cell cycle proteins and senescence markers. These compounds also show synergism in combination with other cancer therapies treating glioma. From the data available, vitamin D analogues emerge as interesting candidates for the future improved treatment of human glioma and possibly also other cancers of the nervous system.     

Vitamin D-independent expression of chick brain calbindin-D28K.

A combination of calbindin-D28K-specific cDNA probes and polyclonal antisera were used to investigate expression of the calbindin-D28K in the vitamin D-deficient avian brain in vivo in response to pharmacological doses of the vitamin D3 metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Serum calcium levels were stimulated (2-fold) and intestinal calbindin-D28K expression (between 10- and 30-fold) by 1,25(OH)2D3 (6.5 nmol/animal) after 12 h. In marked contrast, steady-state whole brain levels of calbindin-D28K as judged by enzyme-linked immunoassay (ELISA) remained constant. Northern gel analysis revealed that three species of calbindin-D28K mRNA (2.0, 2.6 and 3.1 kb) were present a priori in the vitamin D-deficient chick brain and that administration of pharmacological doses (6.5 nmol/animal) of 1,25(OH)2D3 failed to influence their relative abundance. Separate but parallel dot blot hybridization analyses also confirmed that brain calbindin-D28K-mRNA levels were not influenced by 1,25(OH)2D3. These experiments demonstrate at the molecular level that, in contrast to the intestine, the gene encoding calbindin-D28K in the brain is regulated by mechanism(s) or factors which are independent of vitamin D status.     

Synthesis of 1,25-dihydroxyvitamin D3 by rat brain macrophages in vitro

Cultured microglial cells were examined for their ability to metabolize 25-hydroxyvitamin D₃ (25-(OH) D₃). Upon exposure to lipopolysaccharide, microglial cells produced a vitamin D metabolite which comigrated with synthetic 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃) in two different systems of high performance liquid chromatography. This metabolite had the same affinity as synthetic 1,25-(OH)₂D₃ for the chick intestinal 1,25-(OH)₂D₃ receptor. Lipopolysaccharide-stimulated microglial cells incubated with 3 nM of 25-(OH) D₃ synthesized up to 5.76 fmol 1,25-(OH)₂D₃/8 × 10⁵ cells/2 hr. Microglial cells stimulated for 48 hr with interferon-γ also produced a significant amount of 1,25-(OH)₂D₃ (4.17 fmol/8 × 10⁵ cells/2 hr). In contrast, levels of 1,25-(OH)₂D₃ produced by resting microglial cells were barely detectable. It is concluded that activated brain macrophages may be committed to synthesize 1,25-(OH)₂D₃ in vitro. This raises the possibility that activation of microglial cells in vivo may be followed by an increase in the level of 1,25-(OH)₂D₃ in the central nervous system (CNS). These results support the emerging concept that the brain constitutes a target tissue for vitamin D metabolites. © 1994 Wiley-Liss, Inc.     

Effect of 1,25-dihydroxyvitamin D(3) on cultured mesencephalic dopaminergic neurons to the combined toxicity caused by L-buthionine sulfoximine and 1-methyl-4-phenylpyridine

A decrease in intracellular glutathione content may be related to the primary event in Parkinson's disease, so increasing the glutathione level may have a therapeutic benefit. The biologically active form of vitamin D, 1,25-dihydroxyvitamin D(3) [1, 25-(OH)(2)D(3)] has been recently reported to enhance the intracellular glutathione concentration in the central nervous system. Exposing rat cultured mesencephalic neurons for 24 hr to a mixture of L-buthionine sulfoximine (BSO) and 1-methyl-4-phenylpyridium ions (MPP(+)) resulted in a relatively selective damage to dopaminergic neurons. This damage has been accompanied by a reduction of intracellular glutathione levels. Low doses, i.e., 1-100 nM, of 1,25-(OH)(2)D(3) protect cultured dopaminergic neurons against this toxicity, although higher concentrations of this active form of vitamin D have been found to enhance the toxic effect. Generation of reactive oxygen species (ROS) by this toxicity has been attenuated in cultures being pretreated with low concentrations of 1,25-(OH)(2)D(3). Because the hormone increases the intracellular glutathione content in cultures, determining how this hormone suppresses ROS generation may involve the enhancement of the antioxidative system. These data suggest that low doses of 1,25-(OH)(2)D(3) are able to protect mesencephalic dopaminergic neurons against BSO/MPP(+)-induced toxicity that causes a depletion in glutathione content.     

Assessment of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D3 concentrations in male and female multiple sclerosis patients and control volunteers

Populations with insufficient ultraviolet exposure and who consume diets low in vitamin D have low vitamin D status (plasma 25-hydroxyvitamin D (25(OH)D) concentrations) and a reported higher incidence of multiple sclerosis (MS). The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is an effective anti-inflammatory molecule. No research to date has assessed 1,25(OH)2D3 concentrations in individuals with MS. In this study, plasma concentrations of 25(OH)D, 1,25(OH)2D3 and parathyroid hormone (PTH) were measured in 29 individuals with MS and 22 age- and sex-matched control volunteers. There were no significant differences in plasma PTH, 25(OH)D and 1,25(OH)2D3 concentrations between individuals with MS and control volunteers. Women with MS had significantly higher 25(OH)D and 1,25(OH)2D3 concentrations than men with MS (79.1+/-45.4 versus 50.2+/-15.3 nmol/L, P=0.019 and 103.8+/-36.8 versus 70.4+/-28.7 pmol/L, P=0.019, respectively). There was a significant positive correlation between 25(OH)D and 1,25(OH)2D3 concentrations in all subjects (r=0.564, P=0.000), but secondary analysis revealed that the correlation was driven by women with MS (r=0.677, P=0.001). Significant sex differences in vitamin D metabolism were observed and were most marked in individuals with MS, suggesting that vitamin D requirements may differ between the sexes, as well as by underlying disease state.     

Genetic analysis of vitamin D related genes in Canadian multiple sclerosis patients. Canadian Collaborative Study Group

The objective of this study was to investigate genes involved in the metabolism and function of vitamin D as candidate genes for genetic susceptibility to MS. Restriction fragment length polymorphisms and highly polymorphic microsatellite markers within or very close to the 1,25(OH)2D3 receptor (VDR) [12q14], the vitamin D binding protein (DBP) [4q12], and the 25(OH)D2 1alpha-hydroxylase [12q13] loci were analyzed for linkage or association with MS. We found no evidence for linkage or association of these candidate genes with MS in the Canadian population.     

1,25-dihydroxyvitamin D3 treatment decreases macrophage accumulation in the CNS of mice with experimental autoimmune encephalomyelitis

Sunlight, which is required for vitamin D biosynthesis, may be protective in multiple sclerosis (MS), due to the immunoregulatory functions of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), the hormonally active vitamin D metabolite. This hypothesis provided the impetus for the experiments reported here investigating mechanisms whereby 1,25-(OH)2D3 may inhibit murine experimental autoimmune encephalomyelitis (EAE). Severe EAE was induced, 1,25-(OH)2D3 or mock treatment was administered, and clinical disease, histopathological disease, and encephalitogenic cells in the central nervous system (CNS) were analyzed within 24-72 h of the treatment. The mock-treated mice remained paralyzed (stage 3 EAE) while most hormone-treated animals regained the partial use of both hind limbs (stage 2 EAE) within 72 h of treatment. A histopathological examination showed the hormone-treated mice had a 50% decrease in white matter and meningeal inflammation at 72 h post treatment. A flow cytometric analysis of cell surface markers on spinal cord cells recovered 24 h post treatment showed the mock-treated mice with EAE had about 7.0 +/- 2.3 million Mac-1+ cells/cord, whereas the hormone-treated mice had about 2.1 +/- 2.6 million Mac-1+ cells/cord, which was not significantly different from the unmanipulated control mice. Otherwise, the flow cytometric analysis detected no significant differences between the groups with respect to CD4+ or CD8+ T cells or B cells or macrophages in draining lymph nodes or spinal cords. These results are discussed with regard to possible fates for the 5 million Mac-1+ cells that were rapidly lost from the inflamed CNS in the 1,25-(OH)2D3-treated mice, and the possible beneficial effect of hormone treatment in resolving acute MS.     

Chronic vitamin D3 treatment protects against neurotoxicity by glutamate in association with upregulation of vitamin D receptor mRNA expression in cultured rat cortical neurons

The vitamin D receptor (VDR) is believed to mediate different biologic actions of vitamin D3, an active metabolite of vitamin D, through regulation of gene expression after binding to specific DNA-response element (VDRE) on target genes. To further understand roles of both vitamin D3 and VDR in the central nervous system, we examined VDRE binding in nuclear extracts prepared from discrete rat brain regions and cultured rat cortical neurons by electrophoretic mobility shift assay. The highest activity of VDRE binding was found in the cerebellum among other brain regions examined, but sequence specific by taking into consideration the efficient competition with excess unlabeled VDRE but not with mutated VDRE. On in situ hybridization analysis, cells stained for VDR mRNA were abundant in neuron-enriched areas of cerebral cortex, hippocampus and cerebellar cortex in the mouse brain. Chronic treatment of vitamin D3 increased the expression of microtubule-associated protein-2, growth-associated protein-43 and synapsin-1 in cultured rat cortical neurons, suggesting a trophic role of vitamin D3 in differentiation and maturation of neurons. Neuronal cell death by brief glutamate exposure was significantly protected in cultured cortical neurons chronically treated with vitamin D3. Parallel studies showed that VDR mRNA was significantly upregulated 12-24 hr after brief glutamate exposure in cultured neurons chronically treated with vitamin D3, but not in those with vehicle alone. Our results suggest that vitamin D3 may play a role in mechanisms relevant to protective properties against the neurotoxicity of glutamate through upregulation of VDR expression in cultured rat cortical neurons.     

Vitamin D receptor gene silencing effects on differentiation of myogenic cell lines

Introduction: The active form of vitamin D (1,25‐dihydroxy‐vitamin D3) is known to increase fast‐type myosin heavy chain expression in differentiated myogenic cell lines. The mechanisms for this effect are not fully understood. The aim of this study was to determine the role of signals transmitted through the vitamin D receptor (VDR) during differentiation of myoblasts. Methods: Electroporation was used to introduce VDR siRNA molecules into C2C12 and G8 murine myoblast cell lines. Gene and protein expression profiles of VDR‐gene silenced cells were analyzed in vitro. Results: Suppressing VDR expression by RNA interference resulted in inhibition of myogenic differentiation of C2C12 and G8 cell lines at both mRNA and protein levels. Conclusions: Our results suggest that myoblasts require signals transmitted through VDR for differentiation into myocytes and emphasize the importance of VDR expression in skeletal muscles for maintaining muscle volume in the elderly. Muscle Nerve 49 : 700–708, 2014     

Vitamin D inquiry in hippocampal neurons: consequences of vitamin D-VDR pathway disruption on calcium channel and the vitamin D requirement

Vitamin D receptor (VDR) and the enzymes involved in bioactivation of vitamin D, shown to be expressed in the central nervous system, particularly in areas affected by neurodegenerative disorders, especially in hippocampus. We showed that amyloid beta (Aβ) pathology includes VDR protein depletion and vitamin D-VDR pathway disruption either induced by Aβ or by VDR siRNA have very similar effects on cortical neurons. The goal of this study is to show the presence of 25 hydroxy vitamin D3-24 hydroxylase (24OHase) which is essential for vitamin D catabolism in hippocampal and cortical neurons. Additional goal is to compare the expression pattern of VDR and 24OHase both in hippocampal and in cortical neurons and to investigate the effects of VDR suppression in hippocampal neurons in order to see whether similar mechanisms work in hippocampus and cerebral cortex. Primary neuronal cultures were prepared from Sprague–dawley rat embryos. qRT-PCR was performed to determine VDR, 24OHase, and LVSCC-A1C mRNA expression levels. Cytotoxicity levels were determined by ELISA. Our findings illustrate that 24OHase mRNA was present both in hippocampal and in cortical neurons. VDR and 24OHase mRNA were higher in hippocampal neurons than the cortical ones. LVSCC-A1C mRNA levels increased in hippocampal neurons when VDR is down-regulated. Our results indicate that hippocampal neurons response to VDR suppression similar as cortical neurons, regarding calcium channel regulation. Higher gene expression of 24OHase and VDR might indicate “higher requirement of vitamin D” in hippocampus and potential consequences of vitamin D deficiency in cognitive decline, neurodegeneration, and Alzheimer’s disease.     

Administration of 1,25-dihydroxyvitamin D3 results in the elevation of hippocampal seizure threshold levels in rats

Electrical stimulation of the dorsal hippocampal formation of the rat was employed to determine the effect of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), the hormonal form of vitamin D, on induced seizure thresholds. Stereotaxic injection of 100 micrograms or 50 micrograms 1,25-dihydroxyvitamin D3 in 2 microliter propylene glycol into the hippocampus resulted in a significant elevation in seizure threshold in all animals treated. 1,25-dihydroxyvitamin D3-induced increases were noted within 5-10 min and lasted at least 120-180 min after injection when the experiments were terminated. Intravenous injection of 1,25-(OH)2D3 also resulted in a significant elevation of seizure threshold; however, the increase was transient, lasting only 30 min. This effect was specific since 200 micrograms vitamin D3 or 200 micrograms 25-hydroxyvitamin D3 (25-(OH)D3), injected into the hippocampus, had no effect on seizure threshold levels. This investigation represents the first direct demonstration of a role for 1,25-(OH)2D3 in the regulation of seizure activity and suggests, along with the previously demonstrated presence of immunoreactive vitamin D-dependent calcium binding protein and receptors for 1,25-dihydroxyvitamin D3 in the brain, that the vitamin D endocrine system may play a significant role in the physiological mechanisms underlying convulsive disorders.     

Rag-1-dependent cells are necessary for 1,25-dihydroxyvitamin D(3) prevention of experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a demyelinating disease involving genetic and environmental risk factors. Geographic, genetic, and biological evidence suggests that one environmental risk factor may be lack of vitamin D. Here, we investigated how 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) inhibits experimental autoimmune encephalomyelitis (EAE), an MS model. The experiments used adoptive transfer of TCR-transgenic (TCR1) cells specific for myelin basic protein (MBP) peptide into unprimed recipients. When unprimed TCR1 splenocytes were transferred, and the recipients were immunized with peptide, the mock-treated mice developed EAE, but the 1,25-(OH)(2)D(3)-treated recipients remained disease-free. Both groups had TCR1 T cells that proliferated in response to MBP Ac1-11 and produced IFN-gamma but not IL-4 in the lymph node. In the central nervous system (CNS), the mock-treated mice had activated TCR1 T cells that produced IFN-gamma but not IL-4, while the 1,25-(OH)(2)D(3)-treated mice had TCR1 T cells with a non-activated phenotype that did not produce IFN-gamma or IL-4. When activated TCR1 T cells producing IFN-gamma were transferred into unprimed mice, the mock-treated and the 1,25-(OH)(2)D(3)-treated recipients developed EAE. Likewise, the 1,25-(OH)(2)D(3) did not inhibit Th1 cell IFN-gamma production or promote Th2 cell genesis or IL-4 production in vitro. Finally, the 1,25-(OH)(2)D(3) inhibited EAE in MBP-specific TCR-transgenic mice that were Rag-1(+), but not in animals that were Rag-1-null. Together, these data refute the hypothesis that the hormone inhibits Th1 cell genesis or function directly or through an action on antigen-presenting cells, or promotes Th2 cell genesis or function. Instead, the evidence supports a model wherein the 1,25-(OH)(2)D(3) acts through a Rag-1-dependent cell to limit the occurrence of activated, autoreactive T cells in the CNS.