Decreased expression of vitamin D receptor may contribute to the hyperimmune status of patients with acquired aplastic anemia

Acquired aplastic anemia (AA) is an immune‐mediated bone marrow failure syndrome. 1α,25‐Dihydroxyvitamin D₃ [1,25(OH)₂D₃], the biologically active metabolite of vitamin D, is a critical modulator of immune response via binding with vitamin D receptor (VDR). Previous studies have established that 1,25(OH)₂D₃ and VDR were involved in the pathogenesis of some autoimmune diseases. In this study, we evaluated the involvement of 1,25(OH)₂D₃ and VDR on T‐cell responses in AA. Plasma 25(OH)D₃ levels were comparable between patients with AA and healthy controls. Surprisingly, VDR mRNA was significantly lower in untreated patients with AA than in healthy controls. Subsequent in vitro experiments revealed that 1,25(OH)₂D₃ treatment suppressed the proliferation of lymphocytes and inhibited the secretion of interferon‐γ, tumor necrosis factor‐α, and interleukin‐17A, meanwhile promoting the production of transforming growth factor‐β1 in patients with AA. Moreover, 1,25(OH)₂D₃ inhibited the differentiation of type 1 and Th17 cells but induced the differentiation of type 2 and regulatory T cells. Interestingly, VDR mRNA was elevated in healthy controls after 1,25(OH)₂D₃ treatment, but not in patients with AA. In conclusion, decreased expression of VDR might contribute to the hyperimmune status of AA and appropriate vitamin D supplementation could partly correct the immune dysfunction by strengthening signal transduction through VDR in patients with AA.     

Two mutations causing vitamin D resistant rickets: modelling on the basis of steroid hormone receptor DNA-binding domain crystal structures

OBJECTIVE Hereditary vitamin D resistant rickets (HVDRR) has been shown to be due to mutations in the gene encoding the vitamin D receptor (VDR). In two patients with the characteristic phenotype we have investigated the functional defect and sequenced the VDR cDNA. We report two new mutations in the DNA binding domain of the VDR gene and we have used the crystal-lographic structure of the glucocorticold and oeltrogen receptors (GR and ER respectively) as models to explain the stereochemical consequences of these mutations. DESIGN Patient and control cell lines prepared from skin fibroblasts were used to measure binding of 1,25dlhydroxyvltamln D₃ (1,25(OH)₂D₃) and functional responses to this hormone. These cells were also used to Isolate VDR mRNA from which cDNA was prepared and sequenced. VDR cDNA from affected and control patlents was also transfected into receptor defective cells to analyse further functional responses to 1,25(OH)₂D₃. Computer analysis of mutations in the VDR gene was carried out using the glucocorticold and oestrogen receptors as model systems. PATIENTS Two patients with HVDRR from unrelated families. MEASUREMENTS Cytosollc binding and nuclear association of 1,25(OH)₂D₃ were determined in control and affected patients, and functional response to 1,25(OH)₂D₃ was assessed by measurement of 2bhydroxyvltamln D-24-hydroxylase activity (24-hydroxylase). VDR cDNA was sequenced and transfected into VDR-deficient CV-1 cells for further analysis of functional response to 1,25(OH)₂D₃ following cotransfection with a chloramphenicol acetyltransferase (CAT) reporter plasmid. RESULTS Cells from HVDRR patients I and II showed detectable numbers of VDR with normal hormone binding. However, unlike controls, the HVDRR cells did not show induction of 24-hydroxylase activity following treatment with 1,25(OH)₂D₃. Sequencing of cDNA revealed single mutations, in patient I (Phe44 → IIe) and in patient II (Lys42 → Glu). Both these residues are conserved in the steroid/thyroid hormone receptor superfamily and stereochemical analysis has been used to deduce the importance of these amino acids and the deleterious effect of these and other mutations in the DNA-binding domain of the VDR. CONCLUSIONS Two new mutations in the vitamin D receptor which cause hereditary vitamin D resistant rickets have been described and using molecular modelling we have been able to analyse the genesis of this inherited disease at the level of stereochemistry.     

Vitamin D treatment of senescence accelerated mice (SAM-P/6) induces several regulators of stromal cell plasticity

In an attempt to understand the regulation of bone marrow multipotential cells plasticity in vivo, we treated 4-month-old SAM-P/6 mice with a constant infusion of either 18 pmol/24 h of 1,25(OH)₂D₃ or vehicle alone for 6 weeks. In vehicle treated animals 78% ± 4 adipose volume vs. total volume was stained positive with oil red O as compared to only 32 ± 3% in 1,25(OH)₂D₃treated animals (P < 0.001). Furthermore, we aimed to identify the changes in gene expression induced by 1,25(OH)₂D₃in bone marrow cells by analyzing a set of 5440 genes in the NIA 15K Mouse cDNA microarray. Overall, a coordinated regulation of genes which both stimulate osteoblastogenesis and inhibit adipogenesis was observed in 1,25(OH)₂D₃-treated mice when compared to vehicle treated mice. In summary, this study illustrates the anti-adipogenic effect of 1,25(OH)₂D₃in bone cells and identifies some of the possible key signals involved in bone cell plasticity.     

Identification of a novel mutation in hereditary vitamin D resistant rickets causing exon skipping

OBJECTIVE Hereditary vitamin D resistant rickets (HVDRR) is an autosomal recessive disorder resulting in target organ resistance to the actions of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃). In many cases, this disorder has been shown to be due to mutations in the gene encoding vitamin D receptors (VDR). In a patient with characteristic features of this disorder, we investigated the functional defect and sequenced the coding region of the gene for mutations. DESIGN Skin fibroblasts from patient and control were used to measure binding of 1,25(OH)₂D₃ and functional responses to the hormone. These cells were also used to prepare RNA from which cDNA was prepared and sequenced. Furthermore, genomic DNA was prepared from the fibroblasts and the intronlexon boundarles sequenced. PATIENT A child with classic features of HVDRR with alopecia diagnosed as having rickets due to resistance to 1,25(OH)₂D₃. MEASUREMENTS Nuclear association of 1,25(OH)₂D₃ was determined in patient and control cells and the functional response to 1,25(OH)₂D₃ was assessed by measurement of 25-hydroxyvitamln D-24-hydroxylase(24-hydroxylase) activity. VDR cDNA and genomic DNA prepared from patient and control cells were sequenced. RESULTS Cells from the patient with HVDRR had undetectable amounts of VDR compared to control cells and did not show induction of 24-hydroxylase activity following treatment with 1,25(OH), D₃. Sequencing of the VDR coding region after RT-PCR of RNA revealed an absence of exon 4 in patient RNA which was not due to a deletion in genomic DNA but was caused by exon skipping during RNA processing. In addition, the deletion of exon 4 sequences from RNA leads to a frameshift in translation resulting in a premature stop codon. Amplification of genomic DNA around the intron/exon boundary of exon 4 revealed a point mutation in the 5’donor splice site of intron 4. CONCLUSION In this study, we have identified a novel mutation in the gene for vitamin D receptors in a patient with the Characteristic phenotype of hereditary vitamin D resistant rickets. The mutation at the + 5 position in intron 4 is most likely to cause skipping of exon 4 in this patient.     

Inhibition by 1,25 dihydroxyvitamin D3 of chemically induced erythroid differentiation of K562 leukemia cells.

The physiologically active form of vitamin D, 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], was found to inhibit erythroid differentiation of human leukemic K562 cells. Differentiation was induced by 1 mumol/L arabinocytosine (Ara-C), 40 mumol/L tiazofurin, 1 mumol/L aphidicolin, or 1 mumol/L hydroxyurea, and was monitored daily by the appearance of hemoglobin in an increasing proportion of cells. Pretreatment for 48 hours with 2.4 x 10(-8) mol/L 1,25(OH)2D3, a concentration that is also optimal for induction of monocytic differentiation of HL-60 cells, reproducibly inhibited subsequent induction of erythroid differentiation by all of the above inducers, and modified the morphologic changes that Ara-C produced in these cells. The inhibition of hemoglobinization was approximately 50% irrespective of the degree of differentiation produced by the various inducers, but growth inhibition associated with exposure to the inducers was not affected by 1,25(OH)2D3. Similar inhibition of differentiation by 1,25(OH)2D3 was observed in mouse erythroleukemia cells MEL-D1B treated with 5 mmol/L hexamethylenebisacetamide. The inhibitory effect of 1,25(OH)2D3 on erythroid differentiation of K562 cells was abrogated by cyclohexamide (20 micrograms/mL), an inhibitor of protein synthesis. The mRNA for 1,25(OH)2D3 receptor (VDR) was detected in K562 cells, and was downregulated by a 96-hour exposure to 1,25(OH)2D3 or a 48-hour exposure to Ara-C. The presence of VDR mRNA suggests a physiologic role for 1,25(OH)2D3 in K562 cells that are precursors of erythroid cells. This role is perhaps to shift the pathways of differentiation from the erythroid to the monocytic lineage.     

Hormonal control of skeletal and mineral homeostasis

The interactions of parathyroid hormone (PTH), calcitonin (CT) and vitamin D in mineral and skeletal homeostasis are discussed. Evidence is presented that not only does PTH control the renal synthesis of 1,25-dihydroxyvitamin D₃ (1,25-(OH)₂D₃), the biologically active form of vitamin D₃, but also that 1,25-(OH)₂D₃ acts as a feedback inhibitor of PTH secretion. Thus, the plasma concentrations of both 1,25-(OH)₂D₃ and calcium are controlled by the actions of PTH, and both of these metabolites operate as feedback inhibitors of PTH secretion. At the level of bone cell function, present evidence indicates that both PTH and CT influence the size of the osteoclast and osteoblast cell pools and the respective activities of the cells in these pools: PTH increases the size of the osteoclast pool and the activity of the cells in this pool; CT has the opposite effects; and 1,25-(OH)₂D₃ seems to increase the activity of these cells as well as those in the osteoblast pool without altering the size of these pools. These different effects are interpreted in terms of the actions of these three hormonal agents on the concentration of three second messengers, cyclic adenosine monophosphate (AMP), calcium ion and monohydrogen phosphate, within the cell.     

Effects of analogs of 1,25(OH)2 Vitamin D3 on the proliferation and differentiation of the human chronic myelogenous leukemia cell line,RWLeu-4

Summary We evaluated the proliferative and differentiative effects of analogs of 1,25(OH)₂ vitamin D₃ [1,25(OH)₂D₃] on a chronic myelogenous leukemia cell line, RWLeu-4, which is growth-inhibited and differentiates in response to 1,25(OH)₂D₃ (ED₅₀-3-10 nM). Side-chain-fluorinated analogs were more potent (ED₅₀=0.7–2 nM) while most of those with altered saturation of the D ring or side-chain carbon-carbon bonds were equally or less effective than 1,25(OH)₂D₃. However, the two analogs with either two additional double bonds or an extra double and triple bond in the D ring had greater antiproliferatiive activity [1,25(OH)₂-16,23-diene D₃ (ED₅₀=2.7 nM) and 1,25(OH)₂-16-ene-23-yne D₃ (ED₅₀=0.7 nM)]. Since the latter of these has been reported to be less potent at mobilizing calcium than 1,25(OH)₂D₃, it (or a similar compound) may be a candidate for clinical use as an antineoplastic agent.Abbreviations 1,25(OH)₂D₃ 1,25-(OH)₂ vitamin D₃ - CML chronic myelogenous leukemia - NBT nitroblue tetrazolium - ED₅₀ 50% effective dose - IC₅₀ 50% inhibitory concentration     

25-Hydroxyvitamin D-1α Hydroxylase: Studies in Mouse Models and Implications for Human Disease

Genetic mouse models with targeted deletion (“knockout”) of the 25-hydroxyvitamin D-1alpha-hydroxylase gene [1α(OH)ase^−/−], as well as with targeted deletion of the VDR gene, when exposed to different dietary regimens, have provided considerable insight into the molecular regulation of skeletal physiology by the 1,25(OH)₂D/VDR system. These regimens induced different phenotypic changes and demonstrated that parathyroid gland size and the development of the cartilaginous growth plate were each co-ordinately regulated by calcium and by 1,25(OH)₂D, and that parathyroid hormone (PTH) secretion and mineralization of bone reflected ambient calcium (and phosphorus) levels rather than the direct actions of the 1,25(OH)₂D/VDR system. In contrast, increased calcium absorption, optimal osteoblastogenesis, and baseline bone formation were observed to be modulated by 1,25(OH)₂D/VDR signaling. These bone anabolic effects of endogenous 1,25(OH)₂D were evident in neonatal mice as well as in older animals, and exogenous 1,25(OH)₂D₃ was also found to stimulate trabecular and cortical bone formation in neonatal double homozygous 1α(OH)ase^−/−PTH^−/− mice. Furthermore, the anabolic effect of exogenously administered PTH appeared to be partly dependent on the stimulation of endogenous 1,25(OH)₂D. Genetic mouse models have also been employed to study extra-skeletal actions modulated by the 1,25(OH)₂D/VDR system. For example, increased blood pressure, activation of the renin/angiotensin system, myocardial hypertrophy, and cardiac dysfunction were observed in 1αOHase^−/− mice, and these alterations could be prevented by treatment with 1,25(OH)₂D₃. These models allow controlled examination of the regulation of both skeletal and extra-skeletal pathophysiology associated with 1,25(OH)₂D deficiency which appear to be relevant to humans, and facilitate studies to prevent and treat these disorders by active vitamin D forms.     

Third F. Raymond Keating, Jr., Memorial Symposium--parathyroid hormone, calcitonin and vitamin D: clinical considerations. II. Vitamin D--1973

The recent advances in our understanding of the functional metabolism of vitamin D to 25-hydroxyvitamin D (25-(OH)D) and subsequently to 1,25-dihydroxyvitamin D (1,25-(OH)₂D) are presented with a review of current views on the regulation of vitamin D metabolism at the 25-hydroxylation and 1-hydroxylation stages. It seems clear that, physiologically, the latter regulation is governed by parathyroid hormone (PTH) under conditions of hypocalcemia and by serum inorganic phosphorus levels under conditions from normal to hypercalcemia. The molecular mechanism of the regulation of the renal hydroxylations remains unknown.The metabolism of 25-(OH)D₃ to 24,25-(OH)₂D₃ and further to 1,24,25-(OH)₃D₃ has been established. The significance of these reactions is unknown, but 1,24,25-(OH)₃D₃ preferentially stimulates intestinal calcium transport.The mechanism whereby 1,25-(OH)₂D₃ stimulates intestinal calcium transport and the receptors of this metabolite in intestine are discussed. In addition, the application of the metabolites of vitamin D to clinical problems has been considered and 1α-(OH)D₃, an important analog of 1,25-(OH)₂D₃, has been introduced as a potentially important therapeutic compound.     

1,25‐dihydroxyvitamin D3 modulates Th17 polarization and interleukin‐22 expression by memory T cells from patients with early rheumatoid arthritis

Objective

To examine the immunologic mechanism by which 1,25‐dihydroxyvitamin D₃ (1,25[OH]₂D₃) may prevent corticosteroid‐induced osteoporosis in patients with early rheumatoid arthritis (RA), with a focus on T cell biology.

Methods

Peripheral blood mononuclear cells (PBMCs) and CD4+CD45RO+ (memory) and CD4+CD45RO− (non‐memory) T cells separated by fluorescence‐activated cell sorting (FACS) from treatment‐naive patients with early RA were stimulated with anti‐CD3/anti‐CD28 in the absence or presence of various concentrations of 1,25(OH)₂D₃, dexamethasone (DEX), and 1,25(OH)₂D₃ and DEX combined. Levels of T cell cytokines were determined by enzyme‐linked immunosorbent assay and flow cytometry.

Results

The presence of 1,25(OH)₂D₃ reduced interleukin‐17A (IL‐17A) and interferon‐γ levels and increased IL‐4 levels in stimulated PBMCs from treatment‐naive patients with early RA. In addition, 1,25(OH)₂D₃ had favorable effects on tumor necrosis factor α (TNFα):IL‐4 and IL‐17A:IL‐4 ratios and prevented the unfavorable effects of DEX on these ratios. Enhanced percentages of IL‐17A– and IL‐22–expressing CD4+ T cells and IL‐17A–expressing memory T cells were observed in PBMCs from treatment‐naive patients with early RA as compared with healthy controls. Of note, we found no difference in the percentage of CD45RO+ and CD45RO− cells between these 2 groups. Interestingly, 1,25(OH)₂D₃, in contrast to DEX, directly modulated human Th17 polarization, accompanied by suppression of IL‐17A, IL‐17F, TNFα, and IL‐22 production by memory T cells sorted by FACS from patients with early RA.

Conclusion

These data indicate that 1,25(OH)₂D₃ may contribute its bone‐sparing effects in RA patients taking corticosteroids by the modulation of Th17 polarization, inhibition of Th17 cytokines, and stimulation of IL‐4.

     

Parathyroid hormone down-regulates 1,25-dihydroxyvitamin D receptors (VDR) and VDR messenger ribonucleic acid in vitro and blocks homologous up-regulation of VDR in vivo

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3) is a known up-regulator of 1,25(OH)2D3 receptor (VDR) both in vitro and in vivo. However, a 5- to 10-fold increase in plasma 1,25(OH)2D3 induced by dietary calcium deficiency does not result in up-regulation of intestinal VDR, and kidney VDR is down-regulated. Under certain physiological stresses, an increase in plasma PTH precedes increased plasma 1,25(OH)2D3. Therefore, the present study examined the effect of PTH on VDR regulation in vitro in ROS 17/2.8 cells and in vivo in male Holtzman rats. Treatment of ROS cells with PTH (0-5 nM) resulted in a dose and time-dependent decline in VDR from 95 +/- 9 to 35 +/- 5 fmol/mg protein at 18 h of exposure. The ED50 for PTH was 1 nM. This decline in VDR protein was attended by a 50% decline in VDR messenger RNA (mRNA). The PTH-mediated down-regulation of VDR occurred without affecting the affinity of VDR for 1,25(OH)2D3 as determined by Scatchard analysis. Also, the effect of PTH on VDR regulation was specific since cell glucocorticoid receptor concentration was not affected by PTH treatment. In accompanying experiments, 1,25(OH)2[3H]D3 treatment of ROS cells was shown to result in a 3- to 4-fold increased expression of VDR and VDR mRNA. The simultaneous addition of PTH and 1,25(OH)2[3H]D3 resulted in inhibition of the 1,25(OH)2[3H]D3-mediated up-regulation of VDR and VDR mRNA. Similarly, PTH also inhibited heterologous up-regulation of VDR and VDR mRNA induced by retinoic acid. In in vivo experiments, rats infused for 5 days with 1,25(OH)2D3 (1.5 ng/h) increased their expression of intestinal VDR, kidney VDR, and kidney 24-hydroxylase by 31, 336, and 4000%, respectively. Coinfusion of PTH (1.8 IU/h) along with 1,25(OH)2D3 completely inhibited the 1,25(OH)2D3-mediated increases in intestinal VDR and kidney 24-hydroxylase and reduced the 1,25(OH)2D3-mediated up-regulation of kidney VDR by more than half. These data suggest that PTH is a potent down-regulator of VDR and that PTH and 1,25(OH)2D3 have opposing effects on the expression of certain genes.     

Dexamethasone enhances vitamin D-24-hydroxylase expression in osteoblastic (UMR-106) and renal (LLC-PK1) cells treated with 1alpha,25-dihydroxyvitamin D3

Chronic glucocorticoid therapy causes rapid bone loss and clinical osteoporosis. We previously found that dexamethasone, a potent glucocorticoid, increased renal expression of vitamin D-24-hydroxylase, which degrades such vitamin D metabolites as 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ (1,25[OH]₂D₃). We therefore investigated the mechanisms of this increase in UMR-106 osteoblast-like cells and LLC-PK₁ kidney cells. To induce 24-hydroxylase expression, 1,25(OH)₂D₃ (10⁻⁷ M) and dexamethasone were added simultaneously to the medium of LLC-PK₁ cells, and 24 h before dexamethasone treatment, 1,25(OH)₂D₃ was added to the medium of UMR-106 cells. Dexamethasone dose dependently increased 24-hydroxylase mRNA and enzymatic activity in 1,25(OH)₂D₃-treated LLC-PK₁ and UMR-106 cells. Maximal stimulation was observed with 10⁻⁶ M dexamethasone in both cell lines. The addition of 10⁻⁶ M dexamethasone significantly increased the abundance of 24-hydroxylase mRNA by 24 and 8 h in 1,25(OH)₂D₃-treated LLC-PK₁ and UMR-106 cells, respectively. Stimulation for dexamethasone in UMR-106 cells persisted for up to 48 h. Dexamethasone stimulation of 24-hydroxylase mRNA expression in UMR-106 cells was abolished by pretreatment with cycloheximide, an inhibitor of protein synthesis. Northern and Western analyses indicated that 10⁻⁶ M dexamethasone markedly increased the abundance of c-fos mRNA at 20 min and c-fos protein concentration at 60 min in 1,25(OH)₂D₃-treated UMR-106 cells but only slightly induced the abundance of c-jun mRNA. The addition of phorbol 12-myristate 13-acetate increased mRNA expression for both c-fos and 24-hydroxylase in 1,25(OH)₂D₃-treated UMR-106 cells. The effect of dexamethasone on 24-hydroxylase mRNA expression was blocked by RO31-8220, a specific inhibitor of protein kinase C. Thus, dexamethasone in the presence of 1,25(OH)₂D₃ enhances expression of 24-hydroxylase in UMR-106 osteoblastic cells via new protein synthesis. The mechanism of this effect appears to involve activation of the AP-1 site by increased c-fos protein.     

1,25-Dihydroxyvitamin D3 selectively and reversibly impairs T helper-cell CNS localization

Pharmacologic targeting of T helper (T_H) cell trafficking poses an attractive opportunity for amelioration of autoimmune diseases such as multiple sclerosis (MS). MS risk is associated with vitamin D deficiency, and its bioactive form, 1,25-dihydroxyvitamin D3 [1,25(OH)₂D₃], has been shown to prevent experimental autoimmune encephalomyelitis, a mouse model of MS, via an incompletely understood mechanism. Herein, we systematically examined 1,25(OH)₂D₃ effects on T_H cells during their migration from the lymph nodes to the CNS. Our data demonstrate that myelin-reactive T_H cells are successfully generated in the presence of 1,25(OH)₂D₃, secrete proinflammatory cytokines, and do not preferentially differentiate into suppressor T cells. These cells are able to leave the lymph node, enter the peripheral circulation, and migrate to the s.c. immunization sites. However, T_H cells from 1,25(OH)₂D₃-treated mice are unable to enter the CNS parenchyma but are instead maintained in the periphery. Upon treatment cessation, mice rapidly develop experimental autoimmune encephalomyelitis, demonstrating that 1,25(OH)₂D₃ prevents the disease only temporarily likely by halting T_H cell migration into the CNS.Multiple sclerosis (MS) is an immune-mediated demyelinating disorder of the central nervous system (CNS). Whereas its etiology is unknown, a number of environmental and genetic factors contribute to the risk of developing MS (1). Epidemiologic studies demonstrate a strong correlation between MS risk and vitamin D deficiency (2). Higher levels of circulating 25-hydroxyvitamin D₃ have been recently shown to correlate with a reduction in the number of new T2 and gadolinium-enhancing lesions (3). Moreover, the bioactive form of vitamin D, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], can completely prevent and treat established experimental autoimmune encephalomyelitis (EAE) (4), a murine MS model. Nonetheless, the mechanism of 1,25(OH)₂D₃^’s in vivo action remains incompletely understood.The pathogeneses of MS and EAE require localization of self-reactive T cells to the CNS. Myelin-specific effector T cells are primed in the lymph nodes (LNs) and subsequently travel a complex route and arrive at the CNS barriers. There, a sequential interaction with a multitude of endothelial adhesion molecules and chemokines leads to T helper (T_H) cell migration into the CNS parenchyma. Several ligand/receptor pairs have been implicated in the pathogeneses of MS and EAE (5, 6). Therefore, manipulation of molecules involved in T-cell trafficking poses an attractive opportunity for therapeutic interventions. Recent studies have highlighted a role for vitamin D in immune cell migration (7–10), but whether 1,25(OH)₂D₃ affects trafficking of lymphocytes in MS and/or EAE has not been investigated.A recent study using conditional deletion of the vitamin D receptor (VDR) has demonstrated that 1,25(OH)₂D₃ prevents EAE by acting directly on the encephalitogenic T_H cells (11). We hypothesized that 1,25(OH)₂D₃ could be inhibiting encephalitogenic T cells via regulation of molecules involved in their trafficking into the CNS. Herein, we provide unique evidence that 1,25(OH)₂D₃ prevents EAE by modulating the migratory phenotype of the pathogenic T_H cells without affecting their priming or effector functions. Our findings further emphasize that 1,25(OH)₂D₃ is a reversible immune modulator, as treatment cessation leads to rapid disease onset. These findings may have important implications for the use of vitamin D as an adjunct treatment of immune-mediated diseases such as MS.     

Treatment of resting zone chondrocytes with bone morphogenetic protein-2 induces maturation into a phenotype characteristic of growth zone chondrocytes by downregulating responsiveness to 24,25(OH)2D3 and upregulating responsiveness to 1,25-(OH)2D3

To determine if bone morphogenetic protein-2 (BMP-2) can induce the endochondral maturation of resting zone (RC) chondrocytes, confluent fourth-passage cultures of these cells were pretreated for 24, 36, 48, 72, or 120 h with recombinant human BMP-2. At the end of pretreatment, the media were replaced with new media containing 10⁻¹⁰–10⁻⁸ M 1,25-(OH)₂D₃ or 10⁻⁹–10⁻⁷ M 24,25-(OH₂)D₃, and the cells incubated for an additional 24 h. This second treatment was chosen, because prior studies had shown that the more mature growth zone (GC) chondrocytes and RC cells respond to 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ in distinctly different ways with respect to the parameters examined. The effect of BMP-2 pretreatment on cell maturation was assessed by measuring alkaline phosphatase specific activity (ALPase). In addition, changes in matrix protein production were assessed by measuring collagen synthesis, as well as [³⁵S]-sulfate incorporation into proteoglycans. When RC cells were pretreated for 72 or 120 h with BMP-2, treatment with 1,25-(OH)₂D₃ caused a dose-dependent increase in ALPase specific activity and collagen synthesis, with no effect on proteoglycan sulfation. RC cells pretreated with 1,25-(OH)₂D₃ responded like RC cells that had not received any pretreatment. RC cells normally respond to 24,25-(OH)₂D₃; however, RC cultures pretreated for 72 or 120 h with BMP-2 lost their responsiveness to 24,25-(OH)₂D₃. These results indicate that BMP-2 directly regulates the differentiation and maturation of RC chondrocytes into GC chondrocytes. These observations support the hypothesis that BMP-2 plays a significant role in regulating chondrocyte maturation during endochondral ossification.     

Two distinct cell lines derived from a human osteosarcoma

Summary Two cell lines were established from a human osteosarcoma transplanted into athymic nude mice after the second (O9N2) and fifth passages (HuO9). Both cell lines expressed 1,25(OH)₂D₃-responsive alkaline phosphatase activity and produced tumors in the dorsum of nude mice that were histologically similar to the original tumor. However, the morphological and growth characteristics of the two cell lines differed. O9N2 cells were large and polygonal, whereas HuO9 cells showed spindle shapes. HuO9 cells had a higher growth rate and saturation density than O9N2 cells. The c-myc oncogene was amplified 4-to 8-fold in HuO9 cells but not in O9N2 cells. Both cell lines had a homozygous internal deletion, lacking the 7.4-kb HindIII fragment in the Rb gene. The results suggest the importance of the c-myc oncogene in the growth and morphological control of human osteosarcoma cells and of the Rb gene in the pathogenesis of the tumor.Abbreviations used ALP alkaline phosphatase - PBS phosphatebuffered saline - 1,25(OH)₂D₃ 1,25-dihydroxyvitamin D₃ - Rb gene retinoblastoma gene     

Specific binding of 1,25 dihydroxyvitamin D3 in lymphocytes

We examined ten cellular or tissue sources of lymphocytes for specific binding of 1,25(OH)₂D₃, the hormonally active form of vitamin D₃. A specific-binding protein was found in three of these sources. Scatchard analysis of cytosol from a follicular lymphoma cell line revealed binding sites with a Kd of 7.0 × 10^?11 and a receptor concentration of 6.6 fmol/mg protein. Sucrose density centrifugation of ³H-1,25(OH)₂D₃ labeled cytosol showed a 3.75 peak which was absent in cytosols incubated with excess nonradioactive 1,25(OH)₂D₃. The relative amounts of vitamin D₃ metabolites required to displace 50% of the specifically bound ³H-1,25(OH)₂D₃ were 1,25(OH)₂D₃: 1,24,25(OH)₃D₃: 25(OH)D₃: 24,25(OH)₂D₃ = 1: 180: 1000: 2700. Excess vitamin D₃, cortisol, and estradiol failed to displace ³H-1,25(OH)₂D₃. Scatchard analysis of spleen cytosol from a patient with prolymphocytic transformation of chronic lymphocytic leukemia demonstrated a binding protein with a Kd of 1.2 × 10^?10 and a receptor concentration of 0.2 fmol/mg protein. DNA cellulose binding confirmed the presence of the specific-binding protein in this cytosol. Specific binding of ³H-1,25(OH)₂D₃ was also quantitated in a cell line from a patient with Burkitt's lymphoma with a Kd of 0.3 × 10^?10 and a receptor concentration of 29.6 fmol/mg protein. No specific binding of ³H-1,25(OH)₂D₃ was observed in lymphocytes from seven other malignant and nonmalignant sources. These results are the first to demonstrate a specific-binding protein for 1,25(OH₂D₃ in lymphocytes from tissue and from these specific cell lines. The presence of this protein in some lymphocytes but not others may reflect the state of activation of the lymphocytes.     

Vitamin D receptor levels and binding are reduced in aged rat intestinal subcellular fractions

The hormonal form of vitamin D, 1α,25(OH)₂-vitaminD₃ [1α,25(OH)₂D₃], stimulates signal transduction pathways in intestinal cells. To gain insight into the relative importance of the vitamin D receptor (VDR) in the rapid hormone responses, the amounts and localization of the VDR were evaluated in young (3 months) and aged (24 months) rat intestinal cells. Immune-fluorescence and Western blot studies showed that VDR levels are diminished in aged enterocytes. Confocal microscopy assays revealed that the VDR and other immune-reactive proteins have mitochondrial, membrane, cytosol and perinuclear localization. Western blot analysis using specific antibodies detected the 60 and 50 kDa bands expected for the VDR in the cytosol and microsomes and, to a lesser extent, in the nucleus and mitochondria. Low molecular weight immune-reactive proteins were also detected in young enterocytes subcellular fractions. Since changes in hormone receptor levels appear to constitute a common manifestation of the ageing process, we also analyzed 1α,25(OH)₂D₃ binding properties and VDR levels in subcellular fractions from young and aged rats. In competition binding assays, employing [³H]-1α,25(OH)₂D₃ and 1α,25(OH)₂D₃, we have detected specific binding in all subcellular fractions, with maximum binding in mitochondrial and nuclear fractions. Both, VDR protein levels and 1α,25(OH)₂D₃ binding, were diminished with ageing. Age-related declines in VDR may have important consequences for correct receptor/effector coupling in the duodenal tissues and may explain age-related declines in the hormonal regulation of signal transduction pathways that we previously reported.