IBMS/ECTS 2001 - PROGRAM and ABSTRACTS
POSTER PRESENTATIONS
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Vitamin D
HYPOVITAMINOSIS 1,25(OH)2D3 IN PATIENTS SUFFERING FROM CHRONIC DISORDERS.
J. Payer1*, Z. Killinger1, J. Rovenský2, H. Kratochvilová1, P. Ondrejka3
11st Department Internal Medicine, University Hospital, Comenius University, Bratislava, Slovakia
2The Research Institute of Rheumatic Diseases, Piest'any, Slovakia
3The Ministry of Inferior Hospital, Bratislava, Slovakia
Vitamin D deficiency may be one reason for the onset and development of osteoporosis. The aim of the present study was to determine the occurrence rates of hypovitaminosis D in a non-selected group of individuals presenting with common medical conditions, hospitalized over long periods of time at the medical department. Concentrations of 1,25 (OH)2D3 were measured in 89 patients (38 males and 51 females) aged 70 years in average. Thirty-eight patients were tested in the spring, the remaining 51 patients in the autumn. A significant reduction of vitamin D3 levels were observed in patients tested in the autumn (p<0.001). The reason for this surprising observation may have been the few sunny days and the long hospital stays during the sampling period, differences in the composition of both groups, as well as the fact that 1,25(OH)2D3 is a parameter reflecting the actual vitamin D3 levels rather than that of its stock in the organism. The results obtained suggesting hypovitaminosis D in a non-selected group of elderly patients hospitalized with common diseases at the medical department, even after the summer season, suggest the need for general year-around supplementation of this vitamin regardless of the risk factors.
DEFINING HYPOVITAMINOSIS D IN THE ELDERLY: EXAMINATION OF PARATHYROID STATUS
M. J. McKenna
St. Michael's Hospital, Dun Laoghaire, Ireland
Background: Hypovitaminosis D is defined as a low circulating concentration of serum 25(OH)D. There is much disagreement on the threshold level for the definition as a consequence, in part, of substantive differences in methodologies for assay of serum 25(OH)D. An alternative approach is to consider parathyroid hormone (PTH) status as a surrogate for vitamin D status given the paradigmatic standing of secondary hyperparathyroidism in the evolution of privational vitamin D deficiency.
Methods: An analysis of reports of PTH status worldwide in elderly populations, that employed immunometric assays, was conducted. Elderly cohorts were subdivided according to country of residence, housebound or non-housebound, and post-therapy with low-dose vitamin D for more than 6-months.
Results: Between 1991 and 1999, 39 studies of PTH status (including 68 elderly groups) and 11 studies of low-dose vitamin D therapy (400 to 1000 iu for 6 months to 3 years) were identified. PTH status was similar in housebound groups regardless of country of residence but higher than non-housebound groups, which in turn were higher than post-therapy groups. PTH response to vitamin D therapy was inversely related to baseline PTH status in spite of large differences in vitamin D status.
Conclusions: (1) PTH status is highest in housebound elderly groups and lowest in post-therapy groups regardless of country of residence; (2) Following low-dose vitamin D therapy for at least 6 months, PTH status in the elderly approximates the serum PTH range in young adults; (3) It should be noted that calcium supplementation has an enabling role in the correction of secondary hyperparathyroidism in these studies of vitamin D therapy; (4) Defining hypovitaminosis D in the elderly is best accomplished by ascertaining the serum 25(OH)D response to low-dose vitamin D therapy (about 800 iu daily for at least 6 months) in calcium replete subjects; this approach surmounts the problem of differences among assays for serum 25(OH)D; (5) In our experience, the threshold of serum 25(OH)D for defining hypovitaminosis D exceeds 75 nmol/L which is higher than currently accepted thresholds.
DECREASED SEASONAL VARIATION IN VITAMIN D LEVELS IN MEDICAL OUTPATIENTS IN MIAMI, FLORIDA (UNITED STATES)
S. Levis1*, A. Gomez2, C. Jimenez2, L. S. Veras2, B. W. Hollis3, S. Lai4, B. A. Roos1
1University of Miami School of Medicine, GRECC/VA Medical Center, State of Florida Teaching Nursing Home at Miami Jewish Home & Hospital for the Aged, Miami, FL, USA
2Jackson Memorial Hospital/University of Miami School of Medicine/VA Medical Center, Miami, FL, USA
3Medical University of South Carolina, Charleston, SC, USA
4Johns Hopkins University, Baltimore, MD, and State of Florida Teaching Nursing Home at Miami Jewish Home & Hospital for the Aged, Miami, FL, USA
Moderate vitamin D deficiency has been associated with bone loss and increased fracture risk, even in the absence of osteomalacia. In the US, most reports of vitamin D levels originate in northern latitudes, areas that have few hours of sunlight during the winter months. These reports demonstrate lower vitamin D levels at the end of winter and higher levels at the end of summer, reflecting lower rates of vitamin D synthesis in winter due to decreased sun exposure. We here report winter and summer 25(OH) vitamin D levels in winter and summer in medical outpatients in Miami, Florida (latitude 25.46 N), an area that because of its southern location enjoys sunny, warm weather even during the winter months.
Methods: 25(OH) vitamin D levels were measured in the same individuals attending an outpatient general medicine clinic at the local county hospital during March 2000 and again in September 2000. None of the participants was taking medications or had diseases known to affect vitamin D.
Results: 103 individuals participated in the study, 71 women (68.9%) and 32 men (31.1%). Their mean age was 60.0±12.1 (SD) years. Mean 25(OH) vitamin D levels in March were 23.58±8.93 (SD) and in September were 26.84±10.14 (SD). There was a 12% decrease in 25(OH) vitamin D levels in winter. The seasonal difference was significant (p<0.0001).
Conclusions: Although medical outpatients living in Miami experience significant seasonal variation in 25(OH)vitamin D levels, only a 12% decrease occurs in winter, compared to up to 30% in the northern US. Living in this southern location could be protective from the pronounced drop in vitamin D levels and known associated rise in PTH levels that result in higher rates of bone loss in winter already described in northern regions of the US.
BONE CELL REGULATION OF VITAMIN D METABOLISM
P. H. Anderson1,2*, S. Iida1, A. J. Moore1, B. K. May3, M. Cochran1, H. A. Morris1,2
1Institute of Medical and Veterinary Science, Adelaide, Australia
2Department of Physiology, Adelaide University, Adelaide, Australia.
3Department of Biochemistry, Adelaide University, Adelaide, Australia.
Activation of vitamin D to 1,25dihydroxyvitamin D (1,25D) by 25-hydroxyvitamin D 1alpha-hydroxylase (CYP1) and catabolism by 25 hydroxyvitamin D 24-hydroxylase (CYP24) in bone cells have been identified for 20 years. However, bone cell physiology of CYP1 and CYP24 gene expression remains largely unknown. We have compared the relationship between 1,25D status and dietary calcium (Ca) levels on CYP1 and CYP24 mRNA expression levels in kidney and bone tissues. Vitamin D-insufficient female Sprague-Dawley rats were raised on a vitamin D-deficient, 1% Ca diet and housed in a UV-free environment from birth. The mean serum 25-hydroxyvitamin D and 1,25D levels in these animals were 9.5 nanomol/L and 34 picomol/L respectively at 6 months of age. Vitamin D-replete and vitamin D-insufficient rats either continued on the 1% Ca diet or were fed a 0.1% Ca diet for a further 3 months. Rats were sacrificed and femora and kidneys were removed. One femur was processed for histology and total RNA was extracted from the second femur and kidneys by phenol/chloroform methods. Real-Time RT-PCR quantified mRNA. Vitamin D-insufficient rats fed 1% Ca demonstrated eucalcaemia and normal mineralisation as indicated by dynamic bone histology, in contrast to hypocalcaemia and mineralisation defects in vitamin D-insufficient rats fed 0.1% Ca. Kidney CYP1 mRNA expression increased 90-fold in vitamin D-insufficient fed 0.1% Ca rats compared to vitamin D-replete rats fed 0.1% Ca (p<0.001). In bone, CYP1 mRNA levels increased 3-fold in rats fed 1% Ca compared to rats fed 0.1% Ca, independent of vitamin D status (p<0.01). Vitamin D-insufficiency suppressed kidney CYP24 mRNA independent of dietary Ca. However, bone CYP24 mRNA levels were maintained and were co-ordinately expressed with bone CYP1 mRNA (R2=0.89). These data indicate tissue-specific control of gene expression for these enzymes. Vitamin D status is a major determinant of CYP1 expression in the kidney whereas dietary Ca levels determine CYP1 expression in bone.
THE C-3 EPIMERIZATION PATHWAY IS INVOLVED IN WHOLE PROCESS OF METABOLISM OF VITAMIN D3
M. Kamao1*, S. Tatematsu1, S. Hatakeyama2, K. Ozono3, N. Kubodera4, T. Okano1
1Kobe Pharmaceutical University, Kobe, Japan
2Nagasaki University, Nagasaki, Japan
3Osaka Medical Center for Maternal and Child Health, Osaka, Japan
4Chugai Pharmaceutical Co. Ltd., Tokyo, Japan
It is well established that 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] is metabolized into side-chain cleaved products through the C-24 and C-23 oxidation pathways. However, recently, it was demonstrated that 1alpha,25(OH)2D3 was also metabolized into its epimer of hydroxyl group at C-3 of the A-ring, 1alpha,25-dihydroxy-3-epi-vitamin D3 [1alpha,25(OH)2-3-epi-D3] in vitro and in vivo. In the present study, we investigated the C-3 epimerization of major metabolites of vitamin D3, 25-hydroxyvitamin D3 [25(OH)D3] and 24R,25-dihydroxyvitamin D3 [24,25(OH)2D3] in cell culture system to clarify whether C-3 epimerization pathway is common to vitamin D compounds. In addition, we studied about metabolism of C-3 epimer of 25(OH)D3 and 1alpha,25(OH)2D3. 10 microM of 25(OH)D3, 24,25(OH)2D3 or 1alpha,25(OH)2D3 were incubated with UMR-106 (rat osteosarcoma), Caco-2 (human colon adenocarcinoma), HepG2 (human hepatoblastoma) and LLC-PK1 (porcine kidney) cells for 48hr. Metabolites were purified by HPLC and identified by LC-MS and 1H-NMR analyses. Metabolism of 25(OH)-3-epi-D3 and 24,25(OH)2-3-epi-D3 were also studied by the same method. In four cell lines, 25(OH)D3 and 24,25(OH)2D3 were metabolized into 25(OH)-3-epi-D3 and 24,25(OH)2-3-epi-D3, respectively. Although the differences existed in the amount of each C-3 epimer formed with different cell types. UMR-106, Caco-2 and HepG2 cells generated 25(OH)-3-epi-D3 from 25(OH)D3 as a major metabolite. LLC-PK1 cells gave a rise of 24,25(OH)2D3 as a major metabolite of 25(OH)D3. The production of 25(OH)-3-epi-D3 from 25(OH)D3 was found in similar amounts of 1alpa,25(OH)2-3-epi-D3 from 1alpha,25(OH)2D3. However, the production of 24,25(OH)2-3-epi-D3 from 24,25(OH)2D3 was low. These findings suggested that the rate of 3-epimerization was affected by side-chain structure. Biological activities of C-3 epimer of 25(OH)D3, 24,25(OH)2D3 and 1alpha,25(OH)2D3 evaluated by vitamin D receptor binding affinity, transcriptional activity, inhibiting proliferation and inducing differentiation of human promyelocytic leukemia cells (HL-60) were lower than 25(OH)D3, 24,25(OH)2D3 and 1alpha,25(OH)2D3. In addition, 25(OH)-3-epi-D3 and 1alpha,25(OH)2-3-epi-D3 were metabolized into their C-24 hydroxide, 24,25(OH)2-3-epi-D3 and 1alpha,24,25(OH)3-3-epi-D3. Our results suggest that the C-3 epimerization pathway is involved in metabolism of vitamin D3 widely, and generated C-3 epimers (3alpha form) are further metabolized through the C-24 oxidation pathway.
TARGETED ABLATION OF THE 25-HYDROXYVITAMIN D-1-ALPHA-HYDROXYLASE: AN ANIMAL MODEL OF PSEUDO VITAMIN D DEFICIENCY RICKETS
O. Dardenne*, J. Prud'homme, A. Arabian, F. H. Glorieux, R. St-Arnaud
Genetics Unit, Shriners Hospital for Children and Depts. of Surgery and Human Genetics, McGill University, Montreal, Quebec, Canada
Pseudo vitamin D deficiency rickets (PDDR) is a rare autosomal recessive disease caused by mutations in the cytochrome P450 enzyme, 25-hydroxyvitamin D-1-alpha-hydroxylase (1-OHase). This enzyme catalyzes the final metabolic conversion of vitamin D from the inactive 25-hydroxyvitamin D metabolite to the active, hormonal form: 1-alpha,25-dihydroxyvitamin D [1,25(OH)2D]. Patients with the disease exhibit failure to thrive and growth retardation, as well as rickets and osteomalacia. Serum biochemistry is characterized by hypercalcemia, secondary hyperparathyroidism, and undetectable levels of 1,25(OH)2D. In order to create an animal model for PDDR, we have inactivated the 1-OHase gene in mice following homologous recombination in embryonic stem cells. The engineered mutation deleted the heme-binding domain of the cytochrome P450 enzyme, generating a null allele, as evidenced by the undetectable levels of circulating 1,25(OH)2D in homozygote mutant animals. Heterozygous animals are fertile and show no discernable phenotype. Homozygous knockout animals showed retarded growth, documented by decreased weight gain and shorter femurs. Serum analysis of homozygote mutant animals confirmed that they were hypocalcemic, hypophosphatemic, hyperparathyroidic and that they displayed elevated serum alkaline phosphatase. Histological analysis of the bone from 3 week-old mutant animals confirmed the evidence of rickets. Histomorphometric analysis revealed increased osteoid volume, osteoid surface, and osteoid thickness in 1-OHase-ablated animals. Growth plate thickness, number of osteoblasts, and osteoblast surface were also greater in homozygote mutant mice. At the age of 8 weeks (young adult), femur from knockout mice presented a more severe disorganization in the architecture of the growth plate and marked osteomalacia. These results show that we have successfully inactivated the 1-OHase gene in mice and established a valid animal model of PDDR.
INFLUENCE OF DONOR AGE AND SKELETAL SITE OF ORIGIN ON VDR MRNA EXPRESSION AND PROTEIN LEVELS AND IN RESPONSE TO 1,25(OH)2D3 IN HUMAN OSTEOBLASTIC CELLS
M. Garcia1*, P. Martinez1, I. Del Valle1, L. Gomez1, J. L. Castrillo2, P. Esbrit3, M. E. Martinez1
1Biochemistry Division La Paz Hospital
2Molecular Biological Center (CBM)
3Bone and Mineral Metabolism Laboratory (Fundacion Jimenez Diaz)
Age-related osteopenia is known to occur differently throughout the skeleton. We previously found differences in human osteoblastic cells (hOB) maturation and in response to vitamin D, related to donor age and skeletal site. We assessed the influence of donor age and bone structure on vitamin D receptor (VDR) expression and protein levels, before and after 1,25(OH)2D3 in hOB in primary cultures.
Human OB were isolated from trabecular bone from donors undergoing knee (trabecular bone) (n= 22, 4 < 50 yr, 18 > 50 yr) or hip (cortical bone)
(n= 16, 6 < 50 yr, 10 > 50 yr) arthroplastia. Human OB were cultured for different time periods, with or without 1,25(OH)2D3 at 10-8-10-6molar. Total RNA and cell protein were extracted by the Trizol method, using RT-PCR and Western-blot to analize VDR mRNA and protein levels, respectively.
Treatment with 10-8molar 1,25(OH)2D3 induced a stepwise increase in VDR protein levels within 72 hours. This metabolite also increased VDR mRNA 2 up to 24 hours. However, the latter time period further increase of this mRNA did not occur. This suggest that stabilization of VDR protein after 1,25(OH)2D3 occurs, perhaps by an increase in the half-life of this receptor. In basal condition, VDR expression (p<0.001) and VDR protein (p<0.05) decreased with aging, only in cortical bone. Hip hOB showed lower levels of both VDR expression (p<0.05) and protein (p<0.05), than knee hOB in older donors. An increase (p<0.05) in VDR protein levels was induced by 10-8 and 10-6molar 1,25(OH)2D3 in all groups studied. However, this effect decreased with aging (p<0.05) at each 1,25(OH)2D3concentration.
Conclusions: Age-related decrease in VDR expression and protein levels in hOB occurs mainly in cortical bone. Treatment with 1,25(OH)2D3 induced an increase in VDR protein levels, but its efficacy decreases with aging.
THE FOK I POLYMORPHISM OF VITAMIN D RECEPTOR GENE DETERMINES BONE MINERAL DENSITY IN CZECH POSTMENOPAUSAL WOMEN
K. Zajíèková1, I. Zofková1*, R. Bahbouh2
1Institute of Endocrinology, Prague, Czech Republic
2Faculty of Medicine, Charles University, Prague, Czech Republic
Vitamin D receptor gene locus has been related to bone mineral density with discordant findings in different populations. In our study, we examined the association between restriction fragment length polymorphisms / Fok I, Apa I, Taq I, Bsm I/ of vitamin D receptor and bone mineral density in 114 Czech postmenopausal women, who averaged 62,5±8,9 years of age.
Methods. Polymorphisms of vitamin D receptor gene were assessed by polymerase chain reaction and restriction endonuclease digestion with Fok I, Apa I, Taq I and Bsm I enzymes. Bone mineral density was measured by dual-energy X-ray absorptiometry (g/cm2). A possible relation of age- and body mass-adjusted bone density with different genotypes was detected by the ANOVA test.
Results. The prevalence of each genotype in the study population was FF 34, Ff 49, ff 17 (per cent). Women with ff genotype had significantly lower mean bone mineral density than those with Ff genotype (p=0,0459), whereas the difference in bone density betweeen ff and FF genotypes did not reach statistical significance. Heterozygote subjects (Ff) had the highest levels of bone mineral density. Conversely, there was no relationship between the remaining polymorphisms and bone mineral density.
To summarize, the Fok I genotypes of vitamin D receptor gene may contribute to postmenopausal bone loss in Czech women.
A LOW CALCIUM DIET INCREASES PLASMA MEMBRANE Ca-ATPASE AND SODIUM/CALCIUM EXCHANGER ACTIVITIES FROM CHICK ENTEROCYTES.
V. Centeno*, A. Alisio, N. Tolosa de Talamoni
Laboratorio de Metabolismo Fosfocálcico y Vitamina D "Dr. Cañas", Facultad de Ciencias Médicas, Universidad Nacional de Córdoba, Córdoba, Argentina
The calcium ion exit from the enterocyte to the lamina propria occurs mainly through the plasma membrane Ca-ATPase or Ca pump and the sodium/calcium exchanger (Na/Ca exchanger). The aim of this study was to determine the effect of a low Ca diet on the activity of these two transporters in chick enterocytes with different degree of differentiation. The experimental design was the following: one-day-old chicks were fed a normal diet for 20 days, and then, they were divided in two groups: 1) chicks fed a normal diet, and 2) chicks fed a low Ca diet (0.1% Ca). After 10 days, the second group was adapted to the mineral deficiency as judged by lower serum Ca and P and higher activity of intestinal alkaline phosphatase than the control one. Calcium uptake studies by enterocytes with different degree of maturation were performed in order to assay the activities of the Ca pump and the Na/Ca exchanger. The data showed that Ca pump activity in mature cells from the apical villus was higher in the low Ca group than in the normal one, whereas the activity in the immature cells was not altered. The Na/Ca exchanger activity in immature enterocytes from the normal group was higher than in more differentiated cells. The activity of the Na/Ca exchanger in mature cells was also increased by the low Ca diet. Since the serum calcitriol levels were 4-fold in the low Ca group as compared to the normal group, the effect of mineral deficiency on the activity of these two transporters could be attributed to the increased synthesis of calcitriol.
In conclusion, a low Ca diet increases the Na/Ca exchanger and Ca pump activities in chick enterocytes from the apical villus presumably through mechanisms triggered by calcitriol.
VITAMIN A ANTAGONIZES CALCIUM RESPONSE TO VITAMIN D IN MAN
S. Johansson*, H. Melhus
Uppsala University, Uppsala, Sweden
For unknown reasons, the highest incidence of osteoporosis is found in Northern Europe. In these populations the sunlight exposure is limited and the vitamin A intake high. The interaction between vitamin A and D has been the subject of several in vitro and animal studies. We have studied the acute effects of vitamin A and D on calcium homeostasis in nine healthy human subjects. We compared the effect of (i) 2 microg 1,25(OH)2D3 (vitamin D), (ii) 15 mg retinyl palmitate (vitamin A), (iii) vitamin A + D, and (iv) placebo in a double-blind crossover study. The subjects took vitamin preparations at 22.00 and the following day blood samples were collected five times from 8.00 to 16.00.
Serum levels of 1,25(OH)2D3 and retinyl esters increased (1,7-fold and 8,3-fold respectively, p<0.01), and there was no difference after single and combined intake. As expected, S-calcium increased (2.3%, p<0.01) and S-PTH decreased (-32%, p<0.05) after vitamin D intake. In contrast, vitamin A intake resulted in a significant decrease in S-calcium when taken alone (-1.0%, p<0.05) and diminished the calcium response to vitamin D after the combined intake (1.4%, p<0.01). S-PTH was unaffected by vitamin A. No significant changes in serum levels of the degradation product of C-telopeptide of type I collagen (CrossLaps), or U-calcium/ creatinine levels were found. In conclusion, an intake of vitamin A corresponding to about one serving of liver antagonizes the rapid intestinal calcium response to physiological levels of vitamin D in man.
THE TYROSINE KINASE C-SRC IS REQUIRED FOR 1,25(OH)2-VITAMIN D3 SIGNALLING TO THE NUCLEUS IN MUSCLE CELLS
C. Buitrago, R. L. Boland*, A. Russo de Boland
Dept. Biologia, Bioquimica & Farmacia - Universidad Nacional del Sur, Bahia Blanca, Buenos Aires, Argentina
We have recently shown that the hormonal form of vitamin D3, 1,25(OH)2-vitamin D3 [1,25(OH)2D3], stimulates the enzymatic activity of the nonreceptor protein tyrosine kinase c-Src in skeletal muscle cells. In this study we show that intracellular and extracellular Ca2 chelation with BAPTA and EGTA, respectively, blocked hormone stimulation of c-Src activity/dephosphorylation, indicating that the calcium messenger system is an upstream activator of c-Src. Tyrosine phosphorylation and stimulation of the growth-related mitogen-activated protein (MAP) kinase by 1,25(OH)2D3 was shown to be dependent on activation of c-Src, since pretreatment with the c-Src specific inhibitor PP1 or muscle cell transfection with an antisense oligodeoxynucleotide directed against c-Src mRNA, markedly reduced hormone stimulation of MAPK phosphorylation. Evidence was obtained indicating that MAPK is then translocated to the cell nucleus in active phosphorylated form and induces the expression of c-myc oncoprotein, as the MAPK kinase (MEK) inhibitor PD 98059 abolished stimulation of c-myc synthesis by 1,25(OH)2D3. In addition, the hormone rapidly stimulated tyrosine phosphorylation of c-myc. In cells pretreated with PP1, the 1,25(OH)2D3-induced increase in tyrosine phosphorylation of c-myc was suppressed. Taken together, these results demonstrate that 1,25(OH)2D3 stimulates proliferation-associated signalling pathways in skeletal muscle cells and implicate c-Src kinase as mediator of this response.
TUMOR NECROSIS FACTOR ALPHA GENE TRANSCRIPTION IS REPRESSED BY THE VITAMIN D3 RECEPTOR-MEDIATED INTERACTION WITH THE KAPPA3 ELEMENT IN THE PROMOTER REGION
A. Takeuchi1*, M. Yoshikawa1, T. Yoshizawa2, S. Kato2, S. Sharma3, T. Okano1
1Kobe Pharmaceutical University, Kobe, Japan
2Institute of Molecular and Cellular Bioscience, The University of Tokyo, Tokyo, Japan
3Women and Infant' Hospital, Brown University, Providence, USA
The active form of vitamin D3, 1alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3) exhibits suppressive effects on the immune system. Of significance are the observations that only activated lymphocytes express vitamin D receptor (VDR) and that 1,25(OH)2D3 treatment of activated T cells results in partial growth inhibition as well as in transcriptional repression of cytokine genes, including IL-2, IFN-gamma, and GM-CSF. Nuclear factor of activated T cells (NFAT) plays a key role in the inducible expression of cytokines in T cells. We have reported that NFAT is a molecular target for 1,25(OH)2D3-mediated immunosuppressive effects, which are VDR/retinoid X receptor alpha (RXR)-dependent. Herein, we examined the effect of 1,25(OH)2D3 on TNFalpha gene transcription. VDR alone or VDR/RXR heterodimers were found to be specifically bound to a synthetic oligonucleotide corresponding to the kappa3 element in the TNFalpha promoter, which includes a variant NFAT binding site. Mutating the inverting sequences between the binding regions for NFAT proteins abolished this specific binding. In vitro inhibition of NFAT complex formation was observed when VDR or VDR/RXR were added to DNA binding reactions. Transient co-expression of VDR and RXR was capable of inhibiting TNFalpha promoter-mediated expression of a reporter gene in activated Jurkat cells treated with 1,25(OH)2D3. Given these observations, we further assessed the effect of 1,25(OH)2D3 on mRNA expression of TNFalpha. Our results demonstrated that the expression of TNFalpha and IL-2 in murine T cells was down-regulated by 1,25(OH)2D3. The VDR null mutant (VDR-KO) mice showed impaired VDR-mediated action of 1,25(OH)2D3. We thus examined the immunomodulatory effects by 1,25(OH)2D3 using T cells from VDR-KO mice. Although nuclear extracts from activated T cells from wild type and VDR-KO mice readily formed a DNA-protein complex with the kappa3 element, the complex formation was significantly inhibited only in 1,25(OH)2D3-treated cells of wild type mice. No inhibition of TNFalpha mRNA expression by 1,25(OH)2D3 was observed in VDR-KO mice. Moreover, 1,25(OH)2D3 treatment inhibited the transcriptional activity of TNFalpha promoter in wild type mice, whereas it failed in VDR-KO mice. These results suggest that 1,25(OH)2D3 system is a transcriptional repressive modulator of the TNFalpha promoter activity.
CALCITRIOL INCREASES BONE TURNOVER BUT NOT MINERAL DENSITY IN THE STREPTOZOTOCIN-INDUCED DIABETIC RATS
J. Del Pino-Montes1*, G. E. Benito2, M. P. Fernández-Salazar2, M. Martín3, E. Martín3, M. J. García-Rodriguez1, M. Fernández-Puente1, J. R. García-Talavera3, J. J. Calvo2
1U. Metabolismo óseo, Hospital Universitario, Salamanca, Spain
2Dept. Fisiología y Farmacología, Universidad de Salamanca, Spain
3S.Medicina Nuclear, Hospital Universitario, Salamanca, Spain
Diabetes mellitus is known to cause osteopenia associated with a low bone turnover. Active metabolites of vitamin D has shown an improvement of metabolic imbalance on bone, by several mechanisms. The aim of this work was to study the effect of high doses of calcitriol on bone mineral density (BMD) and several bone turnover markers and phosphocalcic metabolism in diabetic rats.
Methods: Female 3-month old Wistar rats (180-200 g) were used. Diabetes was induced with streptozotocin (STZ) (65 mg/Kg). Three groups of rats were studied for two months: control (C), diabetic (D) and diabetic treated (DT) with calcitriol (250 ng three times a week p.o.). BMD (DXA) at lumbar spine and tibiae (epiphysis and diaphysis), bone turnover markers (Osteocalcin and D-pyr) and mineral metabolism parameters (blood calcium, phosphate and PTH, and urine calcium and phosphate) were measured. ANOVA followed by Scheffé test were applied.
Results: Calcitriol treatment increases urinary calcium in DT group, but no significant changes in calcium and phosphorus plasma levels could be observed. This is associated to a compensatory decrease of plasma PTH in DT group. BMC and BMD were similar in both groups of diabetic rats; however there is an increase in bone turnover in DT compared with D groups (see table).
It is concluded that calcitriol treatment increases bone turnover and urine calcium without affecting BMD in STZ-induced diabetic rats.
| blood Ca | blood P | blood PTH | urine Ca | osteocalcin | Pyr/Creat | |
| mg/dl | mg/dl | pg/ml | mg/dl | ng/ml | nM/mM | |
| D | 8.9±0.4 | 5.0±1.1 | 143±26 | 1.6±0.4 | 1.5±0.1 | 29.1±4.8 |
| DT | 8.3±0.8 | 4.9±1.1 | 91±33a | 10.1±0.6b | 2.1±0.04b | 43.4±8.7 |
| Table: Values expressed as mean±SEM, (n=6). a: statistically significant differences (p<0.05) as compared to D. b: statistically significant differences (p<0.01) as compared to D |
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