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S. A. Haining, J. H. Galloway, B. L. Brown, and D. F. Guilland-Cumming

ABSTRACT

It has recently been proposed that the action of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on bone metabolism may be mediated by changes in phospholipid metabolism. The effects of vitamin D metabolites on the incorporation of radiolabelled precursors into corresponding phospholipid classes were investigated using cells arising from cultured explants of normal human bone with osteoblast-like characteristics. Treatment with 1,25-(OH)2D3 increased the incorporation of serine, measured as the ratio of [3H]serine in phosphatidylserine (PS) to [14C]ethanolamine in phosphatidylethanolamine (PE), in a time- and dose-dependent manner. The maximum effect on PS/PE of 141·6 ± 5·9% over control (P = 0·022) was observed at a dose of 0·1 nmol 1,25-(OH)2D3/l, maintained for 24 h. Incubations with 25-hydroxyvitamin D3 (0·1 μmol/l) and 24,25-dihydroxyvitamin D3 (10 nmol/l) had no effect. Supraphysiological doses (0·1 μmol/l) of 1,24,25- and 1,25,26-trihydroxyvitamin D3 showed similar effects to those of 1,25-(OH)2D3, emphasizing the importance of 1α-hydroxylation. Incorporation of [14C]choline into phosphatidylcholine, calculated as a ratio to PE, was not affected by treatment with vitamin D metabolites. However, [3H]inositol uptake into phosphatidylinositol was almost doubled when compared with control uptake within 2 h of treatment with 1,25-(OH)2D3 (0·1 μmol/l). This may be of relevance, considering the importance of phosphoinositide metabolism in influencing the intracellular calcium concentration. These results support a role for 1,25-(OH)2D3 in the modulation of phospholipid metabolism in human bone cells, which in turn may be involved in the action of 1,25-(OH)2D3 in bone mineralization.

J. Endocr. (1988) 116,435–441

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HJ Armbrecht, MA Boltz, TL Hodam, and VB Kumar

Non-transformed rat intestinal epithelial cell (IEC) lines were used to study the action of 1,25-dihydroxyvitamin D(3) (1,25(OH)2D) in the intestine. The capacity of 1,25(OH)2D to increase the expression of the cytochrome P450 component of the vitamin D 24-hydroxylase (CYP24) was determined in IEC-6 and IEC-18 cell lines. In IEC-6 cells, which are derived from crypt cells isolated from the whole small intestine, 1,25(OH)2D markedly increased expression of CYP24 protein and mRNA within 12 h. In contrast, in IEC-18 cells, which are derived from crypt cells from the ileum only, 1,25(OH)2D did not increase expression of CYP24 until 24-48 h. The maximal levels of CYP24 mRNA seen in the IEC-18 cells were only 31% of the maximal levels seen in the IEC-6 cells. In the presence of 1,25(OH)2D, phorbol esters rapidly increased CYP24 mRNA levels in IEC-18 cells from almost undetectable to levels seen in IEC-6 cells. Protein kinase inhibitors abolished the stimulation by 1,25(OH)2D and by phorbol esters in both cell lines. Stimulation of mRNA levels by phorbol esters required new protein synthesis but stimulation by 1,25(OH)2D did not. These studies demonstrated that the rapid action of 1,25(OH)2D in IEC-6 cells is related to the activation of protein kinase C, an event which is missing in the IEC-18 cells. This differential response to 1,25(OH)2D probably takes place at a post-receptor site, since the number of vitamin D receptors in each cell line was found to be similar.

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T Michigami, H Yamato, H Suzuki, Y Nagai-Itagaki, K Sato, and K Ozono

In patients with humoral hypercalcemia of malignancy (HHM), serum levels of 1,25-dihydroxyvitamin D (1,25(OH)(2)D) are generally low, although the pathophysiology of the impaired vitamin D metabolism is not fully understood. In the present study, we have investigated vitamin D metabolism in our newly developed rat model of HHM in which a human infantile fibrosarcoma producing parathyroid hormone-related protein (PTHrP), named OMC-1, was inoculated s.c. into athymic nude rats. In OMC-1-bearing rats, the serum concentration of 1,25(OH)(2)D was markedly reduced when the animals exhibited severe hypercalcemia (Ca > or =15 mg/dl), while it was rather elevated in those with mild hypercalcemia. To further examine whether serum Ca levels affect 1,25(OH)(2)D concentration, we administered bisphosphonate YM529 to OMC-1-bearing rats when they exhibited severe hypercalcemia. The restoration of the serum Ca level by administration of YM529 was accompanied by a marked increase in the 1,25(OH)(2)D level, suggesting that the serum Ca level itself plays an important role in the regulation of the 1,25(OH)(2)D level in these rats. On the other hand, when the OMC-1-bearing rats were treated with a neutralizing antibody against PTHrP, serum 1,25(OH)(2)D levels remained low despite the reduction in serum Ca levels. Expression of 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) in kidney was decreased in OMC-1-bearing rats with severe hypercalcemia, and markedly enhanced after treatment with bisphosphonate. This enhancement in 1 alpha-hydroxylase expression was not observed after treatment with the antibody against PTHrP. These results suggest that PTHrP was responsible for the enhanced expression of 1 alpha-hydroxylase in YM529-treated rats, and that hypercalcemia played a role in reducing the serum 1,25(OH)(2)D level in OMC-1-bearing rats by suppressing the PTHrP-induced expression of the 1 alpha-hydroxylase gene.

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J. Levy, I. Zuili, N. Yankowitz, and S. Shany

ABSTRACT

The role of oestradiol-17β in the induction of specific cytosolic receptors for 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) was examined in the immature rat uterus.

An acrylamide gel electrophoretic analysis was developed to separate the specific receptor for 1,25(OH)2D3 from the plasma binding protein for vitamin D3 metabolites. Employing this sensitive method the presence of receptors for 1,25(OH)2D3 in the mature rat uterus was evident. Such receptors were not found in the uterus of saline-treated immature rats. However, oestradiol administration caused an induction of these receptors in the immature rat uterus, together with a significant increase in the uterine weight, progesterone receptor level and peroxidase activity.

These results suggest a mechanism for oestradiol regulation of calcium metabolism in the uterus at times of high demand for this cation during the gestation period.

J. Endocr. (1984) 100, 265–269

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S. C. MANOLAGAS, C. M. TAYLOR, and D. C. ANDERSON

A method developed initially for the detection of high-affinity binding of glucocorticoids in the cytosol from foetal rat calvaria has been adapted for metabolites of vitamin D. Consistent displacement of [3H]1,25-dihydroxycholecalciferol ([3H]1,25(OH)2D3) by unlabelled 1,25(OH)2D3 was obtained with an apparent dissociation constant (K d) of 2·3 × 10−9 mol/l. The specificity of this binding was examined by competition experiments. Displacement of labelled 1,25(OH)2D3 by a 100-fold excess of unlabelled metabolites, expressing the fall with unlabelled 1,25(OH)2D3 as 100%, was as follows: 25-hydroxycholecalciferol (25(OH)D3), 61%; 24,25-dihydroxycholecalciferol, 29%; cholecalciferol, 3%. These are similar to results for the chick mucosa nuclear 1,25(OH)2D3 receptor. No displacement was obtained with corticosterone, testosterone, oestradiol or progesterone. When [3H]25(OH)D3 was used as ligand, a displacement curve with unlabelled 25(OH)D3 indicated only binding with a greater K d (approximately 10−7 mol/l). These data suggest a direct action of 1,25(OH)2D3 on bone which is similar to that of steroid hormones on their target tissues.

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R Yacobi, R Koren, U A Liberman, C Rotem, L Wasserman, and A Ravid

Abstract

Renal cell carcinoma is a chemotherapy-resistant tumor which is relatively responsive to immunotherapy. Immunotherapeutic regimes employ interferons or interleukin 2 with or without lymphokine-activated killer cells. Secondary cytokines, induced by interleukin 2 or interferon, may have an important impact on their anti-neoplastic activity. Notable among them is tumor necrosis factor (TNFα). We assessed the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the susceptibility of the human renal cell carcinoma cell line SK-RC-29 to the cytotoxic and cytostatic actions of TNFα, interferon a and lymphokine-activated killer cells. Using uptake of the vital dye neutral red as an indicator of viable cell number, we found that addition of 1,25(OH)2D3 (100 nm) to TNFα (30 ng/ml)-treated cultures resulted in a 2·6 ± 0·2-fold (mean ± s.e.) increase in the cytotoxic effect of the cytokine. The potentiating effect of 1,25(OH)2D3 was dose-dependent, and significant at concentrations equal to or higher than 10 nm. Another dihydroxylated vitamin D metabolite, 24,25(OH)2D3, had no effect on TNFα action. The cytotoxic effect of TNFα increased whereas the potentiation by 1,25(OH)2D3 decreased with cell density in culture. 1,25(OH)2D3, in contrast to its potentiating effect on TNFα action, did not modulate the cytostatic effect of interferon α or the susceptibility of SK-RC-29 to killing by lymphokine-activated killer cells. The findings reported here may explain some of the in vivo anti-tumor activity of 1,25(OH)2D3 and provide a rationale for the employment of active vitamin D analogs during immune anti-cancer therapy.

Journal of Endocrinology (1996) 149, 327–333

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G. R. Williams and G. A. Brent

The retinoids, vitamin D3 and thyroid hormone exert diverse and complex tissue-specific actions by a common mechanism within the cell nucleus. These hormones, like the classical steroid hormones, glucocorticoid and oestrogen, bind to nuclear receptor proteins and modify transcriptional activity of target genes. The receptors are members of the steroid/thyroid hormone nuclear receptor superfamily of structurally homologous ligand-responsive transcription factors which activate or repress expression of hormone-responsive target genes (Evans, 1988; Green & Chambon, 1988; Moore, 1990; O'Malley, 1990; Moore & Brent, 1991).

The receptors for 3,5,3′-l-tri-iodothyronine (T3Rs), 1,25(OH)2-vitamin D3 (VDRs) and all-trans retinoic acid (RARs) form a subclass of homologous and functionally related proteins within the steroid superfamily. The receptors can bind to DNA in the absence of ligand (Brent, Dunn, Harney et al. 1989; Graupner, Wills, Tzukerman et al. 1989), they reside in the nucleus and their response elements possess

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Ankana Ganguly, Jennifer A Tamblyn, Alexandra Shattock, Annsha Joseph, Dean P Larner, Carl Jenkinson, Janesh Gupta, Stephane R Gross, and Martin Hewison

Early pregnancy is characterised by elevated circulating levels of vitamin D binding protein (DBP). The impact of this on maternal and fetal health is unclear but DBP is present in the placenta, and DBP gene variants have been linked to malplacentation disorders such as preeclampsia. A functional role for DBP in the placenta was investigated using trophoblastic JEG3, BeWo and HTR8 cells. All three cells lines showed intracellular DBP, with increased expression and nuclear localisation of DBP in cells treated with the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D). When cultured in serum from mice lacking DBP (DBP-/-), JEG3 cells showed no intracellular DBP indicating uptake of exogenous DBP. Inhibition of the membrane receptor for DBP, megalin, also suppressed intracellular DBP. Elimination of intracellular DBP with DBP-/- serum or megalin inhibitor suppressed matrix invasion by trophoblast cells, and was associated with increased nuclear accumulation of G-actin. Conversely, treatment with 1,25D enhanced matrix invasion. This was independent of the nuclear vitamin D receptor but was associated with enhanced ERK phosphorylation, and inhibition of ERK kinase suppressed trophoblast matrix invasion. When cultured with serum from pregnant women, trophoblast matrix invasion correlated with DBP concentration, and DBP was lower in first trimester serum from women who later developed preeclampsia. These data show that trophoblast matrix invasion involves uptake of serum DBP and associated intracellular actin binding and homeostasis. DBP is a potential marker of placentation disorders such as preeclampsia and may also provide a therapeutic option for improved placenta and pregnancy health.

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M. C. Eliam, M. Baslé, Z. Bouizar, J. Bielakoff, M. Moukhtar, and M. C. de Vernejoul

ABSTRACT

Isolated osteoclasts obtained from young chickens fed a normal (+ Ca) or deficient ( − Ca) calcium and vitamin D diet for 3 weeks, were studied for their ability to bind salmon calcitonin (sCT). Osteoclasts obtained from −Ca chickens, when incubated with 0·1 μmol sCT/l, doubled cyclic (c)AMP production and retracted from a glass support, as observed by scanning electron microscopy. The presence of receptors was also demonstrated by autoradiography and competition analysis of 125I-labelled sCT binding. The number of receptors per cell was 0·9 ± 0·1 × 104. In contrast, osteoclasts obtained from + Ca chickens did not increase cAMP production and did not retract in the presence of 0·1 μmol sCT/l. No specific binding of 125I-labelled sCT could be demonstrated on these osteoclasts.

Plasma levels of calcium and calcitonin were measured in +Ca and − Ca chickens. The plasma concentration of calcium was markedly lower at 3 weeks in −Ca than in +Ca chickens. The plasma concentration of calcitonin was decreased in − Ca chickens compared with +Ca chickens at the first week and kept decreasing during the 3 weeks.

These results strongly support the hypothesis that calcium and vitamin D intake regulate plasma calcitonin levels in chickens, and that calcitonin receptors can be detected on chicken osteoclasts only when blood calcium is decreased by a diet deficient in calcium and vitamin D.

J. Endocr. (1988) 119, 243–248

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I Nemere, D Yazzie-Atkinson, DO Johns, and D Larsson

An earlier study revealed that 24R,25-dihydroxyvitamin D(3) (24R,25(OH)(2)D(3)) inhibits the rapid actions of 1,25(OH)(2)D(3) on stimulation of calcium transport in perfused duodena, as well as activation of protein kinases C and A. In the present work, a specific binding protein (24,25-BP) has been identified and partially characterized. Percoll-gradient resolution of differential centrifugation fractions from mucosal homogenates revealed the highest levels of specific [(3)H]24R,25(OH)(2)D(3) binding to be in lysosomes (approximately eight to tenfold greater than in basal lateral membrane fractions). Incubation of isolated enterocytes with 6.5 nM [(3)H]24R,25(OH)(2)D(3) for 10 s also demonstrated targeting of the steroid to lysosomal fractions. Using freshly isolated lysosomal fractions, time course studies indicated maximal specific binding after a 2-h incubation on ice. Western analyses revealed that the serum transport protein, DBP (vitamin D binding protein), was absent from both lysosomal and basal lateral membrane fractions. Protein dependence studies demonstrated linear binding between 0.05 and 0.155 mg of lysosomal protein. Saturation analyses yielded K(d)=7.4+/- 1.8 nM, B(max)=142+/-16 fmol/mg protein for lysosomes, and K(d)=8.5 nM, B(max)=149+/-25 fmol/mg protein for basal lateral membranes. Hill analyses of lysosomal binding yielded a Hill coefficient of 0.57+/-0.11, indicative of negative cooperativity. Studies with lysosomal proteins revealed a 81%+/-7% competition of 24S,25(OH)(2)D(3) with [(3)H]24R,25(OH)(2)D(3) for binding (P>0.05, relative to competition with 24R,25(OH)(2)D(3)), while 25(OH)D(3) and 1,25(OH)(2)D(3) yielded 53%+/-13% and 39%+/-11% competition respectively (each, P<0.05, relative to competition with 24R,25(OH)(2)D(3)). The apparent affinity of 24S,25(OH)(2)D(3) for 24,25-BP led to testing of the metabolites effectiveness in the perfused duodenal loop system. Vascular perfusion with 130 pM 1,25(OH)(2)D(3) stimulated (45)Ca transport to 2.5-fold above control levels after 40 min, while simultaneous perfusion with 6.5 nM 24S,25(OH)(2)D(3) and 130 pM 1,25(OH)(2)D(3) abolished the stimulatory activity completely. Purification of the 24,25-BP by chromatography revealed a single protein band upon SDS-PAGE and silver staining of 66 kDa. The combined results suggest that 24R,25(OH)(2)D(3) may mediate its hormonal activities through a specific binding protein.