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E. M. W. Maunder, A. V. Pillay, and A. D. Care

ABSTRACT

An i.v. injection of calcitriol (1,25-(OH)2D3) had no effect within 2·5 h on plasma concentrations of calbindin-D9k (vitamin D-induced calcium-binding protein; CaBP) in hypocalcaemic pigs with inherited vitamin D-dependent rickets type I or in their normocalcaemic siblings or half-siblings. Three days later the plasma concentration of CaBP had doubled in the hypocalcaemic pigs, but was unaltered in the normocalcaemic siblings and half-siblings. Following daily i.v. injections of 1,25-(OH)2D3 for a further 5 days (days 4–8) plasma concentrations of CaBP increased in both the hypocalcaemic (days 4–8) and normocalcaemic (day 8) pigs, the effect being more rapid and greater in the hypocalcaemic 1,25-(OH)2D3-deficient animals. An i.v. injection of 1,25-(OH)2D3 to pure Yucatan pigs also had no effect on plasma concentrations of CaBP within 1·5 h, but in the following 1 h there was some indication of an increase in plasma CaBP levels.

In contrast to the normal pigs, insulin-induced hypoglycaemia did not lead to a peak in plasma CaBP concentrations in the hypocalcaemic pigs. There was also no change in the plasma concentrations of 1,25-(OH)2D3 associated with the peak in plasma CaBP following insulin-induced hypoglycaemia in normocalcaemic pigs. These results suggest that changes in plasma concentrations of 1,25-(OH)2D3 are not directly involved in mediating the increase in plasma CaBP which follows hypoglycaemia induced by insulin in normal pigs, although 1,25-(OH)2D3 probably plays a permissive role.

J. Endocr. (1987) 115, 129–134

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B. Escoubet, C. Silve, S. Balsan, and C. Amiel

ABSTRACT

It is accepted that renal phosphate wasting is the basis of hypophosphataemia in vitamin D-resistant hypophosphataemic rickets (VDRR). Abnormal renal adaptation to phosphate deprivation has also been reported in these patients. We studied sodium-dependent phosphate transport and its modulation by phosphate deprivation in skin fibroblasts cultured from healthy subjects and patients with VDRR. Control fibroblasts exhibited high-affinity sodium-dependent phosphate transport (77 ± 12 μmol/l) which resembled the ubiquitous transport of renal and non-renal cells. Phosphate deprivation (incubation in low phosphate medium) increased the maximal velocity (V max) of the transport by 2·7-fold after 24 h, with no change in the affinity. The increase in V max was dependent on gene transcription and protein synthesis. The sodium-dependent phosphate transport exhibited in fibroblasts from VDRR patients did not significantly differ from that of control subjects, except that the V max of the phosphate transport was higher in cells from patients with VDRR under normal and phosphate-deprivation conditions, although the difference was significant only after 24 h of phosphate deprivation (V max: 22·6 ± 2·4 pmol/mg protein per s in VDRR vs 16 ± 3·6 pmol/mg protein per s in controls, P < 0·05).

These data demonstrate that sodium-coupled phosphate transport in human skin fibroblasts has the properties of ubiquitous sodium-phosphate co-transport and show that this transport is not deficient in patients with VDRR. Indeed paradoxically the V max was 40% higher in VDRR than in control subjects after 24 h of phosphate deprivation. The transport must be either different from that of kidney cells responsible for the phosphate leak, or differently modulated. Therefore, skin fibroblasts cannot be used to determine the molecular defect responsible for the renal phosphate leak in VDRR patients.

Journal of Endocrinology (1992) 133, 301–309

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PM Bourlon, B Billaudel, and A Faure-Dussert

Because 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) is known to activate the biosynthesis of numerous proteins in various tissues, experiments were undertaken to compare the influence of 1,25(OH)2D3 in vitro on both the secretion and biosynthesis of insulin in islets of Langerhans from both 4-week vitamin D3-deficient rats and normal rats. Islets were either incubated or perifused after a 6-h induction period in the presence of various concentrations of 1, 25(OH)2D3 from 10(-12) M, which was inactive in controls, to 10(-6) M. Experiments were performed in the presence of a non-labelled amino acid mixture, to favour protein synthesis. Tritiated tyrosine was added as tracer during glucose stimulation. The newly synthesised proteins, labelled with [3H]tyrosine, were extracted by an acid-alcohol method and separated by gel chromatography adapted for low-molecular-weight proteins. Even in the presence of the amino acid mixture, the insulin response of the islets to 16.7 mM glucose was decreased by vitamin D3 deficiency and improved by 1,25(OH)2D3. This beneficial effect did not occur in basal conditions, but only during glucose stimulation, and was observed in both phases of insulin release. Moreover, these effects disappeared in the presence of 5x10(-4 )M cycloheximide, a protein biosynthesis inhibitor. Islets from vitamin D3-deficient rats exhibited a general decrease in the amount of de novo biosynthesised proteins and of [3H]tyrosine-labelled insulin and proinsulin fractions. A 6-h period of 1,25(OH)2D3 induction significantly improved the amount of de novo biosynthesised proteins, and particularly of newly synthesised insulin in response to a 2-h glucose stimulation. Calculation of the rate of conversion of newly synthesised proinsulin-like material to insulin as the [3H]insulin/[3H]proinsulin-like material ratio provided evidence for a dose-dependent increase, induced by 1, 25(OH)2D3, that could exceed that of normal islets. These data support the hypothesis that 1,25(OH)2D3 in vitro not only facilitated the biosynthetic capacity of the beta cell - which was highly induced during a 16.7-mM glucose stimulation, via a global activation of islets protein biosynthesis - but also produced an acceleration of the conversion of proinsulin to insulin.

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R Gniadecki and J Serup

Abstract

KH 1060 is a 20-epi analogue of 1,25-dihydroxyvitamin D3 and a potent agonist of the vitamin D receptor. Our recent finding that it stimulates glycosaminoglycan synthesis and transforming growth factor-β1 (TGF-β1) expression in normal skin provided a rationale for investigating its influence on the process of wound healing. Normal and betamethasone-impaired granulation tissue formation was studied in a polytetrafluoroethylene dead space model in hairless mice. The application of KH 1060 increased the indexes of fibroplasia and cellularity ([3H]thymidine incorporation and DNA concentration) of the betamethasone-impaired granulation tissue. Collagen production and deposition, measured as hydroxyproline synthesis and concentration in the granulation tissue, were also increased. The effect of KH 1060 on normal connective tissue repair was less pronounced; DNA and hydroxyproline concentrations in granulation tissue were unchanged. KH 1060 strongly stimulated the expression of TGF-β1 in betamethasone-impaired granulation tissue. Thus, it effectively reversed the deleterious effect of betamethasone on granulation tissue. The hyperproliferative response to this vitamin D analogue might be related to the direct stimulation of the vitamin D receptors in the granulation tissue, while the increased collagen synthesis and deposition was probably caused indirectly, via stimulation of TGF-β1.

Journal of Endocrinology (1994) 141, 411–415

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A Ravid, E Rubinstein, A Gamady, C Rotem, UA Liberman, and R Koren

In addition to its known effects on keratinocyte proliferation and differentiation, the hormonal form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), has been shown to protect keratinocytes from UV- and chemotherapy-induced damage. Epidermal keratinocytes contain both the machinery needed to produce 1,25(OH)(2)D(3) and vitamin D receptors. The activation of the stress-activated protein kinases (SAPKs), such as c-Jun N-terminal kinase (JNK) and p38, is an early cellular response to stress signals and an important determinant of cell fate. This study examines whether modulation of these SAPKs is associated with the effects of 1,25(OH)(2)D(3) on keratinocytes under stress. HaCaT keratinocytes were exposed to heat shock, hyperosmotic concentrations of sorbitol, the epidermal growth factor receptor tyrosine kinase inhibitor AG1487, the pro-inflammatory cytokine tumor necrosis factor alpha, and H(2)O(2). These stresses activated both SAPKs. Pretreatment with 1,25(OH)(2)D(3) inhibited the activation of JNK by all stresses and the activation of p38 by heat shock, AG1478 and tumor necrosis factor alpha. Under the same conditions, treatment with 1,25(OH)(2)D(3) protected HaCaT keratinocytes from cytotoxicity induced by exposure to H(2)O(2) and hyperosmotic shock. The effect of 1,25(OH)(2)D(3) was dose-dependent, already apparent at nanomolar concentrations, and time-dependent, maximal after a 24-h pre-incubation. We suggest that inhibition of SAPK activation may account for some of the well-documented protective effects of 1,25(OH)(2)D(3) on epidermal cells during exposure to UV or chemotherapy and may also be related to the anti-inflammatory actions of the hormone in skin.

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R Bland, R L Sammons, M C Sheppard, and G R Williams

Abstract

3,5,3′-Tri-iodothyronine (T3), 1α,25(OH)2-vitamin D3 (D3) and retinoids activate related nuclear receptors which interact by heterodimerisation to regulate gene expression. Actions of each hormone are discrete and may be specified by changes in the relative concentrations of their receptors (T3R, vitamin D receptor (VDR), retinoic acid receptor (RAR), retinoid X receptor (RXR)). T3, D3 and retinoids are essential for skeletal development and maintenance and we have previously shown complex interactions amongst their signalling pathways in osteosarcoma cells. In these studies we demonstrate that similar T3R, VDR, RAR and RXR proteins are co-expressed in both osteoblast lineage cell primary cultures and osteosarcoma cells by Western blotting. We investigated whether hormone interactions in bone result from changes in receptor stoichiometry. Cells were treated with combinations of T3, D3, 9-cis retinoic acid (9-cis RA) and all-trans retinoic acid (RA) that are known from previous studies to produce complex cell specific responses. No alteration in expression of any receptor protein was seen in response to any hormone combination in three phenotypically distinct osteosarcoma cell lines. Thus, in contrast to studies of overexpressed receptors in vitro, changes in the physiological concentrations of endogenous T3R, VDR, RAR and RXR do not specify discrete hormone actions in osteoblastic cells. Other unidentified factors are likely to modulate hormone action in these bone cells.

Journal of Endocrinology (1997) 154, 63–74

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BR Pal, T Marshall, C James, and NJ Shaw

There is no consensus between Authors on the definition of a replete or deficient vitamin D state. Our aim was to describe a suitable method that could be used to compare vitamin D data in subject groups with small or large numbers. Two hundred and forty indigenous asymptomatic, non-pregnant adult subjects recruited from a single-consultation outpatient attendance with normal biochemistry, represented a sample of our inner city district population. 25-hydroxyvitamin D (25,OHD3) levels were measured to illustrate the effects of season, sex and ethnic group on vitamin D levels and subjected to distribution analysis. This method quantifies as a percentage the distribution of 25,OHD3 concentrations (observed concentration, OC) in pooled group data. The data can be expressed as distribution frequency domains or cumulative frequency ogives (0-100%) or transformed into discrete linear probits, amenable to regression analysis. An estimate of the OC50 (mid-point) and upper (either OC75 or OC95) or lower (either OC25 or OC5) range or at any other frequency between subject groups can be compared. A marked difference in 25,OHD3 levels between Asian and non-Asian asymptomatic adult subjects was seen during both seasons. 25,OHD3 deficiency was defined as at or below the OC25 for the non-Asian group (for both sexes: winter < 13.36 ng/ml, summer <13.38 ng/ml). The majority of Asians of both sexes were 25,OHD3 deficient (winter 94%, summer 82%). The distribution analysis provides an easy technique to compare 25,OHD3 status of different subject groups, allowing the description of populations using either longitudinal or cross-sectional data. This method may offer a way of describing 25,OHD3 deficiency between observers worldwide.

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B. L. Nyomba, R. Bouillon, and P. De Moor

ABSTRACT

Vitamin D metabolites and vitamin D-binding protein (DBP) were measured in non-diabetic rats and in rats made diabetic with streptozotocin. The animals were studied in the intact state, after gonadectomy and during pregnancy. In male non-diabetic rats the serum concentrations of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and DBP decreased after orchidectomy and were restored by treatment with testosterone. In female non-diabetic rats, these parameters increased after ovariectomy. Increased 1,25-(OH)2D3 and decreased DBP concentrations were found during pregnancy in non-diabetic rats.

After the induction of diabetes in intact rats of both sexes, the concentration of DBP decreased, but a significant decrease in the concentration of 1,25-(OH)2D3 was found in male animals only. After ovariectomy, however, 1,25-(OH)2D3 decreased also in female diabetic rats.

Both orchidectomy and insulin deficiency depressed serum concentrations of 1,25-(OH)2D3 (−22 and −45% respectively) and DBP (−14 and −29% respectively), but the effects of insulin deficiency were greater than those of androgen withdrawal. Moreover, the testosterone concentration was twofold lower in intact male diabetic rats than in non-diabetic animals. Insulin, but not testosterone treatment, however, restored DBP and 1,25-(OH)2D3 concentrations in diabetic rats, and insulin was effective in intact as well as in gonadectomized animals.

This study shows that insulin deficiency decreases the concentrations of DBP and 1,25-(OH)2D3 in the rat, and that these decreases are facilitated by androgens, but counteracted by oestrogens.

J. Endocr. (1987) 115, 295–301

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S. K. Abbas, A. D. Care, H. Van Baelen, and R. Bouillon

ABSTRACT

A radioimmunoassay for ovine vitamin D-binding protein (DBP) has been developed. This assay can also effectively measure DBP in goat plasma. A suitable ovine DBP antiserum raised in a rabbit produced a single monospecific line of precipitation when reacted against purified sheep DBP and sheep plasma. The preliminary purification of 125I-labelled ovine DBP was carried out using adsorption chromatography, and the final purification immediately before addition to the assay tubes was achieved by high-pressure liquid chromatography. Displacement of 125I-labelled ovine DBP by dilutions of sheep and goat plasma or standard DBP gave parallel curves, and only weak competition was observed with calf and pig plasma. The assay detected as little as 26 pmol DBP/1 with intra- and interassay coefficients of variation of 3 and 14% respectively. The mean plasma concentration of DBP in nine pregnant sheep (110–120 days of gestation) was 8·7 ± 0·3 (s.e.m.) μmol/l. These levels were significantly (P<0·02; paired t-test) higher than those in matched fetal plasma (6·7 ±0·4 μmol/l) obtained in utero through a catheter in a carotid artery. Plasma DBP concentrations in pregnant sheep were also significantly (P<0·02) higher than in five normal non-pregnant sheep (6·8 ± 0·5 μmol/l). The mean concentrations of total 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) in maternal and fetal plasma were 92·0 ±8·7 pmol/l and 152·5±18·0 pmol/l respectively (P<0·05). The free 1,25-(OH)2D3 index, a measure of the level of free 1,25-(OH)2D3 in plasma, was calculated as the ratio between the molar concentrations of total 1,25-(OH)2D3 and DBP. The mean value of the free 1,25-(OH)2D3 index in the fetus (2·3 × 10−5) was significantly (P<0·01) higher than that in the dam (1·1 × 10−5), thus indicating a gradient of free 1,25-(OH)2D3 during the latter part of ovine pregnancy with a mean fetal/maternal ratio of 2·1.

J. Endocr. (1987) 115, 7–12

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S Y James, A G Mackay, L Binderup, and K W Colston

Abstract

The anti-proliferative effects of the novel vitamin D analogue, EB1089, were assessed in the hormone-dependent breast cancer cell line, MCF-7, in vitro. In the present study, EB1089 was shown to be at least an order of magnitude more potent at inhibiting MCF-7 cell proliferation than the native hormone, lα,25-dihydroxyvitamin D3 (1,25(OH)2D3). Treatment of MCF-7 cell cultures with combinations of oestradiol and EB1089 ranging from 5 × 10−11 m to 5 × 10−9 m revealed the ability of EB1089 to suppress the mitogenic effects of oestradiol in these cells dose-dependently, as determined by [3H]thymidine incorporation and cell counts. EB1089 also exhibited a significant time- and dose-dependent decrease in MCF-7 oestrogen receptor (ER) concentration, as assessed by ligand binding assay. A fourfold reduction of ER levels by 5 × 10−9 m EB1089 relative to control ER levels was observed, whilst 5 × 10−9 m 1,25(OH)2D3 produced a significant but less dramatic decrease in ER levels. In addition, reduction of ER protein in EB1089-treated cell cultures was also demonstrated using an oestrogen receptor enzyme immunoassay.

The interaction of EB1089 and anti-oestrogens on the oestradiol-stimulated growth of MCF-7 cells was investigated. The treatment of cell cultures with 5 × 10−10 m EB1089 in combination with the pure anti-oestrogen, ICI 182,780 (5 × 10−8 m), and in the presence of between 5 × 10−10 m and 5 × 10−9 m oestradiol, produced an augmented inhibition of MCF-7 cell proliferation compared with the actions of either compound alone.

This study demonstrates that EB1089 is a potent anti-proliferative agent of breast cancer cells in vitro, and that part of its mechanism to inhibit cell growth may involve the modulation of ER expression, such that the responsiveness of cells to the growth-stimulatory effects of oestradiol is diminished. This new vitamin D analogue has potential in the treatment of breast cancer.

Journal of Endocrinology (1994) 141, 555–563