The effect of disodium ethane-1-hydroxy-1,1-diphosphonate on the metabolism of calcitriol in chicks.

Decreased intestinal absorption of Ca2+ occurs in response to treatment with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP). The effect is due to decreased 1-hydroxylation of calcidiol (25-hydroxycholecalciferol) in the kidney. In an attempt to establish whether impairment of vitamin D metabolism at steps beyond kidney hydroxylation occurs due to treatment with EHDP, chicks were depleted of vitamin D and were treated with calcitriol (1,25-dihydroxycholecalciferol) as their sole source of the vitamin. The chicks were then divided into two groups, one being treated with EHDP while the second group served as control. Intestinal absorption of Ca2+ in the EHDP-treated group was found to be impaired, along with decreases in concentrations of calbindin D28K (the 28,000-Mr vitamin D-dependent Ca2+-binding protein). When the chicks were dosed with [3H]calcitriol, significantly lower concentrations of the sterol were detected in the duodena of EHDP-treated birds. Measurement of levels of receptors for calcitriol in duodena showed no difference between groups, but levels of calcitriol in sera were considerably lower in the EHDP-treated group along with the elevated biliary and urinary excretion of glucuronidated conjugates. It is therefore concluded that treatment with EHDP results in increased catabolism of calcitriol in addition to the known suppression of the renal production of the hormone.     

VDR-mediated gene expression patterns in resting human coronary artery smooth muscle cells

Vitamin D analogs such as paricalcitol and calcitriol that activate the vitamin D receptor (VDR) provide survival benefit for Stage 5 chronic kidney disease (CKD) patients, possibly associated with a decrease in cardiovascular (CV)-related incidents. Phenotypic changes of smooth muscle cells play an important role in CV disease. The role of vitamin D analogs in modulating gene expression in smooth muscle cells is still not well understood. In this study, DNA microarray analysis of approximately 22,000 different human genes was used to characterize the VDR-mediated gene expression profile in human coronary artery smooth muscle cells (CASMC) at rest. Cells in serum free medium were treated with 0.1 microM calcitriol (1alpha,25-dihydroxyvitamin D(3)) or paricalcitol (19-nor-1alpha,25-(OH)(2)D(2)) for 30 h. A total of 181 target genes were identified, with 103 genes upregulated and 78 downregulated (>two fold changes in either drug treatment group with P < 0.01). No significant difference was observed between calcitriol and paricalcitol. Target genes fell into various categories with the top five in cellular process, cell communication, signal transduction, development, and morphogenesis. Twenty-two selected genes linked to the CV system were also impacted. Real-time RT-PCR and/or Western blotting analysis were employed to confirm the expression patterns of selected genes such as 25-hydroxyvitamin D-24-hydroxylase, Wilms' tumor gene 1, transforming growth factorbeta3, plasminogen activator inhibitor-1, thrombospondin-1 (THBS1), and thrombomodulin (TM). This study provides insight into understanding the role of VDR in regulating gene expression in resting smooth muscle cells.     

Glucocorticoids antagonize induction of prolactin-gene expression by calcitriol in rat pituitary tumour cells.

Clonal strains of rat pituitary tumour (GH4C1) cells are known to possess specific intracellular binding sites for calcitriol (1,25-dihydroxycholecalciferol, 1,25-dihydroxyvitamin D3). GH4C1 cells respond to calcitriol by a selective increase in prolactin(PRL)-gene expression. The interaction between calcitriol and glucocorticoids was studied by using this cultured-cell model. It was found that cortisol potently antagonized the induction of PRL mRNA and PRL production by calcitriol. The effects were concentration-dependent and were evident at glucocorticoid concentrations that did not alter basal PRL production. Inhibition was half-maximal at 3.2 nM-cortisol and 0.4 nM-dexamethasone. Calcitriol-induced PRL mRNA fell by more than 50% at 25 h and reached the control level 50 h after treatment with cortisol. The inhibition by cortisol of calcitriol induction of PRL production was selective when compared with effects on other inducers of PRL-gene expression [thyroliberin, epidermal growth factor and phorbol myristate acetate ('12-omicron-tetradecanoylphorbol 13-acetate')]. Potent antagonism by glucocorticoids of vitamin D action on specific gene expression has been demonstrated. Further studies with this cultured-cell model may help to explain the mechanism of this hormonal interaction, which assumes particular importance at major sites of vitamin D action such as the intestine.     

The inflammatory response of keratinocytes and its modulation by vitamin D: the role of MAPK signaling pathways

The hormonal form of vitamin D, calcitriol, and its analogs are known for their beneficial effect in the treatment of inflammatory skin disorders. Keratinocytes play a role in epidermal inflammatory responses invoked by breeching of the epidermal barrier, by infectious agents and by infiltrating immune cells. We studied the role of calcitriol in the initiation of keratinocyte inflammatory response by the viral and injury mimic polyinosinic-polycytidylic acid (poly(I:C)) and in its maintenance by tumor-necrosis-factor α (TNFα) and investigated the role of the mitogen-activated protein kinase cascades in these processes and their regulation by calcitriol. The inflammatory response of human HaCaT keratinocytes to poly(I:C) or TNFα was assessed by measuring mRNA levels of 13 inflammation-related molecules by real-time PCR microarray and by in-depth investigation of the regulation of interleukin 8, intercellular-adhesion-molecule 1, and TNFα expression. We found that while calcitriol had only a minor effect on the keratinocyte response to poly(I:C) and a modest effect on the early response (2 h) to TNFα, it markedly attenuated the later response (16-24 h) to TNFα. The expression of CYP27B1, the enzyme responsible for calcitriol production, was marginally increased by poly(I:C) and markedly by TNFα treatment. This pattern suggests that while allowing the initial keratinocyte inflammatory response to proceed, calcitriol contributes to its timely resolution. Using pharmacological inhibitors we found that while the p38 MAPK and the extracellular signal-regulated kinase have only a minor role, c-Jun N-terminal kinase plays a pivotal role in the induction of the pro-inflammatory genes and its modulation by calcitriol.     

Treatment with 1,25-dihydroxyvitamin D3 reduces impairment of human osteoblast functions during cellular aging in culture

Adequate responses to various hormones, such as 1,25-dihydroxyvitamin D(3) (calcitriol) are a prerequisite for optimal osteoblast functions. We have previously characterized several human diploid osteoblastic cell lines that exhibit typical in vitro aging characteristics during long-term subculturing. In order to study in vitro age-related changes in osteoblast functions, we compared constitutive mRNA levels of osteoblast-specific genes in early-passage (< 50% lifespan completed) with those of late-passage cells (> 90% lifespan completed). We found a significant reduction in mRNA levels of alkaline phosphatase (AP: 68%), osteocalcin (OC: 67%), and collagen type I (ColI: 76%) in in vitro senescent late-passage cells compared to early-passage cells, suggesting an in vitro age-related impairment of osteoblast functions. We hypothesized that decreased osteoblast functions with in vitro aging is due to impaired responsiveness to calcitriol known to be important for the regulation of biological activities of the osteoblasts. Thus, we examined changes in vitamin D receptor (VDR) system and the osteoblastic responses to calcitriol treatment during in vitro osteoblast aging. We found no change in the amount of VDR at either steady state mRNA level or protein level with increasing in vitro osteoblast age and examination of VDR localization, nuclear translocation and DNA binding activity revealed no in vitro age-related changes. Furthermore, calcitriol (10(-8)M) treatment of early-passage osteoblastic cells inhibited their proliferation by 57 +/- 1% and stimulated steady state mRNA levels of AP (1.7 +/- 0.1-fold) and OC (1.8 +/- 0.2-fold). Similarly, calcitriol treatment increased mRNA levels of AP (1.7 +/- 0.2-fold) and OC (3.0 +/- 0.3-fold) in late-passage osteoblastic cells. Thus, in vitro senescent osteoblastic cells maintain their responsiveness to calcitriol and some of the observed in vitro age-related decreases in biological markers of osteoblast functions can be reverted by calcitriol treatment.     

Vitamin D-inducible calcium transport and gene expression in three Caco-2 cell lines

The parental cell line (P) of Caco-2 cells and two clones, BBe and TC7, were studied at 11 days postconfluence to test the facilitated diffusion model of vitamin D-mediated intestinal calcium absorption (CaTx). Nuclear vitamin D receptor (nVDR) and calbindin D(9k) (CaBP) were measured by Western blot; 1,25-hydroxyvitamin D(3) 24-hydroxylase (CYP24), CaBP, plasma membrane Ca-ATPase (PMCA), and Ca transport channel (CaT1) mRNA levels were examined by RT-PCR; and net apical-to-basolateral CaTx was examined after treating cells with vehicle or 10 nM calcitriol for 8 (mRNA levels) or 48 h (protein, CaBP mRNA, CaTx). nVDR level was lowest in BBe (38% P value) and directly related to CYP24 induction (TC7 = P, which were 1.56 times greater than BBe). nVDR was inversely related to the vitamin D-induced levels of CaT1 mRNA, CaBP mRNA, PMCA mRNA, and net CaTx, with the highest induction seen in BBe. Basal CaBP mRNA (86 times greater than P) and protein levels were highest in TC7 cells and were not associated with higher net CaTx, suggesting CaBP may not be rate limiting for CaTx in these cells.     

Characterization of Vitamin D insensitive prostate cancer cells

The antitumor effects of 1,25-dihydroxyvitamin D₃ (calcitriol) are being exploited for prevention and treatment of prostate cancer (CaP). These studies examined the antiproliferative effects of calcitriol in primary cell cultures derived from transgenic adenocarcinoma of mouse prostate (TRAMP) mice chronically treated with calcitriol (20 μg/kg) or vehicle 3×/week from 4 weeks-of-age until palpable tumors developed. This is a report on the response of two representative control (Vitamin D naïve, naïve) and calcitriol-treated (Vitamin D insensitive, VDI) cells to calcitriol. VDI cells were less sensitive to calcitriol based on less cell growth inhibition and less inhibition of DNA synthesis as measured by MTT and BrdU incorporation assays. Similarly, VDI cells were less sensitive to growth inhibition by the vitamin analog, 19-nor-1,25-dihydroxyvitamin D₂ (paricalcitol). There was no change in apoptosis following treatment of naïve and VDI cells with calcitriol. Vitamin D receptor (VDR) expression was up-regulated by calcitriol in both naïve and VDI cells. In addition, calcitriol induced the Vitamin D metabolizing enzyme, 24-hydroxylase (cyp24) mRNA and enzyme activity similarly in naïve and VDI cells as measured by RT-PCR and HPLC, respectively. In summary, VDI cells are less responsive to the antiproliferative effects of calcitriol. Understanding Vitamin D insensitivity will further clinical development of Vitamin D compounds for prevention and treatment of CaP.     

Molecular mechanisms involved in the enhancement of mitochondrial malate dehydrogenase activity by calcitriol in chick intestine

Mitochondrial malate dehydrogenase (mMDH) from the intestine is the NAD-linked oxidoreductase of the tricarboxylic acid cycle with the highest activity and response to vitamin D treatment in vitamin D-deficient chicks (?D). The aim of this study was to elucidate potential molecular mechanisms by which cholecalciferol or calcitriol enhances the activity of this enzyme. One group of animals used was composed of ?D and ?D treated with cholecalciferol or with calcitriol. A second group consisted of ?D and ?D supplemented with high Ca²⁺ diet. A third group included chicks receiving either a normal or a low Ca²⁺ diet. In some experiments, animals were injected with cycloheximide. Data showed that either vitamin D (cholecalciferol or calcitriol) or a low Ca²⁺ diet increases mMDH activity. High Ca²⁺ diet did not modify the intestinal mMDH activity from ?D. The mMDH activity from ?D remained unaltered when duodenal cells were exposed to 10^?8 mol/L calcitriol for 15 min. The enhancement of mMDH activity by calcitriol was completely abolished by simultaneous cycloheximide injection to ?D. mMDH mRNA levels, detected by RT-PCR, indicate that calcitriol did not affect gene expression. In contrast, Western blots show that calcitriol enhanced the protein expression. In conclusion, calcitriol stimulates intestinal mMDH activity by increasing protein synthesis. No response of mMDH activity by rapid effects of calcitriol or activation through increment of serum Ca²⁺ was demonstrated. Consequently, ATP production would be increased, facilitating the Ca²⁺ exit from the enterocytes via the Ca²⁺-ATPase and Na⁺/Ca²⁺ exchanger, which participate in the intestinal Ca²⁺ absorption.     

Lipopolysaccharide negatively modulates vitamin D action by down-regulating expression of vitamin D-induced VDR in human monocytic THP-1 cells

Vitamin D3, an important seco-steroid hormone for the regulation of body calcium homeostasis, promotes immature myeloid precursor cells to differentiate into monocytes/macrophages. Vitamin D receptor (VDR) belongs to a nuclear receptor super-family that mediates the genomic actions of vitamin D3 and regulates gene expression by binding with vitamin D response elements in the promoter region of the cognate gene. Thus by regulating gene expression, VDR plays an important role in modulating cellular events such as differentiation, apoptosis, and growth. Here we report lipopolysaccharide (LPS), a bacterial toxin; decreases VDR protein levels and thus inhibits VDR functions in the human blood monocytic cell line, THP-1. The biologically active form of vitamin D3, 1alpha,25-dihydroxy vitamin D3 [1,25(OH)2D3], induced VDR in THP-1 cells after 24 h treatment, and LPS inhibited 1,25(OH)2D3-mediated VDR induction. However, LPS and 1,25(OH)2D3 both increased VDR mRNA levels in THP-1 cells 20 h after treatment, as observed by real time RT-PCR. Moreover, LPS plus 1,25(OH)2D3 action on VDR mRNA level was additive and synergistic. A time course experiment up to 60 h showed an increase in VDR mRNA that was not preceded with an increase in VDR protein levels. Although the proteasome pathway plays an important role in VDR degradation, the proteasome inhibitor lactacystin had no effect on the LPS-mediated down-regulation of 1,25(OH)2D3 induced VDR levels. Reduced VDR levels by LPS were accompanied by decreased 1,25(OH)2D3/VDR function determined by VDR responsive 24-hydroxylase (CYP24) gene expression. The above results suggest that LPS impairs 1,25(OH)2D3/VDR functions, which may negatively affect the ability of 1,25(OH)2D3 to induce myeloid differentiation into monocytes/macrophages.     

1,25-(OH)2-16ene-23yne-D3 reduces secondary hyperparathyroidism in uremic rats with little calcemic effect

OBJECTIVES: To compare the effects of vitamin D analogs versus calcitriol on serum levels of Ca, P and parathyroid hormone (PTH). A compound better than calcitriol should increase the Ca x P product less than calcitriol for an equivalent decrease in PTH levels. METHODS: Biological activity of 4 vitamin D analogs, 1,25-(OH)(2)-16ene- D(3) (RO(1)), 1,25-(OH)(2)-16ene-23yne-D(3) (RO(2)), 1,25-(OH)(2)-26,27-hexafluoro-16ene-23yne-D(3) (RO(3)) and 1,25-(OH)(2)-16ene-23yne-26,27-hexafluoro-19nor-D(3) (RO(4)) was tested vs. calcitriol in parathyroidectomized rats. In a second set of experiments, the effects of RO(2), RO(4) and calcitriol were studied in 5/6 nephrectomized rats with secondary hyperparathyroidism. RESULTS: In parathyroidectomized rats, all analogs (250 pmol/day) led calcemia to rise after 7 days. In uremic rats, all treatments reduced PTH levels. RO(4) revealed toxicity. RO(2) was as effective as calcitriol in suppressing PTH in a dose dependent manner. Mean plasma ionized calcium did not change from baseline to day 14 and day 28 on RO(2) (250 or 500 pmol/day) whereas it increased significantly on RO(2) (1,000 pmol/day) and calcitriol (125 or 250 pmol/day). Increasing the dose of calcitriol led Ca x P to rise more dramatically than increasing the dose of RO(2), which appears to have a wider therapeutic window than calcitriol. CONCLUSION: 1,25-(OH)(2)-16ene-23yne-D(3) (RO(2)) may represent a novel candidate for the treatment of renal osteodystrophy in humans.     

Inhibitory effect of 22-oxa-1,25-dihydroxyvitamin D3, maxacalcitol, on the proliferation of pancreatic cancer cell lines

Effective chemotherapy for pancreatic cancer is urgently needed. The aim of this study was to compare the anti-proliferative activity on pancreatic cancer cell lines of the vitamin D(3) analog, 22-oxa-1,25-dihydroxyvitamin D(3), maxacalcitol, with that of 1,25-dihydroxyvitamin D(3), calcitriol, with analysis of vitamin D receptor status and the G(1)-phase cell cycle-regulating factors. Antiproliferative effects of both agents were compared using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method and by measuring the tumor size of xenografts inoculated into athymic mice. Scatchard analysis of vitamin D receptor contents, and mutational analysis of receptor complementary DNA were performed. Levels of expression of cyclins, cyclin-dependent kinases and cyclin-dependent kinase inhibitors, p21 and p27, were analysed by western blotting. In vitro, maxacalcitol and calcitriol markedly inhibited the proliferation and caused a G(1) phase cell cycle arrest with the appearance of numerous domes. In vivo, maxacalcitol inhibited the growth of BxPC-3 xenografts more significantly than calcitriol, without inducing hypercalcemia. Responsive cells had abundant functional vitamin D receptors. However, Hs 766T, showing no response to either agent, had the second highest receptor contents with no abnormalities in its primary structure deduced by receptor complementary DNA. In the responsive cells, p21 and p27 were markedly up-regulated after 24h of treatment with both agents. In non-responsive cells, no such changes were observed. In conclusion, maxacalcitol and calcitriol up-regulate p21 and p27 as an early event, which in turn could block the G(1)/S transition and induce growth inhibition in responsive cells, and maxacalcitol may provide a more useful tool for the chemotherapy of pancreatic cancer than calcitriol because of its low toxicity.     

History of the development of new vitamin D analogs: studies on 22-oxacalcitriol (OCT) and 2beta-(3-hydroxypropoxy)calcitriol (ED-71)

In 1981 Suda and his colleagues first reported the new activity of calcitriol namely its ability to differentiate the myeloid leukemia cells into normal monocytes-macrophages. However, the possibility of using calcitriol as an antileukemic drug was not feasible because of its potent calcemic effects. Based on these observations, several pharmaceutical companies initiated the synthesis of vitamin D analogs with the aim to separate the calcemic actions of calcitriol from its actions on regulating the cell growth and differentiation. As a result, numerous noncalcemic analogs with a potential for the treatment of leukemia and other cancers were synthesized. The group at Chugai introduced two characteristic analogs of opposite type namely, 22-oxacalcitriol (OCT) and 2beta-(3-hydroxypropoxy)calcitriol (ED-71) which have been shown to have therapeutic value and are already being used clinically. The work on OCT and ED-71 together with the work on calcipotriol and KH-1060 by Leo Laboratories, and 1alpha,25(OH)(2)-16-ene-23-yne-D(3) by Hoffmann-La Roche, vigorously stimulated research world-wide in the development of vitamin D analogs into pharmaceutical products. More recently new impressive vitamin D analogs such as 3-epi analogs, 19-nor analogs, 18-nor analogs, 2-methyl-20-epi-calcitriol, non-steroidal vitamin D analogs are being developed. The authors are convinced that various vitamin D analogs will become highly effective therapeutic agents at the clinical level in the new century, and also that a new theory on the mechanism of vitamin D action will be generated.     

Demonstration and characterization of 1,25-dihydroxyvitamin D3 receptors in human mononuclear blood cells

Circulating human mononuclear blood cells were studied for the presence of specific 1,25-dihydroxyvitamin D3 (calcitriol) binding macromolecules. Cells were isolated by density gradient centrifugation and characterized by surface markers. Specific reversible high affinity binding by a 3.5 S macromolecule was demonstrated in malignant B-cells and circulating monocytes. In monocytes specific calcitriol binding was found both in the presence and absence of vitamin D3 to saturate the vitamin D3 binding serum protein. No specific calcitriol binding was found in resting B or T lymphocytes. The data suggest a role of calcitriol in the control of mononuclear blood cell proliferation/differentiation.     

Modulation of rat calbindin-D28 gene expression by 1,25-dihydroxyvitamin D3 and dietary alteration

We have used a specific cDNA to the mammalian 28,000 Mr vitamin D-dependent calcium binding protein (calbindin-D28k) to study the regulation of the expression of this mRNA in rat kidney and brain. The effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and dietary alteration on genomic expression were characterized by both Northern and slot blot analysis. Administration of 1,25-(OH)2D3 for 7 days (25 ng/day) to vitamin D-deficient rats resulted in a marked increase in renal calbindin-DmRNA, renal calbindin, and serum calcium. When vitamin D-deficient rats were supplemented for 10 days with calcium (3% calcium gluconate in the water, 2% calcium in the diet) serum calcium levels were similar to the levels observed in the 1,25-(OH)2D3-treated rats. However, in the calcium-supplemented rats the levels of renal calbindin and renal calbindin mRNA were similar to the levels observed in the vitamin D-deficient rats, suggesting that calcium alone without vitamin D does not regulate renal calbindin gene expression in vivo. In dietary alteration studies in vitamin D-replete rats, renal calbindin protein and mRNA increased 2.5-fold in rats fed diets low in phosphate providing evidence that in the rat the nutritional induction of calbindin is accompanied by a corresponding alteration in the concentration of its specific mRNA. Under low dietary calcium conditions, the levels of renal calbindin protein and mRNA were similar to the levels observed in control rats, although 1,25-(OH)2D3 serum levels were markedly elevated, suggesting that factors in addition to 1,25-(OH)2D3 can modulate renal calbindin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)