Regulation Of Vitamin Research Articles

Métabolisme minéral osseux: données récentes et perspectives relatives à l’ostéogenèse

Important data have recently been added to our knowledge of bone mineral metabolism in children. Molecular pathophysiology of several pediatric syndromes has been clarified. Specially, the components of endocrine and metabolic regulations are tightly related with regard to the trophicity of bone. On another hand, the impact of several therapeutics of bone diseases like biphosphonates, parathormone (PTH) or growth hormone on bone anabolism is now strongly emphasized. All these points are important for the becoming of bone pediatric diseases in the adult life. Here we analyze the essential components of mineral metabolism and of its regulation in view of the recent biological data, like PTH/PTHrP (PTH-related peptide)-evoked cell signaling, the role of FGF 23 (Fibroblast growth factor 23) in hypophosphatemia and the regulation of vitamin D metabolism by 1α-hydroxylase. Inter-relation of these regulating elements is present in several genetic diseases and in the Mc Cune Albright syndrome. Relationships between metabolic and endocrine factors are analyzed considering their impact on PTH secretion and osteogenesis.

Response element binding proteins and intracellular vitamin D binding proteins: novel regulators of vitamin D trafficking, action and metabolism*1

Response element binding proteins and intracellular vitamin D binding proteins: novel regulators of vitamin D trafficking, action and metabolism*1

P38 MAPK Activation Selectively Induces Cell Death in K-ras-mutated Human Colon Cancer Cells through Regulation of Vitamin D Receptor

Ras is the most characterized oncogene in human cancer, and yet there are no effective therapeutics to selectively target this oncogene. Our previous work demonstrated the inhibitory activity of the p38 pathway in Ras proliferative signaling in experimental NIH 3T3 cells (Chen, G., Hitomi, M., Han, J., and Stacey, D. W. (2000) J. Biol. Chem. 275, 38973-38980). Here we explore the therapeutic potential of p38 kinase activation in human colon cancer cells with and without endogenous K-ras activation. p38 activation by both adenovirus-mediated gene delivery of constitutively active p38 activator MKK6 and by arsenite selectively induces cell death in K-ras-activated human colon cancer HCT116 cells but not in the K-ras-disrupted HCT116-derived sublines. The cell death-inducing effect of MKK6 is not because of its selective activation of p38 kinase or its downstream transcription factor substrates, ATF-2 or c-Jun, in K-ras-activated cells. Rather, cell death in K-ras-activated cells is linked to the down-regulation of vitamin D receptor (VDR) by an AP-1-dependent mechanism. Forced VDR expression in K-ras-activated cells inhibits p38 activation-induced cell death, and inhibition of endogenous VDR protein expression in K-ras-disrupted cells increased the arsenite-induced toxicity. Analysis of an additional two human colon cancer cell lines with and without K-ras mutation also showed a K-ras- and VDR-dependent toxicity of MKK6. Hence, p38 pathway activation selectively induces cell death in K-ras-mutated human colon cancer cells by mechanisms involving the suppression of VDR activity.

The vitamin K–dependent γ-glutamyl carboxylase gene contains a TATA-less promoter with a novel upstream regulatory element

AbstractThe expression of the vitamin K–dependent γ-glutamyl carboxylase gene in liver is developmentally regulated. Since the gene product catalyzes an essential post-translational modification of the vitamin K–dependent blood coagulation proteins, the regulation of carboxylase expression is critical for hemostasis. We analyzed the activity of the rat carboxylase gene 5′-regulatory DNA sequences in rat hepatoma cell lines at different states of differentiation. These studies demonstrated that the 2.6-kb 5′-flanking sequence has differentiation-dependent transcriptional activity. Transient gene expression assays, examining the effects of nested deletions and site-directed mutagenesis of putative regulatory sequences, together with electrophoretic mobility shift assays (EMSAs) were used to identify sequences critical for the developmentally regulated transcription of the rat carboxylase gene. We identified a DNA sequence (–76 to –65; GTTCCGGCCTTC) not known to bind to transcription factors, yet which functions as an upstream promoter element. In vivo genomic DNA footprinting confirms the presence of nuclear protein–DNA interactions at this site in the endogenous carboxylase gene in differentiated hepatoma cells. Therefore, this DNA sequence has specific nuclear protein–binding activity and functional properties consistent with a regulatory element that plays a critical role in the developmental expression of the carboxylase gene, and hence the regulation of vitamin K–dependent blood coagulation protein synthesis.

Gene expression of vitamin D hydroxylase and megalin in the remnant kidney of nephrectomized rats

Regulation of vitamin D hydroxylase genes in the early stage of chronic renal failure is not fully understood. Using nephrectomized rats, we examined changes in mRNA levels of CYP27B1 (25-hydroxyvitamin D3-1 alpha-hydroxylase), CYP24 (25-hydroxyvitamin D3-24-hydroxylase), and vitamin D receptor in relation to megalin, recently found to participate in renal vitamin D metabolism. A rat model of moderate renal failure was induced by 3/4 nephrectomy. Plasma parameters, including vitamin D metabolite concentrations, were measured at weeks 2, 4 and 8, and poly(A)+ RNA extracted from the remnant kidneys was subjected to Northern blot hybridization. Plasma creatinine concentration at week 2 was 0.40 +/- 0.02 mg/dL in the sham-operated and 0.93 +/- 0.15 mg/dL in the nephrectomized rats, and both values remained constant up to week 8. Plasma concentrations of 25(OH)D3, 1 alpha,25(OH)2D3, and 24,25(OH)2D3 were unchanged between nephrectomized and sham-operated rats at week 8. Intact parathyroid hormone (PTH) increased at week 8 in nephrectomized rats. CYP27B1 mRNA in nephrectomized rats did not vary at week 2, but increased approximately two- and four-fold at weeks 4 and 8, respectively, compared to the sham-operated rats. CYP24 and megalin mRNAs, on the other hand, began to decline as early as at week 2 in nephrectomized rats and kept decreasing throughout the experiment. The expression of vitamin D receptor was modestly but significantly decreased only at week 8. Coordinated and reciprocal alterations of the increase in CYP27B1 mRNA and the decrease in CYP24 mRNA may play a pivotal role in maintaining the plasma level of 1 alpha,25(OH)2D3 in the face of reduced nephron mass and/or megalin expression.

Adaptive responses of 25-hydroxyvitamin D3 1-alpha hydroxylase expression to dietary phosphate restriction in young and adult rats

Regulation of vitamin D metabolism alters with age. The present study is undertaken to investigate if the loss of renal 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) production in response to dietary phosphate (P) restriction in adult rats is due to an alteration in the renal expression of 25-hydroxyvitamin D3 1-alpha hydroxylase (1-OHase). Young (4–6 weeks old) and adult (12–14 weeks old) male Sprague Dawley rats were fed either normal P (NPD) or low P diet (LPD) for 0–5 days. Basal expression of 1-OHase protein was higher in adult rats. Young rats, but not adult rats, significantly increased 1-OHase protein and mRNA expressions in response to LPD in a time-dependent manner. To determine if the stability of renal 1-OHase protein changes with LPD feeding, young and adult rats fed either NPD or LPD for 5 days were injected intravenously with cycloheximide (CHX), a protein synthesis inhibitor. CHX decreased 1-OHase protein expression in young rats fed NPD. However, CHX did not alter 1-OHase protein expression in young rats fed LPD nor in adult rats fed either diet. The results indicate that the stability of renal 1-OHase protein increased with age and that LPD increased its stability only in young rats.

Regulation of vitamin D biosynthesis by calcium

Vitamin D is a secosteroid that is made in the skin by the action of sunlight. Vitamin D is biologically insert and must undergo two successive hydroxylations in the liver and kidney to become the biologically active vitamin D [1alpha,25 (OH)(2)D]. Recently it was appreciated that 1alpha,25 (OH)(2)D not only regulates calcium but also is capable of inhibiting the proliferation and inducing terminal differentiation of variety of cells. The renal production of 1alpha,25 (OH)(2)D is tightly regulated by serum calcium levels through the action of parathyroid hormone.

Vitamin E and transfer proteins.

Recently, the intracellular transport as well as cellular uptake and excretion of alpha-tocopherol, the major representative of vitamin E, have been elucidated. Alpha-tocopherol transfer protein has been identified as the major intracellular transport protein for vitamin E, mediating alpha-tocopherol secretion into the plasma via a non-Golgi-dependent pathway, while other binding proteins seem to play a less important role. New information has accumulated concerning the role of this protein in the transport and supply of vitamin E to tissues such as the central nervous system and the feto-maternal unit. The scavenger receptor class B type I receptor, a membrane-bound protein, is capable of transferring vitamin E into the cell, while the ATP-binding cassette transporter A1 can excrete vitamin E out of the cell. Advances in the area of vitamin E metabolism have shown that alpha-CEHC (2,5,7,8-tetramethyl-2-(2'-carboxyethyl)-6-hydroxychroman) and gamma-CEHC (2,7,8-trimethyl-2-(2'-carboxyethyl)-6-hydroxychroman) are formed by a cytochrome p450-mediated process, important for alpha and gamma-tocopherol excretion. Insights into the regulation of vitamin E transport and metabolism on the cellular level have made enormous advances, showing the complex interplay of influx, trafficking, efflux and metabolism of this crucial antioxidant.

Regulation of the 25-hydroxyvitamin D-1alpha-hydroxylase gene and its splice variant.

The 25-hydroxyvitamin D-1alpha-OHase (1alpha-OHase) is responsible for producing the active form of vitamin D, 1alpha,25-dihydroxyvitamin D. The enzyme not only is expressed in kidneys, but also is expressed in many nonrenal tissues, including skin. In this study, we compared the regulation of the 1alpha-OHase expression in kidney cells and keratinocytes. Using transfected luciferase reporter gene constructs, we compared the activity and regulatory features of the human 1alpha-OHase gene promoter in C-21 human kidney cells (PTH/PTHrP receptor positive) and cultured human keratinocytes (NHKs). We found that two regions, -1,100 bp and -396 bp from the ATG, were highly sensitive to parathyroid hormone (PTH) in C-21 cells but not in NHK. Furthermore, three CRE-like sequences (CLS) were identified within this PTH-sensitive area of the 1alpha-OHase promoter and when deleted they reduced induction of PTH by 50%-95% in C-21 cells. To further investigate the differential regulation profile, we examined the protein products of 1alpha-OHase in kidney and skin. Western blot analysis of whole cell extracts from these tissues with a 1alpha-OHase-specific antibody revealed the predicted 1alpha-OHase protein product of 56 kDa in kidney and a larger protein product of 59 kDa in skin. Using RT-PCR for the 1alpha-OHase in skin and kidney, we detected an insertion between exons 2 and 3 in skin but not in kidney. These results suggest that the regulation of renal and skin 1alpha-OHase gene expression may be tissue specific and possibly produce different splice variants, and that this specificity is likely conferred by differential expression of CRE-binding proteins in different cell types. In conclusion, the differential tissue expression of 1alpha-OHase gene variants and the tissue-specific regulation profile open up a new paradigm in the understanding of the role of 25-hydroxyvitamin D3 1alpha-hydroxylase gene in the regulation of vitamin D physiology.

Characterization of transgenic rats constitutively expressing vitamin D-24-hydroxylase gene

Vitamin D-24-hydroxylase (CYP24) is one of the enzymes responsible for vitamin D metabolism. CYP24 catalyzes the conversion of 25-hydroxyvitamin D 3 [25(OH)D 3] to 24,25-dihydroxyvitamin D 3 [24,25(OH) 2D 3] in the kidney. CYP24 is also involved in the breakdown of 1α,25-dihydroxyvitamin D 3 [1α,25(OH) 2D 3], the active form of vitamin D 3. In this study, we generated transgenic (Tg) rats constitutively expressing CYP24 gene to investigate the biological role of CYP24 in vivo. Surprisingly, the Tg rats showed a significantly low level of plasma 24,25(OH) 2D 3. Furthermore, the Tg rats developed albuminuria and hyperlipidemia shortly after weaning. The plasma lipid profile revealed that all lipoprotein fractions were elevated in the Tg rats. Also, the Tg rats showed atherosclerotic lesions in the aorta, which greatly progressed with high-fat and high-cholesterol feeding. These unexpected results suggest that CYP24 is involved in functions other than the regulation of vitamin D metabolism.

The p38 and JNK Pathways Cooperate to trans-Activate Vitamin D Receptor via c-Jun/AP-1 and Sensitize Human Breast Cancer Cells to Vitamin D3-induced Growth Inhibition

The signaling connection between mitogen-activated protein kinases(MAPKs) and nuclear steroid receptors is complex and remains mostly unexplored. Here we report that stress-activated protein kinases p38 and JNK trans-activate nuclear steroid vitamin D receptor (VDR) gene and increase vitamin D(3)-dependent growth inhibition in human breast cancer cells. Activation of p38 and JNK by an active MAPK kinase 6 stimulates VDR promoter activity independently of the ligand vitamin D(3) and estrogen receptor expression. Moreover, stimulation of the endogenous stress pathways by adenovirus-mediated delivery of recombinant MAPK kinase 6 also activates VDR and sensitizes MCF-7 cells to vitamin D(3)-dependent growth inhibition. Both the p38 and JNK MAPK pathways and the downstream transcription factor c-Jun/AP-1 are required for the VDR stimulation, as revealed by application of their dominant negatives, the specific p38 inhibitor SB203580, and site-directed mutagenesis of the AP-1 element in the VDR promoter. The essential role of the p38 and JNK stress pathways in up-regulation of VDR expression is further confirmed by using the chemical stimulator arsenite. These results establish a signaling connection between the stress MAPK pathways and steroid hormone receptor VDR expression and thereby offer new insights into regulation of cell growth by the MAPK pathways through regulation of vitamin D(3)/VDR activity.

Mechanism and regulation of vitamin B(6) uptake by renal tubular epithelia: studies with cultured OK cells.

The kidneys play an important role in regulating vitamin B(6) body homeostasis, but limited information exists regarding the mechanism of pyridoxine uptake by renal epithelial cells, and no study exists on its regulation. To address these issues, we used the renal opossum-derived tubular epithelial (opossum kidney; OK) cells and found pyridoxine uptake to 1) be temperature and energy dependent, 2) be pH dependent, with a higher uptake at alkaline or neutral buffer pH compared with acidic pH, 3) be Na(+) independent, 4) involve a saturable component (apparent Michaelis- Menten constant of 2.40 +/- 0.23 microM), 5) be inhibited by structural analogs, and 6) be amiloride sensitive. Maintaining OK cells in a vitamin B(6)-deficient growth medium (for 48 h) led to a significant upregulation of pyridoxine uptake. This upregulation was found to be specific for pyridoxine, inhibited by cyclohexamide and actinomycin D, reversible, and mediated via an increase in maximal velocity. Pretreating OK cells with modulates of a Ca(2+)/calmodulin-mediated pathway led to a significant downregulation in pyridoxine uptake via inhibition of maximal velocity. These results demonstrate that pyridoxine uptake by renal tubular epithelial OK cells is via a specialized pH-sensitive carrier-mediated mechanism. This mechanism appears to be regulated by extracellular vitamin B(6) levels and an intracellular Ca(2+)/calmodulin-mediated pathway.

Regulation of vitamin C transport by protein kinase C (PKC)

Regulation of vitamin C transport by protein kinase C (PKC)

Regulation of vitamin D synthesis and vitamin D receptor expression: Relevance for human colon cancer progression

Regulation of vitamin D synthesis and vitamin D receptor expression: Relevance for human colon cancer progression

1,25-dihydroxyvitamin D 3 receptor is upregulated in aortic smooth muscle cells during hypervitaminosis D

Several studies have demonstrated that excess of vitamin D 3 is toxic particularly to vascular tissues. A notable pathological feature is arterial calcification. The nature of the toxic metabolite in hypervitaminosis D and the pathogenesis of arterial calcification are not clearly understood. The present study was undertaken to explore whether arterial calcification is a sequel of increased calcium uptake by arterial smooth muscle mediated by up regulation of vitamin D receptor in the cells in response to elevated circulating levels of vitamin D 3 in serum. The experimental study was performed in 20 New Zealand white female rabbits aged 6 months. Animals in the test group were injected 10,000 IU of cholecalciferol intramuscularly twice a week for one month. Six control animals were given intra-muscular injections of plain cottonseed oil. Animals were sacrificed and aortas were examined for pathological lesions, 1,25-dihyroxyvitamin D 3 (1,25(OH) 2 D 3) receptor levels and 45Ca uptake in smooth muscle cells. Serum samples collected at intervals were assayed for levels of 25-OH-D 3 and calcium. The results showed that in animals given injections of cholecalciferol, serum levels of 25-OH-D 3 were elevated. In four of these animals calcification and aneurysmal changes were seen in the aorta. Histological lesions comprised of fragmentation of elastic fibers as well as extensive loss of elastic layers. 1,25(OH) 2 D 3 receptor levels were up regulated and 45Ca uptake enhanced in aortas of animals which were given excessive vitamin D 3. The evidences gathered suggest that excess vitamin D is arteriotoxic and that the vitamin induces arterial calcification through up regulation of 1,25(OH) 2D 3 receptor and increased calcium uptake in smooth muscle cells of the arteries.

Molecular Mechanism of the Effect of Fructooligosaccharides on Calcium Absorption

Previous reports have demonstrated through the use of animal models that dietary fructooligosaccharides (FOS) exert stimulatory effects on the absorption of the minerals; calcium, magnesium, and iron. Recently, these effects were also clinically confirmed in humans. This paper reviews studies that used the techniques of molecular biology to elucidate the mechanisms by which FOS stimulates calcium absorption for which the small intestine has long been considered a primary site. However, Ohta et al. reported that the large intestine, and the cecum in particular, has an important role. This conclusion was based on the observation that the stimulatory effect of FOS on calcium absorption was not observed in cecectomized rats. The contribution of paracellular and transcellular paths was suggested for the stimulation caused by FOS. Transcellular transport is a multistep process comprising several components and is under the regulation of vitamin D. One component is calbindin-D9k (CaBP), a calcium binding protein. The upstream region of the CaBP gene was analyzed, and a responsive sequence for vitamin D receptor was identified. Mucosal cells were then obtained from FOS-fed rats, and the expression of CaBP protein and mRNA were determined. Dietary FOS decreased the expression of CaBP in the small intestine, but resulted in increased levels in the cecum and colorectum. Since protein and mRNA both demonstrated similar patterns of expression, the effects of FOS can be seen to affect CaBP at the transcription level. Furthermore, the changes in CaBP expression were proportional to both the level of apparent calcium absorption and the amount of FOS ingested. This means that the increase of calcium absorption induced by FOS comprises paracellular and CaBP-mediated transcellular transport. In a hypothetical model, short-chain organic acids produced from FOS by fermentation were proposed as key elements to regulate both the paracellular and the transcellular transport of calcium. However, the molecular mechanisms of this regulation have not yet been clarified.

Regulation of vitamin D-1α-hydroxylase in a human cortical collecting duct cell line

Recent studies have shown that renal expression of 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) is not restricted to proximal tubules. To investigate the significance of this expression, we characterized the regulation of 1alpha-OHase expression and activity in a human cortical collecting duct cell line (HCD). Expression of 1alpha-OHase mRNA and protein was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses. Enzyme activity was quantified using 25-hydroxyvitamin D3 as the substrate; conversion to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and 24,25-dihydroxyvitamin D3 was then determined by thin-layer chromatography. HCD cells expressed mRNA and protein for 1alpha-OHase. However, basal 1,25(OH)2D3 production was lower than that observed in proximal tubule HKC-8 cells. In both cell lines, synthesis of 1,25(OH)2D3 was increased by forskolin, parathyroid hormone, and low calcium medium. Conversely, treatment with 1,25(OH)2D3 itself decreased 1alpha-OHase activity. This effect was more pronounced in HCD cells, which also demonstrated significantly higher levels of 24-hydroxylase activity. The most striking induction of 1alpha-OHase activity was observed in the HCD cells following incubation with lipopolysaccharide, which was coincident with the expression of mRNA for both CD14 and Toll-like receptor 4. These results highlight the capacity for synthesis of 1,25(OH)2D3 in cells from more distal areas of the nephron. However, more sensitive feedback regulation and immune induction of 1alpha-OHase in the HCD cells suggest a more localized role for 1,25(OH)2D3 production in the distal nephron.

Cooperation of liver cells in health and disease.

Cooperation of liver cells in health and disease.

Regulation of vitamin A metabolism-related gene expression.

Cellular retinol-binding protein, type II (CRBPII) is abundantly expressed in the small intestinal epithelial cells and plays a pivotal role in intestinal absorption and metabolism of retinol and beta-carotene. In the 5'-flanking region of rat CRBPII gene, two DR-1 type elements which consist of a direct repeat of the AGGTCA-like motif spaced by a single nucleotide have been identified as putative binding sites for a heterodimer of peroxisome proliferator-activated receptor (PPAR) and retinoid X-receptor (RXR). We found that CRBPII levels were elevated in the residual jejunal segment of rats subjected to jejunal bypass operation, where a concomitant increase in the apoprotein B levels occurred. This result suggested that CRBPII expression was enhanced by a condition where fat absorption was stimulated. Indeed, dietary fat (especially unsaturated fatty acids) has been shown to induce CRBPII gene expression in the jejunum. Nuclear run-on assays revealed that this increase of CRBPII mRNA levels by a high-fat diet was the result of the induction of the gene transcription through the rise in PPARalpha expression level as well as the increase in its ligand levels. Electrophoretic mobility shift assay using the DR-1 type cis-elements of CRBP II gene showed that PPARalpha-RXRalpha heterodimer was capable of binding to these elements, and that nuclear extracts from the jejunum of rats fed the high-fat diet gave greater density of retarded bands than those of rats fed a fat-free diet. We also found that the expression of PPARdelta was rather reduced by dietary fat. Thus, CRBPII gene expression is regulated predominantly by dietary fatty acids.

Regulation of vitamin D-1alpha-hydroxylase and -24-hydroxylase expression by dexamethasone in mouse kidney.

We investigated the effects of dexamethasone on vitamin D-1alpha-hydroxylase and -24-hydroxylase expression and on vitamin D receptor (VDR) content in the kidneys of mice fed either a normal (NCD) diet or a calcium- and vitamin D-deficient (LCD) diet for 2 weeks. For the last 5 days mice received either vehicle or dexamethasone (2 mg/kg per day s.c.). Dexamethasone significantly increased plasma calcium concentrations without changing plasma concentrations of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) in both NCD and LCD groups. Northern blot and enzyme activity analyses in NCD mice revealed that dexamethasone increased renal VDR mRNA expression modestly and greatly increased 24-hydroxylase mRNA abundance and enzyme activity, but did not affect 1alpha-hydroxylase mRNA abundance and enzyme activity. In mice fed an LCD diet, dexamethasone increased renal VDR mRNA expression 1.5-fold, decreased 1alpha-hydroxylase mRNA abundance (52%) and activity (34%), and markedly increased 24-hydroxylase mRNA abundance (16-fold) and enzyme activity (9-fold). Dexamethasone treatment did not alter functional VDR number (B(max) 125-141 fmol/mg protein) or ligand affinity (K(d) 0.13-0.10 nM) in LCD mice. Subcutaneous injections of 1,25(OH)(2)D(3) (0.24 nmol/kg per day for 5 days) into NCD mice strongly increased renal 24-hydroxylase mRNA abundance and enzyme activity, while there was no effect of dexamethasone on renal 24-hydroxylase expression in these mice. This may be due to overwhelming induction of 24-hydroxylase by 1,25(OH)(2)D(3). These findings suggest that glucocorticoid-induced osteoporosis is caused by direct action of the steroids on bone, and the regulatory effect of glucocorticoids on renal 25-hydroxyvitamin D(3) metabolism may be less implicated in the initiation and progression of the disease.