Parathyroid hormone down-regulates 1,25-dihydroxyvitamin D receptors (VDR) and VDR messenger ribonucleic acid in vitro and blocks homologous up-regulation of VDR in vivo

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3) is a known up-regulator of 1,25(OH)2D3 receptor (VDR) both in vitro and in vivo. However, a 5- to 10-fold increase in plasma 1,25(OH)2D3 induced by dietary calcium deficiency does not result in up-regulation of intestinal VDR, and kidney VDR is down-regulated. Under certain physiological stresses, an increase in plasma PTH precedes increased plasma 1,25(OH)2D3. Therefore, the present study examined the effect of PTH on VDR regulation in vitro in ROS 17/2.8 cells and in vivo in male Holtzman rats. Treatment of ROS cells with PTH (0-5 nM) resulted in a dose and time-dependent decline in VDR from 95 +/- 9 to 35 +/- 5 fmol/mg protein at 18 h of exposure. The ED50 for PTH was 1 nM. This decline in VDR protein was attended by a 50% decline in VDR messenger RNA (mRNA). The PTH-mediated down-regulation of VDR occurred without affecting the affinity of VDR for 1,25(OH)2D3 as determined by Scatchard analysis. Also, the effect of PTH on VDR regulation was specific since cell glucocorticoid receptor concentration was not affected by PTH treatment. In accompanying experiments, 1,25(OH)2[3H]D3 treatment of ROS cells was shown to result in a 3- to 4-fold increased expression of VDR and VDR mRNA. The simultaneous addition of PTH and 1,25(OH)2[3H]D3 resulted in inhibition of the 1,25(OH)2[3H]D3-mediated up-regulation of VDR and VDR mRNA. Similarly, PTH also inhibited heterologous up-regulation of VDR and VDR mRNA induced by retinoic acid. In in vivo experiments, rats infused for 5 days with 1,25(OH)2D3 (1.5 ng/h) increased their expression of intestinal VDR, kidney VDR, and kidney 24-hydroxylase by 31, 336, and 4000%, respectively. Coinfusion of PTH (1.8 IU/h) along with 1,25(OH)2D3 completely inhibited the 1,25(OH)2D3-mediated increases in intestinal VDR and kidney 24-hydroxylase and reduced the 1,25(OH)2D3-mediated up-regulation of kidney VDR by more than half. These data suggest that PTH is a potent down-regulator of VDR and that PTH and 1,25(OH)2D3 have opposing effects on the expression of certain genes.     

Hydrocortisone and deflazacort induce different effects on vitamin D receptor level increase produced by 1,25-dihydroxyvitamin D3 in rat osteoblast-like UMR-106 cells

OBJECTIVE: The homologous upregulation produced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on vitamin D receptor (VDR) levels, and the effects produced by the heterologous agents hydrocortisone or deflazacort, alone or in conjunction with this vitamin D metabolite, were studied in rat osteoblastic UMR-106 osteosarcoma cells. METHODS: VDR were determined by binding analysis (Bmax and dissociation constant). VDR mRNA expression levels were measured by Northern blot analysis. RESULTS: Incubation with 10 nM 1,25(OH)2D3 produced a significant increase in Bmax with respect to ethanol-treated cells (100.2 +/- 13.2 vs 11.4 +/- 4.8 fmol 3H-1,25(OH)2D3 bound/mg protein) together with a significant increase in VDR mRNA expression (483 +/- 170% vs 100%). The addition of 10 nM hydrocortisone to 1,25(OH)2D3 produced a significant decrease in Bmax (from 100.2 +/- 13.2 to 44 +/- 5.6), with mRNA levels similar to those of basal conditions (116 +/- 25% vs 100%). However, the addition of 10 nM deflazacort did not reduce the activation in Bmax produced by 1,25(OH)2D3 (92.4 +/- 16 vs 100.2 +/- 13.2), maintaining the increase in mRNA levels (430 +/- 10% vs 483 +/- 170%). If 10 nM hydrocortisone or 10 nM deflazacort was added to UMR-106 cells without 1,25(OH)2D3, a similar increase was observed in Bmax with respect to basal conditions (20.4 +/- 1.3 or 20.9 +/- 1.6 vs 11.4 +/- 4.8 in control cells), but hydrocortisone did not produce any significant variation in mRNA VDR levels, while deflazacort itself produced an increase in VDR mRNA expression. CONCLUSION: Our findings of different actions produced by hydrocortisone and deflazacort on the increase of VDR levels produced by 1,25(OH)2D3 could explain some of the different actions produced by both antiinflammatory medications on bone metabolism.     

Regulation of the murine renal vitamin D receptor by 1,25-dihydroxyvitamin D3 and calcium

Renal vitamin D receptor (VDR) is required for 1,25-dihydroxyvitamin D3-[1,25(OH)2D3]-induced renal reabsorption of calcium and for 1,25(OH)2D3-induced 1,25(OH)2D3 24-hydroxylase. The long-term effect of vitamin D and dietary calcium on the expression of renal VDR was examined in the nonobese diabetic mouse. Vitamin D-deficient and vitamin D-replete mice were maintained on diets containing 0.02%, 0.25%, 0.47%, and 1.20% calcium with or without 50 ng of 1,25(OH)2D3 per day. Vitamin D-replete mice on a 1.20% calcium diet had renal VDR levels of 165 fmol/mg protein. Calcium restriction caused renal VDR levels to decrease to <30 fmol/mg protein in vitamin D-deficient mice and to approximately 80 fmol/mg protein in vitamin D-replete mice. When dietary calcium was present, 50 ng of 1,25(OH)2D3 elevated the VDR levels 2- to 10-fold, depending on vitamin D status and the level of calcium. In the absence of either vitamin D or calcium, the VDR mRNA was expressed at a basal level. 1,25(OH)2D3 supplementation caused relative VDR mRNA to increase 8- to 10-fold in the vitamin D-deficient mouse when dietary calcium was available. This increase was completely absent in the calcium-restricted mice. This in vivo study demonstrates that 1,25(OH)2D3 and calcium are both required for renal VDR mRNA expression above a basal level, furthering our understanding of the complex regulation of renal VDR by 1,25(OH)2D3 and calcium.     

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.     

Intestinal resistance to 1,25 dihydroxyvitamin D in mice heterozygous for the vitamin D receptor knockout allele

We tested the hypothesis that low vitamin D receptor (VDR) level causes intestinal vitamin D resistance and intestinal calcium (Ca) malabsorption. To do so, we examined vitamin D regulated duodenal Ca absorption and gene expression [transient receptor potential channel, vallinoid subfamily member 6 (TRPV6), 24-hydroxylase, calbindin D(9k) (CaBP) mRNA, and CaBP protein] in wild-type mice and mice with reduced tissue VDR levels [i.e. heterozygotes for the VDR gene knockout (HT)]. Induction of 24-hydroxylase mRNA levels by 1,25 dihydroxyvitamin D(3) [1,25(OH)(2) D(3)] injection was significantly reduced in the duodenum and kidney of HT mice in both time-course and dose-response experiments. TRPV6 and CaBP mRNA levels in duodenum were significantly induced after 1,25(OH)(2) D(3) injection, but there was no difference in response between wild-type and HT mice. Feeding a low-calcium diet for 1 wk increased plasma PTH, renal 1alpha-hydroxylase (CYP27B1) mRNA level, and plasma 1,25(OH)(2) D(3), and this response was greater in HT mice (by 88, 55, and 37% higher, respectively). In contrast, duodenal TRPV6 and CaBP mRNA were not higher in HT mice fed the low-calcium diet. However, the response of duodenal Ca absorption and CaBP protein to increasing 1,25(OH)(2) D(3) levels was blunted by 40% in HT mice. Our data show that low VDR levels lead to resistance of intestinal Ca absorption to 1,25(OH)(2) D(3), and this resistance may be due to a role for the VDR (and VDR level) in the translation of CaBP.     

Responsiveness of the intestinal 1,25-dihydroxyvitamin D3 receptor to magnesium depletion in the rat.

In contrast to man, the rat exhibits hypercalcemia during the course of magnesium depletion. To investigate the role of the vitamin D (D) endocrine system in the induction of hypercalcemia, circulating D metabolites, the binding properties of the duodenal 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] receptor (VDR), and 45Ca transport studies were undertaken in magnesium-replete rats or after 10 days of magnesium depletion in animals presenting the following D status: D depletion and hypo- or normocalcemia (achieved by oral calcium supplementation), D3 or 1,25-(OH)2D3 repletion. Magnesium depletion did not influence serum calcium in hypo- or normocalcemic D depleted rats, but increased serum calcium in animals receiving D3 (P less than 0.002) or 1,25-(OH)2D3 (P less than 0.0001), suggesting that the D3 endocrine system is necessary to mediate the rise in extracellular calcium and that dietary calcium alone is not sufficient to significantly increase extracellular calcium in the hypomagnesemic rat. The data also show that 25-hydroxyvitamin D formation was not perturbed, but circulating 1,25-(OH)2D3 concentrations were reduced by 10 days of magnesium depletion (P less than 0.0001) even in animals infused with 1,25-(OH)2D3, suggesting increased clearance of the hormone. The kinetic data of the duodenal VDR revealed maximum binding sites ranging from 1018-1500 fmol/mg DNA and Kd ranging from 0.17-0.38 nM, with no significant between-group difference in magnesium-sufficient animals. Ten days of magnesium depletion did not significantly influence VDR affinity in any of the groups, but significantly increased receptor number in hypocalcemic D-depleted rats from 1190 +/- 154 to 2748 +/- 430 fmol/mg DNA (P less than 0.004). Calcium transport studies in D-replete animals indicate that intestinal calcium transport is influenced by the progressive depletion in magnesium, with time-related increases coinciding with the in vivo increase in circulating ionized calcium (day 6 of magnesium depletion). However, despite persistent elevated serum ionized calcium, calcium transport declined only to predepletion levels on days 8 and 10 of magnesium depletion. To investigate the influence of the D3 endocrine system on 45Ca absorption, D-depleted rats sufficient or depleted in magnesium were injected with 1,25-(OH)2D3, either acutely (to reveal its membrane effects) or 16 and 5 h before death (to reveal its genomic effect). The data reveal a reduced response in magnesium-depleted rats to acute 1,25-(OH)2D3 injection (P less than 0.0002), but similar responses when the hormone was injected 16 and 5 h before the experiment.(ABSTRACT TRUNCATED AT 400 WORDS)     

1,25-dihydroxyvitamin D3 production and vitamin D3 receptor expression are developmentally regulated during differentiation of human monocytes into macrophages

It has been well established that human mononuclear phagocytes have the capacity to produce 1,25-dihydroxy-vitamin D3 [1,25(OH)3D3] and express the vitamin D receptor (VDR). However, 1 alpha-hydroxylase activity and VDR receptor expression during differentiation of monocytes (MO) into mature macrophages (MAC) have not been previously examined. The in vitro maturation of blood MO can serve as a model for the in vivo transformation of immature blood MO into MAC. Here, when cultured in the presence of serum, MO undergo characteristic changes in morphology, antigenic phenotype, and functional activity consistent with their differentiation into MAC. We serially measured 1,25(OH)2D3 and 24,25- dihydroxyvitamin D3 [24,25(OH)2D3] synthesis, specific [3H]-1,25(OH)2D3 binding, and VDR mRNA levels during in vitro maturation of MO into MAC and correlated these functions with maturation-associated changes in the phenotype (MAX.1 and CD71) and secretory repertoire (interleukin-1 beta [IL-1 beta], neopterin) of the cells. MO showed only little conversion of 25-(OH)D3 into 1,25(OH)2D3 (1.4 +/- 0.4 pmol/10(6) cells/6 h, n = 5) that increased gradually during maturation into MAC at day 8 of culture (5.3 +/- 4.3 pmol/10(6) cells/6 h, n = 5). Interferon-gamma (IFN-gamma) increased baseline 1,25(OH)2D3-synthesis approximately twofold during all phases of differentiation. The time course of increased 1,25(OH)2D3-synthesis correlated with enhanced secretion of neopterin and expression of MAX.1 and CD71. The addition of exogenous 1,25(OH)2D3 did not influence constitutive 1,25(OH)2D3 synthesis, but IFN-gamma-stimulated production was suppressed to baseline levels. Exogenous 1,25(OH)2D3 also stimulated 24,25(OH)2D3 synthesis in freshly isolated MO (from 1.0 +/- 0.8 pmol/6 h to 5.6 +/- 0.9 pmol), whereas matured MAC showed no 24,25(OH)2D3 synthesis. Furthermore, we examined the expression of the VDR during the differentiation process. VDR mRNA and protein were constitutively expressed in MO, whereas VDR was downregulated in mature MAC on both the mRNA and protein levels. Homologous upregulation of VDR protein by 1,25(OH)2D3 occurred in MO and, to a lesser degree, in MAC. In contrast, VDR mRNA concentrations were not influenced by 1,25(OH)2D3. Taken together, our results show that MO into MAC differentiation in vitro is associated with (1) an enhanced capacity to synthesize 1,25(OH)2D3, (2) a loss of 24,25(OH)2D3-synthesizing activity, and (3) a decrease in the expression of VDR mRNA and protein. Because 1,25(OH)2D3 was shown to induce differentiation of MO into MAC, our data sugest an autoregulatory mechanism of MO/MAC generation by 1,25(OH)2D3.     

Vitamin D-influenced gene expression via a ligand-independent, receptor-DNA complex intermediate.

A lingering question regarding the regulation of target gene expression by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has been the delineation of vitamin D receptor (VDR)-DNA binding and transactivation. This report confirms that initial VDR-DNA interaction occurs in a ligand-independent fashion. An electrophoretic mobility-shift analysis demonstrated that VDR, derived from extracts of the small intestines of vitamin D-deficient rats, is capable of binding a vitamin D response element (DRE). Additional mobility-shift studies using either porcine-derived VDR or recombinant rat VDR from insect cells revealed DRE-binding capability in the absence of 1,25-(OH)2D3. The reactions were performed in various salt environments, with the maximum of porcine VDR-DRE and rat VDR-DRE binding detected at 100 mM and 150 mM KCl, respectively. The addition of 1,25-(OH)2D3 to an identical set of reaction mixtures resulted in increased DRE binding with greater affinities exhibited by both VDR types. These two phenomena were confirmed upon examination of an elution profile of VDR bound to DRE-linked Sepharose. When a linear KCl gradient was used for elution without the addition of 1,25-(OH)2D3, the peak of VDR was 205 mM KCl; the presence of exogenous hormone shifted the maximum VDR elution to a position corresponding to 265 mM KCl. Based on these data and previous reports on VDR-mediated transactivation, we propose a model for 1,25-(OH)2D3-influenced target gene expression.     

Glucocorticoid regulation of 1,25(OH)2vitamin D3 receptors: divergent effects on mouse and rat intestine

We have investigated the possibility that glucocorticoids alter responsiveness to vitamin D by regulating the 1,25(OH)2D3 receptor in rat intestine. In contrast to the mouse where glucocorticoids caused receptors to decline, rats treated with glucocorticoids showed a substantial increase ( approximately 50%) in the number of intestinal 1,25(OH)2D3 receptors. This resulted from an increase in receptor content with no change in affinity for 1,25(OH)2D3. Receptor stimulation was even greater in vitamin D-deplete rats. Moreover, adrenalectomy led to a significant decline in receptor number. Although the properties of the receptor are similar in rat and mouse intestine, the divergent response to glucocorticoids emphasizes major differences between species in the regulation of 1,25(OH)2D3 receptor number.     

Evidence of an age-related decrease in intestinal responsiveness to vitamin D: relationship between serum 1,25-dihydroxyvitamin D3 and intestinal vitamin D receptor concentrations in normal women.

Although aged rats reportedly have reduced intestinal vitamin D receptor (VDR) concentrations, it is unclear whether an analogous age-related defect occurs in man. Thus, we assessed the interrelationship among serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], calcium absorption and intestinal VDR in 44 healthy, ambulatory women, ages 20-87 yr. Fractional calcium absorption was measured after oral administration of 45Ca (20 mg CaCl2 as carrier); serum 1,25-(OH)2D3, by the calf thymus binding assay; and serum intact PTH, by a two-site immunochemiluminometric assay. Vitamin D receptor concentration was measured, by a new immunoradiometric assay, in biopsy specimens taken from the second part of the duodenum during gastroduodenoscopy in 35 of the women. Despite an age-related increase in serum PTH (r = 0.48; P less than 0.001) and in serum 1,25-(OH)2D3 concentration (r = 0.32; P less than 0.05), intestinal VDR concentration decreased with age (r = -0.38; P = 0.03) and fractional calcium absorption did not change with age. Although a contribution of decreased 25-hydroxyvitamin D 1 alpha-hydroxylase activity to the blunting of the increase in serum 1,25-(OH)2D3 concentration late in life is not excluded, the data are far more consistent with impaired intestinal responsiveness to 1,25-(OH)2D3 action. This defect could lead to compensatory increases in PTH secretion and 1,25-(OH)2D3 production which maintain calcium absorption and serum ionic calcium, but at the expense of increased bone loss.     

Contrasting effects of exogenous 1,25-dihydroxyvitamin D [1,25-(OH)2D] versus endogenous 1,25-(OH)2D, induced by dietary calcium restriction, on vitamin D receptors

The biological actions of 1,25-dihydroxyvitamin D [1,25-(OH)2D] are mediated by specific binding of the hormone with an intracellular vitamin D receptor, which ultimately regulates expression of genes within the target tissues. The quantity of vitamin D receptors varies between target tissues and within target tissues, depending on the physiological state of the animal. One factor that can modulate tissue vitamin D receptor content is 1,25-(OH)2D. In the present study performed in male rats, exogenous administration of 36 ng 1,25-(OH)2D3/day for 7 days increased plasma 1,25-(OH)2D concentrations 5-fold above those in control rats (to 261 +/- 17 pg/ml). Compared with those in control rats, 1,25-(OH)2D3 treatment resulted in a 1.5-fold increase in duodenal vitamin D receptor content (351 +/- 16 vs. 520 +/- 21 fmol/mg protein) and a 3-fold increase in renal vitamin D receptor content (60.3 +/- 5.2 vs. 193.8 +/- 22.7 fmol/mg protein). The effects of endogenously produced 1,25-(OH)2D on tissue vitamin D receptor content were studied by feeding rats either a 0.02% or 1% calcium diet for 2, 7, 14, or 21 days. Rats fed the low calcium diet exhibited plasma 1,25-(OH)2D concentrations similar to (day 7) or exceeding (days 14 and 21) those achieved by exogenous administration of 1,25-(OH)2D3, yet duodenal vitamin D receptor content was not up-regulated by dietary calcium restriction at any time point. The renal vitamin D receptor content of calcium restricted rats was 20-38% lower (P less than 0.05) than that in rats fed a calcium-replete diet 7, 14, and 21 days after initiation of the dietary treatments. These data suggest that under physiological conditions, increased plasma concentrations of 1,25-(OH)2D do not result in up-regulation of tissue vitamin D receptor concentrations, and that dietary calcium restriction must induce some factor(s) that results in down-regulation of vitamin D receptors in the kidney.     

VDR起始密码子区突变功能与大肠肿瘤易患性的关系

目的探讨维生素D受体（VDR）基因FokⅠ变异的意义及对大肠肿瘤易患性的影响。方法利用瞬时转染技术在不同时间给予肠道COS-7细胞不同浓度的1,25-二羟维生素D3[1,25-（OH）2VD3]处理,检测转染VDR野生型和突变型真核表达载体后的细胞对下游靶基因CYP450转录水平的影响。结果将野生型和突变型VDR质粒共转染于COS-7细胞中,在1,25-（OH）2VD3孵育24、36、48 h后,3个浓度组（10、20、50μmol/L）的CYP3A4mRNA转录水平分别与对照组和空载体对照组比较,差异均具有统计学意义,且转染FokⅠ突变型的CYP3A4 mR-NA转录水平高于野生型。结论 FokⅠ突变使CYP3A4蛋白转录激活能力显著升高,可能影响大肠肿瘤发生的易患性。     

Dietary phosphorus deprivation induces 25-hydroxyvitamin D(3) 1alpha-hydroxylase gene expression

Dietary phosphorus deprivation causes hypophosphatemia and an increase in serum 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] concentrations. To determine the molecular mechanisms of this regulation, the effects of dietary phosphorus deprivation and hypophysectomy on 25-hydroxyvitamin D(3) 1alpha-hydroxylase (1alpha-hydroxylase) protein and messenger RNA (mRNA) expression were examined in rats. A low phosphorus diet (LPD) for 4 days resulted in hypophosphatemia and an increase in serum 1,25-(OH)(2)D(3) levels. This increase was caused by the induction of 1alpha-hydroxylase protein and mRNA expression (4- and 10-fold increases, respectively). Administration of the LPD or normal phosphorus diet to hypophysectomized (HPX) rats resulted in hypophosphatemia and suppression of 1alpha-hydroxylase gene expression, indicating that hypophosphatemia itself is not sufficient to induce 1alpha-hydroxylase mRNA expression. Administration of GH to HPX rats fed LPD could partially restore 1alpha-hydroxylase mRNA expression, whereas supplementation with insulin-like growth factor I, T(3), estrogen, or corticosterone had no effect. We also examined Phex gene expression in the bone, because the clinical features of X-linked hypophosphatemia resemble those of HPX rats. Phex mRNA expression, however, was not altered in HPX rats. In conclusion, we demonstrated that the increase in serum 1,25-(OH)(2)D(3) levels caused by dietary phosphorus deprivation is due to the induction of 1alpha-hydroxylase mRNA expression, and this increase is mediated in part by a GH-dependent mechanism.     

Calcium transporter 1 and epithelial calcium channel messenger ribonucleic acid are differentially regulated by 1,25 dihydroxyvitamin D3 in the intestine and kidney of mice

We examined the expression of calcium transporter 1 (CaT1) and epithelial calcium channel (ECaC) mRNA in the duodenum and kidney of mice. Intestinal CaT1 mRNA level increased 30-fold at weaning, coincident with the induction of calbindin-D(9k) expression. In contrast, renal CaT1 and ECaC mRNA expression was equal until weaning when ECaC mRNA is induced and CaT1 mRNA levels fall 70%. Long- and short-term adaptation to changes in dietary calcium (Ca) level and 1,25 dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] injection strongly regulated duodenal calbindin D(9k) and CaT1 mRNA. Following a single dose of 1,25(OH)(2)D(3), induction of CaT1 mRNA occurred rapidly (within 3 h, peak at 6 h of 9.6 +/- 0.8-fold) and preceded the induction of intestinal Ca absorption (significantly increased at 6 h, peak at 9 h). Neither renal CaT1 nor ECaC mRNA were strongly regulated by dietary calcium level or 1,25(OH)(2)D(3) injection. Our data indicate that CaT1 and ECaC mRNA levels are differentially regulated by 1,25(OH)(2)D(3) in kidney and intestine and that there may be a specialized role for CaT1 in kidney in fetal and neonatal development. The rapid induction of intestinal CaT1 mRNA expression by 1,25(OH)(2)D(3), and the marked induction at weaning, suggest that CaT1 is critical for 1,25(OH)(2)D(3)-mediated intestinal Ca absorption.     

Hereditary 1,25-dihydroxyvitamin D resistant rickets due to a mutation causing multiple defects in vitamin D receptor function

Hereditary vitamin D-resistant rickets (HVDRR) is an autosomal recessive disease caused by mutations in the vitamin D receptor (VDR). We studied a young Saudi Arabian girl who exhibited the typical clinical features of HVDRR, but without alopecia. Analysis of her VDR gene revealed a homozygous T to C mutation in exon 7 that changed isoleucine to threonine at amino acid 268 (I268T). From crystallographic studies of the VDR ligand-binding domain, I268 directly interacts with 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and is involved in the hydrophobic stabilization of helix H12. We recreated the I268T mutation and analyzed its effects on VDR function. In ligand binding assays, the I268T mutant VDR exhibited an approximately 5- to 10-fold lower affinity for [(3)H]1,25(OH)(2)D(3) compared with the wild-type (WT) VDR. The I268T mutant required approximately a 65-fold higher concentration of 1,25(OH)(2)D(3) to be equipotent in gene transactivation. Both retinoid X receptor heterodimerization and coactivator binding were reduced in the I268T mutant. Analogs of 1,25(OH)(2)D(3) have been proposed as potential therapeutics for patients with HVDRR. Interestingly, in protease sensitivity assays, treatment with the potent vitamin D analog, 20-epi-1,25(OH)(2)D(3), stabilized I268T mutant proteolytic fragments better than 1,25(OH)(2)D(3). Moreover, 20-epi-1,25(OH)(2)D(3) restored transactivation of the I268T mutant to levels exhibited by WT VDR treated with 1,25(OH)(2)D(3). In conclusion, we describe a novel mutation, I268T, in the VDR ligand-binding domain that alters ligand binding, retinoid X receptor heterodimerization, and coactivator binding. These combined defects in VDR function cause resistance to 1,25(OH)(2)D(3) action and result in the syndrome of HVDRR.     

Peripheral blood monocyte vitamin D receptor levels are elevated in patients with idiopathic hypercalciuria

Idiopathic hypercalciuria (IH) is the most common cause of calcium oxalate nephrolithiasis. Increased intestinal calcium absorption and bone resorption and decreased tubule calcium reabsorption may be caused by elevated serum 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] in some patients but not in those with normal serum 1,25(OH)(2)D(3) levels. Because 1,25(OH)(2)D(3) exerts its biological actions through binding to the cellular vitamin D receptor (VDR), the present study was undertaken to test the hypothesis that VDR levels are elevated in IH patients.Ten male IH calcium oxalate stone-formers were paired with controls matched in age within 5 yr and lacking a history of stones or family history of stones. Blood was obtained for serum, peripheral blood monocytes (PBMs) were separated from lymphocytes and other mononuclear cells, and PBM VDR content was measured by Western blotting.The PBM VDR level was 2-fold greater in IH men at 49 +/- 21 vs. 20 +/- 15 fmol/mg protein, mean +/- sd; P < 0.008. Serum 1,25(OH)(2)D(3) levels were not higher than controls (48 +/- 14 vs. 39 +/- 11 pg/ml; P < 0.068). In conclusion, PBM VDR levels are elevated in IH calcium oxalate stone-formers. The elevation could not be ascribed to increased serum 1,25(OH)(2)D(3) levels. These results suggest that the molecular basis for IH involves a pathological elevation of tissue VDR level, which may elevate intestinal calcium absorption and bone resorption and decrease renal tubule calcium reabsorption. The mechanism for increased VDR in IH patients with normal serum 1,25(OH)(2)D(3) levels is unknown.     

Vitamin D metabolism in peripheral blood mononuclear cells is influenced by chewing "betel nut" (Areca catechu) and vitamin D status

CONTEXT: Vitamin D deficiency, common in South Asians, is a risk factor for metabolic syndrome, type 2 diabetes, and ischemic heart disease. Vitamin D receptor (VDR) activation depends on activated vitamin D [1,25-dihydroxyvitamin D (1,25(OH)(2)D)] concentration, reflecting opposing actions of 25-hydroxyvitamin D-1alpha-hydroxylase [1-alpha(OH)ase] for formation and 25(OH)D-24-hydroxylase [24(OH)ase] for catabolism. We previously reported that circulating 1,25(OH)(2)D contributed to determination of VDR-protein levels and VDR genotype was a determinant of both VDR mRNA and VDR-protein in South Asians. OBJECTIVE: We hypothesized that chewing betel nut, an addictive habit common throughout South Asian communities, contributes to hypovitaminosis-D by modulating the enzymes regulating circulating 1,25(OH)(2)D concentration. DESIGN: Peripheral blood mononuclear cell (PBMC) 1-alpha(OH)ase and 24(OH)ase mRNA concentrations were measured and examined in relation to cross-sectional data on the vitamin-D axis, diet, smoking, and betel usage, including PBMC VDR-RNA and VDR-protein content in a pilot study of 33 healthy British Bangladeshis. RESULTS: PBMC 24(OH)ase mRNA correlated positively and serum 1,25(OH)(2)D negatively with betel quids per day (r = 0.49, P = 0.006 and r = -0.486, P = 0.006, respectively). Independent determinants for 24(OH)ase included betel quids per day (P < 0.0001) and serum 25-OHD (P = 0.024). Independent determinants for serum 1,25(OH)(2)D were gender, smoking, and betel quids per day. PBMC 1-alpha(OH)ase mRNA correlated inversely with VDR mRNA (r = -0.44; P = 0.013); its independent determinants were serum 1,25(OH)(2)D and VDR TaqI and BsmI polymorphisms (P = 0.03-0.0001). CONCLUSIONS: Betel chewing is a more powerful independent determinant of increased 24(OH)ase expression and of decreased serum calcitriol than serum 25-OHD, supporting the hypothesis that this habit could aggravate the effects of vitamin-D deficiency.     

Synergistic enhancement by 12-O-tetradecanoylphorbol-13-acetate and dibutyryl cAMP of 1alpha,25-dihydroxyvitamin D3 action in human promyelocytic leukemic HL-60 cells.

We have reported that dibutyryl cAMP (dbcAMP), an activator of cAMP-dependent protein kinase (PKA), potentiated the effects of 1alpha,25-dihydroxyvitamin D3(1,25-(OH)2D3)-induced 24-hydroxylation activity in HL-60 cells by increasing 1,25-(OH)2D3 receptor (VDR). The present study demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent phorbol ester, also potentiated the effect of 1,25-(OH)2D3 on HL-60 cells and that TPA and dbcAMP acted in a synergistic manner to enhance the effect of 1,25-(OH)2D3. It is interesting that TPA induced 24-hydroxylation activity far more efficiently than dbcAMP, in addition to their effects in increasing VDR. TPA increased basal levels of c-fos mRNA to the maximum by 1 h after the treatment, whereas dbcAMP failed to affect c-fos gene expression. Together with the previous data indicating the presence of AP-1-like sequence in the promoter of 24-hydroxylase gene, it was suggested that TPA potentiated the effect of 1,25-(OH)2D3 through an activation of c-fos gene expression. This notion was further supported by the data showing that TPA and dbcAMP also acted in a synergistic manner to activate c-fos gene expression. Neither TPA nor dbcAMP affected c-jun early response gene in the HL-60 clone used in the present study. The present study suggested that the activation of early c-fos response gene by TPA might be another mechanism to enhance the effect of 1,25-(OH)2D3, besides up-regulation of VDR.