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.     

Modulation of chick intestinal and renal calbindin gene expression by dietary vitamin D3, 1,25-dihydroxyvitamin D3, calcium and phosphorus

Synthetic oligonucleotide probes complementary to chick calbindin-28 kDa-mRNA were used to study the latter's regulation and relationship to calbindin in the chick. The effects of vitamin D3 sources and dietary alteration on the genomic expression were characterized by Northern blot and solution hybridization. Intestinal calbindin and its mRNA were almost absent in vitamin D-deficient chicks and were not affected by dietary alteration. Renal calbindin and its mRNA were lower in the vitamin D-deficient than in vitamin D3- or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-fed chicks. In the same animal, renal calbindin mRNA and calbindin were higher than intestinal. In vitamin D3-fed chicks, dietary calcium (Ca) or phosphorus (P) restriction induced, and high dietary Ca inhibited, intestinal calbindin and its mRNA synthesis. In the same chicks, dietary P restriction induced renal calbindin mRNA and calbindin synthesis. In 1,25-(OH)2D3-fed chicks, dietary P restriction induced and high dietary Ca inhibited the synthesis of intestinal and renal calbindin. The results suggest that: (a) most of the changes in renal and intestinal calbindin could be attributed to the changes in the mRNA; (b) the adaptation to dietary Ca and P alterations requires vitamin D metabolites; (c) high dietary Ca affects intestinal and renal calbindin-mRNA and calbindin via mechanisms independent of kidney 1-hydroxylase; and (d) plasma Ca and renal calbindin or its mRNA tend to change together in vitamin D-deficient or vitamin D3-fed, but not in 1,25(OH)2D3-fed chicks.     

Vitamin D and human health: lessons from vitamin D receptor null mice

The vitamin D endocrine system is essential for calcium and bone homeostasis. The precise mode of action and the full spectrum of activities of the vitamin D hormone, 1,25-dihydroxyvitamin D [1,25-(OH)(2)D], can now be better evaluated by critical analysis of mice with engineered deletion of the vitamin D receptor (VDR). Absence of a functional VDR or the key activating enzyme, 25-OHD-1alpha-hydroxylase (CYP27B1), in mice creates a bone and growth plate phenotype that mimics humans with the same congenital disease or severe vitamin D deficiency. The intestine is the key target for the VDR because high calcium intake, or selective VDR rescue in the intestine, restores a normal bone and growth plate phenotype. The VDR is nearly ubiquitously expressed, and almost all cells respond to 1,25-(OH)(2)D exposure; about 3% of the mouse or human genome is regulated, directly and/or indirectly, by the vitamin D endocrine system, suggesting a more widespread function. VDR-deficient mice, but not vitamin D- or 1alpha-hydroxylase-deficient mice, and man develop total alopecia, indicating that the function of the VDR and its ligand is not fully overlapping. The immune system of VDR- or vitamin D-deficient mice is grossly normal but shows increased sensitivity to autoimmune diseases such as inflammatory bowel disease or type 1 diabetes after exposure to predisposing factors. VDR-deficient mice do not have a spontaneous increase in cancer but are more prone to oncogene- or chemocarcinogen-induced tumors. They also develop high renin hypertension, cardiac hypertrophy, and increased thrombogenicity. Vitamin D deficiency in humans is associated with increased prevalence of diseases, as predicted by the VDR null phenotype. Prospective vitamin D supplementation studies with multiple noncalcemic endpoints are needed to define the benefits of an optimal vitamin D status.     

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.     

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.     

Calcium transport in perfused duodena from normal chicks: enhancement within fourteen minutes of exposure to 1,25-dihydroxyvitamin D3

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3) on calcium transport was studied in vascularly perfused duodena of normal, vitamin D-replete chicks. Addition of 130 pM 1,25(OH)2D3 to the perfusate resulted in a significant increase in 45Ca transport from the lumen to the vascular effluent within 14 min; the transport rate rose to 140% of levels in comparable preparations exposed for 40 min to vehicle. No effects of 1,25(OH)2D3 were noted on the back flux or transfer of 45Ca from the vascular effluent to the lumen. Vascular perfusion with 100 microM colchicine, an antimicrotubular agent, abolished the rapid lumen-to-vascular effluent effect of 1,25(OH)2D3 on 45Ca transport, relative to preparations exposed to the secosteroid and 100 microM lumicolchicine, (a light inactivated analog of colchicine). Colchicine did not, however, alter basal 45Ca transport rates. Addition of 130 pM 1,25(OH)2D3 to the lumenal compartment of normal chicks or vascular perfusion of duodena from vitamin D-deficient birds failed to increase 45Ca transport above control levels. Perfusion of duodena from normal chicks with 650 pM 1,25(OH)2D3 further increased calcium transport to 170% of levels observed in preparations treated with 130 pM steroid, and 210% of levels in controls. Although 15 nM vitamin D3 had no effect, in one series of experiments 125 nM 25-hydroxyvitamin D3 elicited vascular calcium levels that were 185% of controls at 40 min. These results suggest that 1,25(OH)2D3 can act in vitamin D-replete animals to produce rapid unidirectional calcium transport responses (through unknown mechanisms), as well as by interaction with intestinal nuclear receptors in D-deficient animals to promote induction of protein(s) that support long acting calcium transport responses.     

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 enhances the effect of refeeding on steady state preproinsulin messenger ribonucleic acid levels in rats

To elucidate the regulatory mechanism of vitamin D action on insulin biosynthesis and secretion, we examined preproinsulin (ppI) mRNA levels in the pancreas of normal rats, vitamin D-deficient rats, and rats supplemented with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] or calcium (Ca) for 3 days. The ppI mRNA levels determined by dot blot analysis in vitamin D-deficient, 1,25-(OH)2D3-replete, and Ca-replete rats were 39.1%, 68.7%, and 66.7%, respectively, of values in normal rats. These results concur with previously reported levels of insulin secretion in the perfused rat pancreas. The reduced level of ppI mRNA should lead to a decrease in insulin biosynthesis and, thus, impair insulin secretion in vitamin D-deficient rats. The observed partial recovery of ppI mRNA levels through supplementation of 1,25-(OH)2D3 or Ca may be one mechanism by which insulin secretion is restored in rats after 1,25-(OH)2D3 or Ca repletion. We examined further the time course of ppI mRNA accumulation in rats after a single administration of 1,25-(OH)2D3. When fasting was continued for an additional 24-h period after an overnight fast, ppI mRNA levels were not changed significantly in either vitamin D-deficient or replete rats. However, in the rats that were pair-fed after overnight fasting, ppI mRNA levels in 1,25-(OH)2D3-replete rats increased at 8 and 24 h, whereas ppI mRNA in vitamin D-deficient rats increased only at 24 h. Moreover, the increment at 24 h was significantly larger in 1,25-(OH)2D3-replete rats than in vitamin D-deficient rats. We conclude that 1,25-(OH)2D3 enhances steady state levels of ppI mRNA only under conditions of refeeding and during feeding.     

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.     

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.     

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.     

Effect of vitamin D deficiency and 1,25-dihydroxycholecalciferol treatment on epidermal calcium-binding protein (ECaBP) RNA activity

We have previously reported that the amount of epidermal calcium binding protein (ECaBP) in the skin decreases in the absence of vitamin D. Since vitamin D influences epidermal differentiation, and the synthesis of ECaBP may vary with cell differentiation, it was necessary to know whether vitamin D acts directly on the translational or post-translational level of ECaBP synthesis or indirectly by its action on epidermopoiesis. The cell-free translation technique was used to demonstrate the presence of mRNA coding for ECaBP. The activity of this mRNA has been evaluated in the skin of vitamin D-fed and in vitamin D-deficient rats with or without treatment with 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃). Vitamin D deficiency decreased the ECaBP mRNA activity. The latter was selectively increased in animals given a single dose of 1,25(OH)₂D₃. These results suggest that 1,25(OH)₂D₃ stimulates the production of ECaBP mRNA or stabilizes this mRNA.     

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.     

1 alpha,25-dihydroxyvitamin D3 regulates in vivo production of the third component of complement (C3) in bone.

We previously reported that 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] specifically stimulates production of the third component of complement (C3) by murine osteoblastic cells and marrow-derived stromal cells (ST2) in vitro. In the present study we examined tissue-specific production of C3 in vivo in vitamin D-deficient mice, some of which received supplemental 1 alpha,25-(OH)2D3. Western blot analysis indicated that the C3 protein band in bone was undetectable in vitamin D-deficient mice, but became distinct 48 h after 1 alpha,25-(OH)2D3 administration. The mRNA expression of C3 in bone was also undetectable in vitamin D-deficient mice and appeared as early as 24 h after 1 alpha,25-(OH)2D3 administration. mRNA expression apparently preceded the appearance of C3 protein. In contrast, there was no significant difference in the expression of hepatic C3 mRNA among normal mice fed laboratory chow and vitamin D-deficient mice with and without 1 alpha,25-(OH)2D3 administration. The serum concentration of C3 in vitamin D-deficient mice was almost identical to that in normal mice and was unchanged after 1 alpha,25-(OH)2D3 administration. 1 alpha,25-(OH)2D3 receptor (VDR) mRNAs were detected in the kidney and intestine, whereas no appreciable mRNA expression of VDR occurred in the liver. Osteopontin mRNA was expressed in response to 1 alpha,25-(OH)2D3 in the kidney, but not in the intestine. Immunohistochemical studies showed that in normal mice, the C3 protein was located mainly in the periosteal regions of calvaria and on the surfaces of bone trabeculae in the tibial metaphyses. These results demonstrate that 1 alpha,25-(OH)2D3 tissue-specifically regulates in vivo production of C3 in bone. The production of bone C3 cannot be attributed to the presence of VDR alone, and we speculate that other tissue-specific factors are required.     

Modulation of quail intestinal and egg shell gland calbindin (Mr 28,000) gene expression by vitamin D3, 1,25-dihydroxyvitamin D3 and egg laying.

The effects of vitamin D3 sources, egg production and egg cycle on the genomic expression of calbindin (Mr 28,000) in the intestine and egg shell gland (ESG) of quail were characterized by Northern blot and solution hybridization, using synthetic oligonucleotide probe. In vitamin D3- or 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-fed quail, onset of egg production induced duodenal and ESG calbindin mRNA and calbindin synthesis. Duodenal calbindin mRNA was slightly higher during the period of shell calcification as compared with the period during which shells were not formed (ESG inactivity). ESG calbindin mRNA was markedly higher during the period of shell calcification than of ESG inactivity. Increasing dietary intake of [3H]1 alpha-hydroxyvitamin D3 increased the duodenal, but not ESG, content of 1,25-(OH)2D3 and calbindin. Duodenal calbindin and its mRNA were absent in vitamin D-deficient quail and were not affected by egg laying. ESG calbindin in the vitamin D-deficient quail was not affected by egg laying, but calbindin mRNA increased in the vitamin D-deficient birds during shell calcification. The results suggest that: (a) intestinal calbindin mRNA and calbindin are induced and/or regulated, either directly or indirectly, by 1,25-(OH)2D3; (b) intestinal calbindin and its mRNA are further induced at the onset of egg laying by an additional stimulator besides 1,25-(OH)2D3; (c) 1,25-(OH)2D3 is required for the expression of the latter stimulator; (d) ESG calbindin mRNA and calbindin are induced in egg-laying birds by a stimulator associated with the egg cycle; and (e) the induction of ESG calbindin mRNA does not need vitamin D metabolites, but 1,25-(OH)2D3 is required for the translation of the mRNA.     

The role of the vitamin D receptor in regulating vitamin D metabolism: a study of vitamin D-dependent rickets, type II

In vitro studies and animal experiments suggest that the production of 1,25-dihydroxyvitamin D [1,25-(OH)(2)D] and 24,25-(OH)(2)D is reciprocally controlled by 1,25-(OH)(2)D. To investigate the role of the vitamin D receptor (VDR) in controlling vitamin D metabolism in humans, we studied 10 patients with vitamin D-dependent rickets type II due to a defective VDR. After a period of high dose calcium therapy, 7 of the patients had normal serum calcium, phosphorus, alkaline phosphatase, and plasma PTH levels (PTH-N), and 3 showed increased serum alkaline phosphatase and plasma PTH (PTH-H). Serum calcium, phosphorus, alkaline phosphatase, PTH, vitamin D metabolites, urinary calcium/creatinine, and renal phosphate threshold concentration were compared with unaffected family members that comprised the control group. Vitamin D metabolites were measured before and after an oral load of 50,000 U/m(2) cholecalciferol. Compared with the control group, 1,25-(OH)(2)D levels were significantly higher and 24,25-(OH)(2)D levels were lower in the PTH-N group and even more so in the PTH-H group. 1alpha-Hydroxylase (1-OHase) and 24-OHase activities were estimated by the product/substrate ratio. In the PTH-N group, 1-OHase activity was higher and 24-OHase activity was lower than in controls. In the PTH-H group, 1-OHase activity was even higher, probably due to an additive effect of PTH. Thus, 1,25-(OH)(2)D-liganded VDR is a major control mechanism for vitamin D metabolism, and PTH exerts an additive effect. Assessment of the influence of 1,25-(OH)(2)D shows reciprocal control of enzyme activity in man, suppressing 1-OHase and stimulating 24-OHase activity.     

Time course study of insulin secretion after 1,25-dihydroxyvitamin D3 administration

Vitamin D3 is known to be involved in pancreatic endocrine function. The rapidity of action of the biologically active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], was studied over time (from 0-72 h) on pancreatic insulin secretion by the subsequently isolated perfused pancreas of vitamin D-deficient rats pair-fed with vitamin D-deficient control rats treated with vehicle alone. At 8 h after 1,25(OH)2D3 administration (1.3 nmol), augmentation of the insulin secretion in response to 16.6 mM glucose had already significantly appeared and reached a maximum at 14 h, and then markedly decreased to pretreatment baseline values by 36 h. In a separate experiment using 20 mM arginine as a stimulus, insulin secretion from the isolated perfused pancreas also showed a significant increase at 8 h and demonstrated a maximum response at 14 h after 1,25(OH)2D3 administration, followed by gradual decrease to 72 h. The prevailing levels of serum parameters, including calcium, phosphorus, and glucose, seemed not to be involved in this mechanism, since these were not correlated to the amount of insulin secretion by the subsequently isolated perfused pancreas. Also the observed rapid effects of 1,25(OH)2D3 on insulin secretion appear not to be related to a rapid effect of the secosteroid on increased dietary/caloric intake. These results clearly establish both the dependence of and rapid dynamics response of the perfused pancreas to the potentiating effects of in vivo administered 1,25(OH)2D3 on either glucose- or arginine-mediated insulin secretion from the perfused pancreas.