Klotho, a gene related to a syndrome resembling human premature aging, functions in a negative regulatory circuit of vitamin D endocrine system

The klotho gene encodes a novel type I membrane protein of beta-glycosidase family and is expressed principally in distal tubule cells of the kidney and choroid plexus in the brain. These mutants displayed abnormal calcium and phosphorus homeostasis together with increased serum 1,25-(OH)2D. In kl-/- mice at the age of 3 wk, elevated levels of serum calcium (10.9 +/- 0.31 mg/dl vs. 10.0 +/- 0.048 mg/dl in wild-type mice), phosphorus (14.7 +/- 1.1 mg/dl vs. 9.7 +/- 1.5 mg/dl in wild type) and most notably, 1,25-(OH)2D (403 +/- 99.7 mg/dl vs. 88.0 +/- 34.0 mg/dl in wild type) were observed.Reduction of serum 1,25-(OH)2D concentrations by dietary restriction resulted in alleviation of most of the phenotypes, suggesting that they are downstream events resulting from elevated 1,25-(OH)2D. We searched for the signals that lead to up-regulation of vitamin D activating enzymes. We examined the response of 1alpha-hydroxylase gene expression to calcium regulating hormones, such as PTH, calcitonin, and 1,25-(OH)2D3. These pathways were intact in klotho null mutant mice, suggesting the existence of alternate regulatory circuits. We also found that the administration of 1,25-(OH)2D3 induced the expression of klotho in the kidney. These observations suggest that klotho may participate in a negative regulatory circuit of the vitamin D endocrine system, through the regulation of 1alpha-hydroxylase gene expression.     

1,25-Dihydroxycholecalciferol effects on plasma calcitonin levels and calcitonin mRNA in normal or partially vitamin D-depleted rats

The physiological effect of 1,25-(OH)2D3 on the regulation of calcitonin (CT) secretion was studied by measuring plasma CT levels and CT mRNAs extracted from thyroid glands of normal (D+) or partially vitamin D-depleted rats (D-). In both groups, acute 1,25-(OH)2D3 administration of 0.1 microgram/kg b.w. yielded an early drop in plasma calcium concentrations (around 0.6-1 mg/dl) with a maximum decrease 15 min after treatment. In spite of this hypocalcemia, a significant rise in plasma CT levels was observed within 5 min in D+ animals and within 30 min in D- animals after injection of the vitamin D metabolite. Nevertheless, the increased CT secretion was not associated with a marked and sustained rise in CT mRNA levels measured by dot-blot hybridization or CT mRNA activity evaluated by translation assay. By contrast to the observations made previously using supra-physiological doses of the vitamin D metabolites, no clear-cut effect on CT mRNA levels was found with lower doses. If we hypothesized that 1,25-(OH)2D3 plays a physiological role in CT secretion, our results suggest that this rapid control could be exerted at a post-translational level may be via an increase in the cytoplasmic ionized calcium concentration of C-cells.     

Control of DNA polymerase-alpha activity in regenerating rat liver by calcium and 1 alpha,25(OH)2D3

The late G1 surge of DNA polymerase-alpha activity and the initiation of DNA replication in the hepatocytes of partial hepatectomy-induced regenerating liver were severely reduced when the mitogenic partial hepatectomy was carried out in the hypocalcemic and 1,25(OH)2D3 (1 alpha,25-dihydroxycholecalciferol)-deficient environment of parathyroidectomized (PTX) or thyroparathyroidectomized (TPTX) rats. These inhibitions were prevented in TPTX rats by a postpartial hepatectomy injection of 1,25(OH)2D3, which also restored blood calcium to normocalcemic levels. Inhibition of active DNA polymerase-alpha accumulation and initiation of DNA synthesis in TPTX rats were also completely prevented by prefeeding the rats a low phosphorus diet, which stopped the lowering of the blood levels of calcium and 1,25(OH)2D3 following parathyroid removal. These studies indicate that the rise of DNA polymerase-alpha activity and the initiation of DNA replication in regenerating liver are controlled by cellular processes that rely on normal blood levels of calcium and 1,25(OH)2D3. Because DNA polymerase-alpha is the third DNA replication enzyme (the others are ribonucleotide reductase and thymidylate synthase) that has been shown to depend on parathyroid hormone and/or the circulating levels of calcium and 1,25(OH)2D3 that it controls, the authors concluded that the processes dependent on calcium and 1,25(OH)2D3 are parts of a mechanism that coordinately activates the DNA-replicating enzymes. The possibility that cyclic adenosine monophosphate (cAMP)-dependent protein kinases are involved in this replication mechanism is considered.     

Effect of 1,25(OH)2D3 on the relative contributions of skeletal 45Ca and intestinal 40Ca to blood calcium in normal and thyroparathyroidectomized dogs

The effects of gradually increasing doses of 1,25(OH)2D3 on plasma calcium and 45Ca radioactivity were studied in young dogs that had been extensively prelabelled with 45Ca. The effects of orally and intravenously administered 1,25(OH)2D3 were evaluated in normal and thyroparathyroidectomized dogs fed a normal diet. In normal dogs when 1,25(OH)2D3 increased the plasma calcium within the normal range (2.9-3.1 mmol/L) there was no significant increase in plasma 45Ca. In thyroparathyroidectomized dogs, oral or intravenous 1,25(OH)2D3 increased the low blood calcium to a normal level (1.8-2.9 mmol/L) without significantly increasing plasma 45Ca. In normal and thyroparathyroidectomized dogs, any 1,25(OH)2D3-induced increase in plasma calcium above the normal range was associated with a significant increase in 45Ca, indicating mobilization of bone calcium. Intravenous administration of 1,25(OH)2D3 in the normal or thyroparathyroidectomized dogs had a much larger effect than oral doses in mobilizing bone 45Ca when inducing a similar level of hypercalcemia. The major physiological effect of 1,25(OH)2D3 in the low or normal range of plasma calcium is on intestinal absorption of calcium without a significant effect on mobilizing bone calcium. The pharmacological effect of 1,25(OH)2D3 in vivo is to mobilize bone calcium as well as dietary calcium into blood.     

Evidence for calcium-dependent control of 1,25-dihydroxyvitamin D3 production by rat kidney proximal tubules

The role of calcium in the parathyroid hormone-mediated increase in 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) production was evaluated using isolated proximal tubules from rats fed a low calcium diet (0.002% Ca) for 14 days. Tubules were prepared by collagenase digestion and centrifugation through Percoll. Tubules from rats fed a low calcium diet produced 1,25-(OH)2D3 at rates 10 times that of tubules from rats fed normal calcium diet (1.2% Ca). In vitro 1,25-(OH)2D3 biosynthesis was highly dependent upon extracellular calcium with inhibition in the absence of medium calcium and maximal production at 0.25 mM medium calcium (0.9 +/- 0.25 versus 15.1 +/- 2.3 nmol/mg protein/5 min, p less than 0.03). Inhibition of 1,25-(OH)2D3 production was partly due to depressed ATP content (0 versus 1.2 mM calcium, 6.8 +/- 0.6 versus 12.7 +/- 0.6 nmol/mg protein, p less than 0.006). EGTA reduced 1,25-(OH)2D3 synthesis and total cell calcium and ATP production. Ruthenium red blocked the inhibitory effects of EGTA on 1,25-(OH)2D3 production. Barium (1.0 mM) inhibited 1,25-(OH)2D3 production (7.2 +/- 0.5 versus 3.4 +/- 0.3, p less than 0.001) without altering ATP production. The calcium ionophore A23187 increased 1,25-(OH)2D3 production in a calcium-dependent manner. It is concluded that parathyroid hormone-mediated increases in 1,25-(OH)2D3 production, as during low calcium diet, require extracellular calcium. Extracellular calcium maintains mitochondrial calcium at optimal concentrations for normal ATP production, a requirement for 25-hydroxyvitamin D3-1-hydroxylase (25-OH-D3-1-hydroxylase) activity. Inhibition of 25-OH-D3-1-hydroxylase activity by barium without an alteration of ATP suggests calcium may also control 1,25-(OH)2D3 production independent of its effects on oxidative phosphorylation, perhaps through a direct interaction with one or more components of the 25-OH-D3-1-hydroxylase.     

Effect of dietary calcium, phosphate and vitamin D deprivation on the pharmacokinetics of 1,25-dihydroxyvitamin D3 in the rat

The in vivo regulation of circulating 1,25(OH)2D3 concentrations by vitamin D status and by dietary calcium and phosphate deficiency was studied. Adult rats were cannulated in the jugular vein and the clearance of physiological doses of 1,25(OH)2D3 monitored. In vitamin D-replete rats we investigated the effects of dietary calcium and phosphate deficiency on the elimination half life of 1,25(OH)2D3 The results showed no effect of dietary phosphate deficiency on the elimination half life of 1,25(OH)2D3. Dietary calcium deficiency resulted in a small increase of the 1,25(OH)2D3 elimination half life (P = 0.04) (normal diet: 16.3 +/- 1.8 hrs, n = 6; -Ca diet: 18.6 +/- 1.1 hrs, n = 5; -P diet: 16.0 +/- 1.4 hrs, n = 6; mean +/- SD). The experiments with the vitamin D deficient rats showed a marked increase in the elimination half life of 1,25(OH)2D3 (36.4 +/- 6.8 hrs, n = 7), when compared to the rats on the normal diet (P = 0.001). From the experiments in the vitamin D replete rats one can infer that regulation of circulating 1,25(OH)2D3 concentrations by dietary calcium or phosphate takes place at the production site and not by changes in elimination rate. However, vitamin D status appears to regulate circulating 1,25(OH)2D3 concentrations also through an effect on the elimination rate.     

Calcitonin secretion during postnatal development in the rat: beta-adrenergic agents and vitamin D3 metabolites

The possible contribution of catecholamines and vitamin D3 metabolites to the high plasma calcitonin (CT) levels in suckling baby rats is unknown. So, in vivo and in vitro (using a perifusion system) effects of beta-adrenergic agents and vitamin D3 metabolites on CT release were studied in the rat during the postnatal development. In 13-day-old rats, the increase in plasma CT levels induced by isoproterenol injection (0.1 micrograms/kg b.w.) was inhibited by a previous administration of propranolol. A significant decrease in plasma CT levels was observed after propranolol injection in baby rats (0.68 +/- 0.05 ng/ml vs. 0.93 +/- 0.01 ng/ml). A daily injection of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3; 25 pmoles/rat/day during 4 days) induced a marked rise in plasma calcium (16.1 +/- 0.2 mg/dl), and a great decrease in thyroidal CT contents (approximately 70% of control values) in 13-day-old rats while no change was noted with 24,25-dihydroxycholecalciferol (24,25-(OH)2D3). A negative correlation between plasma calcium and thyroidal CT stores was found in suckling and in weaning rats treated with different doses of 1,25-(OH)2D3, suggesting an indirect effect of 1,25-(OH)2D3 on CT secretion. The mobilization of the thyroidal CT content was greater in weaning than in suckling rats in response to a given hypercalcemia. In vitro, 5 X 10(-5) M isoproterenol induced a rapid increase in CT secretion rate while 1,25-(OH)2D3 inhibited the rise in CT release induced by 3.0 mM calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of 25-hydroxyvitamin D3 in renal slices from the X-linked hypophosphatemic (Hyp) mouse: abnormal response to fall in serum calcium

The effect of the X-linked Hyp mutation on 25-hydroxyvitamin D3 (25-OH-D3) metabolism in mouse renal cortical slices was investigated. Vitamin D replete normal mice and Hyp littermates fed the control diet synthesized primarily 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3); only minimal synthesis of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) was detected in both genotypes and 1,25-(OH)2D3 formation was not significantly greater in Hyp mice relative to normal littermates, despite hypophosphatemia and hypocalcemia in the mutants. Calcium-deficient diet fed to normal mice reduced serum calcium (p less than 0.01), increased renal 25-hydroxyvitamin D3-1-hydroxylase (1-OHase) activity (p less than 0.05), and decreased 25-hydroxyvitamin D3-24-hydroxylase (24-OHase) activity (p less than 0.05). In contrast, Hyp littermates on the calcium-deficient diet had decreased serum calcium (p less than 0.01), without significant changes in the renal metabolism of 25-OH-D3. Both normal and Hyp mice responded to the vitamin D-deficient diet with a fall in serum calcium (p less than 0.01), significantly increased renal 1-OHase, and significantly decreased renal 24-OHase activities. In Hyp mice, the fall in serum calcium on the vitamin D-deficient diet was significantly greater than that observed on the calcium-deficient diet. Therefore the ability of Hyp mice to increase renal 1-OHase activity when fed the vitamin D-deficient diet and their failure to do so on the calcium-deficient diet may be related to the resulting degree of hypocalcemia. The results suggest that although Hyp mice can respond to a disturbance of calcium homeostasis, the in vivo signal for the stimulation of renal 1-OHase activity may be set at a different threshold in the Hyp mouse; i.e. a lower serum calcium concentration is necessary for Hyp mice to initiate increased synthesis of 1,25(-OH)2D3.     

Determination of vitamin D and its metabolites in plasma from normal and anephric man

A multiple assay capable of reliably determining vitamins D(2) and D(3) (ergocalciferol and cholecalciferol), 25(OH)D(2) (25-hydroxyvitamin D(2)) and 25(OH)D(3) (25-hydroxyvitamin D(3)), 24,25(OH)(2)D (24,25-dihydroxyvitamin D), 25,26(OH)(2)D (25,26-dihydroxyvitamin D) and 1,25(OH)(2)D (1,25-dihydroxyvitamin D) in a single 3-5ml sample of human plasma was developed. The procedure involves methanol/methylene chloride extraction of plasma lipids followed by separation of the metabolites and purification from interfering contaminants by batch elution chromatography on Sephadex LH-20 and Lipidex 5000 and by h.p.l.c. (high-pressure liquid chromatography). Vitamins D(2) and D(3) and 25(OH)D(2) and 25(OH)D(3) are quantified by h.p.l.c. by using u.v. detection, comparing their peak heights with those of standards. 24,25(OH)(2)D and 25,26(OH)(2)D are measured by competitive protein-binding assay with diluted plasma from vitamin D-deficient rats. 1,25(OH)(2)D is measured by competitive protein-binding assay with diluted cytosol from vitamin D-deficient chick intestine. Values in normal human plasma samples taken in February are: vitamin D 3.5+/-2.5ng/ml; 25(OH)D 31.6+/-9.3ng/ml; 24,25(OH)(2)D 3.5+/-1.4ng/ml; 25,26(OH)(2)D 0.7+/-0.5ng/ml; 1,25(OH)(2)D 31+/-9pg/ml (means+/-s.d.). Values in two normal human plasma samples taken in February after 1 week of high sun exposure are: vitamin D 27.1+/-7.9ng/ml; 25(OH)D 56.8+/-4.2ng/ml; 24,25(OH)(2)D 4.3+/-1.6ng/ml; 25,26(OH)(2)D 0.5+/-0.2ng/ml. Values in anephric-human plasma are: vitamin D 2.7+/-0.8ng/ml; 25(OH)D 36.4+/-16.5ng/ml; 24,25(OH)(2)D 1.9+/-1.3ng/ml; 25,26(OH)(2)D 0.6+/-0.3ng/ml; 1,25(OH)(2)D was undetectable.     

Dietary phosphate deprivation increases 1,25-dihyroxyvitamin D3 synthesis in rat kidney in vitro

A sensitive radioreceptor assay has been used to measure in vitro 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) synthesis in vitamin D-replete rats. Incubation of kidney cortical slices with 25-hydroxyvitamin D3 produced a product which co-migrated on high performance liquid chromatography with authentic 1,25(OH)2D3 in two different solvent systems and displaced 1,25(OH)2D3 from its intestinal receptor. In addition, mass spectral analysis of the product produced a mass fragmentation consistent with that of authentic 1,25(OH)2D3. Endogenous renal cortical 1,25(OH)2D3 content in phosphate-deprived rats averaged 1.1 +/- 0.3 pmol/g (n = 11), which was significantly greater than the renal cortical 1,25(OH)2D3 content of age-matched rats eating a normal diet which averaged 0.44 +/- 0.21 pmol/g (n = 8, p less than 0.001). After incubation, net 1,25(OH)2D3 synthesis in renal slices from phosphate-deprived rats averaged 51 pmol/g/h, about 13-fold greater than the mean of 3.8 pmol/g/h observed in renal slices from rats eating the normal diet. These results indicate that the elevated plasma 1,25(OH)2D3 levels observed in rats during dietary phosphate deprivation are due to increased renal synthesis of the hormone.     

1,25(OH)2D3 only affects long-term levels of plasma Ca2+ but not the rapid minute-to-minute plasma Ca2+ homeostasis in the rat.

Results from our lab have shown previously that parathyroid hormone (PTH) is not the key factor in the rapid regulation of plasma Ca2+. The possible role of 1,25(OH)2D3 in the rapid minute-to-minute regulation of plasma Ca2+, as addressed by a possible rapid non-genomic action of 1,25(OH)2D3, was therefore studied in vivo in rats. The rapid calcemic recovery from induction of hypocalcemia by a brief EGTA infusion was examined in vitamin D-depleted rats with intact parathyroid glands and in vitamin D depleted rats 1 h after parathyroidectomy (PTX). The influence of different levels of plasma 1,25(OH)2D3 on the rapid calcemic recovery from hypocalcemia was examined in PTX rats treated with 1,25(OH)2D3 for two days at two different doses of 0.2 microg/day, 0.05 microg/day or vehicle, and in PTX rats being BNX for two days, as well. Additionally, the long-term effect of 1,25(OH)2D3 on plasma Ca2+ homeostasis was examined. Plasma Ca2+ recovered significantly (P<0.05) 10 min after discontinuing EGTA in vitamin D-depleted rats with or without parathyroid glands. Plasma Ca2+ increased significantly (P<0.05) and at the same rate after induction of hypocalcemia in PTX rats with different levels of plasma 1,25(OH)2D3. The final levels of plasma Ca2+ obtained were set by 1,25(OH)2D3 in a dose-related manner. 1,25(OH)2D3 did not affect the rapid calcemic recovery from EGTA induced hypocalcemia, but only had an effect on the long-term plasma Ca2+ homeostasis in the rat.     

Peripheral effects of PTH are not altered after thyroid surgery in euthyroid patients

BACKGROUND: We have previously found decreased serum levels of both ionized calcium and 1,25(OH)2D and an increase in serum phosphate levels at 1 year after hemithyroidectomy. However, basal and stimulated parathyroid hormone (PTH) secretions were not altered. To investigate whether the observed biochemical changes after unilateral thyroid surgery may be due to a relative end-organ resistance to PTH, we studied the peripheral effects of infused hPTH-(1-34) in 6 patients preoperatively and 3 months after hemithyroidectomy. METHODS: Serum levels of TSH, FT4 and FT3 were measured pre- and postoperatively. hPTH-(1-34) was infused at 0.9 IU/kg/h during 6 h. Blood samples for analysis of ionized calcium, intact PTH, phosphate, 25(OH)D, 1,25(OH)2D and urinary samples for calcium, phosphate and nephrogenous(n)-cAMP analysis were taken at baseline, when the infusion was discontinued after 6 h and at 24 h. RESULTS: Three months after hemithyroidectomy, serum levels of FT3 were decreased and TSH levels increased. Pre- and postoperative hPTH-(1-34) infusions induced increased serum levels of ionized calcium, 1,25(OH)2D, increased urinary excretion of phosphate and elevated n-cAMP levels. The changes in the studied biochemical variables during the hPTH-(1-34) infusions did not differ between the two study occasions. CONCLUSION: By using a 6-hour hPTH-(1-34) infusion protocol, we have shown that the peripheral PTH effect is not altered by a slight reduction in thyroid hormone levels at 3 months after hemithyroidectomy.     

Interrelations of calcium-regulating hormones during normal pregnancy.

Profound changes in calcium metabolism occur during pregnancy. The mother has to make available extra calcium for fetal requirements while ensuring that her plasma and bone calcium concentrations are satisfactorily maintained. In a cross-sectional study plasma concentrations of the major calcium-regulating hormones--namely, calcitonin, parathyroid hormone, 25-hydroxyvitamin D (25-OHD), and 1,25-dihydroxyvitamin D (1,25-(OH)2D)--were measured to establish their interrelations during normal pregnancy. The major changes observed were increases in the circulating concentrations of 1,25-(OH)2D and calcitonin. Concentrations of parathyroid hormone and 25-OHD remained within the normal range. The increased concentrations of 1,25-(OH)2D enable the increased physiological need for calcium to be met by enhancing intestinal absorption of this element. The simultaneous rise in calcitonin opposes the bone-resorbing activities of 1,25-(OH)2D, thereby protecting the integrity of the maternal skeleton. Maternal calcium homeostasis is thus maintained yet the requirements of the fetus are fulfilled.     

Serum osteocalcin levels do not change during rapidly induced hypercalcemia in healthy subjects.

Since osteocalcin has been suggested to play a role in calcium homeostasis, we investigated its serum levels in 6 healthy subjects during a rapid calcium infusion. Serum levels of intact parathyroid hormone (PTH), 25-hydroxyvitamin D [25-(OH) D3] and 1,25-dihydroxyvitamin D [1,25-(OH)2 D3] were also determined. The calcium infusion increased plasma-ionized calcium levels from 1.25 +/- 0.04 to 1.54 +/- 0.07 mmol/l at 30 min (p less than 0.05). Concomitantly, serum levels of intact PTH declined from 2.1 +/- 0.9 to 0.2 +/- 0.3 mmol/l (p less than 0.05). In contrast, serum osteocalcin levels did not change. Further, during calcium infusion, serum levels of 1,25-(OH)2 D3 decreased from 81 +/- 17 to 75 +/- 15 pmol/l (p less than 0.05) whereas serum levels of 25-(OH) D3 did not change. The results therefore suggest that calcium per se does not influence osteocalcin secretion.     

Effect of 1,25-dihydroxyvitamin D3 on pancreatic B and D cell function

To investigate the effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on pancreatic B and D cell function in normal rats, 1 microgram of 1,25(OH)2D3 was administered intravenously 20 hours before the experiment. The plasma 1,25(OH)2D3 and calcium concentrations were significantly elevated, and plasma insulin levels also increased in 1,25(OH)2D3-administered rats compared with controls. Glucose-induced insulin and somatostatin release from the isolated pancreas perfused with lower calcium, however, was the same between the 1,25(OH)2D3-administered group and the controls. On the other hand, when the isolated pancreas was perfused with higher calcium, the glucose-induced insulin release was significantly increased in the 1,25(OH)2D3-administered group, while no significant difference in somatostatin release was observed in any group. These results suggest that the sensitivity of pancreatic B cells to glucose perfused with more calcium may increase when 1,25(OH)2D3 has been previously administered. In addition, 1,25(OH)2D3 does not seem to affect the somatostatin release from the pancreatic D cells.     

The effects of betamethasone on plasma and intestinal calcium binding protein and on 25-hydroxyvitamin D3 metabolism in the pig

Betamethasone (50 micrograms/kg body weight/day) given to young pigs reduced calcium absorption, growth and plasma vitamin D dependent calcium binding protein (CaBP) concentration. No changes occurred in plasma 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and intestinal CaBP concentrations. 1,25(OH)2D3 (0.1 microgram/kg body weight/day) given with betamethasone increased calcium absorption although growth and plasma CaBP concentrations remained low. Intestinal CaBP levels remained unchanged. Plasma CaBP concentrations were not consistently related to intestinal CaBP or calcium absorption in the presence of betamethasone. We conclude that betamethasone-induced depression of calcium absorption was not mediated by alterations in intestinal CaBP, but the mechanism remains obscure.     

Hereditary resistance to 1,25-dihydroxyvitamin D: clinical and radiological improvement during high-dose oral calcium therapy

A 6-year-old boy, of consanguinous parents, presented with severe rickets and alopecia; he was found to have hypocalcaemia and elevated circulating 1,25-dihydroxyvitamin D [1,25-(OH)2D] levels. He showed no calcaemic response to 1,25-(OH)2D3 or ergocalciferol given for 3 or more months in daily doses as high as 48 micrograms and 6 X 10(6) IU, respectively. Analyses with cultured skin fibroblasts revealed a normal capacity and affinity for 1,25-(OH)2D3 in soluble extracts ('cytosol') and in nuclei of intact cells but no detectable response of 25-(OH)D3 24-hydroxylase to 1,25-(OH)2D3 in high concentration. Treatment with high doses of calcium (3-4 g elemental calcium orally per day) produced a striking clinical and radiological improvement. We conclude that high oral doses of calcium can replace many of the actions of calciferols. Therapy with high doses of calcium should be tried in similarly affected cases that appear totally or partially unresponsive to calciferols.     

In vivo administration of 1,25-dihydroxyvitamin D3 suppresses the expression of RANKL mRNA in bone of thyroparathyroidectomized rats constantly infused with PTH

It is known that pharmacological or toxic doses of vitamin D induce bone resorption both in vivo and in vitro, whereas physiological doses of the vitamin have a protective effect on bone in vivo. To investigate the discrepancies of the dose-dependent effect of vitamin D on bone resorption, we examined the in vivo effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] on the expression of the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) and osteoprotegerin (OPG) mRNAs in bone of thyroparathyroidectomized (TPTX) rats infused with or without parathyroid hormone (PTH). Continuous infusion of 50 ng/h of PTH greatly increased the expression of RANKL mRNA in bone of TPTX rats. Expression of OPG mRNA was not altered by PTH infusion. When graded doses of 1,25(OH)(2)D(3) was daily administered orally for 14 days to normocalcemic TPTX rats constantly infused with PTH, 0.01 and 0.1 microg/kg of 1,25(OH)(2)D(3) inhibited the PTH-induced RANKL mRNA expression, but 0.5 microg/kg of the vitamin did not inhibit it. Regulator of G protein signaling-2 (RGS-2) gene expression was suppressed by 1,25(OH)(2)D(3) dose-dependently, but PTH/PTHrP receptor mRNA expression was not altered. Bone morphometric analyses revealed that 1,25(OH)(2)D(3) suppressed PTH-induced osteoclast number in vivo. These results suggest that pharmacological or toxic doses of 1,25(OH)(2)D(3) stimulate bone resorption by inducing RANKL, but a certain range of physiological doses of the vitamin inhibit PTH-induced bone resorption, the latter mechanism appeared to be mediated, at least in part, by the suppression of the PTH/PTHrP receptor-mediated signaling.     

Semaphorin 3B is a 1,25-Dihydroxyvitamin D3-induced gene in osteoblasts that promotes osteoclastogenesis and induces osteopenia in mice

The vitamin D endocrine system is important for skeletal homeostasis. 1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] impacts bone indirectly by promoting intestinal absorption of calcium and phosphate and directly by acting on osteoblasts and osteoclasts. Despite the direct actions of 1,25(OH)(2)D(3) in bone, relatively little is known of the mechanisms or target genes that are regulated by 1,25(OH)(2)D(3) in skeletal cells. Here, we identify semaphorin 3B (SEMA3B) as a 1,25(OH)(2)D(3)-stimulated gene in osteoblastic cells. Northern analysis revealed strong induction of SEMA3B mRNA by 1,25(OH)(2)D(3) in MG-63, ST-2, MC3T3, and primary osteoblastic cells. Moreover, differentiation of these osteogenic cells enhanced SEMA3B gene expression. Biological effects of SEMA3B in the skeletal system have not been reported. Here, we show that osteoblast-derived SEMA3B alters global skeletal homeostasis in intact animals and osteoblast function in cell culture. Osteoblast-targeted expression of SEMA3B in mice resulted in reduced bone mineral density and aberrant trabecular structure compared with nontransgenic littermates. Histomorphometry studies indicated that this was likely due to increased osteoclast numbers and activity. Indeed, primary osteoblasts obtained from SEMA3B transgenic mice stimulated osteoclastogenesis to a greater extent than nontransgenic osteoblasts. This study establishes that SEMA3B is a 1,25(OH)(2)D(3)-induced gene in osteoblasts and that osteoblast-derived SEMA3B impacts skeletal biology in vitro and in vivo. Collectively, these studies support a putative role for SEMA3B as an osteoblast protein that regulates bone mass and skeletal homeostasis.     

24,25(OH)2D3 attenuates the calcemic effect of 1,25(OH)2D3 in rats with reduced renal mass

The present study was undertaken to evaluate the effect of 24,25(OH)2D3 on serum calcium concentration in rats with reduced renal mass. Adult 5/6 nephrectomized male rats were divided into four groups: (i) control rats, (ii) rats treated with 1,25(OH)2D3, (iii) rats treated with 24,25(OH)2D3, and (iv) rats treated with 1,25(OH)2D3 and 24,25(OH)2D3. After 4 days, serum calcium in the 1,25(OH)2D3-treated group was 7.13 +/- 0.32 meq/liter (P less than 0.001 vs control). With the combination of 1,25(OH)2D3 and 24,25(OH)2D3 serum calcium was higher than that in control, 6.25 +/- 0.5 meq/liter (P less than 0.001 vs control), but lower than that in rats receiving 1,25(OH)2D3 alone (P less than 0.05). No change in serum calcium was seen in animals treated with 24,25(OH)2D3 alone. On the eighth day serum calcium in the 1,25(OH)2D3-treated group, 6.52 +/- 0.25, was higher than in the 1,25(OH)2D3 + 24,25(OH)2D3 group, 5.87 +/- 0.17 meq/liter, P less than 0.05, P less than 0.001 vs control. In both 1,25(OH)2D3- and 1,25(OH)2D3 + 24,25(OH)2D3-treated rats, hypercalciuria of similar magnitude occurred on the fourth and eighth day of treatment. No change in urinary calcium was seen in the control and 24,25(OH)2D3-treated rats. Thus, in 5/6 nephrectomized rats combined administration of 1,25(OH)2D3 and 24,25(OH)2D3 attenuates the calcemic response to 1,25(OH)2D3 without changes in urinary calcium excretion. These observations suggest that the effect of 24,25(OH)2D3 on serum calcium is different in 5/6 nephrectomized rats as compared to normal rats, in which an augmentation of serum calcium was observed following administration of both vitamin D metabolites. The effect of 24,25(OH)2D3 on serum calcium in rats with reduced renal mass may result from a direct effect of 24,25(OH)2D3 on the bone.