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.     

Dietary and serum phosphorus regulate fibroblast growth factor 23 expression and 1,25-dihydroxyvitamin D metabolism in mice

Fibroblast growth factor-23 (FGF-23) is a novel circulating peptide that regulates phosphorus (Pi) and vitamin D metabolism, but the mechanisms by which circulating FGF-23 itself is regulated are unknown. To determine whether the serum FGF-23 concentration is regulated by dietary intake of Pi, we fed wild-type (WT), Npt2a gene-ablated (Npt2a(-/-)), and Hyp mice diets containing varying Pi contents (0.02-1.65%). In WT mice, increases in dietary Pi intake from 0.02-1.65% induced a 7-fold increase in serum FGF-23 and a 3-fold increase in serum Pi concentrations. Across the range of dietary Pi, serum FGF-23 concentrations varied directly with serum Pi concentrations (r(2) = 0.72; P < 0.001). In Npt2a(-/-) mice, serum FGF-23 concentrations were significantly lower than in WT mice, and these differences could be accounted for by the lower serum Pi levels in Npt2a(-/-) mice. The serum concentrations of FGF-23 in Hyp mice were 5- to 25-fold higher than values in WT mice, and the values varied with dietary Pi intake. Fgf-23 mRNA abundance in calvaria was significantly higher in Hyp mice than in WT mice on the 1% Pi diet; in both groups of mice, fgf-23 mRNA abundance in calvarial bone was suppressed by 85% on the low (0.02%) Pi diet. In WT mice fed the low (0.02%) Pi diet, renal mitochondrial 1alpha-hydroxylase activity and renal 1alpha-hydroxylase (P450c1alpha) mRNA abundance were significantly higher than in mice fed the higher Pi diets and varied inversely with serum FGF-23 concentrations (r(2) = 0.86 and r(2) = 0.64; P < 0.001, respectively). The present data demonstrate that dietary Pi regulates the serum FGF-23 concentration in mice, and such regulation is independent of phex function. The data suggest that genotype-dependent and dietary Pi-induced changes in the serum FGF-23 concentration reflect changes in fgf-23 gene expression in bone.     

Expression and activity of vitamin D-metabolizing cytochrome P450s (CYP1alpha and CYP24) in human nonsmall cell lung carcinomas.

Extrarenal 25-hydroxyvitamin D3-1alpha-hydroxylase is believed to play a major role in the pathogenesis of hypercalcemia associated with various types of granulomatous and lymphoproliferative diseases and certain solid tumors. In this paper, we describe the cloning of the cytochrome P450 component of the extrarenal enzyme from a human nonsmall cell lung carcinoma, SW 900. The cytochrome P450 for the extrarenal 1alpha-hydroxylase has an amino acid sequence identical to that of the cytochrome P450 component of the CYP1alpha, the renal form of the enzyme, and appears to be a product of the same gene. CYP1alpha messenger RNA (mRNA) and 1alpha-hydroxylase enzyme activity were detected in two (SW 900, SK-Luci-6) of a series of five nonsmall cell lung carcinoma cell lines. All five lung cell lines were cultured with the same medium under the same conditions, but only two of the five expressed 1alpha-hydroxylase enzyme; two others (WT-E, Calu-1) expressed high levels of the reciprocally regulated enzyme, 25-hydroxyvitamin D3-24-hydroxylase, with its specific cytochrome P450 component, CYP24. Although under basal conditions the lung cell line SW 900 expressed only CYP1alpha and showed 1alpha-hydroxylase enzyme activity, when treated with small concentrations of 1alpha,25-dihydroxyvitamin D3 or high concentrations of 25-hydroxyvitamin D3, it began to express CYP24 and exhibit 24-hydroxylase enzyme activity. Somewhat surprisingly, SW 900 cells still had detectable CYP1alpha mRNA some 24 h after vitamin D treatment despite the fact that 1alpha-hydroxylase enzyme activity was unmeasurable. These data are consistent with the emerging hypothesis that vitamin D through its active form does not directly turn off CYP1alpha mRNA production but, rather, strongly stimulates CYP24, thereby masking CYP1alpha activity. The factor(s) responsible for the basal expression of CYP1alpha in SW 900 and SK-Luci-6 is currently unknown.     

Early lethality in Hyp mice with targeted deletion of Pth gene

Although increased circulating levels of PTH with mild hypocalcemia has been reported in Hyp mice, hyperparathyroidism in X-linked hypophosphatemic rickets is postulated to arise from the standard use of phosphate salts, which induce chronic stimulation of PTH secretion. In this study, we sought to examine the role of PTH in the metabolic derangements associated with Hyp by generating hemizygous hypophosphatemic (Hyp/Y) mice homozygous for the Pth-null allele (Pth(-/-);Hyp/Y). Early postnatal lethality was observed in the Pth(-/-);Hyp/Y mice. Within the first 6 h, postpartum serum phosphorus increased to levels comparable to those in the Pth(-/-) mice, whereas in Hyp mice, it decreased during the first 48 h after birth. Serum calcium concentration started low after birth and remained reduced in both Pth(-/-);Hyp/Y and Pth(-/-) mice although more profoundly so in the former group, whereas in Hyp/Y mice, the levels were initially lower than but reached wild-type levels by 24 h. Circulating PTH levels in Hyp/Y mice were higher than wild-type levels throughout the first 48 h after birth and continued to be so well into adulthood. Twice-daily administration of PTH 1-34 to Pth(-/-);Hyp/Y newborn mice increased serum calcium levels and prevented their early demise. The findings here indicate that the cause of death in the Pth(-/-);Hyp/Y mice is severe hypocalcemia. A potential role for fibroblast growth factor 23 in promoting secondary hyperparathyroidism by suppressing renal 25-hydroxyvitamin D(3)-1alpha-hydroxylase (Cyp27b1) activity while increasing that of renal 25-hydroxyvitamin D(3) 24-hydroxylase (Cyp24) is proposed. Hyperparathyroidism, therefore, is an integral component in the pathophysiology of Hyp, and likely X-linked hypophosphatemic rickets and serves to prevent severe hypocalcemia in mice and perhaps in patients afflicted with the disorder.     

Abnormal 24-hydroxylation of 25-hydroxyvitamin D in the X-linked hypophosphatemic mouse

The effect of extracellular phosphate on the control of 25-hydroxyvitamin D3 24-hydroxylase was studied in normal mice and littermates with X-linked hypophosphatemic rickets (Hyp). 24-Hydroxylase activity and plasma concentrations of 24,25-dihydroxyvitamin D3 were significantly higher in Hyp mice than in normal mice when both groups were fed a normal diet containing 1.22% calcium (Ca) and 0.8% phosphorus (Pi). The differential in 24-hydroxylase activity was exaggerated when serum phosphate was reduced in normal mice by means of a low Pi diet or increased in Hyp mice by means of a high Pi diet. Differences in 24-hydroxylase activity between the two groups of mice were also demonstrated in the presence of varying Pi concentrations in vitro. Thus, in both Hyp and normal mice, 24-hydroxylase activity is influenced in a qualitatively similar manner by serum Pi. Plasma concentrations of 1,25-dihydroxyvitamin D3 were the same in normal and Hyp mice. The data are consistent with the hypothesis that control the renal metabolism of 25-hydroxyvitamin D3 in Hyp mice is reset such tht 24-hydroxylase activity is inappropriate high for the prevailing serum phosphate over a wide range of concentrations.     

Effect of the X-linked Hyp mutation and vitamin D status on induction of renal 25-hydroxyvitamin D3-24-hydroxylase

The present study was undertaken to evaluate the response of Hyp mice to regulators known to inhibit renal 25-hydroxyvitamin D3-1-hydroxylase (1-hydroxylase) and stimulate renal 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase). Renal mitochondrial metabolism of 25-hydroxyvitamin D3 (25OHD3) was initially examined in vitamin D- and calcium-deprived normal and mutant mice (with no detectable 24-hydroxylase) treated with either calcium, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], or both calcium + 1,25-(OH)2D3. In normal mice, 1,25-(OH)2D3 treatment was more effective than calcium in turning off 1-hydroxylase and turning on 24-hydroxylase activity; serum calcium, however, was similarly increased by both treatments. Although calcium + 1,25-(OH)2D3 did not result in a further change in 25OHD3 metabolism in normal mice, a further elevation in serum calcium was apparent. In Hyp mice, treatment with calcium + 1,25-(OH)2D3 resulted in a greater decrease in 1-hydroxylase and a greater increase in 24-hydroxylase and in serum calcium than treatment with either agent alone. In spite of similar serum calcium levels in both genotypes, 24-hydroxylase was 20-fold, 3-fold, and 8-fold greater in Hyp mice relative to normals treated with calcium, 1,25-(OH)2D3, and calcium + 1,25-(OH)2D3, respectively. Kinetic studies revealed that the maximum velocity (Vmax) for induced 24-hydroxylase was 6-fold greater than normal in Hyp mice whereas the apparent Michaelis-Menten constant (Km) was not different in the two groups of calcium + 1,25-(OH)2D3-treated mice. The effect of 1,25-(OH)2D3 treatment on the above serum and renal parameters was also examined in vitamin D replete normal and Hyp mice. A sharp rise in serum phosphate was observed in 1,25-(OH)2D3-treated Hyp mice whereas normal littermates experienced marked hypercalcemia in response to treatment. Renal 24-hydroxylase was significantly stimulated by 1,25-(OH)2D3 treatment in both normal and Hyp mice and genotype differences were not apparent. The present study demonstrates that vitamin D- and calcium-deprived Hyp mice are more responsive to signals which induce 24-hydroxylase than normal littermates; Vmax for induced 24-hydroxylase is 6-fold greater in Hyp mice than in normal littermates whereas apparent Km is unchanged; the inhibitory control of 1-hydroxylase appears to be intact in the mutant strain; induced 24-hydroxylase is similar in vitamin D replete normal and Hyp mice; and vitamin D status can thus modify the response of both genotypes to treatment with 1,25-(OH)2D3.(ABSTRACT TRUNCATED AT 400 WORDS)     

A possible role of vitamin D receptors in regulating vitamin D activation in the kidney.

The vitamin D endocrine system is regulated reciprocally by renal 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylases. Previously, we reported that renal proximal convoluted tubules, the major site of 1 alpha, 25-dihydroxyvitamin D3 production, have vitamin D receptors. In the presence of vitamin D receptors, renal proximal convoluted tubules cannot maintain the state of enhanced production of 1 alpha, 25-dihydroxyvitamin D3. To clarify this discrepancy, we proposed a working hypothesis for the reciprocal control of renal 25-hydroxyvitamin D3 1 alpha- and 24-hydroxylase activities. In rat models of enhanced renal production of 1 alpha, 25-dihydroxyvitamin D3, expression of vitamin D receptors and 25-hydroxyvitamin D3 24-hydroxylase mRNAs was strikingly suppressed in renal proximal convoluted tubules but not in the cortical collecting ducts. In vitamin D-deficient rats with up-regulated renal 25-hydroxyvitamin D3 1 alpha-hydroxylase activity, expression of vitamin D receptor mRNA in renal proximal convoluted tubules was also down-regulated, indicating that the down-regulation of vitamin D receptor mRNA is not the result of the enhanced production of 1 alpha, 25-dihydroxyvitamin D3. In Japanese quail models with up-regulated renal 25-hydroxyvitamin D3 1 alpha-hydroxylase activity by sex steroids, expression of vitamin D receptor mRNA was also down-regulated in the kidney but not in the duodenum. These results suggest that the down-regulation of vitamin D receptors plays a critical role in production of 1 alpha, 25-dihydroxyvitamin D3 in renal proximal convoluted tubules.     

Renal expression of the sodium/phosphate cotransporter gene, Npt2, is not required for regulation of renal 1 alpha-hydroxylase by phosphate

Several reports have suggested that the regulation of renal 1,25-dihydroxyvitamin D [1,25-(OH)(2)D] synthesis by extracellular phosphate (Pi) is dependent on normal transepithelial Pi transport by the renal tubule. Mice homozygous for the disrupted Na/Pi cotransporter gene Npt2 (Npt2(-/-)) exhibit renal Pi wasting, an approximately 85% decrease in renal brush border membrane Na/Pi cotransport, hypophosphatemia, and an increase in serum 1,25-(OH)(2)D concentration. We undertook 1) to determine the mechanism for the increased circulating levels of 1,25-(OH)(2)D in Npt2(-/-) mice and 2) to establish whether renal 1alpha-hydroxylase was appropriately regulated by dietary Pi in the absence of Npt2 gene expression. On a control diet, the 2.5-fold increase in the serum 1,25-(OH)(2)D concentration in Npt2(-/-) mice, relative to that in Npt2(+/+) littermates, is associated with a corresponding increase in renal mitochondrial 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) activity and messenger RNA (mRNA) abundance. A low Pi diet elicits an increase in serum 1,25-(OH)(2)D concentration, renal 1alpha-hydroxylase activity, and mRNA abundance in Npt2(+/+) and Npt2(-/-) mice to similar levels in both mouse strains. A high Pi diet has no effect on serum 1,25-(OH)(2)D concentration, renal 1 alpha-hydroxylase activity, or mRNA abundance in Npt2(+/+) mice, but normalizes these parameters in Npt2(-/-) mice. In addition, renal 24-hydroxylase mRNA abundance is significantly reduced in Npt2(-/-) mice compared with that in Npt2(+/+) mice under all dietary conditions. In summary, we demonstrate that 1) increased renal synthesis of 1,25-(OH)(2)D is responsible for the increased serum 1,25-(OH)(2)D concentration in Npt2(-/-) mice; and 2) renal 1alpha-hydroxylase gene expression is appropriately regulated by dietary manipulation of serum Pi in both Npt2(+/+) and Npt2(-/-) mice. Thus, intact renal Na/Pi cotransport is not required for the regulation of renal 1alpha-hydroxylase by Pi.     

25-hydroxyvitamin D(3)-1alpha-hydroxylase expression in normal and pathological parathyroid glands

Active vitamin D, 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)], plays a pivotal role in calcium homeostasis and bone metabolism. Circulating levels of 1,25(OH)(2)D(3) are thought to be dependent mainly on the activity of the renal cytochrome P450 enzyme 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase), which is potently induced by PTH. However, 1alpha-hydroxylase activity or expression has also been reported at several extrarenal sites, at which local synthesis of 1,25(OH)(2)D(3) appears to fulfill autocrine or paracrine functions. This includes tissues such as placenta and brain that also express LRP-2/megalin, an endocytic receptor for multiple ligands, which is involved in the renal uptake of the substrate for 1alpha -hydroxylase, 25-hydroxyvitamin D(3). We have previously demonstrated LRP-2/megalin in parathyroid cells, and here we present results from RT-PCR and immunohistochemical analyses showing coincident expression of 1alpha-hydroxylase in normal and pathological parathyroid tissue. With real-time quantitative RT-PCR analysis, the expression of 1alpha-hydroxylase mRNA was higher in the majority of parathyroid adenomas and secondary hyperplastic glands but lower in parathyroid carcinomas, compared with normal parathyroid tissue. The findings imply that in addition to feedback control by circulating 1,25(OH)(2)D(3) levels, parathyroid cells may also be influenced by local 1alpha -hydroxylase activity with possible growth regulatory and differentiating effects.     

Investigation of the mechanism for abnormal renal 25-hydroxyvitamin D3-1-hydroxylase activity in the X-linked Hyp mouse

Renal mitochondria from mutant hypophosphatemic male mice (Hyp/Y) fed a vitamin D-deficient, low calcium diet synthesize significantly less 1,25-dihydroxyvitamin D3 than mitochondria from normal male (+/Y) littermates on the same diet. Kinetic studies reveal that maximum velocity (Vmax) for 25-hydroxyvitamin D3-1-hydroxylase (1-hydroxylase) is lower in Hyp/Y relative to +/Y mice (0.21 +/- 0.02 vs. 1.06 +/- 0.12 pmol/mg protein X min) whereas the apparent Michaelis-Menten constant (Km) for the reaction is not different in both genotypes (0.55 +/- 0.05 vs. 0.50 +/- 0.08 microM). The presence of an inhibitor for 1-hydroxylase activity in renal mitochondria of Hyp/Y mice was ruled out by estimating enzyme activity in mixtures of renal mitochondria from +/Y and Hyp/Y mice. Phosphate in the incubation medium stimulated 1-hydroxylase activity in +/Y mitochondria. In Hyp/Y mice, the stimulation achieved was smaller in magnitude and the added phosphate did not restore mutant 1-hydroxylase activity to normal. The vitamin D-deficient, low calcium diet led to a significant and comparable increase in serum PTH and urinary excretion of cAMP in +/Y and Hyp/Y, suggesting that the mutant strain had an appropriate PTH response to the diet-induced fall in serum calcium. Furthermore, the fractional excretion index of phosphate which is significantly greater in Hyp/Y than +/Y mice fed the control diet increased 3-fold in both genotypes fed the vitamin D-deficient, low calcium diet. These results suggest that the abnormal renal 1-hydroxylase response in Hyp mice is not the result of generalized renal resistance to PTH in the mutant strain and suggest that the defect in Hyp/Y mice may reside at a regulatory step subsequent to cAMP production.     

Chronic inhibition of ERK1/2 signaling improves disordered bone and mineral metabolism in hypophosphatemic (Hyp) mice

The X-linked hypophosphatemic (Hyp) mouse carries a loss-of-function mutation in the phex gene and is characterized by hypophosphatemia due to renal phosphate (Pi) wasting, inappropriately suppressed 1,25-dihydroxyvitamin D [1,25(OH)?D] production, and rachitic bone disease. Increased serum fibroblast growth factor-23 concentration is responsible for the disordered metabolism of Pi and 1,25(OH)?D. In the present study, we tested the hypothesis that chronic inhibition of fibroblast growth factor-23-induced activation of MAPK signaling in Hyp mice can reverse their metabolic derangements and rachitic bone disease. Hyp mice were administered the MAPK inhibitor, PD0325901 orally for 4 wk. PD0325901 induced a 15-fold and 2-fold increase in renal 1α-hydroxylase mRNA and protein abundance, respectively, and thereby higher serum 1,25(OH)?D concentrations (115 ± 13 vs. 70 ± 16 pg/ml, P < 0.05), compared with values in vehicle-treated Hyp mice. With PD0325901, serum Pi levels were higher (5.1 ± 0.5 vs. 3 ± 0.2 mg/dl, P < 0.05), and the protein abundance of sodium-dependent phosphate cotransporter Npt2a, was greater than in vehicle-treated mice. The rachitic bone disease in Hyp mice is characterized by abundant unmineralized osteoid bone volume, widened epiphyses, and disorganized growth plates. In PD0325901-treated Hyp mice, mineralization of cortical and trabecular bone increased significantly, accompanied by a decrease in unmineralized osteoid volume and thickness, as determined by histomorphometric analysis. The improvement in mineralization in PD0325901-treated Hyp mice was confirmed by microcomputed tomography analysis, which showed an increase in cortical bone volume and thickness. These findings provide evidence that in Hyp mice, chronic MAPK inhibition improves disordered Pi and 1,25(OH)?D metabolism and bone mineralization.     

Variable phosphate-mediated regulation of vitamin D metabolism in the murine hypophosphatemic rachitic/osteomalacic disorders

The familial hypophosphatemic (vitamin D-resistant) disorders are a variety of genetic and acquired syndromes that exhibit unexpected biochemical heterogeneity, manifest as variably abnormal or apparently normal regulation of 1,25-dihydroxyvitamin D [1,25-(OH)2D] production. Recently, we observed that Hyp and Gy mice, murine homologs of X-linked hypophosphatemic rickets, exhibit similarly disparate regulation of vitamin D metabolism. While Gy mice under basal conditions maintain an appropriate elevation (relative to hypophosphatemia) of renal 25-hydroxyvitamin D (25OHD)-1 alpha-hydroxylase, Hyp mice manifest only normal, not increased, enzyme activity. Whether such diversity results from maintenance of phosphate (P)-regulated 1,25-(OH)2D production in Gy mice and loss of this function in Hyp mice or from other variations remains unknown. Therefore, we examined the integrity of P-regulated enzyme activity in the Gy and Hyp mice by testing the effects of enzyme inhibition and alteration of the serum phosphorus concentration on 1,25-(OH)2D production. Initially, we discovered that inhibition of renal 25OHD-1 alpha-hydroxylase suppressed enzyme function in Hyp mice, but did not prevent expression of P-mediated activity in Gy mice. In this regard, while administration of a high calcium diet or 1,25-(OH)2D (0.4 ng/h, sc, for 48 h) resulted in a comparable inhibition of enzyme activity in Hyp (5.9 +/- 0.5 vs. 2.8 +/- 0.7 fmol/mg.min) and normal mice (4.4 +/- 0.6 vs. 2.0 +/- 0.2 fmol/mg.min), similar treatment did not effect complete inhibition of 25OHD-1 alpha-hydroxylase in Gy (10.3 +/- 0.6 vs. 4.9 +/- 0.3 fmol/mg.min) or P-depleted mice (10.2 +/- 0.5 vs. 5.1 +/- 0.4 fmol/mg.min). In accord with the apparent persistence of P-mediated stimulation of enzyme function in Gy mice, dietary P repletion in this mutant resulted in a serum phosphorus concentration similar to that of normal mice and decreased enzyme activity (4.0 +/- 0.8 fmol/mg.min) to a level no different from that expressed in controls (3.4 +/- 0.3 fmol/mg.min). However, in the absence of apparent P-mediated stimulation of enzyme activity, identical treatment of Hyp mice increased the serum phosphorus level comparably, but paradoxically enhanced 25OHD-1 alpha-hydroxylase (3.1 +/- 0.4 vs. 11.7 +/- 2.0 fmol/mg.min). Collectively, these data indicate that enhanced renal 25OHD-1 alpha-hydroxylase expressed in Gy mice and probably in related human diseases results from normally maintained P regulation of enzyme activity, an action absent or mutated in the genetically distinct Hyp mouse.     

IGF-I and GH stimulate Phex mRNA expression in lungs and bones and 1,25-dihydroxyvitamin D(3) production in hypophysectomized rats.

OBJECTIVE: X-linked hypophosphatemia, a renal phosphate (Pi)-wasting disorder with defective bone mineralization, is caused by mutations in the PHEX gene (a Pi-regulating gene with homology to endopeptidases on the X chromosome). We wondered whether changes in Phex and neprilysin (NEP) (another member of the family of zinc endopeptidases) mRNA expression could be observed in relation to vitamin D and Pi metabolism during GH- and IGF-I-stimulated growth of hypophysectomized rats. DESIGN: Animals were infused s.c. for 2 days with vehicle, 200 mU (67 microg) GH or 300 microg IGF-I/rat per 24 h. We determined serum osteocalcin and osteocalcin mRNA in bone, Phex mRNA in bone and lungs, serum 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) and serum Pi levels, and renal expression of 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase), of 25-hydroxyvitamin D(3)-24-hydroxylase (24-hydroxylase) and of the Na-dependent Pi-cotransporter type I and II (Na(d)Pi-I and -II). RESULTS: As compared with vehicle-treated controls, body weight and tibial epiphyseal width significantly increased in GH- and IGF-I-treated animals. Serum osteocalcin and osteocalcin mRNA levels in bone, Phex mRNA in bone and lungs, serum 1,25-(OH)(2)D(3) and renal 1alpha-hydroxylase mRNA rose concomitantly, whereas expression of NEP in lungs was barely affected and renal 24-hydroxylase mRNA decreased. Na(d)Pi-I and -II gene expression in the kidney and serum Pi levels remained unchanged. CONCLUSIONS: Our findings suggest a coordinate regulation of Phex mRNA expression in lungs and bone and vitamin D metabolism during GH- and IGF-I-stimulated growth.     

Abnormal adenosine 3'.5'-monophosphate stimulation of renal 1,25-dihydroxyvitamin D production in hyp mice: evidence that 25-hydroxyvitamin D-1 alpha-hydroxylase dysfunction results from aberrant intracellular function

Previously we have established that abnormal regulation of renal 25-hydroxyvitamin D (25OHD)-1 alpha-hydroxylase in Hyp mice involves the PTH-adenylate cyclase component of enzyme activation. However, it remains unknown if the muted effects of PTH result from 1) abnormal second messenger production or 2) an intracellular defect limiting enzyme activation. To distinguish between these possibilities, we compared cAMP stimulation of renal 25OHD-1 alpha-hydroxylase in normal, phosphate-depleted normal, and Hyp mice. Administration of N6-monobutyryl cAMP iv (200 mg/kg/day) increased enzyme activity in normal (4.1 +/- 1.7 vs. 40.7 +/- 7.0 fmol/mg kidney.min) and phosphate-depleted mice (13.3 +/- 1.8 vs. 78.2 +/- 10.4) to a level significantly greater than that achieved in Hyp mice (7.4 +/- 1.1 vs. 22.7 +/- 3.6). Moreover, similar to our observations after PTH stimulation, the apparent abnormal cAMP effect did not result from an altered time course of enzyme activation or a rightward shift in the dose response. Collectively, these data indicate that abnormal regulation of 1,25-dihydroxyvitamin D production in Hyp mice results from aberrant intracellular regulation of 25OHD-1 alpha-hydroxylase, a defect probably related to deranged phosphate transport in the renal tubule.     

Abnormal parathyroid hormone-related peptide stimulation of renal 25-hydroxyvitamin D-1-hydroxylase in Hyp mice: evidence for a generalized defect of enzyme activity in the proximal convoluted tubule

Previous investigations have established that hypophosphatemic (Hyp) mice exhibit diminished PTH/cAMP stimulation of 25-hydroxyvitamin D (25[OH]D)-1-hydroxylase activity in the renal proximal convoluted tubule. Whether similar muted enzyme responsiveness occurs secondary to provocation by other hormones/metabolic factors that operate by a different mechanism in the same cell system, however, remains unknown. In order to investigate this possibility, we compared renal 25(OH)D-1-hydroxylase activity of normal and Hyp mice upon stimulation with PTH-related peptide (PTHrP), a factor which may affect enzyme function in the PCT by a PTH-independent mechanism. Administration of 1-34 PTHrP, 3.0 micrograms/day sc, increased enzyme activity in normal mice (4.9 + 0.63 vs. 50.3 +/- 6.2 fmol/mg kidney.min) to a level significantly greater than that achieved in the Hyp mice (6.9 + 0.86 vs. 14.5 +/- 0.91 fmol/mg kidney.min). Moreover, similar to our observations after PTH stimulation, abnormal PTHrP effects did not result from an altered time course of enzyme activation or a shift in the dose response. Thus, the 25(OH)D-1-hydroxylase activity increased linearly to a maximum at 24 h in both animal models with a slope greater in normals than in mutants (P less than 0.05). Further, administration of PTHrP in graded amounts (0-9.0 micrograms/day) elicited a curvilinear response in normals and Hyp mice, but the mutants exhibited significantly less function (54 +/- 8.6%) at all doses tested. Additional studies revealed that the muted effects of PTHrP occurred via a PTH-independent mechanism. In this regard, we observed that simultaneous infusion of maximally effective doses of PTH and PTHrP in normal and Hyp mice resulted in an additive increment of 25(OH)D-1-hydroxylase activity. This observation that PTH and PTHrP influence renal 25(OH)D-1-hydroxylase by apparently different mechanisms indicates that the muted effects of these agents on enzyme activity in the Hyp-mouse results from a generalized defect in the proximal convoluted tubule.     

Normal calcitonin stimulation of serum calcitriol in patients with X-linked hypophosphatemic rickets.

Patients with X-linked hypophosphatemic rickets (XLH) have normal or low calcitriol concentrations despite manifesting hypophosphatemia, a known stimulus of 25-hydroxyvitamin D-1 alpha-hydroxylase activity. In accord, administration of pharmacological doses of PTH results in a markedly blunted stimulation of calcitriol levels. In the murine homolog of the human disorder, the Hyp mouse, regulation of 25-hydroxyvitamin D-1 alpha-hydroxylase activity is defective in response to hypophosphatemia and PTH administration, but not in response to calcitonin administration. In the current study we administered calcitonin to controls and patients with XLH to test the hypothesis that calcitonin-stimulatable 25-hydroxyvitamin D-1 alpha-hydroxylase activity is normal in patients with XLH. We found that calcitriol concentrations increased in both groups to a similar degree (78.5 +/- 20.9 pmol/L in patients and 49.9 +/- 19.7 pmol/L in controls) and with a similar time course. Our results indicate that the complex and incomplete defect in the regulation of 25-hydroxyvitamin D-1 alpha-hydroxylase observed in Hyp mice also exists in humans.     

Leptin corrects increased gene expression of renal 25-hydroxyvitamin D3-1 alpha-hydroxylase and -24-hydroxylase in leptin-deficient, ob/ob mice

Leptin, the ob gene product secreted by adipocytes, controls overall energy balance. We investigated leptin effects on bone metabolism using male leptin-deficient obese (ob/ob) mice, which had lower bone mineral density (BMD) and shorter femurs than lean (?/+) controls. Serum concentrations of calcium, phosphate, tartrate-resistant acid phosphatase (a bone resorption marker) and alkaline phosphatase, and urinary calcium and phosphate excretion were significantly elevated in ob/ob mice, whereas urinary concentrations of deoxypyridinoline did not differed between ob/ob and control mice. Because ob/ob mice develop severe hypogonadism, testosterone was administered to these mice for 10 wk (5 mg/kg, sc, twice weekly); this did not affect femoral BMD. Control and ob/ob mice showed similar vitamin D-receptor densities in bone and kidney; the obese mice had marked increases in serum 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] and in mRNA expression and activities of renal 25-hydroxyvitamin D(3)-1 alpha-hydroxylase (CYP27B1) and -24-hydroxylase (CYP24) compared with control mice. Leptin administration to ob/ob mice (4 mg/kg body weight, ip, every 12 h for 2 d) greatly reduced mRNAs and activities of 1 alpha-hydroxylase and 24-hydroxylase. Elevated concentrations of serum calcium, phosphate, and 1,25-(OH)(2)D(3) were normalized by leptin treatment. Thus, leptin suppresses renal gene overexpression for 1 alpha-hydroxylase and 24-hydroxylase and corrects increased serum concentrations of calcium and phosphate in ob/ob mice. Therefore, low BMD in leptin-deficient mice appears to be related to stimulation of bone resorption by 1,25-(OH)(2)D(3).