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Endocrinology Vol. 145, No. 8 3804-3812
Copyright © 2004 by The Endocrine Society

Evidence for Abnormal Translational Regulation of Renal 25-Hydroxyvitamin D-1{alpha}-Hydroxylase Activity in the Hyp-Mouse

Baozhi Yuan, Yina Xing, Ron L. Horst and Marc K. Drezner

Department of Medicine (B.Y., Y.X., M.K.D.), University of Wisconsin and Geriatrics Research Education and Clinical Center, William F. Middleton Veterans Administration Hospital, Madison, Wisconsin 53792; and National Animal Disease Center (R.L.H.), U.S. Department of Agriculture, Agricultural Research Service, Ames, Iowa 50010

Address all correspondence and requests for reprints to: Marc K. Drezner, M.D., H4/554 Clinical Science Center, University of Wisconsin Medical School, 600 Highland Avenue, Madison, Wisconsin 53792-5148. E-mail: mkd{at}medicine.wisc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hyp-mice exhibit abnormal regulation of 25-hydroxyvitamin D [25(OH)D]-1{alpha}-hydroxylase activity. Previous observations suggest such aberrant modulation is posttranscriptional. To investigate this possibility further, we examined whether hyp-mice manifest abnormal translation of 25(OH)D-1{alpha}-hydroxylase mRNA. We compared phosphate, parathyroid, and calcitonin effects on renal 25(OH)D-1{alpha}-hydroxylase protein as well as mRNA and enzyme activity in normal and hyp-mice. We assayed protein by Western blots, mRNA by real-time RT-PCR, and enzyme activity by measuring 1,25-dihydroxyvitamin D production. Although phosphate-depleted mice exhibited enhanced enzyme function, with significantly increased mRNA and protein expression, hyp-mice comparably increased mRNA but failed to augment enzyme activity, concordant with an inability to increase protein expression. PTH stimulation increased mRNA and protein expression as well as enzyme activity in normal mice but in hyp-mice, despite effecting mRNA enhancement, did not increment enzyme function or protein. The inability of hypophosphatemia and PTH to increase 25(OH)D-1{alpha}-hydroxylase activity and protein expression in hyp-mice was not universal because calcitonin stimulation was normal, suggesting proximal convoluted tubule localization of the defect. These data, in accord with absent undue enhancement of protein expression in hyp-mice treated with protease inhibitors, establish that abberrant regulation of vitamin D metabolism results from abnormal translational activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
ABNORMAL VITAMIN D METABOLISM is a characteristic abnormality in patients with X-linked hypophosphatemia (XLH), a prototypic vitamin D-resistant disease. Studies of the hyp-mouse, a murine homologue of XLH, indicate this abnormality is due primarily to aberrant regulation of renal 1,25 dihydroxyvitamin D [1,25(OH)2D] production (1, 2, 3, 4, 5, 6, 7, 8). These investigations suggest that 25- hydroxyvitamin D [25(OH)D]-1{alpha}-hydroxylase in mutants is abnormally regulated by factors that influence enzyme function in the renal proximal convoluted tubule, such as PTH (6), PTH-related peptide (8), cAMP (7), and phosphate (P) (1). In contrast, calcitonin (CT)-stimulated 25(OH)D-1{alpha}-hydroxylase activity in the proximal straight tubule is normally responsive (9). At present, the mechanism underlying such localized enzyme unresponsiveness in XLH remains unclear. Although recent studies indicate that modulation of renal 25(OH)D-1{alpha}-hydroxylase activity occurs secondary to transcriptional regulation of the 1{alpha}-hydroxylase gene, CYP27B1 (10, 11, 12), we have previously reported that the abnormal regulation of 25(OH)D-1{alpha}-hydroxylase in the proximal convoluted tubule of hyp-mice manifests despite normal PTH- and P-mediated control of gene expression (13). These observations affirm the presence of a dissociation between mRNA expression and renal 25(OH)D-1{alpha}-hydroxylase activity in hyp-mice and suggest that abnormal modulation of enzyme function occurs secondary to a posttranscriptional defect.

Thus, in this study, we investigated whether the aberrant P- and PTH-mediated regulation of vitamin D metabolism in hyp-mice results from abnormal translation of 25(OH)D-1{alpha}-hydroxylase mRNA. In the experiments performed, we assessed renal enzyme activity, mRNA expression, and 25(OH)D-1{alpha}-hydroxylase protein content in response to hypophosphatemia and PTH (as well as CT) in hyp-mice and age-matched normals, using a sensitive in vitro assay of murine enzyme function, real-time RT-PCR for assessment of 25(OH)D-1{alpha}-hydroxylase cytochrome P450 mRNA, and Western blotting to measure 25(OH)D-1{alpha}-hydroxylase protein content in renal tissue.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Normal C57BL/6J mice were mated with C57BL/6J heterozygous hyp-mice (Jackson Animal Laboratories, Bar Harbor, ME) as previously described (1). Male and female weanling hyp-mice obtained from the resultant litters were identified and selected for study at 3 wk of age by genotyping and/or serum phosphorus levels. Equal numbers of male and female normal littermates were chosen for investigation. All mice received a diet contain 0.6% of calcium and phosphorus (Teklad Co., Madison, WI) and deionized water ad libitum from the time of weaning until study 4–6 wk later. Care of mice met or exceeded the standards set forth by the National Institutes of Health in the Guidelines for the Care and Use of Laboratory Animals (NIH publication 86–23, revised 1985). The University of Wisconsin Animal Care and Use Committee approved all procedures. Experimental mice were euthanized by ip injection of sodium pentobarbital.

P effects
In initial experiments, we compared the serum phosphorus levels and renal 25(OH)D-1{alpha}-hydroxylase activity as well as enzyme mRNA and protein content in hyp-mice, hypophosphatemic under basal conditions, with those of age- and sex-matched normal and P-depleted normal mice. Whereas normal and hyp-mice received the diet described above, the P-depleted mice were fed an egg white solid-based protein source ration with 0.02% inorganic phosphorus that was identical in all other respects for 1 wk before study. At the time of study, mice (7–9 wk of age) were killed, blood obtained for assay of serum phosphorus, and kidneys excised for measurement of enzyme activity and 25(OH)D-1{alpha}-hydroxylase mRNA and protein.

PTH effects
In subsequent investigations, we examined the effects of PTH on the enzyme activity and 25(OH)D-1{alpha}-hydroxylase mRNA and protein content in age- and sex-matched normal and hyp-mice. To control the uniformity of the applied stimulus, we used surgically implantable Alzet osmotic minipumps (model 2001; Alza Corp., Palo Alto, CA) for continuous sc infusion of bovine PTH 1–34. The PTH was dissolved in physiologic saline (0.9% NaCl) containing 20 mg/ml cysteine hydrochloride. Minipumps were filled with the PTH solution (0.25 µg/µl) and immersed for 24 h before implantation to allow equilibration of pump flow rate. After sodium pentobarbital anesthesia (65 mg/kg ip), a minipump was implanted sc in each mouse through a small skin incision in the dorsal lumbar region. The incision was closed with a wound clip. Normal and hyp-mice, serving as controls for these experiments, were anesthetized and implanted with a minipump containing 0.9% NaCl with 20 mg/ml cysteine hydrochloride. After 12 h of infusion (1 µl/h) the mice were killed and their kidneys excised for measurement of enzyme activity and 25(OH)D-1{alpha}-hydroxylase mRNA and protein.

CT effects
In similar studies we investigated the influence of calcitonin on enzyme activity and 25(OH)D-1{alpha}-hydroxylase mRNA and protein content in age- and sex-matched normal and hyp-mice. Salmon calcitonin in 0.9% NaCl was administered to the animals via osmotic minipumps (as described above) at an infusion rate of 2 IU/h. After 12 h treated mice and appropriate controls were killed and kidneys removed for measurement of enzyme activity and 25(OH)D-1{alpha}-hydroxylase mRNA and protein.

Effects of protease inhibitors on 25(OH)D-1{alpha}-hydroxylase protein content
To determine whether abnormal metabolic and hormone-induced changes in the protein concentration of 25(OH)D-1{alpha}-hydroxylase in hyp-mice are caused by aberrant translation or by altered protein degradation, we examined whether enzyme protein content in hyp-mouse kidney is uniquely altered, compared with that in normal kidney, by inhibiting ubiquitin-mediated proteolysis with a proteasome inhibitor (MG-132) or calcium-activated protease-mediated degradation with a calpain inhibitor (calpain inhibitor-1). MG-132 (10 mg/kg body weight, ip) was administered to normal and hyp-mice every 2 h for 10 h as previously reported and the enzyme protein content, compared with that in untreated mice (14). Similarly, we compared protein content in untreated and calpain inhibitor-1 (10 mg/kg body weight, ip)-treated normal and hyp-mice. The calpain inhibitor was administered to normal and hyp-mice 6 h before killing, as previously reported (15). We quantitated 25(OH)D-1{alpha}-hydroxylase protein in the kidneys from untreated and treated normal and hyp-mice by analysis of Western blots. In these studies, we used ß-actin as the reference protein to control for variations in recovery and application load because this protein has an extraordinary long half-life, which would preclude an effect on concentration by inhibition of protease activity.

Analytical methodology
In vitro assay of murine renal 25(OH)D-1{alpha}-hydroxylase activity.
We assayed maximum velocity of renal 25(OH)D-1{alpha}-hydroxylase activity in kidney homogenates by a modification of our previously described methods (1, 16). In brief, one kidney was excised from anesthetized mice, decapsulated, weighed, and placed in ice-cold buffer [16 mM Tris, 0.19 M, sucrose, 2 mM, magnesium acetate (pH 7.4)] and a 5% (wt/vol) homogenate made with a handheld glass homogenizer. Two milliliters of homogenate were transferred to an Erlenmeyer flask containing 1 ml buffer supplemented with 75 mM succinic acid. Upon addition of 25(OH)D (100 µg), the kidney homogenates were incubated for 15 min at 37 C. The reaction was stopped by the addition of 20 ml methanol/methylene chloride (2:1, vol/vol) and 2 ml buffer. After addition of [3H]1,25(OH)2D3, 10,000 cpm, in 20 µl ethanol, to measure recovery during subsequent purification steps, the reaction mixtures were transferred to 125-ml separatory funnels and 6 ml buffer and 6.5 ml chloroform added. The lipid phase was transferred to glass vials and dried under a stream of nitrogen.

The resultant samples were dissolved in 1 ml acetonitrile and 1 ml water and centrifuged. The supernatant was decanted into a tube containing 0.5 ml of 0.4 M K2HPO4 (pH 10.6) and vortexed. Purification of 1,25(OH)2D3 from these samples was accomplished by a modification of the methods reported by Reinhardt et al. (17). Sep-Pak C-18 cartridges (Waters Corp., Milford, MA) were washed and the samples applied to the columns. The 1,25(OH)2D3 was eluted by sequential washing of the columns with water, methanol/water (70:30), and acetonitrile. The eluents were dried under nitrogen, resolubilized in 0.5 ml hexane/isopropranol (96:4), and applied to prewashed Sep-Pak silica cartridges (Waters Corp.). The 1,25(OH)2D3-containing samples were obtained by serial elution with hexane/isopropranol (96:4), hexane/isopropranol (94:6), and hexane/isopropranol (85:15). After drying the samples under nitrogen the 1,25(OH)2D3 was measured by a RIA (18). Tracer, RIA buffer [50 mM NaH2PO4, 0.1% gelatin, 0.1% sodium azide (pH 6.2)], and antibody were added to tubes containing samples and standards and incubated for 2 h at 20–25 C. Subsequently, tubes were centrifuged, decanted, an aliquot removed to determine recovery of [3H]1,25(OH)2D3, and the remaining samples counted in a {gamma}-counter. Data are expressed as femtomoles per milligram (wet weight) kidney per minute. As previously reported, we determined that the enhanced basal and hormone-stimulated 25(OH)D-24-hydroxylase activity in hyp-mice did not inordinately decrease the measurable 1,25(OH)2D produced during in vitro incubations, thereby validating that the measure of 25(OH)D-1{alpha}-hydroxylase in mutants is comparable with that in normal and P-depleted mice (13).

Real-time PCR assay of mRNA.
We isolated total RNA from decapsulated whole mouse kidneys using the TRIzol protocol (Life Technologies, Inc., Grand Island, NY), as described previously (19), and quantitated 25(OH)D-1{alpha}-hydroxylase cytochrome P450 mRNA concentrations by real-time RT-PCR. RNA concentration in the samples was quantitated using UV spectroscopy at a wavelength of 260 nm. The total RNA was reverse transcribed into cDNA employing random primers from the first-strand cDNA kit (Roche, Indianapolis, IN) before performing real-time PCR. Real-time quantitative PCR was performed on an ABI 7000 using specific fluorescent labeled TaqMan MGB primers (forward: agc agc tcc tgc gac aag aa; reverse: cgt tag caa tcc gca agc a) and a probe (cgc tgt agt ttc tca tca) directed against 25(OH)D-1{alpha}-hydroxylase. An internal control 18s rRNA (Applied Biosystems, Foster City, CA) was amplified in separate tubes. Data were collected quantitatively and the CT number corrected by CT readings of corresponding internal 18s rRNA controls. Data from a minimum of six determinations (mean ± SEM) are expressed in all experiments as fold changes, compared with normal mice.

Western blot assay of 25(OH)D-1{alpha}-hydroxylase protein content.
We compared 25(OH)D-1{alpha}-hydroxylase protein in the kidneys from normal and mutant mice, variably subjected to hormonal/metabolic stimulation, by Western blotting, using a successfully developed modification of methods described by Hewison and colleagues (20, 21). In brief, we prepared protein extracts from murine whole kidneys, which were rinsed free of blood with ice-cold saline. Tissues were homogenized in sucrose buffer containing 20 mM HEPES, 1 mM EDTA, 25 mM sucrose, 0.4 mM phenylmethylsulfonyl fluoride, and 2 µg/ml leupeptin. The homogenate was centrifuged at 1500 x g for 15 min and the pellet containing mitochondrial protein resuspended in the same buffer, aliquoted, and stored at –80 C until use. Protein extract (20 µg) was electrophoresed on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. After washing with Tris buffer (50 mM)-saline (200 mM) solution containing 1% Tween 20, the membrane was serially probed with blocking solution (Tris buffer-saline solution with 1% Tween 20 and 5% dry milk), Tris buffer-saline solution with 1% Tween 20 containing 1:400 specific antirat 25(OH)D-1{alpha}-hydroxylase antibody (20, 21) for 2 h and 1:4000 goat antirabbit IgG antibody conjugated with horseradish peroxidase for 1 h. The immunoblotting signals were detected by subjecting the membrane to treatment with chemifluorescent (ECL plus) detection solution (Amersham, Arlington Heights, IL), scanning the membrane directly with a Storm 860 scanner (Amersham), and analyzing band density using ImageQuant software (version 5.2, Molecular Dynamics, Sunnyvale, CA). The 25(OH)D-1{alpha}-hydroxylase antibody recognizes a 56-kDa protein in murine and rat kidneys. After removal of the antibody by incubation in reprobing solution for 30 min at 50 C, we similarly monitored cytochrome C or ß-actin, using a specific antimouse antibody, as a control for recovery of proteins. In studies shown in Figs. 1Go and 4Go the protein concentrations corrected for cytochrome C or ß-actin content (mean ± SEM) are expressed as fold changes, compared with that in normal mice. Because the protein content in hyp-mice did not significantly differ from that in normal mice under basal conditions, because of limitations in gel size, we expressed the protein concentrations corrected for cytochrome C in Figs. 2Go and 3Go as fold changes, compared with that in respective controls, normal, and hyp-mice.



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  		FIG. 1. Effect of hypophosphatemia on renal 25(OH)D-1{alpha}-hydroxylase activity, mRNA transcripts, and protein content. A, The 1,25(OH)2D3 production, an index of 25(OH)D-1{alpha}-hydroxylase activity, was measured in renal homogenates of age-matched normal, hyp-, and P-depleted mice. In this assay, we used chloroform/methanol (1:2) extraction of kidney homogenates, isolation of the 1,25(OH)2D3 produced in vitro by serial C-18 Sep-Pak and Silica Sep-Pak chromatography, and a RIA for quantifying the 1,25(OH)2D3. Data are expressed as femtomoles 1,25(OH)2D3 produced per milligram kidney per minute. Despite a similar serum phosphorus concentration in hyp- and P-depleted mice, enzyme activity increased significantly (P < 0.05) only in P-depleted mice. B, Using real-time RT-PCR, we quantitated 25(OH)D-1{alpha}-hydroxylase cytochrome P450 mRNA concentrations in RNA isolated from decapsulated whole mouse kidneys of age-matched normal, hyp-, and P-depleted mice. Data for 25(OH)D-1{alpha}-hydroxylase mRNA are presented as fold changes from normal levels by analyzing the CT numbers corrected by CT readings of corresponding internal 18s rRNA controls. The mRNA transcripts in the hyp- and P-depleted mice were significantly increased (P < 0.01) and indistinguishable from one another. C, We measured renal 25(OH)D-1{alpha}-hydroxylase protein content by Western blotting, quantitating chemifluorescent immunoblotting signals with a Storm 860 scanner, and analyzing data using ImageQuant 5.2 software. Signal intensity for each sample was corrected by corresponding cytochrome C intensity. Data for 25(OH)D-1{alpha}-hydroxylase protein are presented as fold change relative to levels in normal kidneys. Although serum phosphorus levels were similar in normal and hyp-mice, renal 25(OH)D-1{alpha}-hydroxylase protein content increased significantly (P < 0.05) only in P-depleted mice.

 


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  		FIG. 4. Effect of protease inhibitors on the renal 25(OH)D-1{alpha}- hydroxylase protein content. A, The effects of MG-132, a proteasomal inhibitor, on renal 25(OH)D-1{alpha}-hydroxylase protein content were assessed using Western blots to quantitate the protein in age-matched and treated normal and hyp-mice. MG-132 treatment, 10 mg/kg ip, significantly (P < 0.01) increased the protein content in both animal models. However, the increment in normal animals was significantly greater (P < 0.05) than that observed in hyp-mice. B, The effects of calpain-1 inhibitor, a calpain protease inhibitor, on renal 25(OH)D-1{alpha}-hydroxylase protein content were assessed using Western blots to quantitate the protein in age-matched and treated normal and hyp-mice. Calpain-1 inhibitor (10 mg/kg, ip) administration resulted in no significant change in the renal 25(OH)D-1{alpha}-hydroxylase protein content in either the normal or hyp-mice.

 


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  		FIG. 2. Effects of PTH on renal 25(OH)D-1{alpha}-hydroxylase activity, mRNA transcripts, and protein content. A, The 1,25(OH)2D3 production, an index of 25(OH)D-1{alpha}-hydroxylase activity, was measured in renal homogenates of age-matched normal and hyp-mice under basal conditions and after PTH stimulation. The assay was performed as detailed in Fig. 1Go. As expected PTH significantly (P < 0.05) increased the renal 25(OH)D-1{alpha}-hydroxylase activity in normal mice but failed to enhance enzyme function in the hyp-mice. B, The 25(OH)D-1{alpha}-hydroxylase mRNA expression in decapsulated kidneys of age-matched normal and hyp-mice, under basal conditions and after PTH stimulation, was measured by real-time RT-PCR. The details of the methodology are presented in the Fig. 1Go legend. The mRNA transcripts in the normal and hyp-mice were similar in the basal state and increased significantly (P < 0.001) to indistinguishable levels after PTH stimulation. C, We measured renal 25(OH)D-1{alpha}-hydroxylase protein content by Western blotting as detailed in the Fig. 1Go legend. PTH stimulation significantly (P < 0.05) increased renal protein content in normal mice but failed to effect enhancement of protein content in the kidneys of hyp-mice.

 


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  		FIG. 3. Effect of CT on renal 25(OH)D-1{alpha}-hydroxylase activity, mRNA transcripts, and protein content. A, The 1,25(OH)2D3 production, an index of 25(OH)D-1{alpha}-hydroxylase activity, was measured in renal homogenates of age-matched normal and hyp-mice under basal conditions and after CT stimulation. The assay was performed as detailed in Fig. 1Go. As expected, CT significantly (P < 0.05) increased the renal 25(OH)D-1{alpha}-hydroxylase activity in normal mice and likewise enhanced enzyme function in the hyp-mice. B, The 25(OH)D-1{alpha}-hydroxylase mRNA expression in decapsulated kidneys of age-matched normal and hyp-mice, under basal conditions and after CT stimulation, was measured by real-time RT-PCR. The details of the methodology are presented in the Fig. 1Go legend. The mRNA expression levels in the normal and hyp-mice were similar in the basal state and increased significantly (P < 0.05) to indistinguishable levels after CT stimulation. C, We measured renal 25(OH)D-1{alpha}-hydroxylase protein content by Western blotting as detailed in the Fig. 1Go legend. CT stimulation significantly (P < 0.05) increased renal protein content in normal mice and hyp-mice to indistinguishable levels.

 
Biochemical measurements
We measured serum phosphorus levels with a Phosphorus Liqui-UV kit (Stanbio Laboratory, Boerne, TX) in samples obtained by precipitation and centrifugation of blood.

Data analysis
Data are expressed as the mean ± SE of at least seven individual determinations. We evaluated the data statistically employing an ANOVA with a Bonferroni correction, when appropriate (22).

Materials
We purchased 25(OH)D from ICN Biomedicals (Costa Mesa, CA), [3H]-1,25(OH)2D (92 Ci/mmol) from Amersham Corp. PTH and CT were obtained from Sigma-Aldrich (St. Louis, MO), MG-132 and calpain inhibitor from Calbiochem (San Diego, CA), the antirat 25(OH)D-1{alpha}-hydroxylase antibody from The Binding Site Ltd. (Birmingham, UK), and the cytochrome C and ß-actin antibodies from Research Diagnostics (Flanders, NJ).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
P effects
At the time of study, hyp- and P-depleted normal mice had comparable serum phosphorus concentrations (3.3 ± 0.2 and 4.0 ± 0.4 mg/dl, respectively), values significantly less (P < 0.001) than that of normals (7.8 ± 0.2 mg/dl). Moreover, similar to our previous report, consistent with decreased serum phosphorus levels, P-depleted normal mice exhibited a significant 3-fold increment of renal 25(OH)D-1{alpha}-hydroxylase activity, compared with that of normal mice, whereas hyp-mice manifesting hypophosphatemia of similar magnitude displayed no significant enhancement of enzyme function (Fig. 1Go). The increased enzyme activity in P-depleted normal mice occurred in concert with a 2.0-fold increment in 25(OH)D-1{alpha}-hydroxylase mRNA transcripts, whereas a similar increment in mRNA transcripts (2.1-fold) occurred in hyp-mice, despite the failure to alter enzyme activity (Fig. 1Go). However, the contrasting effects of hypophosphatemia on enzyme activity in P-depleted normal and hyp-mice occurred concordant with similar divergent effects on 25(OH)D-1{alpha}-hydroxylase protein content, marked by a 2.7-fold enhancement in P-depleted normal mice and no stimulation in hyp-mice (Fig. 1Go).

PTH effects
As previously reported, PTH stimulation enhanced renal 25(OH)D-1{alpha}-hydroxylase activity approximately 3.5-fold in normal mice but failed to elicit a significant increase in hyp-mice (Fig. 2Go). The disparity occurred despite a comparable and substantial PTH-mediated increment in mRNA transcripts in both normal and hyp-mice (6.8 ± 1.3 vs. 8.4 ± 1.4-fold) (Fig. 2Go). In contrast, the disparate PTH-mediated effects on enzyme activity occurred in accord with an incongruent PTH-mediated stimulation of 25(OH)D-1{alpha}- hydroxylase protein content, which occurred only in normal mice (1.6 ± 0.1 vs. 1.08 ± 0.2; P < 0.01) (Fig. 2Go).

CT effects
Unlike PTH, administration of calcitonin, as previously reported, increased 25(OH)D-1{alpha}-hydroxylase activity comparably in normal and hyp-mice (Fig. 3Go). Moreover, in response to this stimulation, the mRNA transcripts in both normal and hyp-mice increased to similar levels (Fig. 3Go). In addition, the 25(OH)D-1{alpha}-hydroxylase protein content likewise increased concordantly in both animal models (Fig. 3Go).

Effects of proteinase inhibitors on P-mediated regulation of 25(OH)D-1{alpha}-hydroxylase protein content
To determine whether the discordant hormonal/metabolic regulation of 25(OH)D-1{alpha}-hydroxylase protein content in hyp-mice occurs at the translational level or is due to increased protein turnover, we studied the effects of proteasomal and calpain protease inhibitors on renal protein levels in normal and mutant mice. As shown in Fig. 4Go, MG-132 inhibition of proteasomal activity significantly increased renal protein content in normal and hyp-mice. Most importantly, however, the increment in 25(OH)D-1{alpha}-hydroxylase protein in the kidneys of hyp-mice was not greater than that in normal mice, indicating that proteasomal activity under basal conditions was not enhanced. Indeed, MG-132 treatment resulted in a significantly lesser increment of protein in the hyp-mouse kidney (Fig. 4Go). In contrast, calpain inhibitor-1 inhibition of calpain protease activity did not significantly increase protein content in the kidneys of normal or hyp-mice, consistent with a limited effect of calpain proteases on the steady-state levels of 25(OH)D-1{alpha}-hydroxylase protein (Fig. 4Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Characteristic phenotypic abnormalities in the hyp-mouse, a murine homolog of XLH, include a 50% reduction in Na+-dependent P transport across the renal brush border membrane (23), resultant hypophosphatemia (24), and rickets and osteomalacia of varying severity (25). Previous studies have also documented that abnormal regulation of renal 1,25(OH)2D production occurs in this animal model (1, 2, 3, 4, 5, 6, 7, 8). These investigations favor the possibility that 25(OH)D-1{alpha}-hydroxylase activity in mutants is abnormally regulated by factors that influence enzyme function in the renal proximal convoluted tubule, such as P and PTH, whereas CT-stimulated activity presumably in the proximal straight tubule is normally responsive (9). Although recent studies established that PTH, CT, 1,25(OH)2D, and P alter renal 25(OH)D-1{alpha}-hydroxylase activity in normal animals secondary to transcriptional regulation of the 1{alpha}-hydroxylase gene, CYP27B1 (10, 11, 12, 26, 27, 28), in previous studies we discovered that abnormal PTH- and P-mediated modulation of renal 25(OH)D-1{alpha}-hydroxylase in hyp-mice manifests despite normal control of CYP27B1 expression (13). Our concurrent observations that CT-mediated enzyme function is normal in hyp-mice and occurs in concert with normally up-regulated gene transcription, limited the dissociation between mRNA expression and renal 25(OH)D-1{alpha}-hydroxylase activity in the mutant mice to the proximal convoluted tubule (13). Although these findings suggested that the defect in 25(OH)D-1{alpha}-hydroxylase activity in hyp-mice likely resulted from a posttranscriptional abnormality such as aberrant mRNA translation, enhanced protein degradation, or deranged posttranslational modification, they failed to define the precise cause.

In contrast, the results of our present investigations provide firm evidence that the defect in renal 25(OH)D-1{alpha}- hydroxylase activity in hyp-mice results from abnormal mRNA translation. In this regard, we confirmed that despite normal P- and PTH-mediated control of CYP27B1 expression and consequent normal up-regulation of 25(OH)D-1{alpha}- hydroxylase mRNA transcripts in hyp-mice, the mutant animals, in contrast to their normal counterparts, failed to substantially alter renal 25(OH)D-1{alpha}-hydroxylase activity in response to these stimuli (Figs. 1Go and 2Go). However, in accord with the dissociation between mRNA expression and renal 25(OH)D-1{alpha}-hydroxylase activity, hypophosphatemia in hyp-mice failed to substantially increase renal 25(OH)D-1{alpha}-hydroxylase protein content, whereas P-depleted normal mice, with similar serum phosphorus levels, exhibited a greater than 2-fold enhancement of protein (Fig. 1Go). Similarly, PTH stimulation of normal mice significantly increased 25(OH)D-1{alpha}-hydroxylase protein content but failed to alter protein in hyp-mice (Fig. 2Go). Coupled with the observation that MG-132 and calpain-inhibitor-1 inhibition of proteasomal and calpain proteases do not result in a significantly greater renal protein content in mutant kidneys (Fig. 4Go), thus excluding the possibility of increased protease activity, the data clearly indicate that abnormal P- and PTH-mediated regulation of 25(OH)D-1{alpha}-hydroxylase protein results from aberrant translation, which underlies the defective regulation of enzyme activity in hyp-mice.

Although these data confirm that hyp-mice manifest abnormal translational regulation of 25(OH)D-1{alpha}-hydroxylase, they do not establish whether the defect is universal or confined to the proximal convoluted tubule. However, because we observed that CT-mediated enzyme function, which occurs in the proximal straight tubule, is normal in hyp-mice and occurs in concert not only with normally up-regulated gene transcription but also 25(OH)D-1{alpha}-hydroxylase protein as well (Fig. 3Go), the observed abnormality is undoubtedly limited to the proximal convoluted tubule, the site of abnormal phosphate transport in the mutants.

Although our observations suggest that P, PTH, and CT normally mediate 25(OH)D-1{alpha}-hydroxylase mRNA in the hyp-mice, these observations are seemingly inconsistent with the data of Azam et al. (29), who reported that disordered regulation of vitamin D metabolism in hyp-mice, considerably older (approximately 28.5 wk) than those used in our experiments, is due to abnormal metabolic/hormone-mediated gene transcription. However, comparison of their data to those we are reporting is confounded by several salient considerations that seem independent of the age difference. First, although similar to our observations, they found that hyp-mice in the basal state had an increased 25(OH)D-1{alpha}-hydroxylase mRNA abundance, they assumed that this was inconsistent with a posttranscriptional abnormality because renal enzyme activity in the mutants was approximately 4-fold elevated above that in normal mice. However, their discovery of increased renal 25(OH)D-1{alpha}-hydroxylase activity in the hyp-mice is inconsistent with the normal enzyme function reported in hyp-mice of wide-ranging age by numerous groups (1, 3, 4, 5, 6, 7, 8, 9, 13, 30, 31, 32), including those in which authors of the manuscript had previously participated (5), and with normal serum 1,25(OH)2D levels in mice of variable age and patients with XLH (2, 33, 34, 35, 36, 37, 38).

Second, in their studies hyp-mice were subjected to phosphate depletion and loading in contrast to the normal diet used in our investigations. As a consequence, although decreased 25(OH)D-1{alpha}-hydroxylase activity and mRNA abundance were found in hyp-mice after P depletion, consistent with abnormal transcriptional regulation underlying the disordered regulation of vitamin D metabolism, the experiments documenting these alterations were poorly controlled. Thus, P depletion of hyp-mice, equivalent to acute P depletion superimposed on a chronically P-depleted animal model, not surprisingly resulted in serum P levels far below those in wild-type mice (1.89 ± 0.03 vs. 9.83 ± 0.06 mg/dl) and less than those consistently achieved on P depletion of wild-type mice (3.59 ± 0.03 mg/dl). Moreover, the profound P challenge occurred in an organism unable to compensate the challenge with enhanced renal P transport (39). Hence, the changes observed may represent the effects of profound cellular ATP deficiency and not those expected from a modest hypophosphatemic challenge (40, 41, 42). Finally, the increased enzyme activity and mRNA abundance found in P-loaded hyp-mice, compared with that in wild-type mice, occurred in experiments wherein the P loading failed to down-regulate 25(OH)D-1{alpha}-hydroxylase activity in wild-type mice, despite the well-known suppressive effects of this pertubation (43). With these considerations in mind, it seems reasonable to conclude that the identification of a transcriptional abnormality as central to the defect in vitamin D metabolism in hyp-mice remains unconfirmed.

In fact, although our current study was designed to assess the presence of a translational abnormality, we performed measurements of 25(OH)D-1{alpha}-hydroxylase activity and mRNA as before (13). Thus, in our current studies, employing the sensitive, reproducible, and accurate real-time RT-PCR method to measure CYP27B1 mRNA transcripts (44), we observed that the effects of P, PTH, and CT in normal and hyp-mice were magnitudinally quite similar to those previously found with an Rnase protection methodology (13). Thus, we have reproducibly documented that a posttranscriptional defect underlies the abnormal vitamin D metabolism in hyp-mice previously reported.

The operative event(s) in the hyp-mouse, which dictates abnormal translation and the mechanism disrupting mRNA translation, remain unknown. Nevertheless, speculation abounds that the defect in vitamin D metabolism in hyp-mice is caused by abnormal Phex function and consequent excess phosphatonin (45), the presence of which may directly influence vitamin D metabolism in the proximal convoluted tubule cells. In accord with this possibility (Karaplis, A., personal communication) found that transgenic mice overexpressing FGF-23, a documented phosphatonin, manifest up-regulation of 25(OH)D-1{alpha}-hydroxylase mRNA transcripts but decreased enzyme activity due to a translational abnormality. Alternatively, several groups have suggested that the abnormal phosphate transport in the proximal convoluted tubular cells, and the resultant alterations in the intracellular milieu may directly and independently decrease expression of 25(OH)D-1{alpha}-hydroxylase activity, despite the hypophosphatemic stimulus and possibly due to the inability of the renal tubular cells to reciprocally increase P transport. In accord with this possibility, we previously reported that phosphonoformic acid-induced renal P wasting and consequent P depletion in mice, occurring in the absence of compensatory changes in renal P transport, does not increase 1,25(OH)2D production (46).

Regardless of the controlling operative event, the recent recognition that translational regulation is paramount in regulation of singular or group protein synthesis in a variety of diverse physiological circumstances indicates that anyone of a host of mechanisms currently poorly understood may underlie the observed defect in the hyp-mouse (14, 47, 48, 49, 50, 51, 52, 53, 54, 55). Most likely, however, the existence of an uncompensated hypophosphatemic milieu in the proximal convoluted tubule cells of hyp-mice and resultant aberrant protein phosphorylation may serve as the primary mechanism underlying the abnormal translation. In this regard, the physical bridging of ribosomes to mRNA in mammalian cells is coordinated primarily by two related modular scaffolding phosphoproteins, eIF4GI and eIF4GII (14, 47). Limited protein phosphorylation, therefore, may have profound consequences on 25(OH)D- 1{alpha}-hydroxylase mRNA translation. Although such regulation has been recognized as affecting the entire complement of mRNAs within a cell, recent reports indicate that selective targeting of a single species of mRNA occurs secondary to differential sensitivity of an mRNA to a component of the translation system, most notably eIF4E (48, 49). Although this mechanism has not been established in hyp-mice, such an occurrence may explain the paradoxical relationship between the serum phosphorus concentration and enzyme function in many renal P wasting disorders of man including not only XLH but also Fanconi syndrome, autosomal dominant hypophosphatemia, X-linked recessive hypophosphatemia, and Dent disease, each of which has abnormal renal P transport but only some of which have increased phosphatonin levels.

Nevertheless, recent studies of the Npt2 knockout mouse raise significant questions regarding the role that renal P transport may play in the regulation of vitamin D metabolism. Tenenhouse et al. (56) reported normal P regulation of renal 25(OH)D-1{alpha}-hydroxylase activity in this animal model with absent Npt2-mediated renal P transport. However, these data may have limited applicability to understanding the abnormal vitamin D metabolism in XLH because it is well known that compensatory developmental changes often confound studies in knockout mice with complete absence of a protein function. Thus, further studies are clearly necessary to establish whether the aberrant translational regulation of 25(OH)D-1{alpha}-hydroxylase is secondary to a P-mediated or an as-yet-unknown mechanism.

In any case, we believe that our studies establish that the apparent abnormal regulation of renal 25(OH)D-1{alpha}-hydroxylase activity in hyp-mice results from translational dysregulation in the proximal convoluted tubule cells. Whereas the cause of this abnormality remains unknown, the contemporary pathophysiological model of XLH must now include the concept that phosphatonin not only decreases the Npt2 concentration and renal P reabsorption in XLH, but also either directly or indirectly impairs translation of 25(OH)D-1{alpha}- hydroxylase mRNA transcripts. Such information may help in the progress to identify the phosphatonin(s) operative in XLH.


   Footnotes
 
Portions of this work have appeared in abstract form in J Bone Miner Res (2003;17S:S282–S283).

Abbreviations: CT, Calcitonin; 1,25(OH)2D, 1,25 dihydroxyvitamin D; 25(OH)D, 25-hydroxyvitamin D; P, phosphate; XLH, X-linked hypophosphatemia.

Received February 12, 2004.

Accepted for publication April 27, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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