Hyp Mice Research Articles

MEPE, the Gene Encoding a Tumor-Secreted Protein in Oncogenic Hypophosphatemic Osteomalacia, Is Expressed in Bone

The MEPE (matrix extracellular phosphoglycoprotein) gene is a strong candidate for the tumor-derived phosphaturic factor in oncogenic hypophosphatemic osteomalacia (OHO). X-linked hypophosphatemia (XLH) is phenotypically similar to OHO and results from mutations in PHEX, a putative metallopeptidase believed to process a factor(s) regulating bone mineralization and renal phosphate reabsorption. Here we report the isolation of the murine homologue of MEPE, from a bone cDNA library, that encodes a protein of 433 amino acids, 92 amino acids shorter than human MEPE. Mepe, like Phex, is expressed by fully differentiated osteoblasts and down-regulated by 1,25-(OH)2D3. In contrast to Phex, Mepe expression is markedly increased during osteoblast-mediated matrix mineralization. Greater than normal Mepe mRNA levels were observed in bone and osteoblasts derived from Hyp mice, the murine homologue of human XLH. Our data provide the first evidence that MEPE/Mepe is expressedby osteoblasts in association with mineralization.

Characterization of PHEX endopeptidase catalytic activity: identification of parathyroid-hormone-related peptide107-139 as a substrate and osteocalcin, PPi and phosphate as inhibitors.

Mutations in the PHEX gene (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) are responsible for X-linked hypophosphataemia, and studies in the Hyp mouse model of the human disease implicate the gene product in the regulation of renal phosphate (P(i)) reabsorption and bone mineralization. Although the mechanism for PHEX action is unknown, structural homologies with members of the M13 family of endopeptidases suggest a function for PHEX protein in the activation or degradation of peptide factors involved in the control of renal P(i) transport and matrix mineralization. To determine whether PHEX has endopeptidase activity, we generated a recombinant soluble, secreted form of human PHEX (secPHEX) and tested the activity of the purified protein with several peptide substrates, including a variety of bone-related peptides. We found that parathyroid-hormone-related peptide(107-139) is a substrate for secPHEX and that the enzyme cleaves at three positions within the peptide, all located at the N-terminus of aspartate residues. Furthermore, we show that osteocalcin, PP(i) and P(i), all of which are abundant in bone, are inhibitors of secPHEX activity. Inhibition of secPHEX activity by osteocalcin was abolished in the presence of Ca(2+). We suggest that PHEX activity and mineralization may be controlled in vivo by PP(i)/P(i) and Ca(2+) and, in the latter case, the regulation requires the participation of osteocalcin.

Phosphate wasting in oncogenic osteomalacia: phex is normal and the tumor-derived factor has unique properties

Oncogenic osteomalacia (OOM) is characterized by renal phosphate wasting and abnormal metabolism of vitamin D, somewhat similar to the phenotype of X-linked hypophosphatemic rickets (HYP). DNA from OOM tumor cells was analyzed for mutations in the PHEX gene, which is mutated in HYP. Screening for mutations by single-strand conformation polymorphism analysis and subsequent sequencing of all the exons revealed no mutations. Conditioned media from long-term cultures of OOM tumor cells were used to further characterize the physical properties of the phosphate-regulating factor and its mechanism of action. Inhibition of OK 3B2 cell renal phosphate transport by conditioned media was dose-dependent and maximal after 20 h. This time course differed from that of parathyroid hormone (PTH). The bioactivity was stable to mild acid and alkali treatment and freeze drying and was retained in the aqueous phase following organic solvent extraction. The activity was not suppressed by heat or by treatment with trypsin but was suppressed by the protease papain and had an apparent molecular weight of <5000. No change was detected in the expression of type II sodium/phosphate cotransporter (NaPi) mRNA in OK 3B2 cells in response to conditioned media, unlike the reduction seen in Hyp mice. In the presence of colchicine or cytochalasin D, the inhibitory response to conditioned media was reduced, similar to the effect of these agents on the response to PTH. Cycloheximide also suppressed the inhibitory response of conditioned media, but not the response to PTH. These studies indicate that mutations in the PHEX gene are unlikely to be responsible for OOM and suggest that the tumor-derived factor that inhibits phosphate uptake is a small protein that does not downregulate type II NaPi mRNA, and requires an intact cytoskeleton and protein synthesis for activity.

Osteomalacia in hyp mice is associated with abnormal phex expression and with altered bone matrix protein expression and deposition.

To explore how the loss of Phex function contributes to the pathogenesis of osteomalacia, we examined the abnormalities of mineralization, Phex, and bone matrix protein expression occurring in Hyp mice in vivo and in ex vivo bone marrow cell cultures. The results in vivo show that mineralization was decreased significantly in Hyp mouse bone. Phex protein was identifiable in osteoblasts and osteocytes in wild-type mice, but not in Hyp mice. In Hyp mice, osteocalcin, bone sialoprotein, and vitronectin expression were down-regulated, whereas biglycan and fibrillin-1 expression were up-regulated in osteocytes and bone matrix relative to those in their wild-type counterparts. Parallel studies ex vivo demonstrated that cells derived from 18-day Hyp mouse bone marrow cell cultures had a 3'-Phex deletion, no Phex protein expression, decreased alkaline phosphatase activity, collagen deposition, and calcium accumulation, and reduced osteocalcin, bone sialoprotein, and vitronectin at both the protein and messenger RNA levels. Furthermore conditioned medium from Hyp mouse bone marrow cultures could induce analogous defects in bone marrow cell cultures of wild-type cells. These novel findings indicate that there is an intrinsic osteogenic cell differentiation defect in addition to the known hypomineralization of bone in Hyp mice, which may be inducible by an autocrine/paracrine secreted factor. These results suggest that alterations in the Phex gene may control bone matrix mineralization indirectly by regulating the synthesis and deposition of bone matrix proteins.

Osteomalacia in Hyp Mice Is Associated with Abnormal Phex Expression and with Altered Bone Matrix Protein Expression and Deposition

Osteomalacia in Hyp Mice Is Associated with Abnormal Phex Expression and with Altered Bone Matrix Protein Expression and Deposition

Developmental Expression and Tissue Distribution of Phex Protein: Effect of the Hyp Mutation and Relationship to Bone Markers

Mutations in PHEX, a phosphate-regulating gene with homology to endopeptidases on the X chromosome, are responsible for X-linked hypophosphatemia (XLH). The murine Hyp homologue has the phenotypic features of XLH and harbors a large deletion in the 3' region of the Phex gene. We characterized the developmental expression and tissue distribution of Phex protein, using a monoclonal antibody against human PHEX, examined the effect of the Hyp mutation on Phex expression, and compared neprilysin (NEP), osteocalcin, and parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor gene expression in bone of normal and Hyp mice. Phex encodes a 100- to 105-kDa glycoprotein, which is present in bones and teeth of normal mice but not Hyp animals. These results were confirmed by in situ hybridization (ISH) and ribonuclease protection assay. Phex protein expression in femur and calvaria decreases with age, suggesting a correlation between Phex expression and bone formation. Immunohistochemical studies detected Phex protein in osteoblasts, osteocytes, and odontoblasts, but not in osteoblast precursors. In contrast to Phex, the abundance of NEP messenger RNA (mRNA) and protein is not significantly altered in Hyp bone. Similarly, osteocalcin and PTH/PTHrP receptor gene expression are not compromised in bone of Hyp mice. Our results are consistent with the hypothesis that loss of Phex function affects the mineralizing activity of osteoblasts rather than their differentiation.

The effects of bone marrow transplantation on X-linked hypophosphatemic mice.

The genes responsible for X-linked hypophosphatemic (XLH) vitamin D-resistant rickets and the murine homolog, hypophosphatemic mice (Hyp), were identified as PHEX and Phex (phosphate-regulating gene with homology to endopeptidases on the X chromosome), respectively. However, the mechanism by which inactivating mutations of PHEX cause XLH remains unknown. We investigated the mechanisms by syngeneic bone marrow transplantation (BMT) from wild mice to Hyp mice. The expression of the Phex gene was detected in mouse BM cells. BMT introduced a chimerism in recipient Hyp mice and a significant increase in the serum phosphorus level. The renal sodium phosphate cotransporter gene expression was significantly increased. The effect of BMT on the serum phosphorus level depended on engraftment efficiencies, which represent the dosage of normal gene. Similarly, the serum alkaline phosphatase (ALP) activity was decreased and bone mineral density was increased. Furthermore, the renal expression of 25-hydroxyvitamin D3 24-hydroxylase, which is a key enzyme in the catabolic pathway and is increased in XLH/Hyp, was improved. From these results, we conclude that transplantation of normal BM cells improved abnormal bone mineral metabolism and deranged vitamin D metabolism in Hyp by replacing defective gene product(s) with normal gene product(s). This result may provide strong evidence for clinical application of BMT in metabolic bone disorders.

Blunted effect of parathyroid hormone on adenosine 3′,5′-cyclic monophosphate production is derived from ATP depletion in proximal convoluted tubules of hypophosphatemic mice

Background. To clarify the pathophysiological features associated with phosphate depletion in hypophosphatemic (Hyp) mice, we examined the effect of extracellular phosphate on parathyroid hormone (PTH)-stimulated adenosine 3′,5′-cyclic monophosphate (cAMP) production in the proximal convoluted tubules (PCT). Methods. PTH-Stimulated cAMP production and ATP content were determined by radioimmunoassay and luciferin-luciferase chemiluminescence methods, respectively. Results. The level of cAMP stimulated by PTH at an extracellular phosphate concentration of 0.78 mM was lower in the Hyp mice than in normal mice. The PTH-stimulated cAMP production in the Hyp mice was increased when the extracellular phosphate concentration was raised. In contrast, an increase in extracellular phosphate did not affect the PTH-stimulated cAMP production in the normal mice. Although the ATP content of the PCT was not different between the normal and Hyp mice immediately after microdissection, after 60 min of incubation, it had decreased to a greater extent in Hyp mice than in the normal animals. Raising the extracellular phosphate concentration from 0.78 to 2.3 mM prevented the decrease in ATP content in Hyp mice, and the intracellular ATP content then became comparable to that in the normal control. Conclusions. These results suggest that ATP content in the PCT tended to be decreased in Hyp mice by a decreased phosphate supply and that the blunted effect of PTH on cAMP production in these mice is due to ATP depletion.

Isoproterenol Increases Renal Tubular Reabsorption of Phosphate in X-Linked Hypophosphatemic (Hyp) Mice

Hypophosphatemic (Hyp) mice have a reduced renal retention of phosphate. The β-adrenergic agonist isoproterenol reverses phosphaturia induced by parathyroid hormone in the rat. This study determined whether isoproterenol could raise the renal tubular maximum transport of phosphate (TmP) in Hyp mice. Male 10- to 12-week-old normal and Hyp mice were infused with ³H-inulin and isoproterenol (0.0, 0.25, 0.5 and 1.0 µg/kg/min) in 0.9% NaCl. Glomerular filtration rate, plasma phosphate, urinary phosphate excretion, TmP, urinary sodium excretion, and urinary cyclic adenosine monophosphate were measured. As expected, Hypcontrols (0.0 dose) had a TmP which was significantly below that of normal controls: 1.15±(SEM) 0.6 (n = 9) versus 2.13±0.10 (n = 11) µmol P/ml glomerular filtrate (p<0.001). Isoproterenol at doses of 0.5 and 1.0 µg/kg/min elevated the TmP of Hyp mice to the level of normal controls: 1.94±0.19 (n = 7) and 1.98±0.10 (n = 9) µmol P/ml glomerular filtrate, respectively. These results indicate that the low tubular reabsorption of phosphate in Hyp mice is not due to a fixed low level of phosphate transport, but to decreased stimulation of phosphate transporters, since Hyp mouse kidneys increase phosphate reabsorption with suitable stimulus.

X-linked hypophosphataemia: a homologous disorder in humans and mice.

X-linked hypophosphatemia is an inherited disorder of phosphate (Pi) homeostasis characterized by growth retardation, rickets and osteomalacia, hypophosphataemia, and aberrant renal Pi reabsorption and vitamin D metabolism. Studies in murine Hyp and Gy homologues have identified a specific defect in Na+-Pi cotransport at the brush border membrane, abnormal regulation of 1,25-dihydroxyvitamin D3 (1,25(OH)2D) synthesis and degradation, and an intrinsic defect in bone mineralization. The mutant gene has been identified in XLH patients, by positional cloning, and in Hyp and Gy mice, and was designated PHEX/Phex to signify a PHosphate-regulating gene with homology to Endopeptidases on the X chromosome. PHEX/Phex is expressed in bones and teeth but not in kidney and efforts are under way to elucidate how loss of PHEX/Phex function elicits the mutant phenotype. Based on its homology to endopeptidases, it is postulated that PHEX/Phex is involved in the activation or inactivation of a peptide hormone(s) which plays a key role in the regulation of bone mineralization, renal Pi handling and vitamin D metabolism.

Dietary regulation of renal phosphate transporters in hypophosphatemic mice

X-linked hypophosphatemic rickets (XLH) is known to impair renal adaptive response to Pi restriction. We investigated the effects of dietary Pi on the synthesis of renal sodium-dependent inorganic phosphate (Na/Pi) cotransporters (Npt1 and NaPi-7) in X-linked hypophosphatemic mice (Hyp). The NaPi-7 mRNA level in Hyp mice was reduced to 50% of that of normal mice while the Npt1 mRNA level was unchanged. After feeding a low-Pi diet, the amounts of NaPi-7 protein and mRNA were markedly increased in both normal and Hyp mice. In contrast, after feeding a high-Pi diet, the levels of protein and mRNA were largely decreased in both mice. Immunohistochemical analysis indicated that NaPi-7 staining was largely enhanced in the apical membrane of renal proximal tubular cells in the normal and Hyp mice fed the low-Pi diet. In contrast, NaPi-7 staining was decreased in both groups of rats fed the high-Pi diet. Npt1 immunoreactivity was detected in the apical membrane of proximal convoluted and straight tubular cells in Hyp and normal mice, and was unchanged regardless of dietary Pi manipulation in both mice. Thus, dietary regulation for the synthesis of the two cotransporters is not impaired in Hyp mice.

Genetic screening for X-linked hypophosphatemic mice and ontogenic characterization of the defect in the renal sodium-phosphate transporter.

X-linked hypophosphatemic (Hyp) rickets is characterized by short stature, rickets, and bone abnormalities. Biochemically, hypophosphatemia and decreased renal reabsorption of phosphate are the hallmark of the disorder. Mutation of the PEX gene has been linked to human and murine Hyp rickets. Our study showed that phenotypical changes of this disease could be detected in 6-wk-old mice, but not in 2-wk-old mice. Therefore, we developed a PCR method to identify Hyp mice by detecting a lack of the 3' region of the PEX gene. Serum inorganic phosphate (Pi) levels were decreased, whereas alkaline phosphatase activity was increased in 2- and 6-wk-old Hyp mice. Northern blot showed that renal Na(+)-Pi transporter mRNA levels were decreased by 2.1-fold (1.47 +/- 0.21 densitometric units for normals; 0.68 +/- 1.43 for Hyp mice; p < 0.040) in 2-wk-old Hyp mice and by 1.7-fold (2.41 +/- 0.42 for normals; 1.44 +/- 0.33 for Hyp mice; p < 0.027) in 6-wk-old mice. Western blot showed that levels of immunoreactive renal Na(+)-Pi transporter protein were decreased by 4.5-fold (0.90 +/- 0.10 for normals; 0.22 +/- 0.08 for Hyp mice; p < 0.001) in 2-wk-old Hyp mice; and by 4.9-fold (1.47 +/- 0.19 for normals; 0.30 +/- 0.09 for Hyp mice; p < 0.0001) in 6-wk-old Hyp mice. In addition, levels of Na(+)-Pi transporter mRNA and protein were increased between 2- and 6-wk-old normal mice, but not in Hyp mice. This study demonstrates an easy assay to detect Hyp mutation and characterizes the defect during ontogeny of the Na(+)-Pi transporter in Hyp mice.

Intrinsic mineralization defect in Hyp mouse osteoblasts.

X-linked hypophosphatemia (XLH) is caused by inactivating mutations of PEX, an endopeptidase of uncertain function. This defect is shared by Hyp mice, the murine homologue of the human disease, in which a 3' Pex deletion has been documented. In the present study, we report that immortalized osteoblasts derived from the simian virus 40 (SV40) transgenic Hyp mouse (TMOb-Hyp) have an impaired capacity to mineralize extracellular matrix in vitro. Compared with immortalized osteoblasts from the SV40 transgenic normal mouse (TMOb-Nl), osteoblast cultures from the SV40 Hyp mouse exhibit diminished 45Ca accumulation into extracellular matrix (37 +/- 6 vs. 1,484 +/- 68 counts . min-1 . microgram protein-1) and reduced formation of mineralization nodules. Moreover, in coculture experiments, we found evidence that osteoblasts from the SV40 Hyp mouse produce a diffusible factor that blocks mineralization of extracellular matrix in normal osteoblasts. Our findings indicate that abnormal PEX in osteoblasts is associated with the accumulation of a factor(s) that inhibits mineralization of extracellular matrix in vitro.

Differential expression, abundance, and regulation of Na+-phosphate cotransporter genes in murine kidney.

Three classes of high-affinity Na+-Pi cotransporters are expressed in mammalian kidney. These include Npt1 (type I), Npt2 (type II), and the cellular receptors for gibbon ape leukemia virus (Glvr-1) and amphotropic murine retrovirus (Ram-1) (type III). We defined the tissue distribution as well as the relative renal abundance of Npt1, Npt2, Glvr-1, and Ram-1 mRNAs and examined the effects of low-Pi diet, the Hyp mutation, and growth hormone (GH) on their renal expression by ribonuclease protection assay. In normal mouse kidney, Npt1, Npt2, Glvr-1, and Ram-1 accounted for 15 +/- 1.0, 84 +/- 1.0, 0.5 +/- 0.2, and 0.5 +/- 0.3% of total Na+-Pi cotransporter mRNAs, respectively. Evidence was obtained for low-abundance Npt1 mRNA expression in liver and Npt2 mRNA expression in intestine, whereas Glvr-1 and Ram-1 mRNAs were also detected in bone, intestine, heart, and liver. Npt2 mRNA was localized to proximal tubules in the renal outer cortex, whereas Glvr-1 transcripts were detected throughout the kidney by in situ hybridization. The Hyp mutation elicited a significant reduction in renal Npt1 and Npt2 mRNAs (78 +/- 8 and 57 +/- 3% of normal, respectively), whereas neither low-Pi diet nor GH influenced the renal abundance of Npt1 and Npt2 transcripts. Renal Glvr-1 mRNA expression was significantly increased in Hyp mice and GH-treated mice (145 +/- 6 and 165 +/- 5% of control, respectively), whereas the renal abundance of Ram-1 transcript was unaffected by either the Hyp mutation, low-Pi diet, or GH treatment. In summary, we demonstrate that Npt2 is the predominant Na+-Pi cotransporter in mouse kidney, that Npt2 and Glvr-1 have distinct patterns of renal expression, and that the Hyp mutation modulates the renal expression of Npt1, Npt2, and Glvr-1 mRNAs. Our results suggest that increased renal Glvr-1 mRNA may contribute to GH stimulation of renal Na+-Pi cotransport.

Spermine deficiency in Gy mice caused by deletion of the spermine synthase gene.

Two mouse mutations gyro (Gy) and hypophosphatemia (Hyp) are mouse models for X-linked hypophosphatemic rickets and have been shown to be deleted for the 5' and 3' end of the mouse homolog of PHEX (phosphate regulating gene with homologies to endopeptidases on the X chromosome; formerly called PEX), respectively. In addition to the metabolic disorder observed in Hyp mice, male Gy mice are sterile and show circling behavior and reduced viability. The human SMS (spermine synthase) gene maps approximately 39 kb upstream of PHEX and is transcribed in the same direction. To elucidate the complex phenotype of Gy mice, we characterized the genomic region upstream of Phex. By establishing the genomic structure of mouse Sms, a 160-190 kb deletion was shown in Gy mice, which includes both Phex and Sms. There are several pseudogenes of SMS / Sms in man and mouse. Northern analysis revealed three different Sms transcripts which are absent in Gy mice. Measurement of polyamine levels revealed a marked decrease in spermine in liver and pancreas of affected male Gy mice. Analysis of brain tissue revealed no gross or histological abnormalities. Gy provides a mouse model for a defect in the polyamine pathway, which is known to play a key role in cell proliferation.

Osteocalcin Production in Primary Osteoblast Cultures Derived from Normal and Hyp Mice

Rickets and osteomalacia are characteristic features of the Hyp mouse model of human X-linked hypophosphatemia. Hyp mice demonstrate elevated circulating osteocalcin levels, as well as altered regulation of osteocalcin by 1,25(OH)2D3. Whether this osteocalcin abnormality is intrinsic to the osteoblast, or mediated by the in vivo milieu, has not been established. We therefore characterized osteocalcin production and its regulation by 1,25(OH)2D3 in primary cultures of murine osteoblasts and examined osteocalcin and its messenger RNA in response to 1,25(OH)2D3 in cultures of Hyp mouse-derived osteoblasts. Cell viability and osteocalcin production are optimal when murine cells are harvested within 36 h of age. Murine primary osteoblast cultures mineralize and produce osteocalcin in a maturation-dependent fashion (as demonstrated in other species), and continuous exposure to 1,25(OH)2D3, beginning at day 9 of culture, inhibits osteoblast differentiation and osteocalcin production and prevents mineralization of the culture. However, in contrast to other species, exposure to 1,25(OH)2D3, added later (days 17-25) in culture, does not stimulate osteocalcin but arrests osteocalcin production at current levels. Ambient media levels of osteocalcin were no different in cultures from Hyp mice and their normal litter mates, and the down-regulatory response to 1,25(OH)2D3 was comparable in cultures from normal and Hyp mice. Furthermore, expression of osteocalcin messenger RNA in murine cultures is reduced with exposure to 1,25(OH)2D3, and there is no difference between normal and Hyp cultures in this response. Thus, primary murine osteoblasts manifest a species-specific effect of 1,25(OH)2D3 on osteocalcin production. Furthermore, the increased serum osteocalcin production seen in intact Hyp mice, and the altered response to 1,25(OH)2D3 in Hyp mice, are not observed in osteoblast cultures derived from the mutant strain. These data indicate that abnormalities of osteocalcin described in intact Hyp mice require factors other than those present in cultured cells.

Osteocalcin Production in Primary Osteoblast Cultures Derived from Normal and Hyp Mice

Osteocalcin Production in Primary Osteoblast Cultures Derived from Normal and Hyp Mice

Growth hormone normalizes renal 1,25-dihydroxyvitamin D3-24-hydroxylase gene expression but not Na+-phosphate cotransporter (Npt2) mRNA in phosphate-deprived Hyp mice.

The murine X-linked Hyp mutation is characterized by decreased renal expression of type II Na+-phosphate (Pi) cotransporter (Npt2) mRNA and an abnormal vitamin D response to Pi deprivation. The latter is manifest by an aberrant fall in serum 1,25-dihydroxyvitamin D3 (1,25(OH)2D) levels that is associated with an increase in renal 1,25(OH)2D-24-hydroxylase (24-hydroxylase), the first enzyme in the C-24 oxidation pathway. Because growth hormone (GH) enhances renal Na+-Pi cotransport and permits the adaptive 1,25(OH)2D response in Pi-deprived hypophysectomized rats, we examined the effects of GH on vitamin D metabolism and renal Npt2 mRNA abundance in Hyp mice fed control and low Pi diets. GH significantly decreased renal 24-hydroxylase activity (0.202+/-0.020 to 0.098+/-0.008 pmol/mg of protein/minute, p < 0.05) and mRNA abundance, relative to beta-actin mRNA (299+/-13 to 78+/-14, p < 0.05), in Hyp mice fed the low Pi diet but had no effect on either parameter in mutants fed the control diet. Moreover, after GH treatment, renal 24-hydroxylase gene expression was no longer elevated in Pi-deprived Hyp mice relative to mutants fed control diet. In contrast, GH did not correct the serum concentration of 1,25(OH)2D in Pi-deprived Hyp mice. We also demonstrate that GH did not normalize renal Npt2 mRNA expression, relative to beta-actin mRNA, in Hyp mice fed either control or low Pi diets. The present data demonstrate that normalization of renal 24-hydroxylase gene expression in Pi-deprived Hyp mice by GH is not sufficient to correct the serum concentration of 1,25(OH)2D and is not associated with an alteration in renal Npt2 mRNA expression.

Positional cloning of the PEX gene: new insights into the pathophysiology of X-linked hypophosphatemic rickets.

X-linked hypophosphatemic rickets (HYP) is the most common form of hereditary renal phosphate wasting. The hallmarks of this disease are isolated renal phosphate wasting with inappropriately normal calcitriol concentrations and a mineralization defect in bone. Studies in the Hyp mouse, one of the murine models of the human disease, suggest that there is an approximately 50% decrease in both message and protein of NPT-2, the predominant sodium-phosphate cotransporter in the proximal tubule. However, human NPT-2 maps to chromosome 5q35, indicating that it is not the disease gene. Positional cloning studies have led to the identification of a gene, PEX, which is responsible for the disorder. Further studies have led to identification of the murine Pex gene, which is mutated in the murine models of the disorder. These studies, in concert with other studies, have led to improved understanding of the pathophysiology of HYP and a new appreciation for the complexity of normal phosphate homeostasis.

Skeletal casein kinase activity defect in the HYP mouse.

The Hyp mouse, a model for human X-linked hypophosphatemia (XLH), is characterized by phosphate wasting and defective mineralization. Since osteopontin (OPN) is considered pivotal for biological mineralization, we examined the biosynthesis of OPN in osteoblasts of +/Y and Hyp/Y mice. Immunoprecipitation analyses using a specific antibody to OPN revealed that Hyp/Y and +/Y osteoblasts secrete similar levels of OPN as determined by [35S]-methionine biosynthetic labeling, but a reduced phosphorylation was noted after 32P-PO4 biosynthetic labeling. Northern blot hybridization analysis of +/Y and Hyp/Y mice osteoblast mRNAs, using a cDNA probe for mouse OPN, revealed no difference in the steady state levels of osteopontin mRNA. Analysis of casein kinase II activity in +/Y and Hyp/Y mice osteoblast, kidney, heart and liver membrane fractions revealed that casein kinase II activity in the Hyp/Y mice osteoblasts and kidney is only 35%-50%, respectively, of that of the +/Y mice tissues. The accumulated data are consistent with a post-translation defect in the Hyp/Y mouse osteoblast which results in the under-phosphorylation of osteopontin and subsequent under-mineralization of bone matrix.