Phex mRNA Research Articles

Reduced estrogen signaling contributes to bone loss and cardiac dysfunction in interleukin-10 knockout mice.

Characterization of the interleukin (IL)-10 knockout (KO) mouse with chronic gut inflammation, cardiovascular dysfunction, and bone loss suggests a critical role for this cytokine in interorgan communication within the gut, bone, and cardiovascular axis. We sought to understand the role of IL-10 in the cross-talk between these systems. Six-week-old IL-10 KO mice and their wild type (WT) counterparts were maintained on a standard rodent diet for 3 or 6 months. Gene expression of proinflammatory markers and Fgf23, serum 17β-estradiol (E2), and cardiac protein expression were assessed. Ileal Il17a and Tnf mRNA increased while Il6 mRNA increased in the bone and heart by at least 2-fold in IL-10 KO mice. Bone Dmp1 and Phex mRNA were repressed at 6 months in IL-10 KO mice, resulting in increased Fgf23 mRNA (~4-fold) that contributed to increased fibrosis. In the IL-10 KO mice, gut bacterial β-glucuronidase activity and ovarian Cyp19a1 mRNA were lower (p < 0.05), consistent with reduced serum E2 and reduced cardiac pNOS3 (Ser1119 ) in these mice. Treatment of ileal lymphocytes with E2 reduced gut inflammation in WT but not IL-10 KO mice. In conclusion, our data suggest that diminished estrogen and defective bone mineralization increased FGF23 which contributed to cardiac fibrosis in the IL-10 KO mouse.

Function of PHEX mutations p.Glu145* and p.Trp749Arg in families with X-linked hypophosphatemic rickets by the negative regulation mechanism on FGF23 promoter transcription

X-linked hypophosphatemic rickets (XLH) is characterized by increased circulating fibroblast growth factor 23 (FGF23) concentration caused by PHEX (NM_000444.5) mutations. Renal tubular resorption of phosphate is impaired, resulting in rickets and impaired bone mineralization. By phenotypic-genetic linkage analysis, two PHEX pathogenic mutations were found in two XLH families: c.433 G > T, p.Glu145* in exon 4 and c.2245 T > C, p.Trp749Arg in exon 22. Immunofluorescence showed that the localization of p.Glu145* and p.Trp749Arg mutant and secretory PHEX (secPHEX) changed, with decreased expression. In a HEK293T cell model co-transfected with PHEX, secPHEX, and FGF23, wild-type PHEX, secPHEX, and FGF23 proteins were distributed in the cell membrane or endoplasmic reticulum, while the mutant was located in the nuclear membrane and cytoplasm. qPCR of p.Glu145* revealed decreased PHEX and secPHEX mRNA expression in cells, with no difference in mRNA expression of p.Trp749Arg. Both mutations decreased intracellular PHEX endopeptidase activity. Western blot analysis showed decrease in mutant and secPHEX protein expression and no FGF23 protein expression in single-transfected PHEX and secPHEX cells. In cells co-transfected with FGF23, PHEX and secPHEX mutation promoted FGF23 expression. Dual-luciferase reporter gene was used to detect the effect of PHEX on FGF23 promoter. The dual-luciferase reporter gene showed that after PHEX overexpression, the activity of mutant firefly luciferase was significantly higher than that of wild type. The regulatory mechanism between PHEX and FGF23 is still unclear, but we found that PHEX is a direct transcriptional inhibitor of FGF23 and affects the expression of FGF23. This study verified the pathogenicity of the two variants and revealed the possible regulatory mechanism between PHEX and FGF23.

The Effects of Inorganic Phosphorus Levels on Phosphorus Utilization, Local Bone-Derived Regulators, and BMP/MAPK Pathway in Primary Cultured Osteoblasts of Broiler Chicks.

Understanding the underlying mechanisms that regulate the bone phosphorus (P) utilization would be helpful for developing feasible strategies to improve utilization efficiency of P in poultry. We aimed to investigate the effects of inorganic P levels on P utilization, local bone-derived regulators and bone morphogenetic protein/mitogen-activated protein kinase (BMP/MAPK) pathway in primary cultured osteoblasts of broiler chicks in order to address whether local bone-derived regulators or BMP/MAPK pathway was involved in regulating the bone P utilization of broilers using an in vitro model. The primary cultured tibial osteoblasts of broiler chicks were randomly divided into one of five treatments with six replicates for each treatment. Then, cells were respectively incubated with 0.0, 0.5, 1.0, 1.5, or 2.0 mmol/L of added P as NaH2PO4 for 24 days. The results showed that as added P levels increased, tibial osteoblastic P retention rate, number and area of mineralized nodules, the mRNA expressions of endopeptidases on the X chromosome (PHEX), dentin matrix protein 1 (DMP1), bone morphogenetic protein 2 (BMP2), and the mRNA and protein expressions of matrix extracellular phosphoglycoprotein (MEPE) increased linearly (p < 0.001) or quadratically (p < 0.04), while extracellular signal-regulated kinase 1 (ERK1) mRNA expression and c-Jun N-terminal kinase 1 (JNK1) phosphorylated level decreased linearly (p < 0.02) or quadratically (p < 0.01). Correlation analyses showed that tibial osteoblastic P retention rate was positively correlated (r = 0.452–0.564, p < 0.03) with MEPE and BMP2 mRNA expressions. Furthermore, both number and area of mineralized nodules were positively correlated (r = 0.414–0.612, p < 0.03) with PHEX, DMP1, MEPE, and BMP2 mRNA expressions but negatively correlated (r = −0.566 to −0.414, p < 0.04) with the ERK1 mRNA expression and JNK1 phosphorylated level. These results suggested that P utilization in primary cultured tibial osteoblasts of broiler chicks might be partly regulated by PHEX, DMP1, MEPE, BMP2, ERK1, and JNK1.

Dentoalveolar Defects in the Hyp Mouse Model of X-linked Hypophosphatemia

Mutations in PHEX cause X-linked hypophosphatemia (XLH), a form of hypophosphatemic rickets. Hyp (Phex mutant) mice recapitulate the XLH phenotype. Dental disorders are prevalent in individuals with XLH; however, underlying dentoalveolar defects remain incompletely understood. We analyzed Hyp mouse dentoalveolar defects at 42 and 90 d postnatal to comparatively define effects of XLH on dental formation and function. Phex mRNA was expressed by odontoblasts (dentin), osteocytes (bone), and cementocytes (cellular cementum) in wild-type (WT) mice. Enamel density was unaffected, though enamel volume was significantly reduced in Hyp mice. Dentin defects in Hyp molars were indicated histologically by wide predentin, thin dentin, and extensive interglobular dentin, confirming micro–computed tomography (micro-CT) findings of reduced dentin volume and density. Acellular cementum was thin and showed periodontal ligament detachment. Mechanical testing indicated dramatically altered periodontal mechanical properties in Hyp versus WT mice. Hyp mandibles demonstrated expanded alveolar bone with accumulation of osteoid, and micro-CT confirmed decreased bone volume fraction and alveolar bone density. Cellular cementum area was significantly increased in Hyp versus WT molars owing to accumulation of hypomineralized cementoid. Histology, scanning electron microscopy, and nanoindentation revealed hypomineralized “halos” surrounding Hyp cementocyte and osteocyte lacunae. Three-dimensional micro-CT analyses confirmed larger cementocyte/osteocyte lacunae and significantly reduced perilacunar mineral density. While long bone and alveolar bone osteocytes in Hyp mice overexpressed fibroblast growth factor 23 (Fgf23), its expression in molars was much lower, with cementocyte Fgf23 expression particularly low. Expression and distribution of other selected markers were disturbed in Hyp versus WT long bone, alveolar bone, and cementum, including osteocyte/cementocyte marker dentin matrix protein 1 (Dmp1). This study reports for the first time a quantitative analysis of the Hyp mouse dentoalveolar phenotype, including all mineralized tissues. Novel insights into cellular cementum provide evidence for a role for cementocytes in perilacunar mineralization and cementum biology.

Calcium induces pro-anabolic effects on human primary osteoblasts associated with acquisition of mature osteocyte markers

Calcium, in combination with vitamin D, is an effective treatment for osteoporosis. Since bone mineralisation occurs concurrently with osteoblast to osteocyte transition, we hypothesised that calcium would stimulate this process. The effect of calcium (1.8–11.8mM) was tested on human primary osteoblast (NHBC) differentiation in vitro. Cultures were assayed for cell-associated mineral and gene expression associated with osteoblast differentiation and mineralisation. Treatment with calcium resulted in a striking dose- and time-dependent increase in cell-associated mineralisation. Calcium appeared to promote osteoblast to osteocyte differentiation, as indicated by increased expression of osteocalcin (OCN), E11, dentin matrix protein 1 (DMP1) and SOST mRNA. The expression of the osteoclast inhibitor, osteoprotegerin, was dramatically enhanced by calcium. Calcium also increased the ratio of PHEX mRNA expression relative to that of MEPE, suggesting a mechanism for the pro-anabolic effect. Consistent with this, calcium-dependent mineralisation was reversed in the presence of MEPE–ASARM peptides. This study suggests that calcium promotes osteoblast to osteocyte transition and concurrent matrix mineralisation, at least in part through the PHEX–MEPE axis.

Mutational analysis of patients with FGF23-related hypophosphatemic rickets

X-linked hypophosphatemic rickets (XLHR) caused by mutations in the PHEX gene is considered to be the most frequent cause of fibroblast growth factor 23 (FGF23)-related congenital hypophosphatemic rickets. In previous studies, mutations in the PHEX gene were detected in 60-70% of patients with clinical diagnoses of XLHR. This leads to the question whether current screening methods for mutations in the PHEX gene are inadequate or whether there is a substantial number of patients with other genetic causes of hypophosphatemic rickets. We conducted a genetic analysis of patients with FGF23-related hypophosphatemic rickets to clarify their etiology and evaluate the prevalence of XLHR among this group. We studied 27 patients with familial and sporadic congenital hypophosphatemic rickets in whom serum FGF23 was above 30 pg/ml using an assay for the full-length protein. Exons and exon-intron junctions of genomic DNA of causative genes for FGF23-related hypophosphatemic rickets were sequenced. PHEX mRNA from peripheral blood was analyzed in some patients. Direct sequencing of genomic DNA identified 11 novel and four known mutations in the PHEX gene. Additionally, there was a large PHEX gene deletion in one case and abnormal PHEX mRNA splicing in another. In summary, 26 patients (96%) had XLHR and one patient had autosomal recessive hypophosphatemic rickets 2. XLHR is by far the most prevalent cause of FGF23-related hypophosphatemic rickets. We propose that analysis of PHEX mRNA from peripheral blood would be appropriate for the first screening step in determining the etiology of FGF23-related hypophosphatemic rickets.

A novel Phex mutation in a new mouse model of hypophosphatemic rickets

X-linked hypophosphatemic rickets (XLH) is a dominantly inherited disease characterized by renal phosphate wasting, aberrant vitamin D metabolism, and defective bone mineralization. It is known that XLH in humans and in certain mouse models is caused by inactivating mutations in PHEX/Phex (phosphate-regulating gene with homologies to endopeptidases on the X chromosome). By a genome-wide N-ethyl-N-nitrosourea (ENU)-induced mutagenesis screen in mice, we identified a dominant mouse mutation that exhibits the classic clinical manifestations of XLH, including growth retardation, skeletal abnormalities (rickets/osteomalacia), hypophosphatemia, and increased serum alkaline phosphatase (ALP) levels. Mapping and sequencing revealed that these mice carry a point mutation in exon 14 of the Phex gene that introduces a stop codon at amino acid 496 of the coding sequence (Phex(Jrt) also published as Phex(K496X) [Ichikawa et al., 2012]). Fgf23 mRNA expression as well as that of osteocalcin, bone sialoprotein, and matrix extracellular phosphoglycoprotein was upregulated in male mutant long bone, but that of sclerostin was unaffected. Although Phex mRNA is expressed in bone from mutant hemizygous male mice (Phex(Jrt)/Y mice), no Phex protein was detected in immunoblots of femoral bone protein. Stromal cultures from mutant bone marrow were indistinguishable from those of wild-type mice with respect to differentiation and mineralization. The ability of Phex(Jrt)/Y osteoblasts to mineralize and the altered expression levels of matrix proteins compared with the well-studied Hyp mice makes it a unique model with which to further explore the clinical manifestations of XLH and its link to FGF23 as well as to evaluate potential new therapeutic strategies.

Conditional Disruption of Pkd1 in Osteoblasts Results in Osteopenia Due to Direct Impairment of Bone Formation

PKD1 (polycystin-1), the disease-causing gene for ADPKD, is widely expressed in various cell types, including osteoblasts, where its function is unknown. Although global inactivation of Pkd1 in mice results in abnormal skeletal development, the presence of polycystic kidneys and perinatal lethality confound ascertaining the direct osteoblastic functions of PKD1 in adult bone. To determine the role of PKD1 in osteoblasts, we conditionally inactivated Pkd1 in postnatal mature osteoblasts by crossing Oc (osteocalcin)-Cre mice with floxed Pkd1 (Pkd1(flox/m1Bei)) mice to generate conditional heterozygous (Oc-Cre;Pkd1(flox/+)) and homozygous (Oc-Cre;Pkd1(flox/m1Bei)) Pkd1-deficient mice. Cre-mediated recombination (Pkd1(Delta flox)) occurred exclusively in bone. Compared with control mice, the conditional deletion of Pkd1 from osteoblasts resulted in a gene dose-dependent reduction in bone mineral density, trabecular bone volume, and cortical thickness. In addition, mineral apposition rates and osteoblast-related gene expression, including Runx2-II (Runt-related transcription factor 2), osteocalcin, osteopontin, and bone sialoprotein, were reduced proportionate to the reduction of Pkd1 gene dose in bone of Oc-Cre;Pkd1(flox/+) and Oc-Cre;Pkd1(flox/m1Bei) mice. Primary osteoblasts derived from Oc-Cre;Pkd1(flox/m1Bei) displayed impaired differentiation and suppressed activity of the phosphatidylinositol 3-kinase-Akt-GSK3beta-beta-catenin signaling pathways. The conditional deletion of Pkd1 also resulted in increased adipogenesis in bone marrow and in osteoblast cultures. Thus, PKD1 directly functions in osteoblasts to regulate bone formation.

The role of tissue-nonspecific alkaline phosphatase in the phosphate-induced activation of alkaline phosphatase and mineralization in SaOS-2 human osteoblast-like cells

Tissue-nonspecific alkaline phosphatase (TNAP) plays a key role in mineralization by degrading inorganic pyrophosphate and providing free inorganic phosphate. We have previously reported that TNAP is induced by beta-glycerophosphate and NaH(2)PO(4) in short-term cultures of SaOS-2 human osteoblast-like cells and that PHEX (phosphate-regulating gene with homologies to endopeptidase on the X chromosome) mRNA is also induced after TNAP induction. In the present study, we have investigated the effects of levamisole, a TNAP inhibitor, and phosphonoformic acid (PFA), a type III sodium-phosphate cotransporter inhibitor, on the phosphate-induced expression of TNAP and mineralization. Levamisole inhibited beta-glycerophosphate-induced mineralization, TNAP and PHEX expression, and the increase in enzymatic activity of NPP1 (5'-nucleotide pyrophosphatase phosphodiesterase 1), but did not inhibit NaH(2)PO(4)-induced mineralization. PFA completely inhibited NaH(2)PO(4)-induced mineralization and NPP1 enzymatic activation, and partly inhibited beta-glycerophosphate-induced mineralization, but did not affect the increase in TNAP activity. These results suggest that phosphate derived from TNAP-induced hydrolysis of beta-glycerophosphate yields signals that induce TNAP expression and mineralization, and that PHEX expression may be linked to TNAP expression. However, luciferase assays failed to detect any transcriptional activation of the promoter region of the human TNAP gene by beta-glycerophosphate or NaH(2)PO(4), suggesting that the effects of these phosphates may be indirect.

Degradation of MEPE, DMP1, and Release of SIBLING ASARM-Peptides (Minhibins): ASARM-Peptide(s) Are Directly Responsible for Defective Mineralization in HYP

Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) and DMP1 (dentin matrix protein 1) result in X-linked hypophosphatemic rickets (HYP) and autosomal-recessive hypophosphatemic-rickets (ARHR), respectively. Specific binding of PHEX to matrix extracellular phosphoglycoprotein (MEPE) regulates the release of small protease-resistant MEPE peptides [acidic serine- and aspartate-rich MEPE-associated motif (ASARM) peptides]. ASARM peptides are potent inhibitors of mineralization (minhibins) that also occur in DMP1 [MEPE-related small integrin-binding ligand, N-linked glycoprotein (SIBLING) protein]. It is not known whether these peptides are directly responsible for the mineralization defect. We therefore used a bone marrow stromal cell (BMSC) coculture model, ASARM peptides, anti-ASARM antibodies, and a small synthetic PHEX peptide (SPR4; 4.2 kDa) to examine this. Surface plasmon resonance (SPR) and two-dimensional (1)H/(15)N nuclear magnetic resonance demonstrated specific binding of SPR4 peptide to ASARM peptide. When cultured individually for 21 d, HYP BMSCs displayed reduced mineralization compared with wild type (WT) (-87%, P < 0.05). When cocultured, both HYP and WT cells failed to mineralize. However, cocultures (HYP and WT) or monocultures of HYP BMSCs treated with SPR4 peptide or anti-ASARM neutralizing antibodies mineralized normally. WT BMSCs treated with ASARM peptide also failed to mineralize properly without SPR4 peptide or anti-ASARM neutralizing antibodies. ASARM peptide treatment decreased PHEX mRNA and protein (-80%, P < 0.05) and SPR4 peptide cotreatment reversed this by binding ASARM peptide. SPR4 peptide also reversed ASARM peptide-mediated changes in expression of key osteoclast and osteoblast differentiation genes. Western blots of HYP calvariae and BMSCs revealed massive degradation of both MEPE and DMP1 protein compared with the WT. We conclude that degradation of MEPE and DMP-1 and release of ASARM peptides are chiefly responsible for the HYP mineralization defect and changes in osteoblast-osteoclast differentiation.

Downregulation of osteoblast Phex expression by PTH

Human/murine X-linked hypophosphatemia is a dominant disorder associated with renal phosphate wasting and defective bone mineralization. This disorder results from mutations in the PHEX/ Phex ( Phosphate-regulating gene with homologies to endopeptidases on the X chromosome) gene, which is expressed in fully differentiated osteoblasts. The purpose of the present study was to assess whether PTH, a major regulator of bone development and turnover, modulates osteoblastic Phex expression. The effects of different concentrations of PTH (rat fragment 1–34) were determined on Phex mRNA and protein expression in vitro using MC3T3-E1 osteoblastic cells and mouse primary osteoblasts; and in vivo using 45-day-old mice infused for 3 days with PTH. Phex mRNA levels were quantitated on Northern blots by densitometric analysis relative to GAPDH mRNA levels. Phex protein levels were analyzed by immunoprecipitation of 35S-methionine-labeled osteoblast lysates or by immunoblotting of calvaria membrane extracts using a polyclonal rabbit antiserum raised against a mouse Phex carboxy-terminal peptide. Fully differentiated MC3T3-E1 cells were incubated for 4 to 48 h with increasing concentrations of PTH (10 −11 to 10 −7 M). PTH inhibited Phex mRNA expression in both mineralizing and nonmineralizing osteoblast cultures in a dose- and time-dependent manner with a maximal inhibition at 10 −7 M PTH after 24 h (15 ± 7% of control levels, n = 5, P < 0.001). The PTH-mediated downregulation of Phex mRNA levels was associated with corresponding decreases in Phex protein synthesis and suppression at 10 −7 M PTH. Similar results were obtained with primary osteoblasts isolated from newborn mouse calvaria. Consistent with the in vitro findings, continuous PTH infusion to mice elicited decreases in Phex expression in calvaria. The effect of PTH was also assessed on matrix mineralization by mature MC3T3-E1 cells by measuring 45Ca accumulation in cell layers. PTH (10 −7 M) inhibited the initiation (57 ± 2% of control levels, n = 5, P < 0.001) and the progression of matrix mineralization (75 ± 1% of control levels, n = 5, P < 0.001). In summary, PTH inhibits osteoblastic Phex expression in vitro and in vivo. The downregulation of Phex expression by PTH in vitro is associated with inhibition of matrix mineralization, consistent with a role for Phex in bone mineralization.

1,25-Dihydroxyvitamin D3 Down-regulation of PHEX Gene Expression Is Mediated by Apparent Repression of a 110 kDa Transfactor That Binds to a Polyadenine Element in the Promoter

The PHEX gene encodes an endopeptidase expressed in osteoblasts that inactivates an uncharacterized peptide hormone, phosphatonin, which suppresses bone mineralization as well as renal phosphate reabsorption and vitamin D bioactivation. We demonstrate that 1alpha-25-dihydroxyvitamin D (1,25(OH)2D3), the, active renal vitamin D metabolite, decreases PHEX mRNA in the rat osteoblastic cell line, UMR-106, as well as in mouse calvaria. Promoter/reporter construct analysis of the murine PHEX gene in transfected UMR-106 cells localized the repressive effect of 1,25(OH)2D3 to the -133 to -74 bp region, and gel mobility shift experiments revealed that 1,25(OH)2D3 treatment of the cells diminished the binding of a nuclear protein(s) to a stretch of 17 adenines from bp -116 to -100 in the proximal PHEX promoter. Either overexpression of a dominant-negative vitamin D receptor (VDR) or deletion of this sequence of 17 A-T base pairs abolished the repressive effect of 1,25(OH)2D3 by attenuating basal promoter activity, indicating that this region mediates the 1,25(OH)2D3 response and is involved in basal transcription. South-western blot analysis and DNA affinity purification show that an unidentified 110 kDa nuclear protein binds to the poly(A) element. Because 1,25(OH)2D3-liganded VDR neither binds to the polyadenine region of the PHEX promoter nor directly influences the association of the 110 kDa transfactor, we conclude that 1,25(OH)2D3 indirectly decreases PHEX expression via VDR-mediated repression (or modification) of this novel transactivator. Thus, we have identified a cis-element required for PHEX gene transcription that participates in negative feedback control of PHEX expression and thereby modulates the actions of phosphatonin.

Bone as a source of FGF23: regulation by phosphate?

The identification of FGF23 as a factor involved in several disorders of phosphate regulation and of PHEX as the gene mutated in X-linked Hypophosphatemic Rickets indicates that both these genes may be involved in phosphate homeostasis, although their physiological roles are unclear. In this study, FGF23 mRNA expression was analyzed by real-time RT-PCR and found to be higher in normal human bone than in kidney, liver, thyroid, or parathyroid tissue, while expression in oncogenic osteomalacia tumor tissue was several hundred-fold higher than in bone. Expression of FGF23 mRNA in human osteoblast-like bone cells, quantitated by real-time RT-PCR, increased with increasing extracellular phosphate and was 2-fold higher in cells treated with 2 mM extracellular phosphate compared to 0 mM phosphate treatment. PHEX mRNA expression increased 1.3-fold after treatment with 2 mM phosphate. FGF23 expression in the bone cells increased with increased mineralization over a 20-day treatment period under mineralizing conditions with β-glycerophosphate, while PHEX expression decreased. The results indicate that FGF23 mRNA expression in bone cells is regulated by extracellular phosphate and by mineralization. These results support proposals that bone may be a source of circulating FGF23 and suggest that FGF23 expression by bone is regulated.

Differential regulation of PHEX expression in bone and parathyroid gland by chronic renal insufficiency and 1,25-dihydroxyvitamin D3.

Mutations in the PHEX gene are responsible for X-linked hypophosphatemia, a renal phosphate-wasting disorder associated with defective skeletal mineralization. PHEX is predominantly expressed in bones and teeth and in the parathyroid gland of patients with chronic renal failure and tertiary hyperparathyroidism. The purpose of the present study was to examine the effects of renal insufficiency and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the regulation of PHEX expression in rat tibia and parathyroid gland. In rats fed a high-phosphate (Pi) diet, nephrectomy elicited a significant increase in the serum parathyroid hormone (PTH) concentration that was associated with a significant increase in the abundance of PHEX mRNA and protein in the tibia and a significant increase in PHEX mRNA in the parathyroid gland. In contrast, 1,25(OH)2D3 administration to intact rats fed a control diet elicited a significant decrease in the serum PTH concentration that was accompanied by a significant decrease in PHEX mRNA and protein abundance in the tibia and a significant decrease in PHEX mRNA in the parathyroid gland. In addition, the increases in serum PTH levels and PHEX mRNA in the tibia and parathyroid gland in nephrectomized rats fed a high-Pi diet were blunted by 1,25(OH)2D3. Serum PTH concentration was positively and significantly correlated with tibial PHEX mRNA and protein abundance. In summary, we demonstrate that PHEX expression in the tibia and parathyroid gland is increased by chronic renal insufficiency and decreased by 1,25(OH)2D3 administration and suggest that PTH status may play an important role in mediating these changes in PHEX expression.

Parathyroid hormone-related protein(1-34) regulates Phex expression in osteoblasts through the protein kinase A pathway.

Phex (a phosphate-regulating gene with homologies to endopeptidases on the X chromosome) is expressed predominantly in bone in which it has been implicated in the mineralization process. Multiple factors and hormones, including PTHrP, regulate formation, development, and/or homeostasis of bone. The purpose of the present study was to determine whether PTHrP(1-34) regulates Phex expression and identify the signaling pathway used. Phex mRNA and protein levels were analyzed by RT-PCR and immunoblotting, respectively. In UMR-106 cells, PTHrP(1-34) caused a time- and concentration-dependent decrease in Phex expression. Forskolin, an adenylate cyclase activator, had the same effect. Dibutiryl cAMP also decreased Phex expression, and its effect was blocked by H89, a protein kinase A (PKA) inhibitor. In contrast, 12-O-tetradecanoyl phorbol-13-acetate, a protein kinase C (PKC) activator, increased Phex expression in a time- and dose-dependent manner. This effect was reversed by bisindolylmaleimide Iota, a PKC inhibitor. Bovine PTH(3-34), which activates PKC but not PKA, had no effect. On the contrary, human PTH(1-31), which activates PKA but not PKC, decreased Phex expression. H89 but not bisindolylmaleimide Iota blocked the effect of PTHrP(1-34). PTHrP(1-34) also decreased Phex expression in cultures of fetal rat calvaria cells at d 7 of culture but not at later stages. These data demonstrate that PTHrP(1-34), through PKA, down-regulates Phex expression in osteoblasts.

An Ethyl-Nitrosourea-Induced Point Mutation in Phex Causes Exon Skipping, X-Linked Hypophosphatemia, and Rickets

We describe the clinical, genetic, biochemical, and molecular characterization of a mouse that arose in the first generation (G(1)) of a random mutagenesis screen with the chemical mutagen ethyl-nitrosourea. The mouse was observed to have skeletal abnormalities inherited with an X-linked dominant pattern of inheritance. The causative mutation, named Skeletal abnormality 1 (Ska1), was shown to be a single base pair mutation in a splice donor site immediately following exon 8 of the Phex (phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome) gene. This point mutation caused skipping of exon 8 from Phex mRNA, hypophosphatemia, and features of rickets. This experimentally induced phenotype mirrors the human condition X-linked hypophosphatemia; directly confirms the role of Phex in phosphate homeostasis, normal skeletal development, and rickets; and illustrates the power of mutagenesis in exploring animal models of human disease.

Glucocorticoid regulation of the murine PHEX gene.

The phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) is a member of the neutral endopeptidase family, which is expressed predominantly on the plasma membranes of mature osteoblasts and osteocytes. Although it is known that the loss of PHEX function results in X-linked hypophosphatemic rickets, characterized by abnormal bone matrix mineralization and renal phosphate wasting, little is known about how PHEX is regulated. We therefore sought to determine whether the murine PHEX gene is regulated by glucocorticoids (GCs), which are known to influence phosphate homeostasis and bone metabolism. Northern blot analysis revealed increased PHEX mRNA expression in GC-treated suckling mice (1.5-fold) and in rat osteogenic sarcoma (UMR-106) cells (2.5-fold). An increase was also seen in PHEX promoter activity in transiently transfected UMR-106 cells with GC treatment. Analysis of nested promoter deletions revealed that an atypical GC response element was located between -337 and -315 bp. Mutational analysis and electrophoretic mobility shift assays further identified -326 to -321 bp as a site involved in GC regulation. Supershift analyses and electrophoretic mobility shift assay competition studies indicated that the core binding factor alpha1-subunit transcription factor is able to bind to this region and may therefore play a role in the GC response of the murine PHEX gene.

Overexpression of Phex in Osteoblasts Fails to Rescue the Hyp Mouse Phenotype

Inactivating mutations of Phex, a phosphate-regulating endopeptidase, cause hypophosphatemia and impaired mineralization in X-linked hypophosphatemia (XLH) and its mouse homologue, Hyp. Because Phex is predominantly expressed in bone and cultured osteoblasts from Hyp mice display an apparent intrinsic mineralization defect, it is thought that reduced expression of Phex in mature osteoblasts is the primary cause of XLH. To test this hypothesis, we studied both targeted expression of Phex to osteoblasts in vivo under the control of the mouse osteocalcin (OG2) promoter and retroviral mediated overexpression of Phex in Hyp-derived osteoblasts (TMOb-Hyp) in vitro. Targeted overexpression of Phex to osteoblasts of OG2 Phex transgenic Hyp mice normalized Phex endopeptidase activity in bone but failed to correct the hypophosphatemia, rickets, or osteomalacia. OG2 Phex transgenic Hyp mice did exhibit a small, but significant, increase in bone mineral density and dry ashed weight, suggesting a partial mineralization effect from restoration of Phex function in mature osteoblasts. Similarly, retroviral mediated overexpression of Phex in TMOb-Hyp osteoblasts restored Phex mRNA levels, protein expression, and endopeptidase activity but failed to correct their intrinsic mineralization defect. In addition, we failed to detect the Phex substrate FGF-23 in osteoblasts. Taken together, these in vivo and in vitro data indicate that expression of Phex in osteoblasts is not sufficient to rescue the Hyp phenotype and that other sites of Phex expression and/or additional factors are likely to be important in the pathogenesis of XLH.

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

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. 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). 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. 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.

Dietary phosphorus deprivation induces 25-hydroxyvitamin D(3) 1alpha-hydroxylase gene expression.

Dietary phosphorus deprivation causes hypophosphatemia and an increase in serum 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] concentrations. To determine the molecular mechanisms of this regulation, the effects of dietary phosphorus deprivation and hypophysectomy on 25-hydroxyvitamin D(3) 1alpha-hydroxylase (1alpha-hydroxylase) protein and messenger RNA (mRNA) expression were examined in rats. A low phosphorus diet (LPD) for 4 days resulted in hypophosphatemia and an increase in serum 1,25-(OH)(2)D(3) levels. This increase was caused by the induction of 1alpha-hydroxylase protein and mRNA expression (4- and 10-fold increases, respectively). Administration of the LPD or normal phosphorus diet to hypophysectomized (HPX) rats resulted in hypophosphatemia and suppression of 1alpha-hydroxylase gene expression, indicating that hypophosphatemia itself is not sufficient to induce 1alpha-hydroxylase mRNA expression. Administration of GH to HPX rats fed LPD could partially restore 1alpha-hydroxylase mRNA expression, whereas supplementation with insulin-like growth factor I, T(3), estrogen, or corticosterone had no effect. We also examined Phex gene expression in the bone, because the clinical features of X-linked hypophosphatemia resemble those of HPX rats. Phex mRNA expression, however, was not altered in HPX rats. In conclusion, we demonstrated that the increase in serum 1,25-(OH)(2)D(3) levels caused by dietary phosphorus deprivation is due to the induction of 1alpha-hydroxylase mRNA expression, and this increase is mediated in part by a GH-dependent mechanism.