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B Ecarot

Publications and source records attributed to B Ecarot.

17 recordsLinked to original sources
PubMed2001

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 expressed by osteoblasts in association with mineralization.

Amino Acid Sequence↗
PubMed1999

Evidence for Phex haploinsufficiency in murine X-linked hypophosphatemia.

Mutations in the PHEX gene (phosphate-regulating gene with homology to endopeptidases on the X-chromosome) are responsible for X-linked hypophosphatemia (HYP). We previously reported the full-length coding sequence of murine Phex cDNA and provided evidence of Phex expression in bone and tooth. Here, we report the cloning of the entire 3.5-kb 3'UTR of the Phex gene, yielding a total of 6248 bp for the Phex transcript. Southern blot and RT-PCR analyses revealed that the 3' end of the coding sequence and the 3'UTR of the Phex gene, spanning exons 16 to 22, are deleted in Hyp, the mouse model for HYP. Northern blot analysis of bone revealed lack of expression of stable Phex mRNA from the mutant allele and expression of Phex transcripts from the wild-type allele in Hyp heterozygous females. Expression of the Phex protein in heterozygotes was confirmed by Western analysis with antibodies raised against a COOH-terminal peptide of the mouse Phex protein. Taken together, these results indicate that the dominant pattern of Hyp inheritance in mice is due to Phex haploinsufficiency.

3' Untranslated Regions↗
PubMed1999

1,25-(OH)2D3 down-regulates expression of Phex, a marker of the mature osteoblast.

Mutations in the PHEX/Phex gene, which encodes for a protein with homology to neutral endopeptidases, are responsible for human and murine X-linked hypophosphatemia. The present study examined Phex messenger RNA (mRNA) and protein expression in cultured osteoblasts and its regulation by 1,25-(OH)2D3. Phex mRNA levels were quantitated on Northern blots by densitometric analysis relatively to GAPDH mRNA levels. Immunoreactive Phex protein levels were evaluated by immunoprecipitation using a polyclonal rabbit antiserum raised against a mouse Phex carboxy-terminal peptide. Beta-glycerophosphate-induced matrix mineralization in primary osteoblast cultures was associated with significant increases in Phex mRNA and protein. Phex mRNA and protein levels were low or undetectable in proliferating preosteoblastic MC3T3-E1 cells and dramatically increased concomitantly with initiation of matrix mineralization. The pattern of Phex expression, however, was similar in nonmineralizing cultures grown in the absence of beta-glycerophosphate, indicating that the induction of Phex expression in MC3T3-E1 cells was related to cell differentiation rather than matrix mineralization. 1,25-(OH)2D3 inhibited mineral deposition and down-regulated Phex mRNA and protein expression in a time- and dose-dependent manner. These results indicate that Phex is a marker of the fully differentiated osteoblast and that its expression is stimulated during beta-glycerophosphate-induced mineralization in primary osteoblast cultures and down-regulated by 1,25-(OH)2D3, an inhibitor of matrix mineralization. These findings add support for Phex having an important role in bone mineralization.

Animals↗
PubMed1996

cDNA cloning of the murine Pex gene implicated in X-linked hypophosphatemia and evidence for expression in bone.

The recently identified human PEX gene apparently encodes for a neutral endopeptidase that is mutated in patients with X-linked hypophosphatemia. The 3' and 5' ends of the coding region of PEX have not been cloned, nor has the tissue expression of the gene been identified. Here we report the isolation and characterization of the complete open reading frame of the mouse Pex gene and the demonstration of its expression in bone. Mouse Pex cDNA is predicted to encode a protein of 749 amino acids with 95% identity to the available human PEX sequence and significant homology to members of the membrane-bound metalloendopeptidase family. Northern blot analysis revealed a 6.6-kb transcript in bone and in cultured osteoblasts from normal mice that was not detectable in samples from the Hyp mouse, the murine homolog of human X-linked hypophosphatemia. Pex transcripts were, however, detectable in Hyp bone by RT-PCR amplification. Of particular interest, a cDNA clone from rat incisor shows 93% sequence identity to the 5' end of Pex cDNA, suggesting that Pex may be expressed in another calcified tissue, the tooth. The association of impaired mineralization of bone and teeth and disturbed renal phosphate reabsorption with altered expression of Pex suggests that the Pex gene product may play a critical role in these processes.

Amino Acid Sequence↗
PubMed1996

Fine genetic mapping of the Hyp mutation on mouse chromosome X.

The hypophosphatemic (Hyp) mouse is the murine homolog of hypophosphatemic vitamin-D-resistant rickets (HYP) in human. Despite extensive investigations in the Hyp mouse, the pathophysiology of this X-linked dominant disorder remains unclear. As a first step toward cloning the Hyp gene, we have generated a high-resolution linkage map in the vicinity of the Hyp locus using two independent backcross panels segregating the Hyp mutation, one generated from an interspecific mating between C57BL/6J-Hyp/Hyp and Mus spretus and the other from an intrasubspecific mating between C57BL/6J-Hyp/Hyp and Mus musculus castaneus. Linkage analyses in 1101 backcross progeny using a total of 23 DNA markers favor the following gene order from the centromere: DXMitl3-(DXMit11, DXMit34)-(DXMit36, Alas2)-(Hyp, DXMit8O)-DXMi198-(DXMit28, DXMit33, DXMit7O)-Pdhal-DXMit2O. This study has localized Hyp to a region of approximately 1 cM flanked by the proximal markers DXMit36 and Alas2 and the distal marker DXMit98. One microsatellite marker, DXMit8O, was found to be very tightly linked to Hyp, as it was nonrecombinant with Hyp among all the progeny of both backcrosses corresponding to 1101 meioses.

Animals↗
PubMed1996

Abnormal modulation of serum osteocalcin by dietary phosphate and 1,25-dihydroxyvitamin D3 in the hypophosphatemic mouse.

We evaluated in normal and hypophosphatemic (Hyp) mice whether changes in serum levels of osteocalcin in response to dietary phosphate supplementation, parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) administration were related to perturbations in calcium phosphate homeostasis. In normal mice, serum osteocalcin levels were not altered by phosphate supplementation. In contrast, phosphate supplementation in Hyp mice led to a 2-fold decrease in serum osteocalcin to normal levels after 3 days and to an increase in osteocalcin levels after 14 days. The decrease in osteocalcin was associated with normophosphatemia, severe hypocalcemia, and marked increases in circulating 1,25(OH)2D3 levels, whereas the increase in osteocalcin levels was associated with normophosphatemia and no change in serum calcium and 1,25(OH)2D3. Administration of PTH decreased serum osteocalcin in both genotypes. Infusion of 1,25(OH)2D3 for 3 days elicited increases in serum osteocalcin and calcium levels in normal mice, whereas in Hyp mice it produced significant decreases in osteocalcin levels and no change in serum calcium. However, with a more prolonged infusion of 1,25(OH)2D3, hypercalcemia and increases in serum osteocalcin were induced in mutant mice. Our results suggest that the abnormal osteocalcin response of Hyp mice is not directly attributable to an osteoblast dysfunction but is secondary, at least in part, to perturbations in factors that modulate the osteoblast activity, especially serum calcium and/or PTH.

Analysis of Variance↗
PubMed1995

Effect of 1,25-dihydroxyvitamin D3 treatment on bone formation by transplanted cells from normal and X-linked hypophosphatemic mice.

Bone cells isolated from the Hyp mouse, the murine homologue for hypophosphatemic vitamin D-resistant rickets, produce abnormal bone when transplanted to either normal or phosphate-supplemented Hyp mice. To assess whether correction of the bone formation by mutant cells transplanted into either normal or Hyp mice could be achieved in the presence of supraphysiologic serum concentrations of 1.25-dihydroxyvitamin D3 (1.25-(OH)2D3), recipient mice of both genotypes were infused continuously with 1.25-(OH)2D3 (0.2 micrograms/kg/day). Bone nodules present in transplants recovered after 14 days were characterized by measuring the osteoid thickness and volume. Administration of 1.25-(OH)2D3 to Hyp mice corrected the defective bone formation by normal cells but not by pair-transplanted Hyp cells, despite normalization of serum phosphate levels and 3-fold increases in serum 1.25-(OH)2D3. The osteoid thickness and volume in Hyp transplants into 1.25-(OH)2D3-treated Hyp mice were, however, markedly reduced down to values observed for Hyp transplants into recipient normal mice. Administration of 1.25-(OH)2D3 to normal mice improved further bone formation by mutant cells without affecting that by pair-transplanted normal cells. Administration of 24.25-(OH)2D3 (1 microgram/kg/day) combined with 1.25-(OH)2D3 to recipient mice of both genotypes prevented the sharp fall in serum 24.25-(OH)2D3 but was not more beneficial than 1.25-(OH)2D3 alone for improving bone formation by transplanted Hyp cells. These observations demonstrate an abnormal response of the mutant cells to the extracellular environment and support the concept of an intrinsic osteoblast defect in the Hyp mouse.

24,25-Dihydroxyvitamin D 3↗
PubMed1995

Serum insulin-like growth factor binding protein-3 in the hypophosphatemic mouse: decreased activity and abnormal modulation by dietary phosphate.

The hypophosphatemic mouse, the murine homologue of X-linked hypophosphatemia, is characterized by renal defects in phosphate reabsorption and 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) production and by an osteoblast dysfunction. In view of the potential importance of insulin-like growth factors (IGFs) in the regulation of these processes and the role of IGF-binding proteins (IGFBPs) as modulators of IGF action, we asked whether Hyp mice have alterations in IGFs or IGFBPs. Using specific radioimmunoassays and Western ligand blot analysis, we evaluated serum levels of IGFs (IGF-1 and IGF-II) and IGFBPs, respectively, in normal and Hyp mice. We also examined the effect of dietary phosphatase on these parameters. Serum levels of IGF-1 and IGF-II in Hyp mice were not significantly different from those in normal mice, but IGFBP-3 levels were significantly lower (70% of normal, p < 0.05) in the mutant strain. The other IGFBP species appear unchanged. Phosphate supplementation normalized serum phosphate levels in Hyp mice and elicited a significant decrease in serum IGF-I levels (23%, p < 0.05) and a further deduction in IGFBP-3 (22%, p < 0.02). Phosphate deprivation induced hypophosphatemia IGF-II. The present results indicate that the low serum IGFBP-3 activity in Hyp mice is not related to hypophosphatemia per se. Based on the documented effects of parathyroid hormone (PTH) on IGF-I and IGFBP-3, we propose that the secondary hyperparathyroidism displayed by Hyp mice and its exacerbation by phosphate supplementation may contribute to low IGFBP-3 levels in control Hyp mice and to the decreases in serum IGF-I and IGFBP-3 in phosphate-supplemented Hyp mice.

Analysis of Variance↗
PubMed1994

Phosphate transport by osteoblasts from X-linked hypophosphatemic mice.

Hypophosphatemic vitamin D-resistant rickets is characterized by impaired renal reabsorption of Pi. The underlying mechanism of this abnormality remains unknown. Because the osteoblast is likely a target for the HYP mutation, we investigated the Pi transport activity in osteoblasts isolated from the murine homologue for the human disease, the Hyp mouse. Kinetic analysis of sodium-dependent Pi uptake in quiescent normal and Hyp osteoblasts indicated no significant differences in apparent maximal capacity (Vmax) and apparent affinity (Km) of the carrier for Pi. In rapidly growing cells, higher levels of Pi uptake were observed in mutants cells associated with a 1.4- to 1.7-fold increase in Vmax and no change in Km for Pi. This increase in Pi uptake seemed related to changes in the sodium electrochemical gradient inasmuch as a similar increase was observed in alanine uptake. The adaptive response of sodium-dependent Pi transport to Pi deprivation was not altered in mutant cells relative to normal cells. To test whether the expression of a Pi transport defect was dependent on a humoral factor for its expression, we evaluated the activity of the serum from Hyp mice on Pi transport in osteoblasts from both genotypes. No difference in activity was observed between sera from normal and mutant mice. In summary, cultured osteoblasts derived from Hyp mice did not express impaired sodium-dependent Pi transport when compared with cells from normal mice.

Adaptation, Physiological↗
PubMed1992

Defective bone formation by Hyp mouse bone cells transplanted into normal mice: evidence in favor of an intrinsic osteoblast defect.

The hypophosphatemic (Hyp) mouse is an animal model for human hypophosphatemic vitamin D-resistant rickets. We have reported that bone cells isolated from Hyp mice born to homozygous mutant females produce abnormal bone when transplanted into normal mice. To test whether an environmentally acquired defect of the mutant cells contributed to the impaired bone formation observed in transplants, periostea and osteoblasts from normal and Hyp littermates were transplanted intramuscularly into normal animals. To test more specifically for an hypophosphatemia-induced cell alteration before transplantation, bone cells isolated from phosphate-depleted normal mice were transplanted into normal animals. The bone nodules formed in 2 week transplants were characterized by measuring their osteoid thickness and volume. Impaired bone formation was evidenced in Hyp transplants compared to normal littermate transplants by increased osteoid thickness and volume. In contrast to cells from mutant mice, cells isolated from normal mice with comparable hypophosphatemia produced normal bone. These results indicate that the inability of Hyp osteoblasts to produce normal bone when placed in a normal environment is not the consequence of prior exposure to an altered environmental but likely of an intrinsic cellular abnormality. These observations add further support to the concept that the osteoblast is an important target for the Hyp mutation.

Animals↗
PubMed1992

Effect of dietary phosphate deprivation and supplementation of recipient mice on bone formation by transplanted cells from normal and X-linked hypophosphatemic mice.

The hypophosphatemic (Hyp) mouse is the murine homolog for human hypophosphatemic vitamin D-resistant rickets. We previously reported that bone cells isolated from normal and Hyp mice produced abnormal bone when transplanted intramuscularly into mutant mice. To assess the role of hypophosphatemia on bone formation in transplants, normal and Hyp mouse periostea were pair transplanted into control or phosphate (P)-supplemented Hyp mice and into control or P-deprived normal mice. The bone nodules formed in transplants after 2 weeks were characterized by measuring the thickness of the surrounding osteoid seams and the relative osteoid volume. P restriction in normal recipient mice impaired bone formation by transplanted normal cells and aggravated the defective bone formation by Hyp cells. The osteoid thickness and volume remained significantly higher in Hyp transplants than in normal cotransplants, however. P supplementation of Hyp recipient mice normalized bone formation by transplanted normal cells but not by Hyp cells. However, a marked decrease in osteoid thickness and volume was observed in Hyp transplants down to values observed in normal recipient mice. These results indicate that hypophosphatemia is not the only cause of abnormal bone formation in the Hyp mouse but that an osteoblast dysfunction contributes to the bone disease. These observations further support the concept that the osteoblast may be an important target for the Hyp mutation.

Animals↗
PubMed1992

Abnormal response of osteoblasts from Hyp mice to 1,25-dihydroxyvitamin D3.

To further explore the hypothesis of an osteoblast inappropriate response to 1,25-(OH)2D3 in hypophosphatemic vitamin D-resistant rickets (HYP), osteoblasts were isolated from Hyp mice, the animal model for human HYP, and their response to a physiologic dose of 1,25-(OH)2D3 (10(-10) M) was investigated with respect to alkaline phosphatase (ALP) activity and cell proliferation, and compared to that of normal osteoblasts. Cells in secondary culture were incubated for 72 h while in log phase, with or without 1,25-(OH)2D3, at various medium phosphate (P) concentrations ranging from 0.5 to 4.5 mM. Stimulation of ALP activity and inhibition of cell proliferation was induced by 10(-10)M 1,25-(OH)2D3 in normal cells exposed to medium P concentration corresponding to serum levels observed in normal mice (2.1-2.7 mM P). By contrast, Hyp cells failed to respond to 1,25-(OH)2D3 in that range of P concentrations. Stimulation of ALP activity and inhibition of proliferation of mutant cells were evident at higher medium P concentrations (over 3 mM). 1,25-(OH)2D3 at the supraphysiologic level of 10(-9)M had no consistent effect on ALP activity in normal and Hyp mouse osteoblasts, but inhibited cell proliferation in cultures of both genotypes at all P concentrations tested. These results indicate that extracellular P modulates the action of 1,25-(OH)2D3 on osteoblasts, and that this modulation was altered in osteoblasts from Hyp mice. The failure of Hyp cells to respond to a physiologic dose of 1,25-(OH)2D3 upon normal P concentration may reflect the abnormal response of bone to 1,25-(OH)2D3 observed in Hyp mice and HYP patients.

Alkaline Phosphatase↗
PubMed1991

In vivo osteogenic activity of isolated human bone cells.

Human bone cells were obtained as the outgrowth from cancellous bone fragments pretreated with collagenase and DNase. The osteogenic potential of cells in primary culture was assessed upon intramuscular transplantation into young mice pretreated with cortisone. Transplants were recovered after 2 weeks and examined by light microscopy. Of 34 transplants, 6 showed evidence of osteogenesis and 12 the production of unmineralized matrix. Only cells were observed in the other transplants. In an attempt to find a biochemical marker for osteogenic cells we have assayed medium osteocalcin and alkaline phosphatase activity levels in cultures before transplantation. No correlation was found between the level of expression of the two osteoblast markers and the osteogenic potential of the cells.

Adolescent↗
PubMed1991

Phospholipid changes in the bones of the hypophosphatemic mouse.

The mineral and lipid composition of the bones of 35 day old hypophosphatemic (Hyp/Y) and control (+/Y) mice were compared in order to test the hypothesis that phosphate status has an effect on the complexed acidic phospholipid content of developing bones. The Hyp/Y bones were found to be rachitic and osteomalacic, having significantly reduced mineral content. That mineral was shown by X-ray diffraction to consist of larger/more perfect crystals than that in +/Y animals, indicating either a preference for crystal growth rather than new mineral deposition, or an increased mineral turnover. The increased crystal perfection was confirmed by chemical analyses which showed an increased calcium to phosphorus ratio in the Hyp/Y bones. The bones of Hyp/Y animals had significantly reduced complexed acidic phospholipid contents relative to those of control animals. Since these complexes are believed to play a role in vitro and in vivo mineral deposition, it is suggested that the deficiency of these complexes contributes to the mineralization defect. The magnitude of the complexed acidic phospholipid deficiency in the Hyp/Y animals indicates the importance of phosphate for the formation of these lipids. Although the proportion of phosphatidylserine and phosphatidylinositol and lysophospholipids tended to be reduced in the Hyp/Y bones, the absence of other statistically significant phospholipid abnormalities in the bones and brains of these animals suggests that the lipid defect is not systemic, but is associated with a decrease in phosphate at the site of bone formation.

Animals↗