Role of matrix extracellular phosphoglycoprotein in the pathogenesis of X-linked hypophosphatemia

Abstract

X-linked hypophosphatemia (XLH), a disorder characterized by hypophosphatemia, impaired skeletal mineralization, and aberrant regulation of 1, 25(OH)(2)D(3), is caused by inactivating mutations of Phex, which results in the accumulation of putative phosphaturic factors, called phosphatonins. Matrix extracellular phosphoglycoprotein (Mepe) is a proposed candidate for phosphatonin. The authors found that Hyp mice had increased expression of the MEPE and another phosphaturic factor, Fgf23. To establish MEPE's role in the pathogenesis of the XLH, Mepe-deficient mice were back-crossed onto the Hyp mouse homologue of XLH and phenotypes of wild-type, Mepe(-/-), Hyp, and Mepe(-/-)/Hyp mice were examined. Transfer of Mepe deficiency onto the Phex-deficient Hyp mouse background failed to correct hypophosphatemia and aberrant serum 1,25(OH)(2)D(3) levels. Increased Fgf23 levels in Hyp mice were not affected by superimposed Mepe deficiency. In addition, Mepe-deficient Hyp mice retained bone mineralization defects in vivo, characterized by decreased bone mineral density, reduced mineralized trabecular bone volume, lower flexural strength, and histologic evidence of osteomalacia; however, cultures of Hyp-derived bone marrow stromal cells in the absence of Mepe showed improved mineralization and normalization of osteoblast gene expression profiles observed in cells derived from Mepe-null mice. These results demonstrate that MEPE elevation in Hyp mice does not contribute to the hypophosphatemia associated with inactivating Phex mutations and is therefore not phosphatonin.

Figure 1

Representative nondecalcified bone sections of tibias from wild-type (WT) and mutant mice. (A to D) Goldner-stained sections are shown in the upper panel (×500). In Goldner-stained sections, mineralized bone is blue and unmineralized bone is red–brown in color. WT mice have small amounts of osteoid relative to mineralized bone (A). In contrast, Hyp bone has an increased relative osteoid volume (B). Mepe-null mice did not alter the ratio of osteoid to mineralized bone (C). Superimposed Mepe deficiency in Hyp mice (D) failed to correct the hyperosteoidosis, as evidenced by the persistent increase in relative osteoid volume in Mepe^−/−/Hyp mice. (E to H) Villanueava-stained section viewed under florescent light in the lower panels (×500). Wild-type mice have two distinct double labels indicative of normal mineralization (E). Hyp mouse bone has diffuse labels consistent with impaired mineralization (F). Trabecular bone from Mepe-null mice exhibit an apparent increase in mineral apposition, as evidenced by a greater distance between double labels (G). Similar to Hyp mice, combined Mepe^−/−/Hyp mice have diminished florescent labeling of bone (H), indicating that Mepe deficiency failed to correct the mineralization defect in vivo.

Figure 2

BMD and dry ash weight of femurs in WT and mutant mice. (A) BMD of the femur was assessed with the PIXImus mouse densitometer in 13-wk-old male and female WT, Hyp, Mepe^−/−, and Mepe^−/−/Hyp mice. (B) Dry ash weight was assessed in 13-wk-old male and female WT, Hyp, Mepe^−/−, and Mepe^−/−/Hyp mice. The decrease in mineralization of the skeleton in the Hyp compared with WT was confirmed by significantly reduced bone ash weight and BMD; however, Mepe^−/−/Hyp exhibited no significant increment in bone ash weight and BMD compared with Hyp mice. Values sharing the same superscript are not significantly statistic different at P < 0.05.

Figure 3

Three-dimensional structures measured by μCT of the femurs from 13-wk-old male WT and mutant mice. (A to D) Gross appearance of whole femurs. Femurs derived from Hyp mice are short, widened, and bowed, consistent with rickets/osteomalacia. Superimposed Mepe^−/−/Hyp does not alter the gross appearance of the skeleton. (E to H) Sagittal views of femurs. Femurs of Hyp and Mepe^−/−/Hyp mice show unmineralized growth plates and much less trabecular bones compared with WT mice. There are no significant changes between WT and Mepe knockout mice. (I to L) Three-dimensional imagine of trabecular architecture of femoral metaphysis of distal femurs. (M to P) Three-dimensional imagine of cortical bones in mid-shaft of femurs. Cortical thickness is decreased and cortical porosity is increased in both Hyp and Mepe^−/−/Hyp mice, consistent with excessive unmineralized osteoid on the endosteal surfaces and Haversian channels. [brackets] represent widened growth plate.

Figure 4

Effects of Mepe deficiency on Hyp-derived bone marrow stromal cell maturation and mineralization. (A) Alizarin red-S staining of primary bone marrow stromal cells derived from WT, Hyp, Mepe-null, and combined Mepe-null Hyp mice. Compared with WT, bone marrow stromal cells derived from Hyp mice formed less mineralization nodules, whereas the cells from Mepe^−/− mice formed much more mineralized nodules. Mepe^−/−/Hyp mice formed more mineralized nodules compared with cells from Hyp. (B) Quantification of mineralization. The alizarin red-S stain was extracted with 10% cetylpyridinium chloride and quantified by absorbance measurement at 562 nm as described in Materials and Methods. Numeric values represent the mean ± SEM of six wells. Bone marrow-derived mesenchymal stem cells were culture from WT and mutant mice for 14 d in the presence of ascorbic acid and β-glycerophosphate to induce osteoblast differentiation. Values sharing the same letter superscript are not significantly different at P < 0.05 by one-way ANOVA analysis.