Phosphorylated acidic serine-aspartate-rich MEPE-associated motif peptide from matrix extracellular phosphoglycoprotein inhibits phosphate regulating gene with homologies to endopeptidases on the X-chromosome enzyme activity

Abstract

Inactivating PHEX (phosphate regulating gene with homologies to endopeptidases on the X chromosome) mutations cause X-linked hypophosphatemia in humans and mice (Hyp) through overproduction of fibroblast growth factor 23 (FGF23) a phosphaturic factor, by osteocytes. Matrix extracellular phosphoglycoprotein (MEPE) is also elevated in Hyp and other hypophosphatemic disorders. In addition, the administration of an ASARM (acidic serine-aspartate rich MEPE-associated motif) peptide derived from MEPE causes phosphaturia and inhibits bone mineralization in mice, suggesting that MEPE also plays a role in phosphate homeostasis. Since recent studies found that MEPE binds specifically to PHEX in vitro, we tested the effect of recombinant-MEPE and its ASARM peptide on PHEX enzyme activity in vitro and FGF23 expression in bone marrow stromal cell cultures ex vivo. We found that both recombinant MEPE and synthetic phosphorylated ASARM peptide (ASARM-PO(4)) inhibit PHEX enzyme activities in an in vitro fluorescent-quenched PHEX enzyme activity assay. The ASARM-PO(4) peptide inhibits PHEX enzyme activity in a dose-dependent manner with a K(i) of 128 nM and V(max-i) of 100%. Recombinant MEPE also inhibits PHEX activity (K(i) = 2 nM and V(max-i) = 26%). Long-term bone marrow stromal cell cultures supplemented with 10 microM ASARM-PO(4) peptide resulted in significant elevation of FGF23 transcripts and inhibition of mineralization. These findings suggest that MEPE inhibits mineralization and PHEX activity and leads to increased FGF23 production. The resulting coordination of mineralization and release of a phosphaturic factor by MEPE may serve a physiological role in regulating systemic phosphate homeostasis to meet the needs for bone mineralization.

Figure 1

Kinetics of inhibition of PHEX enzymatic activity by recombinant MEPE. Michaelis–Menton graph (A) and corresponding Lineweaver–Burke plot (B) showing percentage inhibition of secPHEX activity as measured using the quenched fluorescence enzymatic assay (see Materials and Methods). Percentage inhibition represents the percentage change in enzyme velocity per unit time relative to control recombinant secPHEX in the absence of inhibitor peptides (0% inhibition of activity). Recombinant insect expressed MEPE-inhibited secPHEX activity in a dose-dependent and saturable manner with a maximum inhibition (V_max–i) of 25·45% and a K_i of 2·1 nM. Each experimental dose point is statistically significant by one-way ANOVA analysis at P value <0·05.

Figure 2

Kinetics of inhibition of PHEX enzymatic activity by peptides derived from MEPE. (A) Time-course of phosphorylated ASARM peptide (ASARM-PO₄) inhibition of PHEX enzyme activity. Graph shows raw fluorescence data measurements of PHEX fluorogenic enzymatic assay in the absence and in the presence of different concentrations of ASARM-PO₄ peptide. The fluorescence strength in the reactions increased in linear relationship from 0 to 40 min. There was a dose-dependent inhibition of PHEX by ASARM-PO₄ (10–300 nM). (B) Dose-dependent effects of ASARM-PO₄ inhibition of PHEX. Percentage inhibition reflects the inhibition with peptide compared with the enzyme activity without peptide from n=3. (C and D) Kinetics of inhibition of PHEX enzymatic activity inhibition by ASARM-PO₄ peptide. Michaelis–Menton (C) and corresponding Lineweaver–Burke plot (D). RGD and non-phosphorylated ASARM control peptides had no effect on enzyme activity up to micromolar concentrations (data not shown). ASARM-PO₄ inhibited secPHEX activity in a dose-dependent and saturable manner with a maximum inhibition (V_max–i) of 100% and a K_i of 128·7 nM. Each experimental dose point is statistically significant by one-way ANOVA analysis at P value <0·05.

Figure 3

Effects of ASARM-PO₄ on FGF23 expression in BMSC cultures. (A) FGF23 message levels measured by quantitative real-time RT-PCR in BMSC cultures from wild-type (controls), wild-type treated with ASARM-PO₄, and Hyp (positive controls). FGF23 mRNA levels are expressed relative to the levels of the cyclophilin A mRNA. Values represent the mean±S.E.M. (n=3). ASARM-PO₄ significantly stimulated FGF23 mRNA expression. (B) Assessment of ASARM-PO₄ effects on eGFP expression driven by endogenous FGF23 promoter in BMSC cultures. The mineralization nodules in BMSC cultured in differentiation medium for 14 days were viewed under fluorescent microscope (×200). WT control cultures showed no eGFP (left panel), whereas WT BMSCs cultured with the ASARM-PO₄ peptide at 10 μM concentration for 14 days showed demonstrable eGFP-positive cells embedded in the mineralization nodule (middle panel). eGFP expression was less in ASARM-PO₄-treated cultures compared with the abundant eGFP-positive cells in the mineralization nodules in Hyp-derived BMSCs (Hyp), which were used as positive controls (right panel).

Figure 4

Effect of ASARM-PO₄ on BMSC mineralization. Alizarin Red-S staining of mineralized extracellular matrix in BMSC cultures (A) and quantification of mineralization (B). The addition of the ASARM-PO₄ (10 μM) to wild-type BMSCs for 14 days resulted in less mineralization nodules similar to BMSCs derived from HyP mice. Numeric values represent the mean±S.E.M. of Alizarin red-S from a 35 mm plate (n=3).