MEPE-Derived ASARM Peptide Inhibits Odontogenic Differentiation of Dental Pulp Stem Cells and Impairs Mineralization in Tooth Models of X-Linked Hypophosphatemia

Abstract

Mutations in PHEX (phosphate-regulating gene with homologies to endopeptidases on the X-chromosome) cause X-linked familial hypophosphatemic rickets (XLH), a disorder having severe bone and tooth dentin mineralization defects. The absence of functional PHEX leads to abnormal accumulation of ASARM (acidic serine- and aspartate-rich motif) peptide − a substrate for PHEX and a strong inhibitor of mineralization − derived from MEPE (matrix extracellular phosphoglycoprotein) and other matrix proteins. MEPE-derived ASARM peptide accumulates in tooth dentin of XLH patients where it may impair dentinogenesis. Here, we investigated the effects of ASARM peptides in vitro and in vivo on odontoblast differentiation and matrix mineralization. Dental pulp stem cells from human exfoliated deciduous teeth (SHEDs) were seeded into a 3D collagen scaffold, and induced towards odontogenic differentiation. Cultures were treated with synthetic ASARM peptides (phosphorylated and nonphosphorylated) derived from the human MEPE sequence. Phosphorylated ASARM peptide inhibited SHED differentiation in vitro, with no mineralized nodule formation, decreased odontoblast marker expression, and upregulated MEPE expression. Phosphorylated ASARM peptide implanted in a rat molar pulp injury model impaired reparative dentin formation and mineralization, with increased MEPE immunohistochemical staining. In conclusion, using complementary models to study tooth dentin defects observed in XLH, we demonstrate that the MEPE-derived ASARM peptide inhibits both odontogenic differentiation and matrix mineralization, while increasing MEPE expression. These results contribute to a partial mechanistic explanation of XLH pathogenesis: direct inhibition of mineralization by ASARM peptide leads to the mineralization defects in XLH teeth. This process appears to be positively reinforced by the increased MEPE expression induced by ASARM. The MEPE-ASARM system can therefore be considered as a potential therapeutic target.

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“…1C represents the ASARM peptide signal because of the vast excess of ASARM peptide relative to 15 N-labeled SPR4 peptide (5.8-fold molar excess). The 15 N-HSQC-edited spectra with 15 N-labeled SPR4 peptide confirmed this assertion (Fig. 1D).…”

“…1D). The same interactions analyzed using 15 N-HSQC-edited spectra showed vastly reduced SPR4 line-broadening effects (Fig. 1D).…”

“…Broadening and quenching of spectral peaks as measured using 1 H-NMR even at low concentrations of Gd 3ϩ contrast agent "gadodiamide" occurred. D: in contrast, 15 N-HSQC-edited spectra of the same sample run shows vastly reduced 15 N-labeled SPR4 peptide line broadening effects. For further discussion and explanation, see the text.…”

“…The 1 H-NMR spectra of the ASARM peptide and SPR4 peptide binding complex and the effects of gadodiamide (Omniscan) on T2 relaxation are shown. A 5.8-fold molar excess of ASARM peptide (2.73 mM) to 15 N-labeled SPR4 peptide (0.48 mM) was used as previously described (12). Broadening and quenching of spectral peaks as measured using 1 H-NMR even at low concentrations of Gd 3ϩ contrast agent "gadodiamide" occurred.…”

“…NMR studies: 15 N/ 1 H. All peptides (including 15 N isotopically labeled for NMR) were synthesized by Peptide Group (San Diego, CA) as previously described (12,16,18). Confirmation of SPR4 binding to peptides was obtained using two-dimensional 1 H/ 15 N NMR (12) (Fig.…”