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

<div><p>Mutations in <i>PHEX</i> (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 <i>in vitro</i> and <i>in vivo</i> 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 <i>in vitro</i>, 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.</p> </div

Immunohistochemical detection of osteocalcin, DSP and MEPE after treatment with MEPE-ASARM peptides.

<p>SHED cell cultures were maintained in nonmineralizing (NM) or mineralizing (M) conditions in the absence or presence of 20 µM of either phosphorylated (p-ASARM) or nonphosphorylated (np-ASARM) ASARM peptide for 21 days. Immunohistochemistry for osteocalcin, DSP and MEPE was performed on methyl methacrylate sections of 21-day cultures. Osteocalcin immunostaining (top row) is strong in SHEDs (arrows) and nodules (arrowheads) in the M and M+p-ASARM conditions. Immunohistochemistry for DSP shows strong staining in mineralized nodules in both the M and M+np-ASARM conditions. MEPE immunostaining is found in SHEDs and nodules in mineralizing conditions the (M and M+np-ASARM conditions) but is particularly strong in the cultures treated with p-ASARM that do not mineralize (M+p-ASARM). The images presented are representative of all sections examined.</p

Summary of the role of the MEPE-derived ASARM peptide in the etiology of tooth dentin abnormalities in XLH patients.

<p>A: SIBLING proteins containing the ASARM peptide are processed by a multitude of proteolytic enzymes, some of which may release the ASARM peptide or larger protein fragments containing the ASARM peptide into the extracellular matrix (ECM). ASARM and ASARM-containing peptides are inhibitory for mineralization, binding directly to hydroxyapatite (HAP) mineral crystals in bones and teeth. In normal conditions, neutralizing PHEX cleavage of ASARM releases extracellular matrices from this inhibition and mineralization proceeds appropriately. B: In XLH tooth dentin, inactivating mutations in the <i>PHEX</i> gene result in nonfunctional PHEX enzyme that allows HAP crystal-binding, ASARM-containing peptides to accumulate in the dentin thus inhibiting tooth mineralization (pathway 1). Normal PHEX also protects full-length MEPE from cleavage by proteases (cathepsin B), thereby preventing release of mineralization-inhibiting ASARM. In XLH, excessive cleavage of MEPE by proteases (in the absence of functional PHEX) to release the inhibitory ASARM peptide might also contribute to the impaired mineralization of dentin. Finally, ASARM accumulation in XLH may impair dentinogenesis by decreasing odontoblast differentiation and downregulating genes encoding for secreted ECM proteins (pathways 2 and 3), while increasing MEPE expression (pathway 4) which in turn would further exacerbate the XLH hypomineralization tooth phenotype.</p

Light and electron microscopy of the cells, matrix and mineral in 3D SHED cell cultures.

<p>SHED cell cultures maintained in nonmineralizing (NM) or mineralizing (M) conditions in the absence or presence of 20 µM of either phosphorylated (p-ASARM) or nonphosphorylated (np-ASARM) ASARM peptide for 21 days were visualized by light microscopy and by scanning (SEM) and transmission (TEM) electron microscopy. <b>A,B:</b> SEM reveals SHEDs (arrows) well integrated into the collagen scaffold. Mineralization of the cultures appears as nodules within the collagen scaffold (arrowheads) only in the M and the M+np-ASARM conditions. Energy-dispersive X-ray spectroscopy (EDX) for compositional microanalysis of the nodules (performed at the white square) shows major spectral peaks for calcium (Ca) and phosphorus (P) with an acquired Ca/P ratio of 1.67+/−0.05 in both mineralizing conditions where nodules appeared. <b>C:</b> Light microscopy (left panel) and TEM (center and right panel) of the mineralized cultures (M and M+np-ASARM). Mineralized nodules (black box, left panel) are often in close proximity to the SHED cells, and consist of aggregates of multiple, smaller mineralization nodules (arrowheads) and occasional mineralized collagen fibrils (white box center panel, and right panel).</p

Effect of MEPE-ASARM peptides on osteocalcin, DSPP and MEPE expression.

<p>SHED cell cultures were maintained in nonmineralizing (NM) or mineralizing (M) conditions in the absence or presence of 20 µM of either phosphorylated (p-ASARM) or nonphosphorylated (np-ASARM) ASARM peptide for 21 days. Quantitative real-time PCR at day 7, 14 and 21, and Western blotting at day 21, for osteocalcin, DSPP/DSP and MEPE were performed. <b>A,B:</b> Osteocalcin and DSPP/DSP expression are induced under the M condition both at the mRNA and protein levels. This induction is reduced in the presence of both the p-ASARM and np-ASARM peptides. <b>C:</b> mRNA and Western blot analysis for MEPE reveal increased expression of MEPE in all mineralizing conditions from day 7 to day 14 (expression was not detectable at baseline). At day 21, MEPE expression was still strongly upregulated in the M+p-ASARM condition. <i>n</i> = 3, error bars +/− SD, * indicates significant difference (p<0.05) relative to the mineralizing condition without peptides (M).</p

Inhibition of mineralization by phosphorylated MEPE-ASARM peptide in a 3D collagen/tooth slice culture model. A:

<p>Schematic of the 3D culture model. Stem cells from human deciduous exfoliated teeth (SHEDs) were obtained for cell cultures studies from the pulp of caries-free human third molars. Passaged cells (10⁶ SHEDs) seeded into a type I collagen gel scaffold were applied to a human tooth slice with a pulp chamber cavity to mimick the tooth/dentin environment, all of which were supported peripherally by a steel wire mesh to minimize collagen gel contraction. <b>B:</b> Calcium content in the cell/matrix layer determined by flame atomic absorption spectrometry of cultures maintained in NM or M conditions in the absence or presence of p-ASARM or np-ASARM at a concentration of 5, 10 or 20 µM for 21 days. Calcium content significantly decreased in the presence of 20 µM of p-ASARM while the nonphosphorylated form of the peptide had no effect on calcium content of the cultures. <b>C-D:</b> SHED cultures were maintained in nonmineralizing (NM) or mineralizing (M) conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056749#s2" target="_blank">Materials and Methods</a>) in the absence or presence of 20 µM phosphorylated (p-ASARM) or nonphosphorylated (np-ASARM) peptide for 21 days. Mineralized nodules in the extracellular matrix were clearly visible by light microscopy after von Kossa staining in the M and M+np-ASARM. Quantification of mineralized nodules (<b>D</b>) shows that they were essentially undetectable in presence of the p-ASARM peptide, whereas about 40 nodules were counted per slice in the M and M+np-ASARM samples. <i>n</i> = 3, error bars +/− SD, ** indicates significant difference (p<0.01) relative to mineralizing condition without peptide.</p

Effect of MEPE-ASARM peptides on type I collagen and tissue-nonspecific alkaline phosphatase expression. A:

<p>Quantitative real-time PCR (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056749#s2" target="_blank">Materials and Methods</a>) for type I collagen and tissue-nonspecific alkaline phosphatase expression in 3D cultures of SHEDs maintained in nonmineralizing (NM) or mineralizing (M) conditions in the absence or presence of either 20 µM phosphorylated (p-ASARM) or nonphosphorylated (np-ASARM) ASARM peptide for 21 days. mRNA expression levels were upregulated from day 7 to day 14 in M, M+p-ASARM and M+np-ASARM conditions and, at day 21, returned to a level comparable to that of the NM condition, except for alkaline phosphatase which remained high in the M+p-ASARM condition. <i>n</i> = 3, error bars +/− SD, * indicates significant difference (p<0.05) relative to NM condition. <b>B:</b> Sirius Red staining of collagen in deciduous tooth sections from a 3-year-old male XLH patient (upper right panel), permanent tooth sections from a 15-year-old female XLH patient (bottom right panel) and control patient (upper left panel) revealing an intact collagen distribution only where dentin has mineralized in the form of characteristic calcospherites (arrows), and not in the interglobular spaces (asterisks) in the dentin where mineralization is typically impaired in XLH and where matrix degradation occurs. Sirius Red staining of normal dentin (right panel) is generally homogeneous across the tubular dentin. Similarly, von Kossa staining counterstained with toluidine blue in the same XLH permanent tooth sections (bottom left panel) show lack of fusion of calcospherites (arrows) with large non-mineralized interglobular spaces (asterisks) in the dentin. o: odontoblast. pd: predentin. d: dentin. p: pulp.</p

Clinical and biological characteristics of the patients with XLH at the time of tooth extraction.

*<p>(normal range is inferior to 300 IU/L).</p>**<p>DMFT: Decayed Missing Filled permanent Teeth; dft: decayed filled deciduous teeth.</p