Rôle d'EMMPRIN et MMPS dans le développement dentaire, la carie dentaire et la régénération pulpo-dentinaire

Tooth development is regulated by a series of reciprocal inductive signalings between the dental epithelium and mesenchyme, which culminates with the formation of dentin and enamel. EMMPRIN/CD147 is an Extracellular Matrix MetalloPRoteinase (MMP) INducer that mediates epithelial-mesenchymal interactions in cancer and other pathological processes and is expressed in developing teeth. Here we used EMMPRIN knockout (KO) mice to determine the functional role of EMMPRIN on dental tissues formation. We demonstrated that EMMPRIN deficiency results in decreased in MMP-3 and MMP-20 expressions, delayed in basement membrane degradation in tooth germ, delayed in enamel formation well distinguishable in incisor, and in decreased enamel volume and thickness but normal maturation. These results indicate that EMMPRIN is involved in the epithelial-mesenchymal cross-talk during tooth development by regulating the expression of MMPs. Then we tried to investigate the potential role of EMMPRIN in the pulp dentin repair process by comparing the healing of injured pulps of EMMPRIN KO and WT mice. Finally, we evaluated the capacity of grape-seed extracts (known to be natural inhibitors of MMPs and used in new daily mouthrinse) to prevent the degradation of human demineralized dentin matrix by MMP-3.Le développement dentaire est orchestré par une série de signalisations inductives réciproques entre l'épithélium dentaire et le mésenchyme, qui conduit à la formation de la dentine et de l'émail. EMMPRIN/CD147 est un INducteur des MetalloPRoteinases de la Matrice Extracellulaire (MMPs) qui régule les interactions épithélio-mésenchymateuses dans le cancer et d'autres processus pathologiques et est exprimé lors du développement dentaire. Ainsi, nous avons utilisé des souris KO pour EMMPRIN pour déterminer le rôle d'EMMPRIN dans la formation des tissus dentaires. Nous avons démontré que l’absence d’EMMPRIN conduisait dans le germe dentaire à une diminution de l’expression de MMP-3 et de MMP-20, à un retard de la dégradation de la membrane basale, à un retard de la formation de l’émail bien visible dans l'incisive à croissance continue, à une diminution du volume et de l'épaisseur d'émail, mais à une maturation amélaire normale. Ces résultats indiquent qu'EMMPRIN est impliqué dans le dialogue épithélio-mésenchymateuse pendant le développement dentaire, principalement par la régulation de l'expression de certaines MMPS. Nous avons ensuite essayé d'évaluer le rôle potentiel d'EMMPRIN dans le processus de réparation dentaire en comparant la cicatrisation de blessures pulpaires des souris KO pour EMMPRIN à des souris WT. Enfin, dans un souci de transfert vers la clinique, nous avons évalué la capacité d’extraits de pépin de raisin (connu pour être des inhibiteurs naturels de MMPs) à empêcher la dégradation de la matrice dentinaire humaine déminéralisée et traitée par MMP-3

Grape seed extracts inhibit dentin matrix degradation by MMP-3

International audienc

EMMPRIN/CD147 deficiency disturbs ameloblast-odontoblast cross-talk and delays enamel mineralization

International audienc

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

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

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

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

Primer design for RT-PCR analysis.

<p>Primer design for RT-PCR analysis.</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