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

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.     

Regulation of the Cell Type-specific dentin sialophosphoprotein gene expression in mouse odontoblasts by a novel transcription repressor and an activator CCAAT-binding factor

Dentin sialophosphoprotein (DSPP) is an extracellular matrix protein that is cleaved into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) with a highly restricted expression pattern in tooth and bone. Mutations of the DSPP gene are associated with dentin genetic diseases. Regulation of tissue-specific DSPP expression has not been described. To define the molecular basis of this cell-specific expression, we characterized the promoter responsible for the cell-specific expression of the DSPP gene in odontoblasts. Within this region, DNase I footprinting and electrophoretic mobility shift assays delineated one element that contains an inverted CCAAT-binding factor site and a protein-DNA binding site using nuclear extracts from odontoblasts. A series of competitive electrophoretic mobility shift assay analyses showed that the protein-DNA binding core sequence, ACCCCCA, is a novel site sufficient for protein binding. These two protein-DNA binding sequences are conserved at the same proximal position in the mouse, rat, and human DSPP gene promoters and are ubiquitously present in the promoters of other tooth/bone genes. Mutations of the CCAAT-binding factor binding site resulted in a 5-fold decrease in promoter activity, whereas abolishment of the novel protein-DNA binding site increased promoter activity by about 4.6-fold. In contrast to DSPP, expression levels of the novel protein were significantly reduced during odontoblastic differentiation and dentin mineralization. The novel protein was shown to have a molecular mass of 72 kDa. This study shows that expression of the cell type-specific DSPP gene is mediated by the combination of inhibitory and activating mechanisms.     

Mutant DLX 3 disrupts odontoblast polarization and dentin formation

Tricho-dento-osseous (TDO) syndrome is an autosomal dominant disorder characterized by abnormalities in the thickness and density of bones and teeth. A 4-bp deletion mutation in the Distal-Less 3 (DLX3) gene is etiologic for most cases of TDO. To investigate the in vivo role of mutant DLX3 (MT-DLX3) on dentin development, we generated transgenic (TG) mice expressing MT-DLX3 driven by a mouse 2.3 Col1A1 promoter. Dentin defects were radiographically evident in all teeth and the size of the nonmineralized pulp was enlarged in TG mice, consistent with clinical characteristics in patients with TDO. High-resolution radiography, microcomputed tomography, and SEM revealed a reduced zone of mineralized dentin with anomalies in the number and organization of dentinal tubules in MT-DLX3 TG mice. Histological and immunohistochemical studies demonstrated that the decreased dentin was accompanied by altered odontoblast cytology that included disruption of odontoblast polarization and reduced numbers of odontoblasts. TUNEL assays indicated enhanced odontoblast apoptosis. Expression levels of the apoptotic marker caspase-3 were increased in odontoblasts in TG mice as well as in odontoblastic-like MDPC-23 cells transfected with MT-DLX3 cDNA. Expression of Runx2, Wnt 10A, and TBC1D19 colocalized with DLX3 expression in odontoblasts, and MT-DLX3 significantly reduced expression of all three genes. TBC1D19 functions in cell polarity and decreased TBC1D19 expression may contribute to the observed disruption of odontoblast polarity and apoptosis. These data indicate that MT-DLX3 acts to disrupt odontoblast cytodifferentiation leading to odontoblast apoptosis, and aberrations of dentin tubule formation and dentin matrix production, resulting in decreased dentin and taurodontism.In summary, this TG model demonstrates that MT-DLX3 has differential effects on matrix production and mineralization in dentin and bone and provides a novel tool for the investigation of odontoblast biology.     

Dentin sialoprotein and dentin phosphoprotein have distinct roles in dentin mineralization

Dentin sialophosphoprotein (DSPP), a major non-collagenous matrix protein of odontoblasts, is proteolytically cleaved into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Our previous studies revealed that DSPP null mice display a phenotype similar to human autosomal dominant dentinogenesis imperfecta, in which teeth have widened predentin and irregular dentin mineralization resulting in sporadic unmineralized areas in dentin and frequent pulp exposure. Earlier in vitro studies suggested that DPP, but not DSP, plays a significant role in initiation and maturation of dentin mineralization. However, the precise in vivo roles of DSP and DPP are far from clear. Here we report the generation of DPPcKO mice, in which only DSP is expressed in a DSPP null background, resulting in a conditional DPP knockout. DPPcKO teeth show a partial rescue of the DSPP null phenotype with the restored predentin width, an absence of irregular unmineralized areas in dentin, and less frequent pulp exposure. Micro-computed tomography (micro-CT) analysis of DPPcKO molars further confirmed this partial rescue with a significant recovery in the dentin volume, but not in the dentin mineral density. These results indicate distinct roles of DSP and DPP in dentin mineralization, with DSP regulating initiation of dentin mineralization, and DPP being involved in the maturation of mineralized dentin.     

Macrophage inflammatory protein-3alpha and beta-defensin-2 stimulate dentin sialophosphoprotein gene expression in human pulp cells

Macrophage inflammatory protein (MIP)-3alpha and beta-defensin (BD)-2 have antimicrobial activity and chemotactic activity for immature dendritic cells, natural killer cells, and memory T cells. However, it remains unknown if the widespread effects of these peptides also include an influence on the differentiation of mesenchymal cells. Pulp cells have the capacity to differentiate into odontoblasts and to form dentin. The aim of this study was to determine if inflammatory leukocyte products influence the capacity of pulp cells to differentiate. Dentin sialophosphoprotein (DSPP) is a tooth-specific protein being expressed mostly by odontoblast cells. In the present study, we investigated effects of MIP-3alpha and BD-2 on the DSPP and osteopontin (OPN) gene expression in cultures of human pulp-derived fibroblastic cells (HP cells). HP cells expressed mRNA for the CC chemokine receptor (CCR) 6 to which both MIP-3alpha and BD-2 can bind. Real-time PCR showed that MIP-3alpha and BD-2 significantly increased DSPP mRNA levels, although BD-2 increased DSPP mRNA levels less than MIP-3alpha. MIP-3alpha and BD-2 increased OPN mRNA levels very slightly. MIP-3alpha and BD-2 possessed antibacterial activity against Streptococcus mutans and Lactobacillus casei, which are involved in caries, although the antibacterial activity of MIP-3alpha was lower than that of BD-2. These findings suggest the MIP-3alpha and BD-2 have the ability to stimulate odontoblast differentiation in addition to their more traditional role in inflammation and have potential in the removal of bacteria in infected soft dentin and pulp tissues.     

Rescue of odontogenesis in Dmp1-deficient mice by targeted re-expression of DMP1 reveals roles for DMP1 in early odontogenesis and dentin apposition in vivo

Dentin matrix protein 1 (DMP1) is expressed in both pulp and odontoblast cells and deletion of the Dmp1 gene leads to defects in odontogenesis and mineralization. The goals of this study were to examine how DMP1 controls dentin mineralization and odontogenesis in vivo. Fluorochrome labeling of dentin in Dmp1-null mice showed a diffuse labeling pattern with a 3-fold reduction in dentin appositional rate compared to controls. Deletion of DMP1 was also associated with abnormalities in the dentinal tubule system and delayed formation of the third molar. Unlike the mineralization defect in Vitamin D receptor-null mice, the mineralization defect in Dmp1-null mice was not rescued by a high calcium and phosphate diet, suggesting a different effect of DMP1 on mineralization. Re-expression of Dmp1 in early and late odontoblasts under control of the Col1a1 promoter rescued the defects in mineralization as well as the defects in the dentinal tubules and third molar development. In contrast, re-expression of Dmp1 in mature odontoblasts, using the Dspp promoter, produced only a partial rescue of the mineralization defects. These data suggest that DMP1 is a key regulator of odontoblast differentiation, formation of the dentin tubular system and mineralization and its expression is required in both early and late odontoblasts for normal odontogenesis to proceed.     

TGF-beta3 induces ectopic mineralization in fetal mouse dental pulp during tooth germ development

Several members of the transforming growth factor (TGF)-beta superfamily are expressed in developing teeth from the initiation stage through adulthood. Of those, TGF-beta1 regulates odontoblast differentiation and dentin extracellular matrix synthesis. However, the molecular mechanism of TGF-beta3 in dental pulp cells is not clearly understood. In the present study, beads soaked with human recombinant TGF-beta3 induced ectopic mineralization in dental pulp from fetal mouse tooth germ samples, which increased in a dose-dependent manner. Further, TGF-beta3 promoted mRNA expression, and increased protein levels of osteocalcin (OCN) and type I collagen (COL I) in dental pulp cells. We also observed that the expression of dentin sialophosphoprotein and dentin matrix protein 1 was induced by TGF-beta3 in primary cultured dental pulp cells, however, not in calvaria osteoblasts, whereas OCN, osteopontin and osteonectin expression was increased after treatment with TGF-beta3 in both dental pulp cells and calvaria osteoblasts. Dentin sialoprotein was also partially detected in the vicinity of TGF-beta3 soaked beads in vivo. These results indicate for the first time that TGF-beta3 induces ectopic mineralization through upregulation of OCN and COL I expression in dental pulp cells, and may regulate the differentiation of dental pulp stem cells to odontoblasts.     

Immunohistochemical localization of four and a half LIM domains 2 in the odontoblasts of mature human teeth

Four and a half LIM domains 2 (FHL2) participates in cell differentiation and cancer development of various tissues, possessing dual functions either as an activator or as a repressor depending on the protein partners involved. Recent studies show that FHL2 plays an important role in osteoblast differentiation and bone formation. The present study was to investigate the expression and localization of FHL2 in human pulp-dentin complex by immunohistochemistry. Our results showed that in sound mature human teeth, FHL2 was expressed in odontoblasts and some endothelial cells of blood vessels. Moreover, in carious teeth FHL2 immunoreactivity was detected in odontoblasts, odontoblast-like cells and endothelial cells of blood vessels. FHL2 was mainly distributed in cytosol of the odontoblast cell bodies and partly located in nuclei of odontoblasts, but not in the odontoblast processes. Our data suggest a role of FHL2 in odontoblast differentiation and dentin formation both in normal and in carious teeth.