Dentin matrix protein 1 and phosphate homeostasis are critical for postnatal pulp, dentin and enamel formation

Deletion or mutation of dentin matrix protein 1 （DMP1） leads to hypophosphatemic rickets and defects within the dentin. However, it is largely unknown if this pathological change is a direct role of DMP1 or an indirect role of phosphate （Pi） or both. It has also been previously shown that Klotho-deficient mice, which displayed a high Pi level due to a failure of Pi excretion, causes mild defects in the dentinal structure. This study was to address the distinct roles of DMP1 and Pi homeostasis in cell differentiation, apoptosis and mineralization of dentin and enamel. Our working hypothesis was that a stable Pi homeostasis is critical for postnatal tooth formation, and that DMP1 has an antiapoptotic role in both amelogenesis and dentinogenesis. To test this hypothesis, Dmpl-null （Dmpl-/-）, Klotho-deficient （kl/kl）, Dmpl/Klotho-double-deficient （Dmpl-/-/kl/kl） and wild-type （WT） mice were killed at the age of 6 weeks. Combinations of X-ray, microcomputed tomography （I^CT）, scanning electron microscopy （SEM）, histology, apoptosis and immunohistochemical methods were used for characterization of dentin, enamel and pulp structures in these mutant mice. Our results showed that Dmpl-/- （a low Pi level） or kl/kl（a high Pi level） mice displayed mild dentin defects such as thin dentin and a reduction of dentin tubules. Neither deficient mouse line exhibited any apparent changes in enamel or pulp structure. However, the double-deficient mice （a high Pi level） displayed severe defects in dentin and enamel structures, including loss of dentinal tubules and enamel prisms, as well as unexpected ectopic ossification within the pulp root canal. TUNEL assay showed a sharp increase in apoptotic cells in ameloblasts and odontoblasts. Based on the above findings, we conclude that DMP1 has a protective role for odontoblasts and ameloblasts in a pro-apoptotic environment （a high Pi level）.

Nucleus-targeted Dmp1 transgene fails to rescue dental defects in Dmp1 null mice

Dentin matrix protein 1（DMP1） is essential to odontogenesis. Its mutations in human subjects lead to dental problems such as dental deformities, hypomineralization and periodontal impairment. Primarily, DMP1 is considered as an extracellular matrix protein that promotes hydroxyapatite formation and activates intracellular signaling pathway via interacting with avb3 integrin. Recent in vitro studies suggested that DMP1 might also act as a transcription factor. In this study, we examined whether full-length DMP1 could function as a transcription factor in the nucleus and regulate odontogenesis in vivo. We first demonstrated that a patient with the DMP1M1 V mutation, which presumably causes a loss of the secretory DMP1 but does not affect the nuclear translocation of DMP1, shows a typical rachitic tooth defect. Furthermore, we generated transgenic mice expressingNLSDMP1, in which the endoplasmic reticulum（ER） entry signal sequence of DMP1 was replaced by a nuclear localization signal（NLS） sequence, under the control of a 3.6 kb rat type I collagen promoter plus a 1.6 kb intron 1. We then crossbred theNLSDMP1 transgenic mice with Dmp1 null mice to express the NLSDMP1 in Dmp1-deficient genetic background. Although immunohistochemistry demonstrated thatNLSDMP1 was localized in the nuclei of the preodontoblasts and odontoblasts, the histological, morphological and biochemical analyses showed that it failed to rescue the dental and periodontal defects as well as the delayed tooth eruption in Dmp1 null mice. These data suggest that the full-length DMP1 plays no apparent role in the nucleus during odontogenesis.

Glycosylation of dentin matrix protein 1 is a novel key element for astrocyte maturation and BBB integrity

The blood-brain barrier （BBB） is a tight boundary formed between endothelial cells and astrocytes, which separates and protects brain from most pathogens as well as neural toxins in circulation. However, detailed molecular players involved in formation of BBB are not completely known. Dentin matrix protein I （DMP1）-proteoglycan （PG）, which is known to be involved in mineralization of bones and dentin, is also expressed in soft tissues including brain with unknown functions. In the present study, we reported that DMPI-PG was expressed in brain astrocytes and enriched in BBB units. The only glycosylation site of DMP1 is serine89 （S89） in the N-terminal domain of the protein in mouse. Mutant mice with DMP1 point mutations changing S89 to glycine （S89G）, which completely eradicated glycosylation of the protein, demonstrated severe BBB disruption. Another breed of DMP1 mutant mice, which lacked the C-terminal domain of DMP1, manifested normal BBB function. The polarity of S89G-DMP1 astrocytes was disrupted and cell-cell adhesion was loosened. Through a battery of analyses, we found that DMP1 glycosylation was critically required for astrocyte maturation both in vitro and in vivo. S89G-DMP1 mutant astrocytes failed to express aquaporin 4 and had reduced laminin and ZO1 expression, which resulted in disruption of BBB. Interestingly, overexpression of wild-type DMP1-PG in mouse brain driven by the nestin promoter elevated laminin and ZO1 expression beyond wild type levels and could effectively resisted intravenous mannitol-induced BBB reversible opening. Taken together, our study not only revealed a novel element, i.e., DMP1-PG, that reg- ulated BBB formation, but also assigned a new function to DMP1-PG.

Methods for studying tooth root cementum by light microscopy

The tooth root cementum is a thin, mineralized tissue covering the root dentin that is present primarily as acellular cementum on the cervical root and cellular cementum covering the apical root. While cementum shares many properties in common with bone and dentin, it is a unique mineralized tissue and acellular cementum is critical for attachment of the tooth to the surrounding periodontal ligament （PDL）. Resources for methodologies for hard tissues often overlook cementum and approaches that may be of value for studying this tissue. To address this issue, this report offers detailed methodology, as well as comparisons of several histological and immunohistochemical stains available for imaging the cementum-PDL complex by light microscopy. Notably, the infrequently used Alcian blue stain with nuclear fast red counterstain provided utility in imaging cementum in mouse, porcine and human teeth. While no truly unique extracellular matrix markers have been identified to differentiate cementum from the other hard tissues, immunohistochemistry for detection of bone sialoprotein （BSP）, osteopontin （OPN）, and dentin matrix protein 1 （DMP1） is a reliable approach for studying both acellular and cellular cementum and providing insight into developmental biology of these tissues. Histoloeical and immunohistochemical aooroaches Drovide insight on developmental biology of cementum.