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DSPP dosage affects tooth development and dentin mineralization.

ABSTRACT: Dentin Sialoprotein (DSP) and phosphophoryn (PP) are two most dominant non-collagenous proteins in dentin, which are the cleavage products of the DSPP (dentin sialophosphoprotein) precursor protein. The absence of the DSPP gene in DSPP knock-out (KO) mice results in characteristics that are consistent with dentinogenesis imperfecta type III in humans. Symptoms include thin dentin, bigger pulp chamber with frequent pulp exposure as well as abnormal epithelial-mesenchymal interactions, and the appearance of chondrocyte-like cells in dental pulp. To better understand how DSPP influences tooth development and dentin formation, we used a bacterial artificial chromosome transgene construct (BAC-DSPP) that contained the complete DSPP gene and promoter to generate BAC-DSPP transgenic mice directly in a mouse DSPP KO background. Two BAC-DSPP transgenic mouse strains were generated and characterized. DSPP mRNA expression in BAC-DSPP Strain A incisors was similar to that from wild-type (wt) mice. DSPP mRNA expression in BAC-DSPP Strain B animals was only 10% that of wt mice. PP protein content in Strain A incisors was 25% of that found in wt mice, which was sufficient to completely rescue the DSPP KO defect in mineral density, since microCT dentin mineral density analysis in 21-day postnatal animal molars showed essentially identical mineral density in both strain A and wt mice. Strain B mouse incisors, with 5% PP expression, only partially rescued the DSPP KO defect in mineral density, as microCT scans of 21-day postnatal animal molars indicated a reduced dentin mineral density compared to wt mice, though the mineral density was still increased over that of DSPP KO. Furthermore, our findings showed that DSPP dosage in Strain A was sufficient to rescue the DSPP KO defect in terms of epithelial-mesenchymal interactions, odontoblast lineage maintenance, along with normal dentin thickness and normal mineral density while DSPP gene dosage in Strain B only partially rescued the aforementioned DSPP KO defect.

SUBMITTER: Lim D

PROVIDER: S-EPMC8153449 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Tissue-nonspecific alkaline phosphatase (TNAP) is expressed in mineralizing tissues and functions to reduce pyrophosphate (PP(i) ), a potent inhibitor of mineralization. Loss of TNAP function causes hypophosphatasia (HPP), a heritable disorder marked by increased PP(i) , resulting in rickets and osteomalacia. Tooth root cementum defects are well described in both HPP patients and in Alpl(-/-) mice, a model for infantile HPP. In Alpl(-/-) mice, dentin mineralization is specifically delayed in the root; however, reports from human HPP patients are variable and inconsistent regarding dentin defects. In the current study, we aimed to define the molecular basis for changes in dentinogenesis observed in Alpl(-/-) mice. TNAP was found to be highly expressed by mature odontoblasts, and Alpl(-/-) molar and incisor roots featured defective dentin mineralization, ranging from a mild delay to severely disturbed root dentinogenesis. Lack of mantle dentin mineralization was associated with disordered and dysmorphic odontoblasts having disrupted expression of marker genes osteocalcin and dentin sialophosphoprotein. The formation of, initiation of mineralization within, and rupture of matrix vesicles in Alpl(-/-) dentin matrix was not affected. Osteopontin (OPN), an inhibitor of mineralization that contributes to the skeletal pathology in Alpl(-/-) mice, was present in the generally unmineralized Alpl(-/-) mantle dentin at ruptured mineralizing matrix vesicles, as detected by immunohistochemistry and by immunogold labeling. However, ablating the OPN-encoding Spp1 gene in Alpl(-/-) mice was insufficient to rescue the dentin mineralization defect. Administration of bioengineered mineral-targeting human TNAP (ENB-0040) to Alpl(-/-) mice corrected defective dentin mineralization in the molar roots. These studies reveal that TNAP participates in root dentin formation and confirm that reduction of PP(i) during dentinogenesis is necessary for odontoblast differentiation, dentin matrix secretion, and mineralization. Furthermore, these results elucidate developmental mechanisms underlying dentin pathology in HPP patients, and begin to explain the reported variability in the dentin/pulp complex pathology in these patients.

Project description:The periodontal complex is essential for tooth attachment and function and includes the mineralized tissues, cementum and alveolar bone, separated by the unmineralized periodontal ligament (PDL). To gain insights into factors regulating cementum-PDL and bone-PDL borders and protecting against ectopic calcification within the PDL, we employed a proteomic approach to analyze PDL tissue from progressive ankylosis knock-out (Ank-/-) mice, featuring reduced PPi, rapid cementogenesis, and excessive acellular cementum. Using this approach, we identified the matrix protein osteopontin (Spp1/OPN) as an elevated factor of interest in Ank-/- mouse molar PDL. We studied the role of OPN in dental and periodontal development and function. During tooth development in wild-type (WT) mice, Spp1 mRNA was transiently expressed by cementoblasts and strongly by alveolar bone osteoblasts. Developmental analysis from 14 to 240days postnatal (dpn) indicated normal histological structures in Spp1-/- comparable to WT control mice. Microcomputed tomography (micro-CT) analysis at 30 and 90dpn revealed significantly increased volumes and tissue mineral densities of Spp1-/- mouse dentin and alveolar bone, while pulp and PDL volumes were decreased and tissue densities were increased. However, acellular cementum growth was unaltered in Spp1-/- mice. Quantitative PCR of periodontal-derived mRNA failed to identify potential local compensators influencing cementum in Spp1-/- vs. WT mice at 26dpn. We genetically deleted Spp1 on the Ank-/- mouse background to determine whether increased Spp1/OPN was regulating periodontal tissues when the PDL space is challenged by hypercementosis in Ank-/- mice. Ank-/-; Spp1-/- double deficient mice did not exhibit greater hypercementosis than that in Ank-/- mice. Based on these data, we conclude that OPN has a non-redundant role regulating formation and mineralization of dentin and bone, influences tissue properties of PDL and pulp, but does not control acellular cementum apposition. These findings may inform therapies targeted at controlling soft tissue calcification.

Project description:Tooth development is a complex process that is regulated precisely by several signalling pathways and transcription factors. GATA-binding protein 4 (GATA4) is a DNA binding transcription factor, and our previous study showed that GATA4 is a novel regulator of root development. However, it remains unclear whether GATA4 is necessary for odontoblast differentiation and dentin formation. Here, we evaluated the phenotypic changes of Wnt1-Cre;GATA4fl/fl mice. The mutant mice showed defective dentin and short root deformity. The odontoblasts lost polarity instead of exhibiting a shorter height and flattened morphology. Moreover, the expression of several molecules, such as DSPP, COL-1, DCN, and PCNA, were downregulated during mutant tooth development. In vivo, we injected lentivirus to overexpress GATA4 in mice root. The dentin formation and the expression of odonto/osteogenic markers (DSPP, COL-1, DCN) were enhanced in the GATA4 overexpression group. During the in vitro study, the ability of proliferation, migration and odonto/osteogenic differentiation was declined by GATA4 knockdown approach in human dental pulp stem cells (DPSCs). The expression of odonto/osteogenic markers (DSPP, BMP4, RUNX2, OSX, OPN, OCN) was reduced in the shGATA4 group, while overexpressing GATA4 in DPSCs promoted mineralization. Furthermore, an immunoprecipitation-mass spectrometry procedure was used to confirm the interaction between GATA4 and Fructose-1, 6-bisphosphatase 1 (FBP1). We used gain and lose-of-function to delineated the role of GATA4 in regulating FBP1 expression. Knocking down GATA4 in DPSCs resulted in decreased glucose consumption and lactate production. We used small hairpin RNA targeting FBP1 to reduce the expression of FBP1 in DPSCs, which significantly increased glucose consumption and lactate production. Together, the results suggested that GATA4 is important for root formation and odontoblast polarity, as it promotes the growth and differentiation of dental mesenchymal cells around the root and affects the glucose metabolism of DPSCs via the negative regulation of FBP1.

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DSPP dosage affects tooth development and dentin mineralization.

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