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Compounded PHOSPHO1/ALPL deficiencies reduce dentin mineralization.

ABSTRACT: Phosphatases are involved in bone and tooth mineralization, but their mechanisms of action are not completely understood. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) regulates inhibitory extracellular pyrophosphate through its pyrophosphatase activity to control mineral propagation in the matrix; mice without TNAP lack acellular cementum, and have mineralization defects in dentin, enamel, and bone. PHOSPHO1 is a phosphatase found within membrane-bounded matrix vesicles in mineralized tissues, and double ablation of Alpl and Phospho1 in mice leads to a complete absence of skeletal mineralization. Here, we describe mineralization abnormalities in the teeth of Phospho1(-/-) mice, and in compound knockout mice lacking Phospho1 and one allele of Alpl (Phospho1(-/-);Alpl(+/-) ). In wild-type mice, PHOSPHO1 and TNAP co-localized to odontoblasts at early stages of dentinogenesis, coincident with the early mineralization of mantle dentin. In Phospho1 knockout mice, radiography, micro-computed tomography, histology, and transmission electron microscopy all demonstrated mineralization abnormalities of incisor dentin, with the most remarkable findings being reduced overall mineralization coincident with decreased matrix vesicle mineralization in the Phospho1(-/-) mice, and the almost complete absence of matrix vesicles in the Phospho1(-/-);Alpl(+/-) mice, whose incisors showed a further reduction in mineralization. Results from this study support prominent non-redundant roles for both PHOSPHO1 and TNAP in dentin mineralization.

SUBMITTER: McKee MD

PROVIDER: S-EPMC3711567 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Compounded PHOSPHO1/ALPL deficiencies reduce dentin mineralization.

McKee M D MD Yadav M C MC Foster B L BL Somerman M J MJ Farquharson C C Millán J L JL

Journal of dental research 20130521 8

Phosphatases are involved in bone and tooth mineralization, but their mechanisms of action are not completely understood. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) regulates inhibitory extracellular pyrophosphate through its pyrophosphatase activity to control mineral propagation in the matrix; mice without TNAP lack acellular cementum, and have mineralization defects in dentin, enamel, and bone. PHOSPHO1 is a phosphatase found within membrane-bounded matrix vesicles in mineralized ti ...[more]

PMID: 23694930

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Project description:We have previously shown that ablation of either the Phospho1 or Alpl gene, encoding PHOSPHO1 and tissue-nonspecific alkaline phosphatase (TNAP) respectively, lead to hyperosteoidosis, but that their chondrocyte-derived and osteoblast-derived matrix vesicles (MVs) are able to initiate mineralization. In contrast, the double ablation of Phospho1 and Alpl completely abolish initiation and progression of skeletal mineralization. We argued that MVs initiate mineralization by a dual mechanism: PHOSPHO1-mediated intravesicular generation of inorganic phosphate (Pi ) and phosphate transporter-mediated influx of Pi . To test this hypothesis, we generated mice with col2a1-driven Cre-mediated ablation of Slc20a1, hereafter referred to as Pi t1, alone or in combination with a Phospho1 gene deletion. Pi t1(col2/col2) mice did not show any major phenotypic abnormalities, whereas severe skeletal deformities were observed in the [Phospho1(-/-) ; Pi t1(col2/col2) ] double knockout mice that were more pronounced than those observed in the Phospho1(-/-) mice. Histological analysis of [Phospho1(-/-) ; Pi t1(col2/col2) ] bones showed growth plate abnormalities with a shorter hypertrophic chondrocyte zone and extensive hyperosteoidosis. The [Phospho1(-/-) ; Pi t1(col2/col2) ] skeleton displayed significant decreases in BV/TV%, trabecular number, and bone mineral density, as well as decreased stiffness, decreased strength, and increased postyield deflection compared to Phospho1(-/-) mice. Using atomic force microscopy we found that ?80% of [Phospho1(-/-) ; Pi t1(col2/col2) ] MVs were devoid of mineral in comparison to ?50% for the Phospho1(-/-) MVs and ?25% for the WT and Pi t1(col2/col2) MVs. We also found a significant decrease in the number of MVs produced by both Phospho1(-/-) and [Phospho1(-/-) ; Pi t1(col2/col2) ] chondrocytes. These data support the involvement of phosphate transporter 1, hereafter referred to as Pi T-1, in the initiation of skeletal mineralization and provide compelling evidence that PHOSPHO1 function is involved in MV biogenesis. © 2016 American Society for Bone and Mineral Research.

Project description: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.

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:PHOSPHO1 is a bone-specific phosphatase implicated in the initiation of inorganic phosphate generation for matrix mineralization. The control of mineralization is attributed to the actions of tissue-nonspecific alkaline phosphatase (TNAP). However, matrix vesicles (MVs) containing apatite crystals are present in patients with hypophosphatasia as well as TNAP null (Akp2(-/-)) mice. It is therefore likely that other phosphatases work with TNAP to regulate matrix mineralization. Although PHOSPHO1 and TNAP expression is associated with MVs, it is not known if PHOSPHO1 and TNAP are coexpressed during the early stages of limb development. Furthermore, the functional in vivo role of PHOSPHO1 in matrix mineralization has yet to be established. Here, we studied the temporal expression and functional role of PHOSPHO1 within chick limb bud mesenchymal micromass cultures and also in wild-type and talpid(3) chick mutants. These mutants are characterized by defective hedgehog signalling and the absence of endochondral mineralization. The ability of in vitro micromass cultures to differentiate and mineralize their matrix was temporally associated with increased expression of PHOSPHO1 and TNAP. Comparable changes in expression were noted in developing embryonic legs (developmental stages 23-36HH). Micromass cultures treated with lansoprazole, a small-molecule inhibitor of PHOSPHO1 activity, or FGF2, an inhibitor of chondrocyte differentiation, resulted in reduced alizarin red staining (P<0.05). FGF2 treatment also caused a reduction in PHOSPHO1 (P<0.001) and TNAP (P<0.001) expression. Expression analysis by whole-mount RNA in situ hybridization correlated with qPCR micromass data and demonstrated the existence of a tightly regulated pattern of Phospho1 and Tnap expression which precedes mineralization. Treatment of developing embryos for 5 days with lansoprazole completely inhibited mineralization of all leg and wing long bones as assessed by alcian blue/alizarin red staining. Furthermore, long bones of the talpid(3) chick mutant did not express Phospho1 or Tnap whereas flat bones mineralized normally and expressed both phosphatases. In conclusion, this study has disclosed that PHOSPHO1 expression mirrors that of TNAP during embryonic bone development and that PHOSPHO1 contributes to bone mineralization in developing chick long bones.

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Compounded PHOSPHO1/ALPL deficiencies reduce dentin mineralization.

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Compounded PHOSPHO1/ALPL deficiencies reduce dentin mineralization.

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