Dual Role of the Trps1 Transcription Factor in Dentin Mineralization

Kuzynski, Maria; Goss, Morgan; Bottini, Massimo; Yadav, Manisha; Mobley, Callie; Winters, Tony; Poliard, Anne; Kellermann, Odile; Lee, Brendan; Millán, José Luis; Napierala, Dobrawa

doi:10.1074/jbc.m114.550129

Cited by 28 publications

(48 citation statements)

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“…The findings described in this paper enable a mechanistic explanation for three earlier experimental observations: 1) the reduced mineralization and reduced number of MVs in the dentin of [ Phospho1 −/− ; Alpl +/− ] mutant mice (11) ; 2) the reduced mineralization and reduced number of MVs observed in cultured pre-odontoblastic cells deficient in the Trps1 transcription factor, where both PHOSPHO1 and TNAP are downregulated (46) and 3) the synergistic effect of the combined use of PHOSPHO1 and TNAP inhibitors in suppressing vascular smooth muscle cell calcification. (47) All those earlier observations can now be mechanistically understood as follows: the genetic (11, 46) or pharmacological (47) ablation of PHOSPHO1 function impairs MV biogenesis while also diminishing intravesicular P i -generation needed to initiate mineralization as well as causing a downregulation in TNAP expression. (9) In turn, the genetic (4, 9, 46) or pharmacological ablation of TNAP function (47, 48) leads to an increase in extracellular PP i which suppresses extracellular matrix calcification, and also a reduction in perivesicular P i -generation needed for P i T-1 influx (Figure 7).…”

Section: Discussionsupporting

confidence: 60%

“…(47) All those earlier observations can now be mechanistically understood as follows: the genetic (11, 46) or pharmacological (47) ablation of PHOSPHO1 function impairs MV biogenesis while also diminishing intravesicular P i -generation needed to initiate mineralization as well as causing a downregulation in TNAP expression. (9) In turn, the genetic (4, 9, 46) or pharmacological ablation of TNAP function (47, 48) leads to an increase in extracellular PP i which suppresses extracellular matrix calcification, and also a reduction in perivesicular P i -generation needed for P i T-1 influx (Figure 7). The finding reported in this paper that PHOSPHO1 function is involved in MV biogenesis can be exploited pharmacologically for the treatment of arterial calcification and other forms of ectopic ossification.…”

Section: Discussionmentioning

confidence: 99%

“…liposomes), (44) and bacterial capsules (45) and that we recently adapted to the study of MVs. (46) Here, we used AFM analysis to characterize the number, surface morphology and filling of isolated MVs. MVs appeared as flattened globular features (modeled as oblate spheroids) with different sizes and surface morphologies among samples.…”

Section: Discussionmentioning

confidence: 99%

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Skeletal Mineralization Deficits and Impaired Biogenesis and Function of Chondrocyte-Derived Matrix Vesicles in Phospho1–/– and Phospho1/Pit1 Double-Knockout Mice

Yadav

Bottini

Cory

et al. 2016

Journal of Bone and Mineral Research

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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- 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 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 Pit1, alone or in combination with a Phospho1 gene deletion. Pit1col2/col2 mice did not show any major phenotypic abnormalities, while severe skeletal deformities were observed in the [Phospho1−/−; Pit1col2/col2] double knockout mice that were more pronounced than those observed in the Phospho1−/− mice. Histological analysis of [Phospho1−/−; Pit1col2/col2] bones showed growth plate abnormalities with a shorter hypertrophic chondrocyte zone and extensive hyperosteoidosis. The [Phospho1−/−; Pit1col2/col2] skeleton displayed significantly decreases in BV/TV%, trabecular number and bone mineral density, as well as decreased stiffness, decreased strength, and increased post-yield deflection compared to Phospho1−/− mice. Using atomic force microscopy we found that ~80% of [Phospho1−/−; Pit1col2/col2] MVs were devoid of mineral in comparison to ~50 % for the Phospho1−/− MVs and ~25% for the WT and Pit1col2/col2 MVs. We also found a significant decrease in the number of MVs produced by both Phospho1−/− and [Phospho1−/−; Pit1col2/col2] chondrocytes. These data support the involvement of PiT-1 in the initiation of skeletal mineralization and provide compelling evidence that PHOSPHO1 function is involved in MV biogenesis.

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Section: Discussionsupporting

confidence: 60%

Section: Discussionmentioning

confidence: 99%

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Skeletal Mineralization Deficits and Impaired Biogenesis and Function of Chondrocyte-Derived Matrix Vesicles in Phospho1–/– and Phospho1/Pit1 Double-Knockout Mice

Yadav

Bottini

Cory

et al. 2016

Journal of Bone and Mineral Research

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“…Under these conditions, the calcium phosphate deposits in ECM are detected around day 21 of culture in most of the mineralizing cells. In our previous studies, we have shown that these standard osteogenic conditions induce rapid (within 6–8 days) mineralization of 17IIA11 cells, which is accompanied by production of MV (48–50). Therefore, we selected 17IIA11 cells as an in vitro model for studies of mineralization-competent MV biogenesis.…”

Section: Resultsmentioning

confidence: 94%

Phosphate induces formation of matrix vesicles during odontoblast-initiated mineralization in vitro

et al. 2016

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Mineralization is a process of deposition of calcium phosphate crystals within a fibrous extracellular matrix (ECM). In mineralizing tissues, such as dentin, bone and hypertrophic cartilage, this process is initiated by a specific population of extracellular vesicles (EV), called matrix vesicles (MV). Although it has been proposed that MV are formed by shedding of the plasma membrane, the cellular and molecular mechanisms regulating formation of mineralization-competent MV are not fully elucidated. In these studies, 17IIA11, ST2, and MC3T3-E1 osteogenic cell lines were used to determine how formation of MV is regulated during initiation of the mineralization process. In addition, the molecular composition of MV secreted by 17IIA11 cells and exosomes from blood and B16-F10 melanoma cell line was compared to identify the molecular characteristics distinguishing MV from other EV. Western blot analyses demonstrated that MV released from 17IIA11 cells are characterized by high levels of proteins engaged in calcium and phosphate regulation, but do not express the exosomal markers CD81 and HSP70. Furthermore, we uncovered that the molecular composition of MV released by 17IIA11 cells changes upon exposure to the classical inducers of osteogenic differentiation, namely ascorbic acid and phosphate. Specifically, lysosomal proteins Lamp1 and Lamp2a were only detected in MV secreted by cells stimulated with osteogenic factors. Quantitative nanoparticle tracking analyses of MV secreted by osteogenic cells determined that standard osteogenic factors stimulate MV secretion and that phosphate is the main driver of their secretion. On the molecular level, phosphate-induced MV secretion is mediated through activation of extracellular signal-regulated kinases Erk1/2 and is accompanied by re-organization of filamentous actin. In summary, we determined that mineralization-competent MV are distinct from exosomes, and we identified a new role of phosphate in the process of ECM mineralization. These data provide novel insights into the mechanisms of MV formation during initiation of the mineralization process.

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“…Therefore, we focused our further comparative analyses on evaluating Phex and Vdr proteins in mature odontoblasts of WT, Trps1-Tg, and double-Tg mice. For comparison, we included Fam20c, which is also involved in hypophosphatemic rickets but was not affected by Trps1 in vitro (Wang, Wang, Li, et al 2012;Rafaelsen et al 2013;Kuzynski et al 2014).…”

Section: Overexpression Of Trps1 In Secretory Odontoblasts Affects Phmentioning

confidence: 99%

Dspp-independent Effects of Transgenic Trps1 Overexpression on Dentin Formation

et al. 2015

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The Trps1 transcription factor is highly expressed in dental mesenchyme and preodontoblasts, while in mature, secretory odontoblasts, it is expressed at low levels. Previously, we have shown that high Trps1 levels in mature odontoblasts impair their function in vitro and in vivo. Col1a1-Trps1 transgenic (Trps1-Tg) mice demonstrate defective dentin secretion and mineralization, which are associated with significantly decreased Dspp expression due to direct repression of the Dspp gene by Trps1. Here, by crossing Trps1-Tg and Col1a1-Dspp transgenic (Dspp-Tg) mice, we generated Col1a1-Trps1;Col1a1-Dspp double transgenic (double-Tg) mice in which Dspp was restored in odontoblasts overexpressing Trps1. Comparative micro-computed tomography analyses revealed partial correction of the dentin volume and no improvement of dentin mineralization in double transgenic mice in comparison with Trps1-Tg and wild-type (WT) mice. In addition, dentin of double-Tg mice has an irregular mineralization pattern characteristic for dentin in hypophosphatemic rickets. Consistent with this phenotype, decreased levels of Phex, Vdr, and Fam20c proteins are detected in both Trps1-Tg and doubleTg odontoblasts in comparison with WT and Dspp-Tg odontoblasts. This suggests that the Dspp-independent dentin mineralization defects in Trps1-Tg mice are a result of downregulation of a group of proteins critical for mineral deposition within the dentin matrix. In summary, by demonstrating that Trps1 functions as a repressor of later stages of dentinogenesis, we provide functional significance of the dynamic Trps1 expression pattern during dentinogenesis.

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Dual Role of the Trps1 Transcription Factor in Dentin Mineralization

Cited by 28 publications

References 61 publications

Skeletal Mineralization Deficits and Impaired Biogenesis and Function of Chondrocyte-Derived Matrix Vesicles in Phospho1–/– and Phospho1/Pit1 Double-Knockout Mice

Skeletal Mineralization Deficits and Impaired Biogenesis and Function of Chondrocyte-Derived Matrix Vesicles in Phospho1–/– and Phospho1/Pit1 Double-Knockout Mice

Phosphate induces formation of matrix vesicles during odontoblast-initiated mineralization in vitro

Dspp-independent Effects of Transgenic Trps1 Overexpression on Dentin Formation

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