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

Abstract

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.

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

“…The presence of HA in MV isolated from 17IIA11 cells grown under osteogenic conditions for 8 days was further confirmed by examining MV dropped onto a mica substrate and imaged by means of AFM, followed by qualitative evaluations of their internal composition by AFM phase analysis (56). MV AFM phase images showed the presence of internal spots with high phase angle (φ) values surrounded by regions with very low φ values (Fig.…”

“…To further confirm the MV secreted by 17IIA11 cells are mineralization-competent, we used transmission electron microscopy (TEM) with electron diffraction (54, 55) and atomic force microscopy (AFM) phase analyses (56). In our previous studies we determined that mineralization of 17IIA11 cells ECM is visible around day 5 of culture under osteogenic conditions (48–50).…”

“…Although these mechanisms still remain poorly deciphered, two recent studies have identified proteins critical for MV formation in odontoblasts: mouse genetic studies uncovered significantly reduced number of MV in mantle dentin and chondrocytes of Phospho1 −/− ;Alpl +/− mice (36, 56), and our group demonstrated that the Trps1 transcription factor is required for MV secretion from 17IIA11 cells as well as for expression of Phospho1 and Alpl (48). While the mechanism by which these phosphatases support secretion of MV is unknown, we speculate that PHOSPHO1, which uses phospholipids as substrates (75, 76), may affect MV secretion by affecting the fluidity of the plasma membrane.…”

Matrix vesicles from chondrocytes and osteoblasts: Their biogenesis, properties, functions and biomimetic models

“…ENPP1 produces PP i as well as P i from adenosine 5 ′ - triphosphate (ATP), while TNAP hydrolyzes both ATP and PP i to form P i [27]. Phosphate transporter 1 (P i T-1) helps incorporate P i into MVs [28, 29] (Fig. 3A), while annexins may be involved in the binding and transport of Ca 2+ and in the biophysical process that initiates mineralization in the MV lumen [30] (Fig.…”

Biophysical aspects of biomineralization

“…Thus, these studies, using an innovative combination of AFM topography and phase imaging, showed that the genetic ablation of PHOSPHO1 impairs MV biogenesis and NC formation. This result, in turn, suggests that the intra-vesicular production of P i is necessary for the correct Ca 2+ /P i stoichiometry for the formation of the NC (Yadav et al 2016).…”