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. 2016 Jun;31(6):1275-86.
doi: 10.1002/jbmr.2790. Epub 2016 May 17.

Skeletal Mineralization Deficits and Impaired Biogenesis and Function of Chondrocyte-Derived Matrix Vesicles in Phospho1(-/-) and Phospho1/Pi t1 Double-Knockout Mice

Manisha C Yadav  1 , Massimo Bottini  2   3 , Esther Cory  4 , Kunal Bhattacharya  5 , Pia Kuss  1 , Sonoko Narisawa  1 , Robert L Sah  4 , Laurent Beck  6 , Bengt Fadeel  5 , Colin Farquharson  7 , José Luis Millán  1
Affiliations

Skeletal Mineralization Deficits and Impaired Biogenesis and Function of Chondrocyte-Derived Matrix Vesicles in Phospho1(-/-) and Phospho1/Pi t1 Double-Knockout Mice

Manisha C Yadav et al. J Bone Miner Res. 2016 Jun.

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-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.

Keywords: DEVELOPMENT; GENETIC ANIMAL MODELS; GROWTH PLATE; MOLECULAR PATHWAYS.

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Conflict of interest statement

Conflict of interest: All authors report no conflicts of interest.

Figures

Fig. 1
Fig. 1
Conditional ablation of Pit1 in chondrocytes. (A) Immunohistochemistry using anti-PiT-1 antibody on the vertebral bones of 4-month-old WT and Pit1col2/col2 mice showed reduced PiT-1 expression in the hypertrophic chondrocyte region of Pit1col2/col2 mice while PiT-1 expression in skeletal muscle and hematopoietic cells remained unchanged. BM: bone marrow; SM: smooth muscle. Bar=100 µm. B) qRT-PCR of RNA extracted from 5-day-old chondrocytes (n=3) revealed ~30% residual Pit1 gene expression compared to WT chondrocytes (n=3).
Fig. 2
Fig. 2
Phenotypic abnormalities in the skeleton of 1-month-old [Phospho1−/−; Pit1col2/col2] mice. Radiographic images of representative mice showing worsening of the skeletal abnormalities (arrows) in [Phospho1−/−; Pit1col2/col2] mice as compared to the Phospho1−/− mice (n = 10). Arrows show highly bowed long bones and multiple fractures in the spine and limbs in [Phospho1−/−; Pit1col2/col2] mice.
Fig. 3
Fig. 3
Histomorphometric analyses of 15-day-old tibias of Phospho1−/− and [Phospho1−/−; Pit1col2/col2] mice. Von Kossa/van Gieson staining of the tibial section at the knee joint in WT and Pit1col2/col2 mice showed no statistically significant difference in the growth plate (A) as well as the BV/TV and OV/BV ratios in the trabecular bone (B and C) and the secondary ossification center (D). This analysis showed trabecular bone surrounded by widespread, extended osteoid in Phospho1−/− mice (arrows show smaller growth plate (A) and the areas where the osteoid is present). The [Phospho1−/−; Pit1col2/col2] mice show even smaller growth plate (A) and more unmineralized bone in tibia. A-10X, B,C and D-20X magnification (n = 3).
Fig. 4
Fig. 4
µCT analysis of 1-month-old femurs and tibias of WT, Phospho1−/−, Pit1col2/col2 and [Phospho1−/−; Pit1col2/col2] mice. (A) 3D volume renders of the samples (anterior view-femur, anterior view-tibia, side view–full leg, ,) (B) 2D coronal and transaxial cross-sections of femurs and tibias. Scale bar = 1 mm. Both volume renders and cross-sections show highly bowed/ twisted long bones in [Phospho1−/−; Pit1col2/col2] mice compared to WT, Pit1col2/col2 and even Phospho1−/− mice.
Fig. 5
Fig. 5
Determination of the number of MVs and percentage of filled MVs isolated from chondrocytes of each genotype. A) The Phospho1−/− MV preparations show a statistically significant decrease in the total number of produced MVs compared to those isolated from WT chondrocytes. The [Phospho1−/−; Pit1col2/col2] MV preparations also showed a significant decrease in the number of MVs compared to those isolated from WT and Pit1col2/col2 chondrocytes. The difference between the number of MVs isolated from Phospho1−/− and [Phospho1−/−; Pit1col2/col2] chondrocytes was borderline significant (p=0.056). B) We observed a statistically significant decrease in the number of filled MVs in the [Phospho1−/−; Pit1col2/col2] MV samples compared to the WT, Phospho1−/− and Pit1col2/col2 MV samples.
Fig. 6
Fig. 6
Atomic force microscopy (AFM) images were recorded in non-contact (AAC) mode. Height (A) and volume (B) distributions were calculated for MVs isolated from WT, Phospho1−/−, Pit1col2/col2 and [Phospho1−/−; Pit1col2/col2] mice. MV volumes were calculated by assuming MVs as spheroidal structures (see Supplemental Figure 4B). All peaks were fitted by Gaussian curves. C-F) Images of mineral aggregate-unfilled (C and E) and filled (D and F) WT (C, D and F) and Phospho1−/− (E) MVs. From left to right: topography image, amplitude image and three-dimensional reconstruction of topography image (C and D); three-dimensional reconstructions of topography and phase images (E and F). Scale bars = 100 nm.
Fig. 7
Fig. 7
Schematic detailing our current understanding of the biochemical bases for the steps of MV-mediated initiation of skeletal mineralization. Current data are compatible with the following interpretation: MVs initiate mineral deposition by accumulation of Pi generated intravesicularly by the action of PHOSPHO1 on phosphocholine and also via PiT-1-mediated incorporation of Pi generated extravesicularly by TNAP or NPP1. As shown in the current paper, PHOSPHO1 function also appears to be implicated in the biogenesis of MVs. The extravesicular propagation of mineral onto the collagenous matrix is mainly controlled by the pyrophosphatase activity of TNAP that restricts the concentration of this potent mineralization inhibitor to establish a PPi/Pi ratio conducive for controlled calcification. Additionally, osteopontin (OPN), another potent mineralization inhibitor that binds to HA mineral as soon as it is exposed to the extracellular fluid, also restricts the degree of extracellular matrix mineralization. ECM: extracellular matrix; HA: hydroxyapatite OPN: osteopontin.
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