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

SUBMITTER: Yadav MC

PROVIDER: S-EPMC4891278 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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

Yadav Manisha C MC Bottini Massimo M Cory Esther E Bhattacharya Kunal K Kuss Pia P Narisawa Sonoko S Sah Robert L RL Beck Laurent L Fadeel Bengt B Farquharson Colin C Millán José Luis JL

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 20160517 6

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: PHOSPH ...[more]

PMID: 26773408

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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.

Project description:Pathologic calcification of cartilage consists of the formation of basic calcium phosphate (BCP) and/or calcium pyrophosphate dihydrate (CPPD) containing calcium crystals in mature hyaline or articular cartilage and is associated with aging, cartilage injury and likely plays a role in accelerating the pathology of osteoarthritis (OA). The pathways regulating joint calcification, in particular cartilage calcification, are not completely understood, but inflammation and the formation of reactive oxygen species (ROS) are contributory factors. The xanthine oxidase (XO) form of xanthine oxidoreductase (XOR), the key enzyme in xanthine and uric acid metabolism, is a major cellular source of superoxide. We hypothesized that XOR could be implicated in chondrocyte mineralization and cartilage calcification and degradation in OA. We showed both in murine primary chondrocyte and chondrogenic ATDC5 cells, that mineralization was inhibited by two different XOR inhibitors, febuxostat and allopurinol. In addition, XOR inhibition reduced the expression of the pro-mineralizing cytokine interleukin-6 (IL-6). We next generated XOR knock-out chondrocyte cell lines with undetectable XOR expression and XO activity. XOR knock-out chondrocyte cells showed decreased mineralization and reduced alkaline phosphatase (Alp) activity. To assess the precise form of XOR involved, primary chondrocytes of XOR mutant mice expressing either the XDH form (XDH ki) or the XO form (XO ki) were studied. We found that XO ki chondrocytes exhibited increased mineralization compared to XDH ki chondrocytes, and this was associated with enhanced Alp activity, ROS generation and IL-6 secretion. Finally, we found increased XOR expression in damaged vs. undamaged cartilage obtained from OA patients and XOR expression partially co-localized with areas showing pathologic calcification. Altogether, our results suggest that XOR, via its XO form, contribute to chondrocyte mineralization and pathological calcification in OA cartilage.

Dataset Information

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

Publications

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

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