PHOSPHO1 is essential for mechanically competent mineralization and the avoidance of spontaneous fractures☆,☆☆
Research Highlights
►Phospho1−/− mice bones are undermineralized. ►Phospho1−/− long bones undergo plastic deformation and cannot dissipate energy. ►Lower phosphate and carbonate substitutions in Phospho1−/− mineralized matrix. ►Collagen cross-link ratios may indicate a change in lysyl hydroxylase activity.
Introduction
During bone growth, formation and development, the mineralization of the extracellular matrix (ECM) of both chondrocytes and osteoblasts involves the deposition of crystalline hydroxyapatite (HA) within the interior of membrane-limited matrix-vesicles (MVs) [1], [2], [3]. This process is instigated by the accumulation of Ca2+ and inorganic phosphate (Pi) within MVs resulting in the formation of HA crystals. This initial phase is followed by MV membrane rupture/breakdown and the modulation of ECM composition to further promote propagation of HA outside of the MVs [1], [2], [3]. ECM mineralization is a highly regulated process and chondrocytes, osteoblasts and their derived MVs accomplish this by expressing Pi-transporters for Pi uptake [4], [5], annexin V for Ca2+ influx [6] and regulators of inorganic pyrophosphate (PPi) metabolism [7]. Extracellular PPi is a recognized potent mineralization inhibitor in biological fluids [8] and its concentration is regulated by tissue-nonspecific alkaline phosphatase (TNAP) which hydrolyzes PPi in the ECM to establish a Pi/PPi ratio permissive for the initial formation of HA crystals within MVs [9], [10], [11], [12]. Also, nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) ectoplasmically generates PPi from nucleoside triphosphates [13], and the multiple-pass transmembrane protein ANK mediates intracellular to extracellular channeling of PPi [14], [15].
In addition to its PPi hydrolase activity, TNAP also has recognized ATPase activity [16] and disruption of PPi and/or ATP hydrolysis which may contribute to hypophosphatasia (HPP), an inborn error of metabolism resulting in rickets and osteomalacia [17]. Mice deficient in TNAP function (Akp2−/−) phenocopy infantile HPP i.e. their skeleton at birth is mineralized normally but hypomineralization rapidly ensues within 1–2 weeks of postnatal life before death at postnatal day 20 [18], [19]. The failure of bone to mineralize properly after birth in Akp2−/− mice has been associated with PPi accumulating within the ECM and blocking the propagation of HA in the ECM beyond the confines of the MV membrane [20], [21].
An explanation as to why the skeleton of Akp2−/− mice are normally mineralized at birth has focused on the existence of other phosphatases responsible for MV-mediated ECM mineralization and PHOSPHO1 which was identified 10 years ago is a strong candidate for this missing phosphatase. Since its discovery and characterization [16], [22], [23], [24], [25], [26], [27] we have proposed that PHOSPHO1 is, in part, responsible for Pi accumulation (and HA formation) within the MV through its phosphohydrolase activity towards the membrane phospholipids, phosphoethanolamine and phosphocholine [26], [28]. Consequently, due to its cytosolic localization and its known presence and activity within MVs [24], [25], PHOSPHO1 is likely to be partly responsible for the intravesicular HA formation noted in MVs derived from HPP and Akp2−/− chondrocytes and osteoblasts [20], [21]. The critical importance of PHOSPHO1 for skeletal mineralization has been recently suggested by the use of small molecule compounds to inhibit PHOSPHO1 activity in MVs and developing embryonic chick limbs in vivo [25], [27]. Definitive evidence for a mineralization role of PHOSPHO1 was obtained in a comparison of the bone phenotype of Phospho1−/−, Akp2−/− and Phospho1−/−; Akp2−/− double knockout mice [29]. The Akp2−/− and Phospho1−/− mice are both characterized by lower skeletal mineralization whereas the double ablation of PHOSPHO1 and TNAP leads to the complete absence of skeletal mineralization. These data are strongly supportive of independent, non-redundant mechanisms of action of both phosphatases in the mineralization process [18], [29].
Whilst the functional importance of PHOSPHO1 in regulating skeletal mineralization has now been clearly demonstrated [29] it is still unclear as to how PHOSPHO1 fully contributes to the maintenance of bone quality and ultimately, bone strength. Our aim was to analyze the role of PHOSPHO1 during this developmental phase and not at adulthood where any alterations noted in skeletal integrity may be secondary and a consequence of earlier developmental cues. Such information is essential if we are to understand fully the physiological role of PHOSPHO1 in the maintenance of skeletal integrity and explain the pathological long bone bowing and spontaneous greenstick fractures noted in Phospho1−/− mice [29]. In this paper we conclusively demonstrate that PHOSPHO1 is essential for the proper formation of mechanically competent bones able to withstand habitual load.
Section snippets
Mice and tissues
Phospho1-R74X-null mutant (Phospho1−/−) mice were generated by N-ethyl-N-nitrosourea mutagenesis (ENU) as previously described [29]. We chose to study mice at one month of age as the skeletal abnormalities previously described by us [29] present immediately after birth and during juvenile development. For the study of material and mechanical properties and collagen cross-link analysis, 7 wild-type (WT) and 9 Phospho1−/− 30-day old male mice were euthanized and their right tibia and right femur
The long bones of Phospho1−/− mice do not fracture during 3-point bending
Previous analysis of Phospho1−/− mice revealed reduced accumulation of osteoid in the long bones, reduced ash mineral content and reduced bone mineral density (BMD) of cortical bone [29]. Therefore, the consequence of this hypomineralized matrix on the biomechanical properties of Phospho1−/− bones was first determined. Three-point bending analysis indicated that the failure load was not statistically different in the tibia (P = 0.85) and femur (P = 0.60) between the WT and Phospho1−/− mice (Figs. 1
Discussion
The contribution to bone quality of the skeletal-specific phosphatase, PHOSPHO1, is now emerging [27], [29]. When identified over 10 years ago we hypothesized that PHOSPHO1 was involved in the generation of cytosolic Pi to maintain a Pi/PPi ratio permissive for matrix mineralization [22]. This premise has been upheld in this and other recent studies in which the ablation of PHOSPHO1 function in mice results in a number of skeletal abnormalities that include decreased BMD, osteoidosis and altered
Acknowledgments
The authors are grateful to Ms. Jessica Groos for the maintenance of the mouse colonies at the Sanford-Burnham Medical Research Institute and Miss Elaine Seawright and Mr. Juha-Pekka Miettinen for technical help during the completion of these studies.
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This work was funded by grants DE12889, AR47908 and AR53102 from the NIH, USA, a grant from the Thrasher Research Fund and Institute Strategic Program Grant funding from the Biotechnology and Biological Sciences Research Council, UK.
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Conflict of interest: All authors report no conflict of interest.