Abstract
ABSTRACTBiomineralization is a fundamental process key to the development of the skeleton. The phosphatase orphan phosphatase 1 (PHOSPHO1), which likely functions within extracellular matrix vesicles, has emerged as a critical regulator of biomineralization. However, the biochemical pathways that generate intravesicular PHOSPHO1 substrates are currently unknown. We hypothesized that the enzyme ectonucleotide pyrophosphatase/phosphodiesterase 6 (ENPP6) is an upstream source of the PHOSPHO1 substrate. To test this, we characterized skeletal phenotypes of mice homozygous for a targeted deletion of Enpp6 (Enpp6 −/−). Micro‐computed tomography of the trabecular compartment revealed transient hypomineralization in Enpp6 −/− tibias (p < 0.05) that normalized by 12 weeks of age. Whole‐bone cortical analysis also revealed significantly hypomineralized proximal bone in 4‐ but not 12‐week‐old Enpp6 −/− mice (p < 0.05) compared with WT animals. Back‐scattered SEM revealed a failure in 4‐week‐old trabecular bone of mineralization foci to propagate. Static histomorphometry revealed increased osteoid volume (p > 0.01) and osteoid surface (p < 0.05), which recovered by 12 weeks but was not accompanied by changes in osteoblast or osteoclast number. This study is the first to characterize the skeletal phenotype of Enpp6 −/− mice, revealing transient hypomineralization in young animals compared with WT controls. These data suggest that ENPP6 is important for bone mineralization and may function upstream of PHOSPHO1 as a novel means of generating its substrates inside matrix vesicles. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.
Highlights
Biomineralization of the skeleton is a fundamental process indispensable for health and well-being throughout life
The current leading model for intravesicular PHOSPHO1 function involves the chemical transformation of vesicle membrane constituent phospholipids into appropriate enzymatic substrates for PHOSPHO1.(3,16) An as-yet unidentified enzyme with phospholipase A2 activity is first hypothesized to convert membrane-constituent phosphatidylcholine to lysophosphatidylcholine.[9,16] Several vertebrate PLA2 isoforms are known to be expressed by chondrocytes and osteoblasts and were localized to actively mineralizing matrix in cartilage and metaphyseal bone.[17,18] specific candidates that may function as part of the Matrix vesicles (MVs) mechanism have yet to be identified
Immunofluorescence staining with confocal microscopy revealed expression of Ectonucleotide pyrophosphatase/phosphodiesterase 6 (ENPP6) in mature osteoblasts at trabecular and cortical sites, which exhibited strong tissue nonspecific alkaline phosphatase (TNAP) and PHOSPHO1 colocalization (Fig. 1B)
Summary
Biomineralization of the skeleton is a fundamental process indispensable for health and well-being throughout life. The current leading model for intravesicular PHOSPHO1 function involves the chemical transformation of vesicle membrane constituent phospholipids into appropriate enzymatic substrates for PHOSPHO1.(3,16) An as-yet unidentified enzyme (or group of enzymes) with phospholipase A2 activity is first hypothesized to convert membrane-constituent phosphatidylcholine to lysophosphatidylcholine.[9,16] Several vertebrate PLA2 isoforms are known to be expressed by chondrocytes and osteoblasts and were localized to actively mineralizing matrix in cartilage and metaphyseal bone.[17,18] specific candidates that may function as part of the MV mechanism have yet to be identified. This study aims to characterize the skeletal phenotype of the global ENPP6 KO mouse to give insight into its role in bone formation
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