PHOSPHO1 deficiency delays osteogenic differentiation of osteoblasts, impairing mineralization and altering the protein composition of matrix vesicles.

Abstract

Matrix vesicles (MVs), a specialized class of extracellular vesicles, play a central role in skeletal mineralization. Phosphoethanolamine/phosphocholine phosphatase 1 (PHOSPHO1) generates inorganic phosphate (Pi) within the MV lumen and is considered essential for the initiation of mineralization. This study investigated the role of PHOSPHO1 in osteoblast differentiation and MV biology using primary calvarial osteoblasts from 6-day-old wild-type (WT) and Phospho1-/- mice. Cells were cultured under osteogenic conditions for 14 and 21 days, followed by analyses of cell viability, mineralization, gene expression, MV biophysical properties, and proteomic analyses. Phospho1-/- osteoblasts exhibited threefold higher mitochondrial activity than WT cells. Mineral deposition was detected only in WT cultures at day 21, indicating impaired mineralization in the absence of PHOSPHO1. Gene expression analysis revealed increased Alpl and Smpd3, and decreased Enpp1 expression in Phospho1-/- osteoblasts. Consistent with these findings, alkaline phosphatase (ALP) activity in MVs increased 7.5-fold between days 14 and 21 in both genotypes. Atomic force microscopy of MVs derived from Phospho1-/- osteoblasts exhibited a 6.5- and 2.1-fold reduction in vesicle volume and surface roughness, respectively, compared with WT MVs. Proteomic analysis revealed reduced tissue non-specific alkaline phosphatase (TNAP) at day 14, followed by increased TNAP levels at day 21, accompanied by decreased collagen XII and collagen VI content in Phospho1-/- MV preparations. Collectively, these findings indicate that cellular adaptation is insufficient to restore mineralization in the absence of PHOSPHO1. Instead, PHOSPHO1 appears to play a critical role in establishing the molecular and biophysical properties of MVs necessary for mineral nucleation and propagation.