Long-chain polyphosphate in osteoblast matrix vesicles: Enrichment and inhibition of mineralization

Background

Inorganic polyphosphate (polyP) is a fundamental and ubiquitous molecule in prokaryotes and eukaryotes. PolyP has been found in mammalian tissues with particularly high levels of long-chain polyP in bone and cartilage where critical questions remain as to its localization and function. Here, we investigated polyP presence and function in osteoblast-like SaOS-2 cells and cell-derived matrix vesicles (MVs), the initial sites of bone mineral formation.

Methods

PolyP was quantified by 4′,6-diamidino-2-phenylindole (DAPI) fluorescence and characterized by enzymatic methods coupled to urea polyacrylamide gel electrophoresis. Transmission electron microscopy and confocal microscopy were used to investigate polyP localization. A chicken embryo cartilage model was used to investigate the effect of polyP on mineralization.

Results

PolyP increased in concentration as SaOS-2 cells matured and mineralized. Particularly high levels of polyP were observed in MVs. The average length of MV polyP was determined to be longer than 196 P_i residues by gel chromatography. Electron micrographs of MVs, stained by two polyP-specific staining approaches, revealed polyP localization in the vicinity of the MV membrane. Additional extracellular polyP binds to MVs and inhibits MV-induced hydroxyapatite formation.

Conclusion

PolyP is highly enriched in matrix vesicles and can inhibit apatite formation. PolyP may be hydrolysed to phosphate for further mineralization in the extracellular matrix.

General significance

PolyP is a unique yet underappreciated macromolecule which plays a critical role in extracellular mineralization in matrix vesicles.