Phosphorylated Osteopontin ASARM Peptides Inhibit Mineralization in Osteoblast Cultures

Objectives: The SIBLING family of mineral-regulating proteins, which includes matrix extracellular matrix phosphoglycoprotein (MEPE) and osteopontin (OPN), contain an acidic, serine- and aspartate-rich motif (ASARM). Mice with X-linked hypophosphatemic rickets (Hyp mice), caused by inactivating mutations of the Phex gene, have a marked elevation of MEPE-derived ASARM peptides which inhibit mineralization of bone. It is not known whether ASARM peptides of other SIBLING proteins also possess similar mineralization-inhibiting properties. The OPN ASARM motif shares 60% homology with that of MEPE. We investigated the effect of OPN ASARM (OpnASARM) on mineralization of osteoblast cultures.

Methods: Two differentially phosphorylated OpnASARM peptides were synthesized (with 5 phosphoserines [OA5] or 3 phosphoserines [OA3]) and compared with the effects of nonphosphorylated peptide (OA0). MC3T3-E1 osteoblast cultures were treated with these peptides and with recombinant, soluble secreted PHEX enzyme, and effects on cell proliferation, gene expression and mineralization were observed. Peptide binding assays were also performed using synthetic hydroxyapatite.

Results: Osteoblast culture mineralization was dose-dependently inhibited by the two phosphorylated peptides with OA5 being a more potent inhibitor than OA3; OA0 had no effect on mineralization. Cell proliferation (MTT assay) was normal after treatment with all peptides. Collagen deposition was slightly decreased in OA5-treated cultures, but not in OA3- and OA0-treated cultures. Alkaline phosphatase activity and mRNA expression was unaffected by all peptides whereas osteocalcin expression decreased after OA5 treatment. ENPP1 activity and expression were significantly inhibited by OA5 and OA3, but not by OA0. PHEX enzyme rescued the inhibition of mineralization by OA3 (but not by OA5), showing that PHEX modulates OpnASARM activity in a phosphorylation-dependant manner; binding to synthetic hydroxyapatite crystals was also phosphorylation-dependant.

Conclusions: Phosphorylated OpnASARM peptides affect osteoblast gene expression and bind to hydroxyapatite crystals to inhibit mineralization of osteoblast cultures, and PHEX can rescue this inhibition of mineralization. Supported by CIHR.