Pyrophosphate Inhibits Mineralization of Osteoblast Cultures by Binding to Mineral, Up-regulating Osteopontin, and Inhibiting Alkaline Phosphatase Activity

Abstract

Inorganic pyrophosphate (PP i ) produced by cells inhibits mineralization by binding to crystals. Its ubiquitous presence is thought to prevent "soft" tissues from mineralizing, whereas its degradation to P i in bones and teeth by tissue-nonspecific alkaline phosphatase (Tnap, Tnsalp, Alpl, Akp2) may facilitate crystal growth. Whereas the crystal binding properties of PP i are largely understood, less is known about its effects on osteoblast activity. We have used MC3T3-E1 osteoblast cultures to investigate the effect of PP i on osteoblast function and matrix mineralization. Mineralization in the cultures was dose-dependently inhibited by PP i . This inhibition could be reversed by Tnap, but not if PP i was bound to mineral. PP i also led to increased levels of osteopontin (Opn) induced via the Erk1/2 and p38 MAPK signaling pathways. Opn regulation by PP i was also insensitive to foscarnet (an inhibitor of phosphate uptake) and levamisole (an inhibitor of Tnap enzymatic activity), suggesting that increased Opn levels did not result from changes in phosphate. Exogenous OPN inhibited mineralization, but dephosphorylation by Tnap reversed this effect, suggesting that OPN inhibits mineralization via its negatively charged phosphate residues and that like PP i , hydrolysis by Tnap reduces its mineral inhibiting potency. Using enzyme kinetic studies, we have shown that PP i inhibits Tnap-mediated P i release from ␤-glycerophosphate (a commonly used source of organic phosphate for culture mineralization studies) through a mixed type of inhibition. In summary, PP i prevents mineralization in MC3T3-E1 osteoblast cultures by at least three different mechanisms that include direct binding to growing crystals, induction of Opn expression, and inhibition of Tnap activity.

Proteolytic processing of osteopontin by PHEX and accumulation of osteopontin fragments in Hyp mouse bone, the murine model of X-linked hypophosphatemia

“…In binding to the apatitic mineral crystals in bone, available data all indicate that OPN is a potent inhibitor of mineralization, where it is thought to guide crystal growth, shape, size, and alignment in a carefully regulated manner. (32,33) Full-length, phosphorylated OPN potently inhibits osteoblast cell culture mineralization, (19) and OPN-deficient mice show increased mineralization of their bones, (34) data consistent with the mineralization-inhibitory effect of OPN.…”

“…Phosphorylated full-length OPN is a potent mineralization inhibitor of osteoblast cell cultures, (19) as are its phosphorylated peptides, (13,15) with some evidence for an inhibitory function in bone in vivo. (34) Thus, both the full-length protein and its peptides appear to be important in the inhibition of mineralization.…”

“…Although changes in OPN expression could indeed affect mineralization, the phosphorylated state of this OPN is also a key factor in determining its mineralization-inhibiting activity. (15,19) It thus seems that both post-translational changes such as phosphorylation, together with proteolytic processing of OPN, affect its mineral-regulating function.…”

“…Post-translational processing of bone proteins implicated in the regulation of mineralization is considerable, and phosphorylation appears to be particularly important. (16)(17)(18)(19) Another processing step likely to be important in modifying the function of bone and tooth matrix proteins is their proteolytic cleavage by enzymes. (20) Such processing can potentially have effects on mineralization by generating constituent bioactive fragments acting locally within the extracellular matrix (such as the ASARM peptide) (13,14,21) or acting both locally and systemically on mineralization as seen for dentin matrix protein 1 (DMP1) after proteolytic processing by bone morphogenetic protein 1 (BMP1).…”

Pyrophosphate: a key inhibitor of mineralisation

“…Osteopontin is a secreted glycoprotein which limits hydroxyapatite formation and deposition [100,101]. Pyrophosphate can induce osteopontin in osteoblasts via the MAPK signalling pathway [28,102] and both Enpp1 -/-and ank/ank mice display reduced osteopontin expression in osteoblasts and decreased serum levels of the protein [28,44,45].…”