Inositol hexakisphosphate (IP6,
phytic acid) is an endogenous compound present in mammalian cells and tissues. Differentially phosphorylated forms of
inositol are well-documented to have important roles in signal transduction, cell proliferation and differentiation, and IP6 in particular has been suggested to inhibit soft tissue calcification (specifically renal and
vascular calcification) by binding extracellularly to
calcium oxalate and
calcium phosphate crystals. However, the effects of IP6 on bone mineralization are largely unknown. In this study, we used MC3T3-E1 osteoblast cultures to examine the effects of exogenous IP6 on osteoblast function and matrix mineralization. IP6 at physiologic concentrations caused a dose-dependent inhibition of mineralization without affecting cell viability, proliferation or
collagen deposition. Osteoblast
differentiation markers, including tissue-nonspecific
alkaline phosphatase activity,
bone sialoprotein and
osteocalcin mRNA levels, were not adversely affected by IP6 treatment. On the other hand, IP6 markedly increased
protein and
mRNA levels of
osteopontin, a potent inhibitor of crystal growth and matrix mineralization.
Inositol alone (without
phosphate), as well as
inositol hexakis-sulphate, a compound with a high negative charge similar to IP6, had no effect on mineralization or
osteopontin induction. Binding of IP6 to
mineral crystals from the osteoblast cultures, as well as to synthetic
hydroxyapatite crystals, was confirmed by a colorimetric assay for IP6. In summary, IP6 inhibits mineralization of osteoblast cultures by binding to growing crystals through negatively charged
phosphate groups and by induction of inhibitory
osteopontin expression. These data suggest that IP6 may regulate physiologic bone mineralization by directly acting extracellularly, and by serving as a specific signal at the cellular level for the regulation of
osteopontin gene expression.