The role of pyrophosphate/phosphate homeostasis in terminal differentiation and apoptosis of growth plate chondrocytes

Extracellular inorganic phosphate (Pi) concentrations are the highest in the growth plate just before the onset of mineralization. The study reported here demonstrates that Pi not only is required for hydroxyapatite mineral formation but also modulates terminal differentiation and apoptosis of growth plate chondrocytes. Extracellular Pi stimulated terminal differentiation marker gene expression, including the progressive ankylosis gene (ank), alkaline phosphatase (APase), matrix metalloproteinase-13 (MMP-13), osteocalcin, and runx2, mineralization, and apoptosis of growth plate chondrocytes. The stimulatory effect of extracellular Pi on terminal differentiation and apoptosis events of growth plate chondrocytes was dependent on the concentration, the expression levels of type III Na+/Pi cotransporters, and ultimately Pi uptake. A high extracellular Pi concentration was required for the stimulation of apoptosis, whereas lower Pi concentrations were required for the most effective stimulation of terminal differentiation events, including terminal differentiation marker gene expression and mineralization. Suppression of Pit-1 was sufficient to inhibit the stimulatory effects of extracellular Pi on terminal differentiation events. On the other hand, increasing the local extracellular Pi concentration by overexpressing ANK, a protein transporting intracellular PPi to the extracellular milieu where it is hydrolyzed to Pi in the presence of APase, resulted in marked increases of hypertrophic and early terminal differentiation marker mRNA levels, including APase, runx2 and type X collagen, and slight increase of MMP-13 mRNA levels, but decreased osteocalcin mRNA level, a late terminal differentiation markers. In the presence of levamisole, a specific APase inhibitor to prevent hydrolysis of extracellular PPi to Pi, ANK overexpression of growth plate chondrocytes resulted in decreased mRNA levels of hypertrophic and terminal differentiation markers but increased MMP-13 mRNA levels. In conclusion, with extracellular PPi inhibiting and extracellular Pi stimulating hypertrophic and terminal differentiation events, a precise regulation of PPi/Pi homeostasis is required for the spatial and temporal control of terminal differentiation events of growth plate chondrocytes.