Matrix gamma-carboxyglutamic acid protein is a key regulator of PTH-mediated inhibition of mineralization in MC3T3-E1 osteoblast-like cells

As part of its overall function as a major regulator of calcium homeostasis , PTH stimulates bone resorption and inhibits osteoblast-mediated biominera lization. To determine the basis for the inhibitory actions of this hormone , we compared the time course of PTH-dependent inhibition of mineralization in MC3T3-E1 osteoblast-like cells with changes in mRNA levels for several extracellular matrix proteins previously associated either with induction o r inhibition of mineralization. Mineralizing activity was rapidly lost in P TH-treated cells (similar to 30% inhibition after 3 h, 50% inhibition at 6 h). Of the proteins examined, changes in matrix gamma -carboxyglutamic acid protein were best correlated with PTH-dependent inhibition of mineralizati on. Matrix gamma -carboxyglutamic acid protein mRNA was rapidly induced 3 h after PTH treatment, with a 6- to S-fold induction seen after 6 h. Local i n vivo injection of PTH over the calvaria of mice also induced a 2-fold inc rease in matrix gamma -carboxyglutamic acid protein mRNA. Warfarin, an inhi bitor of matrix gamma -carboxyglutamic acid protein gamma -carboxylation, r eversed the effects of PTH on mineralization in MC3T3-E1 cells, whereas vit amin K enhanced PTH activity, as would be expected if a gamma -carboxygluta mic acid-containing protein were required for PTH activity. Levels of the o ther mRNAs examined were not well correlated with the observed changes in m ineralization. Osteopontin, an in vitro inhibitor of mineralization, was in duced approximately 4-fold 12 h after PTH addition. Bone sialoprotein mRNA, which encodes an extracellular matrix component most frequently associated with mineral induction, was inhibited by 50% after 12 h of PTH treatment. Osteocalcin mRNA, encoding the other known gamma -carboxyglutamic acid prot ein in bone, was also inhibited by PTH, but, again, with a significantly sl ower time course than was seen for mineral inhibition. Taken together, thes e results show that the rapid inhibition of osteoblast mineralization induc ed by in vitro PTH treatment is at least in part explained by induction of matrix gamma -carboxyglutamic acid protein.