EFFECTS OF ASCORBIC-ACID ON COLLAGEN MATRIX FORMATION AND OSTEOBLAST DIFFERENTIATION IN MURINE MC3T3-E1 CELLS

Treatment of mouse MC3T3-E1 cells with ascorbic acid initiates the for mation of a collagenous extracellular matrix and synthesis of several osteoblast-related proteins. We recently showed that ascorbic acid dra matically increases alkaline phosphatase and osteocalcin mRNAs and tha t this induction is blocked by inhibitors of collagen triple-helix for mation (Franceschi and Iyer, J Bone Miner Res 7:235). In the present s tudy, the relationship between collagen matrix formation and osteoblas t-specific gene expression is explored in greater detail. Kinetic stud ies revealed that ascorbic acid increased proline hydroxylation in the intracellular procollagen pool within 1 h and stimulated the cleavage of type I collagen propeptides beginning at 2.5 h. Mature alpha(1)(I) and alpha(2)(I) collagen components were first detected at 10 h and c ontinued to increase in both cell layer and culture medium for up to 7 2 h. Ascorbic acid also increased the rate of procollagen secretion fr om cell layers to culture medium. The secretion of another matrix prot ein, fibronectin, was only slightly affected. Alkaline phosphatase or its mRNA was first detected 2-3 days after ascorbic acid addition, but osteocalcin mRNA was not seen until day 6. Two inhibitors of collagen triple-helix formation, ethyl-3,4-dihydroxybenzoate and 3,4-dehydropr oline, inhibited procollagen hydroxylation and alkaline phosphatase in duction. 3,4-Dehydroproline also inhibited the induction of alkaline p hosphatase and osteocalcin mRNAs. Surprisingly, induction was not bloc ked if cells were exposed to ascorbic acid before inhibitor addition. Alkaline phosphatase was also partially inhibited if cells were grown in the presence of purified bacterial collagenase. These results indic ate that the induction of osteoblast markers by ascorbic acid does not require the continuous hydroxylation and processing of procollagens a nd suggest that a stable, possibly matrix-associated signal is generat ed at early times after ascorbic acid addition that allows subsequent induction of osteoblast-related genes.