ABNORMAL DIFFERENTIATION IN MC3T3-E1 PREOSTEOBLASTS EXPRESSING A DOMINANT-NEGATIVE TYPE-I COLLAGEN MUTATION

To examine the effects that an organizing extracellular matrix might h ave on osteoblast precursors, we created MC3T3-E1 cell lines that stab ly incorporated a plasmid that expressed pro alpha 1(I) collagen chain s having a truncated triple helical domain, Cells that had incorporate d the pro alpha 1(I) expression plasmid (pMG155) efficiently secreted molecules with shortened proal(I) chains into culture media. Electron micrographs indicated that expression of the minigene dramatically int erferes with normal type I collagen fibril architecture, The turnover of newly deposited collagenous matrix as measured hy (3)[H]-hydroxypro line release was 29% after a 14 day chase in cells expressing the mini gene compared to essentially no turnover in control cultures, MC3T3-E1 cells in culture normally demonstrate a time dependent reduction of c ell division followed by an increase in osteoblast characteristics. Ce ll number was consistently 20-25% higher than control in MC3T3-E1 cult ures expressing the truncated pro alpha 1(I) gene but ALP activity was only 45% of control, Secretion and steady state mRNA levels for osteo calcin were over fivefold higher than control cultures but expression of other extracellular matrix components was not changed, These findin gs demonstrate that osteoblasts require a normally structured collagen ous matrix for inhibition of cellular proliferation and subsequent upr egulation of ALP, However, in the presence of rapid turnover of osteob last matrix, the gene for osteocalcin may be upregulated in response t o local signals.