Abnormal differentiation in MC3T3-E1 preosteoblasts expressing a dominant-negative type I collagen mutation.

To examine the effects that an organizing extracellular matrix might have on osteoblast precursors, we created MC3T3-E1 cell lines that stably incorporated a plasmid that expressed pro alpha 1 (I) collagen chains having a truncated triple helical domain. Cells that had incorporated the pro alpha 1 (I) expression plasmid (pMG155) efficiently secreted molecules with shortened pro alpha 1 (I) chains into culture media. Electron micrographs indicated that expression of the minigene dramatically interferes with normal type I collagen fibril architecture. The turnover of newly deposited collagenous matrix as measured by 3[H]-hydroxyproline release was 29% after a 14 day chase in cells expressing the minigene compared to essentially no turnover in control cultures. MC3T3-E1 cells in culture normally demonstrate a time dependent reduction of cell division followed by an increase in osteoblast characteristics. Cell number was consistently 20-25% higher than control in MC3T3-E1 cultures expressing the truncated pro alpha 1 (I) gene but ALP activity was only 45% of control. Secretion and steady state mRNA levels for osteocalcin were over fivefold higher than control cultures but expression of other extracellular matrix components was not changed. These findings demonstrate that osteoblasts require a normally structured collagenous matrix for inhibition of cellular proliferation and subsequent upregulation of ALP. However, in the presence of rapid turnover of osteoblast matrix, the gene for osteocalcin may be upregulated in response to local signals.