Expression of matrix metalloproteinases during ascorbate-induced differentiation of osteoblastic MC3T3-E1 cells

The mouse calvarial osteoblast MC3T3-E1 cells released 92 kDa and 68 kDa of gelatinase activities into the conditioned media (CMs) from undifferentiat ed cells. When differentiation was induced by cultivating cells with ascorb ate-2-phosphate (AscP), 68-kDa activity increased significantly in parallel with production of 60-kDa activity. These enzymes required Ca2+ and Zn2+ i ons for their proteolytic activities. The 68-kDa activity was immunological ly identified as latent matrix metalloproteinase 2 (MMP-2). The 92-kDa acti vity was deduced to be latent MMP-9 based on its molecular mass. The 60-kDa activity band was found to possess both gelatin and beta -casein hydrolyzi ng activities, indicating that this activity band might comprise the active form of MMP-2 and latent MMP-13. MC3T3-E1 cells were found to express MMP- 2, MMP-13, and membrane type (MT)1-MMP genes by Northern blotting. MMP-2 wa s expressed constitutively. MMP-13 was up-regulated during the growth with AscP. MT1-MMP expression also was modulated by AscP; at the early stage of differentiation, its messenger RNA (mRNA) level increased and then decrease d gradually to the control level. These changes in the profiles of MMPs obs erved here could be attributed to the maturation of collagenous extracellul ar matrix (ECM) induced by AscP.