THE CALPAIN-CALPASTATIN SYSTEM AND CELLULAR PROLIFERATION AND DIFFERENTIATION IN RODENT OSTEOBLASTIC CELLS

The calpain-calpastatin system, which consists of calpains I and II (t wo ubiquitously distributed. calcium-activated papain like cysteine pr oteases), as well as calpastatin (the endogenous calpain inhibitor), p lays an important role in cell proliferation and differentiation in ma ny tissues. However, its contribution to the regulation of osteoprogen itor or pluripotent stem cell proliferation and differentiation into o steoblasts remains poorly defined. In these studies, rat pluripotent m esodermal cells (ROB-C26) and mouse MC3T3-E1 preosteoblasts were induc ed to differentiate into osteoblasts by long-term culture or in respon se to bone morphogenetic protein (BMP), The occurrence and distributio n of calpain-calpastatin system proteins were determined by immunofluo rescent microscopy, measurement of calcium-dependent proteolytic activ ity, and Western blotting. Treatment of intact MC3T3-E1 cells with an irreversible, membrane-permeable cysteine protease inhibitor attenuate d proliferation and alkaline phosphatase upregulation under differenti ation-enhancing conditions. Calpain II activity increased during diffe rentiation of MC3T3-E1 cells in postconfluent culture. When ROB-C26 ce lls were maintained in long-term culture, neutral protease, calpain I, and calpain II activities increased 2- to 3-fold in the absence of BM P. In the presence of partially purified native BMP, neutral protease and calpain I activities also increased similarly, but calpain II acti vity increased by 10-fold in 3 days. The maximal increase in alkaline phosphatase occurred 4 to 11 days after the calpain II activity had pe aked. Induction of differentiation in long-term MC3T3-E1 cultures was associated with higher calpain II and 70- and 110-kDa calpastatin prot ein levels and lower 17-kDa calpastatin degradation product levels. In conclusion, cysteine protease activity is essential for preosteoblast ic proliferation and differentiation. The calpain-calpastatin system i s regulated during osteoprogenitor proliferation and differentiation, as it is in other cells, and bone morphogenetic protein is a specific regulator of caplain II. (C) 1997 Academic Press.