Calbindin-D-28k is expressed in osteoblastic cells and suppresses their apoptosis by inhibiting caspase-3 activity

The rate of osteoblast apoptosis is a critical determinant of the rate of b one formation. Because the calcium-binding protein calbindin-D-28k has anti -apoptotic properties in neuronal cells and lymphocytes, we searched for th e presence of this protein in osteoblastic cells and investigated whether i t can modify their response to proapoptotic signals. Calbindin-D-28k was ex pressed at low levels in several osteoblastic cell lines and at high levels in primary cultures of murine osteoblastic cells. Transient transfection o f rat calbindin-D-28k cDNA blocked tumor necrosis factor alpha (TNF alpha)- induced apoptosis in osteoblastic MC3T3-E1 cells, as determined by cell via bility and nuclear morphology of cells cotransfected with the green fluores cent protein targeted to the nucleus, whereas transfection of the empty vec tor had no effect. Calbindin-D-28k levels in several stably transfected MC3 T3-E1 lines were directly related to protection from TNF alpha-induced apop tosis, Purified rat calbindin-D-28k markedly reduced the activity of caspas e-3, a critical molecule for the degradation phase of apoptosis, in a cell- free assay. In addition, cell extracts from MC3T3-E1 cells expressing high levels of calbindin-D-28k decreased caspase-3 activity, compared with extra cts from vector-transfected cells. This effect was apparently unrelated to the calcium binding properties of calbindin, as chelation of calcium by EGT A or addition of other calcium-binding proteins such as calbindin-D-9k, S10 0, calmodulin, and osteocalcin, did not affect caspase-3 activity. Last, ca lbindin-D-28k interacts with the active form of caspase-3 as demonstrated b y a GST pull-down assay. These results demonstrate that calbindin-D-28k is a biosynthetic product of osteoblasts with a role in the regulation of apop tosis. They also reveal that the antiapoptotic properties of calbindin-D-28 k may result not only from calcium buffering but also from the ability of t he protein to interact with and to inhibit caspase-3 activity, a property t hat is independent of its calcium binding capability.