Calbindin-D28k is expressed in osteoblastic cells and suppresses their apoptosis by inhibiting caspase-3 activity

J Biol Chem. 2000 Aug 25;275(34):26328-32. doi: 10.1074/jbc.M003600200.


The rate of osteoblast apoptosis is a critical determinant of the rate of bone formation. Because the calcium-binding protein calbindin-D(28k) has anti-apoptotic properties in neuronal cells and lymphocytes, we searched for the presence of this protein in osteoblastic cells and investigated whether it can modify their response to proapoptotic signals. Calbindin-D(28K) was expressed at low levels in several osteoblastic cell lines and at high levels in primary cultures of murine osteoblastic cells. Transient transfection of rat calbindin-D(28k) cDNA blocked tumor necrosis factor alpha (TNFalpha)-induced apoptosis in osteoblastic MC3T3-E1 cells, as determined by cell viability and nuclear morphology of cells cotransfected with the green fluorescent protein targeted to the nucleus, whereas transfection of the empty vector had no effect. Calbindin-D(28k) levels in several stably transfected MC3T3-E1 lines were directly related to protection from TNFalpha-induced apoptosis. Purified rat calbindin-D(28k) markedly reduced the activity of caspase-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 extracts from vector-transfected cells. This effect was apparently unrelated to the calcium binding properties of calbindin, as chelation of calcium by EGTA or addition of other calcium-binding proteins such as calbindin-D(9k), S100, calmodulin, and osteocalcin, did not affect caspase-3 activity. Last, calbindin-D(28k) interacts with the active form of caspase-3 as demonstrated by 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 apoptosis. They also reveal that the antiapoptotic properties of calbindin-D(28k) may result not only from calcium buffering but also from the ability of the protein to interact with and to inhibit caspase-3 activity, a property that is independent of its calcium binding capability.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Apoptosis*
  • Calbindin 1
  • Calbindins
  • Caspase 3
  • Caspases / metabolism*
  • Cell Line
  • Cell-Free System
  • Enzyme Inhibitors / metabolism*
  • Mice
  • Osteoblasts / metabolism*
  • Protein Binding
  • Rats
  • S100 Calcium Binding Protein G / biosynthesis*
  • S100 Calcium Binding Protein G / genetics
  • S100 Calcium Binding Protein G / metabolism*
  • Transfection
  • Tumor Necrosis Factor-alpha / metabolism


  • Calb1 protein, mouse
  • Calb1 protein, rat
  • Calbindin 1
  • Calbindins
  • Enzyme Inhibitors
  • S100 Calcium Binding Protein G
  • Tumor Necrosis Factor-alpha
  • Casp3 protein, mouse
  • Casp3 protein, rat
  • Caspase 3
  • Caspases