title: Calbindin-D28k is expressed in osteoblastic cells and suppresses their apoptosis by inhibiting caspase-3 activity.
abstract: 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.
authors.affiliation: Division of Endocrinology and Metabolism, the Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences, and Central Arkansas Veteterans Health Care System, Little Rock, Arkansas 72205, USA. tmbellido@life.uams.edu
authors.forename: T
authors.initials: T
authors.lastname: Bellido
authors.forename: M
authors.initials: M
authors.lastname: Huening
authors.forename: M
authors.initials: M
authors.lastname: Raval-Pandya
authors.forename: S C
authors.initials: SC
authors.lastname: Manolagas
authors.forename: S
authors.initials: S
authors.lastname: Christakos
mesh_terms: D000818:Animals; D017209:Apoptosis; D064092:Calbindin 1; D064026:Calbindins; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D002474:Cell-Free System; D004791:Enzyme Inhibitors / Q000378:metabolism*; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D064030:S100 Calcium Binding Protein G / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: C576499:Calb1 protein, mouse; C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D004791:Enzyme Inhibitors; D064030:S100 Calcium Binding Protein G; D014409:Tumor Necrosis Factor-alpha; C505409:Casp3 protein, mouse; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases