title: Prevention of glucocorticoid-induced apoptosis in osteocytes and osteoblasts by calbindin-D28k.
abstract: This study show for the first time that calbindin-D28k can prevent glucocorticoid-induced bone cell death. The anti-apoptotic effect of calbindin-D28k involves inhibition of glucocorticoid induced caspase 3 activation as well as ERK activation.
  
  
  BACKGROUND
  Recent studies have indicated that deleterious effects of glucocorticoids on bone involve increased apoptosis of osteocytes and osteoblasts. Because the calcium-binding protein calbindin-D28k has been reported to be anti-apoptotic in different cell types and in response to a variety of insults, we investigated whether calbindin-D28k could protect against glucocorticoid-induced cell death in bone cells.
  
  
  METHODS
  Apoptosis was induced by addition of dexamethasone (dex; 10-6 M) for 6 h to MLO-Y4 osteocytic cells as well as to osteoblastic cells. Apoptosis percentage was determined by examining the nuclear morphology of transfected cells. Caspase 3 activity was evaluated in bone cells and in vitro. SELDI mass spectrometry (MS) was used to examine calbindin-D28k-caspase 3 interaction. Phosphorylation of calbindin-D28k was examined by 32P incorporation as well as by MALDI-TOF MS. ERK activation was determined by Western blot.
  
  
  RESULTS
  The pro-apoptotic effect of dex in MLO-Y4 cells was completely inhibited in cells transfected with calbindin-D28k cDNA (5.6% apoptosis in calbindin-D28k transfected cells compared with 16.2% apoptosis in vector-transfected cells, p < 0.05). Similar results were observed in osteoblastic cells. We found that dex-induced apoptosis in bone cells was accompanied by an increase in caspase 3 activity. This increase in caspase 3 activity was inhibited in the presence of calbindin-D28k. In vitro assays indicated a concentration-dependent inhibition of caspase 3 by calbindin-D28k (Ki = 0.22 microM). Calbindin-D28k was found to inhibit caspase 3 specifically because the activity of other caspases was unaffected by calbindin-D28k. The anti-apoptotic effect of calbindin-D28k in response to dex was also reproducibly associated with an increase in the phosphorylation of ERK 1 and 2, suggesting that calbindin-D28k affects more than one signal in the glucocorticoid-induced apoptotic pathway.
  
  
  CONCLUSIONS
  Calbindin-D28k, a natural non-oncogenic protein, could be an important target in the therapeutic intervention of glucocorticoid-induced osteoporosis.
authors.affiliation: Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.forename: Angela
authors.initials: A
authors.lastname: Porta
authors.forename: Xiaorong
authors.initials: X
authors.lastname: Peng
authors.forename: Kristen
authors.initials: K
authors.lastname: Gengaro
authors.forename: Earlene B
authors.initials: EB
authors.lastname: Cunningham
authors.forename: Hong
authors.initials: H
authors.lastname: Li
authors.forename: Luis A
authors.initials: LA
authors.lastname: Dominguez
authors.forename: Teresita
authors.initials: T
authors.lastname: Bellido
authors.forename: Sylvia
authors.initials: S
authors.lastname: Christakos
mesh_terms: D000818:Animals; D017209:Apoptosis; D064092:Calbindin 1; D064026:Calbindins; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D003907:Dexamethasone / Q000037:antagonists & inhibitors*; D005938:Glucocorticoids / Q000037:antagonists & inhibitors*; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects*; D010011:Osteocytes / Q000166:cytology / Q000187:drug effects*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology; D051381:Rats; D064030:S100 Calcium Binding Protein G / Q000378:metabolism*; D051025:bcl-Associated Death Protein
chemical_list: C494821:Bad protein, rat; C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D002352:Carrier Proteins; D061945:Caspase Inhibitors; D005938:Glucocorticoids; D064030:S100 Calcium Binding Protein G; D051025:bcl-Associated Death Protein; D003907:Dexamethasone; D011493:Protein Kinase C; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases