Prevention of glucocorticoid-induced apoptosis in osteocytes and osteoblasts by calbindin-D28k

J Bone Miner Res. 2004 Mar;19(3):479-90. doi: 10.1359/JBMR.0301242. Epub 2003 Dec 22.


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.

Introduction: 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.

Materials and 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.

Conclusion: Calbindin-D28k, a natural non-oncogenic protein, could be an important target in the therapeutic intervention of glucocorticoid-induced osteoporosis.

Publication types

  • Research Support, Non-U.S. Gov't
  • 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
  • Carrier Proteins / metabolism
  • Caspase 3
  • Caspase Inhibitors
  • Caspases / metabolism
  • Dexamethasone / antagonists & inhibitors*
  • Glucocorticoids / antagonists & inhibitors*
  • Osteoblasts / cytology
  • Osteoblasts / drug effects*
  • Osteocytes / cytology
  • Osteocytes / drug effects*
  • Phosphorylation
  • Protein Kinase C / physiology
  • Rats
  • S100 Calcium Binding Protein G / metabolism*
  • bcl-Associated Death Protein


  • Bad protein, rat
  • Calb1 protein, rat
  • Calbindin 1
  • Calbindins
  • Carrier Proteins
  • Caspase Inhibitors
  • Glucocorticoids
  • S100 Calcium Binding Protein G
  • bcl-Associated Death Protein
  • Dexamethasone
  • Protein Kinase C
  • Casp3 protein, rat
  • Caspase 3
  • Caspases