Prevention of Glucocorticoid-Induced Apoptosis in Osteocytes and Osteoblasts by Calbindin-D28k

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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. 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 by32P 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 μM). 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.

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