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

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

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research (2004-03-26)
Yan Liu, Angela Porta, Xiaorong Peng, Kristen Gengaro, Earlene B Cunningham, Hong Li, Luis A Dominguez, Teresita Bellido, Sylvia Christakos
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. 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. 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. 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. Calbindin-D28k, a natural non-oncogenic protein, could be an important target in the therapeutic intervention of glucocorticoid-induced osteoporosis.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-MAP Kinase (ERK-1, 351-368) antibody produced in rabbit, IgG fraction of antiserum, buffered aqueous solution
Sigma-Aldrich
Monoclonal Anti-MAP Kinase, Activated (Diphosphorylated ERK-1&2) antibody produced in mouse, clone MAPK-YT, ascites fluid