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

Yan Liu, Angela Porta, Xiaorong Peng, Kristen Gengaro, Earlene B. Cunningham, Hong Li, Luis A. Dominguez, Teresita Bellido, Sylvia Christakos

Research output: Contribution to journalArticle

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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 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 μM). Calbindin-D 28k 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.

Original languageEnglish (US)
Pages (from-to)479-490
Number of pages12
JournalJournal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research
Volume19
Issue number3
DOIs
StatePublished - Mar 2004
Externally publishedYes

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Calbindin 1
Osteocytes
Osteoblasts
Glucocorticoids
Apoptosis
Caspase 3
Bone and Bones
Mass Spectrometry
Cell Death
Phosphorylation
Calbindins
Calcium-Binding Proteins
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry

Keywords

  • Apoptosis
  • Calbindin-D
  • Caspase 3
  • Extracellular signal regulated kinase 1 and 2
  • Glucocorticoid-induced osteoporosis

ASJC Scopus subject areas

  • Surgery

Cite this

Prevention of glucocorticoid-induced apoptosis in osteocytes and osteoblasts by calbindin-D28k. / Liu, Yan; Porta, Angela; Peng, Xiaorong; Gengaro, Kristen; Cunningham, Earlene B.; Li, Hong; Dominguez, Luis A.; Bellido, Teresita; Christakos, Sylvia.

In: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, Vol. 19, No. 3, 03.2004, p. 479-490.

Research output: Contribution to journalArticle

Liu, Yan ; Porta, Angela ; Peng, Xiaorong ; Gengaro, Kristen ; Cunningham, Earlene B. ; Li, Hong ; Dominguez, Luis A. ; Bellido, Teresita ; Christakos, Sylvia. / Prevention of glucocorticoid-induced apoptosis in osteocytes and osteoblasts by calbindin-D28k. In: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 2004 ; Vol. 19, No. 3. pp. 479-490.
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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 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 μM). Calbindin-D 28k 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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AU - Peng, Xiaorong

AU - Gengaro, Kristen

AU - Cunningham, Earlene B.

AU - Li, Hong

AU - Dominguez, Luis A.

AU - Bellido, Teresita

AU - Christakos, Sylvia

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N2 - 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 μM). Calbindin-D 28k 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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