Bcl-2 rescues ceramide- and etoposide-induced mitochondrial apoptosis through blockage of caspase-2 activation

Chiou Feng Lin, Chia-Ling Chen, Wen-Tsan Chang, Ming-Shiou Jan, Li-Jin Hsu, Ren-Huang Wu, Yi-Ting Fang, Ming-Jer Tang, Wen Chang Chang, Yee-Shin Lin

Research output: Contribution to journalArticle

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Abstract

Recent studies indicate that caspase-2 is involved in the early stage of apoptosis before mitochondrial damage. Although the activation of caspase-2 has been shown to occur in a large protein complex, the mechanisms of caspase-2 activation remain unclear. Here we report a regulatory role of Bcl-2 on caspase-2 upstream of mitochondria. Stress stimuli, including ceramide and etoposide, caused caspase-2 activation, mitochondrial damage followed by downstream caspase-9 and -3 activation, and cell apoptosis in human lung epithelial cell line A549. When A549 cells were pretreated with the caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-fluoromethyl ketone or transfected with caspase-2 short interfering RNA, both ceramide- and etoposide-induced mitochondrial damage and apoptosis were blocked. Overexpression of Bcl-2 prevented ceramide- and etoposide-induced caspase-2 activation and mitochondrial apoptosis. Furthermore, caspase-2 was activated when A549 cells were introduced with Bcl-2 short interfering RNA or were treated with Bcl-2 inhibitor, which provided direct evidence of a negative regulatory effect of Bcl-2 on caspase-2. Cell survival was observed when caspase-2 was inhibited in Bcl-2-silencing cells. Blockage of the mitochondrial permeability transition pore and caspase-9 demonstrated that Bcl-2-modulated caspase-2 activity occurred upstream of mitochondria. Further studies showed that Bcl-2 was dephosphorylated at serine 70 after ceramide and etoposide treatment. A protein phosphatase inhibitor, okadaic acid, rescued Bcl-2 dephosphorylation and blocked caspase-2 activation, mitochondrial damage, and cell death. Taken together, ceramide and etoposide induced mitochondria-mediated apoptosis by initiating caspase-2 activation, which was, at least in part, regulated by Bcl-2.

Original languageEnglish
Pages (from-to)23758-23765
Number of pages8
JournalJournal of Biological Chemistry
Volume280
Issue number25
DOIs
Publication statusPublished - Jun 24 2005

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Caspase 2
Ceramides
Etoposide
Chemical activation
Apoptosis
Mitochondria
Caspase 9
Small Interfering RNA
Okadaic Acid
Caspase Inhibitors
Phosphoprotein Phosphatases
Cell death
Ketones

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bcl-2 rescues ceramide- and etoposide-induced mitochondrial apoptosis through blockage of caspase-2 activation. / Lin, Chiou Feng; Chen, Chia-Ling; Chang, Wen-Tsan; Jan, Ming-Shiou; Hsu, Li-Jin; Wu, Ren-Huang; Fang, Yi-Ting; Tang, Ming-Jer; Chang, Wen Chang; Lin, Yee-Shin.

In: Journal of Biological Chemistry, Vol. 280, No. 25, 24.06.2005, p. 23758-23765.

Research output: Contribution to journalArticle

Lin, Chiou Feng ; Chen, Chia-Ling ; Chang, Wen-Tsan ; Jan, Ming-Shiou ; Hsu, Li-Jin ; Wu, Ren-Huang ; Fang, Yi-Ting ; Tang, Ming-Jer ; Chang, Wen Chang ; Lin, Yee-Shin. / Bcl-2 rescues ceramide- and etoposide-induced mitochondrial apoptosis through blockage of caspase-2 activation. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 25. pp. 23758-23765.
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AU - Lin, Chiou Feng

AU - Chen, Chia-Ling

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AU - Jan, Ming-Shiou

AU - Hsu, Li-Jin

AU - Wu, Ren-Huang

AU - Fang, Yi-Ting

AU - Tang, Ming-Jer

AU - Chang, Wen Chang

AU - Lin, Yee-Shin

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N2 - Recent studies indicate that caspase-2 is involved in the early stage of apoptosis before mitochondrial damage. Although the activation of caspase-2 has been shown to occur in a large protein complex, the mechanisms of caspase-2 activation remain unclear. Here we report a regulatory role of Bcl-2 on caspase-2 upstream of mitochondria. Stress stimuli, including ceramide and etoposide, caused caspase-2 activation, mitochondrial damage followed by downstream caspase-9 and -3 activation, and cell apoptosis in human lung epithelial cell line A549. When A549 cells were pretreated with the caspase-2 inhibitor benzyloxycarbonyl-Val-Asp(-OMe)-Val-Ala-Asp(-OMe)-fluoromethyl ketone or transfected with caspase-2 short interfering RNA, both ceramide- and etoposide-induced mitochondrial damage and apoptosis were blocked. Overexpression of Bcl-2 prevented ceramide- and etoposide-induced caspase-2 activation and mitochondrial apoptosis. Furthermore, caspase-2 was activated when A549 cells were introduced with Bcl-2 short interfering RNA or were treated with Bcl-2 inhibitor, which provided direct evidence of a negative regulatory effect of Bcl-2 on caspase-2. Cell survival was observed when caspase-2 was inhibited in Bcl-2-silencing cells. Blockage of the mitochondrial permeability transition pore and caspase-9 demonstrated that Bcl-2-modulated caspase-2 activity occurred upstream of mitochondria. Further studies showed that Bcl-2 was dephosphorylated at serine 70 after ceramide and etoposide treatment. A protein phosphatase inhibitor, okadaic acid, rescued Bcl-2 dephosphorylation and blocked caspase-2 activation, mitochondrial damage, and cell death. Taken together, ceramide and etoposide induced mitochondria-mediated apoptosis by initiating caspase-2 activation, which was, at least in part, regulated by Bcl-2.

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