Bcl-2 rescues ceramide- and etoposide-induced mitochondrial apoptosis through blockage of caspase-2 activation.
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.
Lin
Chiou-Feng
CF
Department of Microbiology and Immunology, National Cheng Kung University Medical College, Tainan, Taiwan.
Chen
Chia-Ling
CL
Chang
Wen-Tsan
WT
Jan
Ming-Shiou
MS
Hsu
Li-Jin
LJ
Wu
Ren-Huang
RH
Fang
Yi-Ting
YT
Tang
Ming-Jer
MJ
Chang
Wen-Chang
WC
Lin
Yee-Shin
YS
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D053143:Caspase 2; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D002518:Ceramides / Q000494:pharmacology*; D017931:DNA Primers; D004789:Enzyme Activation; D005047:Etoposide / Q000494:pharmacology*; D006801:Humans; D008928:Mitochondria / Q000187:drug effects* / Q000201:enzymology; D010766:Phosphorylation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology*
D002518:Ceramides; D017931:DNA Primers; D019253:Proto-Oncogene Proteins c-bcl-2; D005047:Etoposide; D053143:Caspase 2; D020169:Caspases