Reduction of caspase-8 and -9 cleavage is associated with increased c-FLIP and increased binding of Apaf-1 and Hsp70 after neonatal hypoxic/ischemic injury in mice overexpressing Hsp70.
OBJECTIVE
Caspase-8 and caspase-9 are essential proteases of the extrinsic and intrinsic apoptotic pathways, respectively. We investigated whether neuroprotection associated with overexpression of heat-shock protein 70 (Hsp70), a natural cellular antiapoptotic protein, is mediated by caspase-8 and caspase-9 signaling in the neonatal mouse brain after hypoxia/ischemia (H/I) injury.


METHODS
Postnatal day 7 transgenic mice overexpressing rat Hsp70 (Hsp70 Tg) and their wild-type (Wt) littermates underwent unilateral common carotid artery ligation followed by 30 minutes of exposure to 8% O2. The expression of apoptotic proteins was quantified by Western blot analysis, and the specific interaction between Hsp70 and apoptotic protease activating factor 1 (Apaf-1) was determined by coimmunoprecipitation.


RESULTS
Hsp70 overexpression reduced cytosolic translocation of cytochrome c without affecting the levels of Apaf-1 and pro-caspase-9 24 hours after H/I. The expression of these apoptotic proteins in the naïve neonatal brains was also not affected by Hsp70 overexpression. Reduced caspase-9 cleavage occurred in Hsp70 Tg mice compared with Wt littermates 24 hours after H/I and correlated with increased binding of Hsp70 and Apaf-1. Increased cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein (FLIP) expression and decreased caspase-8 cleavage were also observed in Hsp70 Tg compared with Wt mice 24 hours after H/I.


CONCLUSIONS
Our results suggest that the extrinsic and intrinsic apoptotic pathways mediate the neuroprotective effects of Hsp70 overexpression in neonatal H/I, specifically by upregulating FLIP and sequestering Apaf-1, leading to reduced cleavage of caspase-8 and caspase-9.
Matsumori
Yasuhiko
Y
Department of Neurological Surgery, University of California, Department of Veterans Affairs Medical Center, San Francisco, CA 94121, USA.
Northington
Frances J
FJ
Hong
Shwuhuey M
SM
Kayama
Takamasa
T
Sheldon
R Ann
RA
Vexler
Zinaida S
ZS
Ferriero
Donna M
DM
Weinstein
Philip R
PR
Liu
Jialing
J
D000818:Animals; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D015153:Blotting, Western; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000096:biosynthesis* / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D005786:Gene Expression Regulation; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D020925:Hypoxia-Ischemia, Brain; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007375:Interleukin-1 / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008928:Mitochondria / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction; D013347:Subcellular Fractions; D015854:Up-Regulation
C506593:Apaf1 protein, mouse; C506594:Apaf1 protein, rat; D053477:Apoptotic Protease-Activating Factor 1; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506387:Cflar protein, mouse; D018840:HSP70 Heat-Shock Proteins; D007375:Interleukin-1; D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; D045304:Cytochromes c; C505526:Casp8 protein, mouse; C505528:Casp8 protein, rat; C506434:Casp9 protein, mouse; C506435:Casp9 protein, rat; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D010100:Oxygen