Intracellular cleavage of osteopontin by caspase-8 modulates hypoxia/reoxygenation cell death through p53.
Osteopontin (OPN) is highly expressed in cancer patients and plays important roles in many stages of tumor progression, such as anti-apoptosis, proliferation, and metastasis. From functional screening of human cDNA library, we isolated OPN as a caspase-8 substrate that regulates cell death during hypoxia/reoxygenation (Hyp/RO). In vitro cleavage assays demonstrate that OPN is cleaved at Asp-135 and Asp-157 by caspase-8. Cellular cleavage of OPN is observed in apoptotic cells exposed to Hyp/RO among various apoptotic stimuli and its cleavage is blocked by zVAD or IETD caspase inhibitor. Further, over-expression of OPN, the form with secretion signal, inhibits Hyp/RO-induced cell death. Caspase cleavage-defective OPN mutant (OPN D135A/D157A) is more efficient to suppress Hyp/RO-induced cell death than wild-type OPN. OPN D135A/D157A sustains AKT activity to increase cell viability through inhibition of caspase-9 during Hyp/RO. In addition, OPN is highly induced in some tumor cells during Hyp/RO, such as HeLa and Huh-7 cells, which is associated with their resistance to Hyp/RO by sustaining AKT activity. Notably, OPN C-terminal cleavage fragment produced by caspase-8 is detected in the nucleus. Plasmid-encoded expression of OPN C-terminal cleavage fragment increases p53 protein level and induces apoptosis of wild-type mouse embryonic fibroblast cells, but not p53(-/-) mouse embryonic fibroblast cells. These observations suggest that the protective function of OPN during Hyp/RO is inactivated via the proteolytic cleavage by caspase-8 and its cleavage product subsequently induces cell death via p53, postulating caspase-8 as a negative regulator of tumorigenic activity of OPN.
Kim
Hyo-Jin
HJ
Creative Research Initiative Acceleration Research, School of Biological Science/Bio-Max Institute, Seoul National University, Seoul 151-747, Korea.
Lee
Ho-June
HJ
Jun
Joon-Il
JI
Oh
Yumin
Y
Choi
Seon-Guk
SG
Kim
Hyunjoo
H
Chung
Chul-Woong
CW
Kim
In-Ki
IK
Park
Il-Sun
IS
Chae
Han-Jung
HJ
Kim
Hyung-Ryong
HR
Jung
Yong-Keun
YK
D017209:Apoptosis / Q000502:physiology*; D015153:Blotting, Western; D053181:Caspase 8 / Q000378:metabolism*; D015687:Cell Hypoxia / Q000502:physiology*; D003720:Densitometry; D006367:HeLa Cells; D006801:Humans; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D016159:Tumor Suppressor Protein p53; D053495:Osteopontin; D053181:Caspase 8
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