Intracellular cleavage of osteopontin by caspase-8 modulates hypoxia/reoxygenation cell death through p53

• Published in: Proceedings of the National Academy of Sciences - PNAS 2009-09, Vol.106 (36), p.15326-15331
• Main Authors: Kim, Hyo-Jin, Lee, Ho-June, Jun, Joon-Il, Oh, Yumin, Choi, Seon-Guk, Kim, Hyunjoo, Chung, Chul-Woong, Kim, In-Ki, Park, Il-Sun, Chae, Han-Jung, Kim, Hyung-Ryong, Jung, Yong-Keun
• Format: Article
• Language: English
• Subjects: Antibodies; Apoptosis; Apoptosis - physiology; Biological Sciences; Blotting, Western; Cancer; Caspase 8 - metabolism; Cell death; Cell extracts; Cell Hypoxia - physiology; Cell lines; Cells; Densitometry; HEK293 cells; HeLa Cells; Humans; Hypoxia; Mutation; Osteopontin - genetics; Osteopontin - metabolism; Proteases; Proteins; Rodents; Tumor Suppressor Protein p53 - metabolism; Tumors; Western blotting
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0903704106

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.