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

doi:10.1073/pnas.0903704106

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

^aCreative Research Initiative Acceleration Research, School of Biological Science/Bio-Max Institute, Seoul National University, Seoul 151–747, Korea;
^bDepartment of Life Science, Gwangju Institute of Science and Technology, Gwangju 500–712, Korea;
^cLG Life Science Research Park, Daejon 305–389, Korea;
^dOntario Cancer Institute, Toronto, Ontario M5G 2M9, Canada;
^eDepartment of Bio-Materials Engineering and Molecular Medicine, School of Medicine, Chosun University, Gwangju 501–759, Korea;
^fSchool of Medicine, Chonbuk National University, Chonbuk 560–180, Korea, and
^gDepartment of Dental Pharmacology, School of Dentistry, Wonkwang University, Chonbuk 570–749, Korea

Edited by Harvey Cantor, Dana-Farber Cancer Institute, Boston, MA, and approved July 20, 2009 (received for review April 3, 2009)

Abstract

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.

Footnotes

¹To whom correspondence should be addressed at:
School of Biological Science/Bio-Max Institute, Seoul National University, Gwanak-ro 599, Gwanak-gu, Seoul 151–747, Korea.
E-mail: ykjung{at}snu.ac.kr
Author contributions: H.-J.K., H.-J.L., and Y.-K.J. designed research; H.-J.K., H.-J.L., J.-I.J., Y.O., S.-G.C., H.K., C.-W.C., and I.-K.K. performed research; I.-S.P., H.-J.C., and H.-R.K. contributed new reagents/analytic tools; H.-J.K., H.-J.L., and Y.-K.J. analyzed data; and H.-J.K. and Y.-K.J. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0903704106/DCSupplemental.