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

Abstract

O steopontin (OPN) is a secreted glycosylated phosphoprotein that is involved in a number of physiological events including bone formation and remodeling (1), immune responses (2, 3), and tumor progression, such as cell proliferation, angiogenesis, metastasis, and anti-apoptosis (4). Especially, OPN is highly up-regulated in cancer patients' plasma, thus it is considered a candidate as a prognostic marker for human cancer diagnosis (4). Multiple cancer-related functions of OPN are mediated by its interaction with integrins or CD44 variants as a cytokine. Generally, secreted OPN acts as an intact protein or fragments cleaved by thrombin; Arg-Gly-Asp (RGD) motif in OPN interacts with integrins (␣ v ␤ 3 , ␣ v ␤ 5 ) and C-terminal region of OPN binds to CD44 variants, which subsequently activates a PI3K-AKT, NIK, or MEKK1 kinase cascade (4, 5). In addition, alternative isoform of OPN is found in cytosol (6) and OPN is detected as a CD44-ERM complex in the cytosolic side of CD44 (7). Further, OPN also associates with polo-like kinase-1 in the nucleus during cell cycle (8). These observations show diverse roles and subcellular localizations of OPN.OPN is also highly induced during hypoxia/reoxygenation (Hyp/ RO), which is closely related to pathological conditions including myocardial ischemia/reperfusion injury, stroke, inflammation, and solid tumors (9, 10). During Hyp/RO, cell death generally occurs after massive generation of reactive oxygen species (ROS) and caspases activation. Several caspases including caspases-8, -9, and -3 were reported to be activated during reoxygenation, which is required for Hyp/RO-induced cell death (11,12). Among these caspases, caspase-8 is a well known receptor-proximal caspase. However, accumulating evidence suggests atypical roles of caspase-8 in nonreceptor-mediated cell deaths (13, 14) and . In addition, caspase-8 deficiency is also detected in human cancers (16,17) and facilitates cellular transfomation (18), showing critical functions of caspase-8 in tumorigenesis and cell death. In the group of hundreds' cellular substrates of various caspases, only a few proteins, such as Bid, p28 Bap31, RIP-1, and plectin, are reported as caspase-8 substrates (19)(20)(21)(22).In this study, we performed genome-wide screening and isolated OPN as a caspase-8 substrate. OPN expression is rapidly increased during Hyp/RO and subsequently cleaved by caspase-8, leading to both inactivation of AKT survival signal and activation of cell death signal via its caspase cleavage fragment in tumor cells. Results OPN Is Cleaved by Caspase-8 in Vitro and in Apoptotic Cells DuringHyp/RO. To unearth caspase substrates, we undertook caspase substrate screening using human cDNA library. Small cDNA pools were transcribed and translated in vitro in the presence of [35 S]methionine and then incubated with recombinant caspases (23). From this analysis, we isolated OPN as a putative substrate of caspase-8. To characterize the cleavage, in vitro translated OPN was incubated with various recombinant active caspases (ca...

Spatiotemporal expression of osteopontin in the striatum of rats subjected to the mitochondrial toxin 3-nitropropionic acid correlates with microcalcification

“…1a). In addition, smaller molecular-weight OPN proteins (50 kDa or 25 kDa), which have been shown to be cleavage products of matrix metalloproteinase activity505152, were found to be increased in striatal tissue at earlier time points (days 3 and 7). However, the roles and significance of these smaller fragments are unknown.…”

Arrestins in Apoptosis

“…Caspase cleavage of several kinases unleashes or abrogates their pro-apoptotic or pro-survival functions, respectively, via changes in activity, subcellular localization, or substrate preferences (Kurokawa and Kornbluth 2009). Caspase cleavage products of diverse proteins contribute to the progression of apoptosis due to loss or gain of function or via dominant-negative action (Kim et al 2009b; Crawford and Wells 2011; Oliver et al 2011). Our experiments revealed a direct role of 1-380 in cytochrome C release, identifying it as an earlier unappreciated active participant in the core mechanism of apoptosis (Kook et al 2013).…”

Osteopontin Is Cleaved at Multiple Sites Close to Its Integrin-binding Motifs in Milk and Is a Novel Substrate for Plasmin and Cathepsin D

“…Another modification that can alter the functionality of OPN is proteolytic processing. Recently, intracellular cleavage of OPN by caspase-8 at Asp 119 -Phe 120 and Asp 141 -Gly 142 has been shown to be a regulatory switch in determining cell death of cancer cells (12). OPN is also a substrate for thrombin and matrix metalloproteinase (MMP)-2, -3, -7, and -9 (13)(14)(15)(16)(17).…”