Original Contribution Cell death induced by 7-oxysterols via lysosomal and mitochondrial pathways is p53-dependent Wei Li a,n , Amit Laskar a , Nargis Sultana a , Ehab Osman a , Moumita Ghosh a , Qianqian Li a , Xi-Ming Yuan a,b a Division of Experimental Pathology, Department of Clinical and Experimental Medicine, Link¨ oping University, Link¨ oping SE-581 85, Sweden b Division of Occupational and Environmental Medicine, Link¨ oping University Hospital, Link¨ oping SE-581 85, Sweden article info Article history: Received 15 April 2012 Received in revised form 25 August 2012 Accepted 10 September 2012 Available online 15 September 2012 Keywords: Apoptosis Atherosclerosis Bax Lysosomes Mitochondria Oxidative stress Free radicals abstract Oxysterol accumulation and p53 expression mainly in macrophages have been associated with cell death and necrotic core formation in human atheroma progression. Oxidative stress and lysosomal membrane permeabilization (LMP) in macrophages are important causes of macrophage apoptosis. However, it is not understood how p53 and oxysterols interact in the process. We show here that 7-oxysterols induce endogenous full-length p53 and phospho-p53 (p53-Ser15) in both nucleus and cytoplasm of THP1 and J774 cells, which is followed by cellular oxidative stress and apoptotic cell death. The role of p53 in 7-oxysterol- mediated cell death is further investigated in temperature sensitive p53-transfected (M1-t-p53) and in p53-deficient (M1) cells. These results reveal that 7-oxysterols induce induction and nuclear translocation of p53 in M1-t-p53 cells, which in turn enhances LMP, mitochondrial translocation of Bax, mitochondrial membrane permeabilization, cytosolic release of cytochrome c, and cell death. Most importantly, the above effects of 7-oxysterols were not observed in p53-deficient M1 cells. The findings reveal that 7-oxysterol- induced cell death occurs via p53-dependent pathways. Subsequent p53 nuclear translocation and induction of wild-type and phosphorylated p53 are early steps in oxysterol-induced lysosomal– mitochondrial pathways involved in cell death. & 2012 Elsevier Inc. All rights reserved. Cell death and subsequent necrotic core formation are prominent features of atherosclerotic lesions and they have been associated with instability and thrombosis of atheroma plaques and acute cardiocer- ebral events. The causative agents responsible for cell death are toxic moieties of oxidized lipids, including cholesterol oxidation products, oxidized low-density lipoproteins (oxLDL), and related oxysterols. Among the oxysterols, 7b-hydroxycholesterol (7bOH) and 7- ketocholesterol (7keto), as well as 7b-hydroperoxycholest-5-en-3b- ol, have been found in human atherosclerotic lesions and in oxLDL, and they are the major toxic components of oxLDL [1–5]. Although 7keto cytotoxicity is diminished when incorporated into acetylated LDL, this does not rule out the possibility that within the environment of the plaque 7keto and other oxysterols may be removed from the lipoprotein particles and concentrated in the macrophage foam cells, so inducing cell death [5]. We demonstrated earlier that oxysterol mixtures, in atheroma- relevant proportions, display synergistic and pro-apoptotic effects [6]. Several apoptotic signaling events have been inter- related to apoptosis induced by oxysterols, including enhanced oxidative stress; mitochondrial pathways [7]; modulated expression of BIM, BAD, and Bcl-xL [7]; and upregulation of p53 [8–11]. Recent studies by our group and others have further established that lysosomal membrane permeabilization (LMP) and the subsequent relocation of lysosomal hydrolases play an important role in apoptosis induced by oxysterols or p53 [12–14]. The tumor suppressor p53 is an essential protein for both cell proliferation and cell death in response to stress signals and it is increased in macrophages of human carotid atheroma lesions [15]. We recently reported that p53 levels in human carotid atherosclerotic lesions (both mutant and wild type, as detected with the p53 antibody clone DO-7) were significantly increased in plaques with necrotic core formation, which is significantly correlated with the expression of cell death-related proteins and lysosomal cathepsin L [15]. In animal models, p53 modulates the process of cell death in atherosclerosis [16,17]. Although the results from both animal models of atherosclero- sis and human lesions suggest that p53-mediated cell death is related to the formation of lipid-rich necrotic cores and contri- butes to the progression of atherosclerotic plaques, the interac- tions of p53 with atheroma-relevant oxysterols in the process of cell death and the underlying mechanisms are unclear. In this study, we first investigated whether various functional forms of p53 induced by oxysterols are involved in 7-oxsyterol- mediated cell death in THP1 and J774 cells. We then investigated Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/freeradbiomed Free Radical Biology and Medicine 0891-5849/$ - see front matter & 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.freeradbiomed.2012.09.007 n Corresponding author. Fax: þ46 13 221529. E-mail address: wei.li@liu.se (W. Li). Free Radical Biology and Medicine 53 (2012) 2054–2061