Cytosolic Hsp60 Is Involved in the NF-kB-Dependent Survival of Cancer Cells via IKK Regulation Jung Nyeo Chun 1 , Boae Choi1, Kyung Wha Lee1, Doo Jae Lee1, Dong Hoon Kang1, Joo Young Lee1, In Sung Song1¤a, Hye In Kim1¤b, Sang-Hee Lee4, Hyeon Soo Kim5, Na Kyung Lee1, Soo Young Lee1,2, Kong- Joo Lee1,3, Jaesang Kim1,2, Sang Won Kang1,2* 1 Division of Life and Pharmaceutical Science and Center for Cell Signaling and Drug Discovery Research, Ewha Womans University, Seoul, Korea, 2 Department of Life Science and College of Natural Science, Ewha Womans University, Seoul, Korea, 3 College of Pharmacy, Ewha Womans University, Seoul, Korea, 4 Division of Electron Microscopic Research, Korea Basic Science Institute, Daejeon, Korea, 5 Department of Anatomy, College of Medicine, Korea University, Seoul, Korea Abstract Cytoplasmic presence of Hsp60, which is principally a nuclear gene-encoded mitochondrial chaperonin, has frequently been stated, but its role in intracellular signaling is largely unknown. In this study, we demonstrate that the cytosolic Hsp60 promotes the TNF-a-mediated activation of the IKK/NF-kB survival pathway via direct interaction with IKKa/b in the cytoplasm. Selective loss or blockade of cytosolic Hsp60 by specific antisense oligonucleotide or neutralizing antibody diminished the IKK/NF-kB activation and the expression of NF-kB target genes, such as Bfl-1/A1 and MnSOD, which thus augmented intracellular ROS production and ASK1-dependent cell death, in response to TNF-a. Conversely, the ectopic expression of cytosol-targeted Hsp60 enhanced IKK/NF-kB activation. Mechanistically, the cytosolic Hsp60 enhanced IKK activation via upregulating the activation-dependent serine phosphorylation in a chaperone-independent manner. Furthermore, transgenic mouse study showed that the cytosolic Hsp60 suppressed hepatic cell death induced by diethylnitrosamine in vivo. The cytosolic Hsp60 is likely to be a regulatory component of IKK complex and it implicates the first mitochondrial factor that regulates cell survival via NF-kB pathway. Citation: Chun JN, Choi B, Lee KW, Lee DJ, Kang DH, et al. (2010) Cytosolic Hsp60 Is Involved in the NF-kB-Dependent Survival of Cancer Cells via IKK Regulation. PLoS ONE 5(3): e9422. doi:10.1371/journal.pone.0009422 Editor: Rafael Linden, Universidade Federal do Rio de Janeiro (UFRJ), Brazil Received December 2, 2009; Accepted January 18, 2010; Published March 23, 2010 Copyright: 2010 Chun et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by 21C Frontier Functional Proteomics Project (FPR08-B1-190) funded by the Ministry of Education, Science & Technology and by KOSEF through the Center for Cell Signaling & Drug Discovery Research (CCS & DDR, R15-2006-020) at Ewha Womans University. This study was also supported in part by a grant of the National R&D Program for Cancer Control, Ministry of Health & Welfare (0420190). J. N. Chun, K. W. Lee, J. Y. Lee, B. A. Choi, and H. I. Kim were supported by Brain Korea 21 grant from the Korea Ministry of Education, Science & Technology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kangsw@ewha.ac.kr ¤a Current address: Medical Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejon, Korea ¤b Current address: CMP Korea Medica Ltd. Seoul, Korea Introduction Mammalian cells express a number of survival genes that play the roles in inhibiting caspase activation, removing harmful oxygen radicals, defending mitochondrial function, and checking cell cycle. Among the transcription factors responsible for the induction of the survival genes, nuclear factor-kB (NF-kB) is a key element that orchestrates the complex cell survival response [1]. In particular, the NF-kB-dependent survival genes include anti- apoptotic genes, such as c-IAPs and c-FLIP, and mitochondrial safeguard genes, such as manganese-superoxide dismutase (MnSOD) and Bcl-2 family members [2,3,4]. A central kinase in NF-kB activation pathway is the inhibitor of kB kinase (IkB kinase or IKK) that phosphorylates the IkB protein in two amino- terminal serine residues, leading to its ubiquitinylation and proteosomal degradation and to the consequent liberation of NF-kB proteins [5]. The extracellular stimuli to activate NF-kB pathway converge to IKK [6]. Therefore, numerous efforts have been made to delineate the regulation of IKK activation. First, the phosphorylation-dependent regulation of IKK activation has been characterized [7]. The phosphorylation of two serine residues (Ser 177/Ser181 in human IKKb) in activation T-loop is essential for activation, while the autophosphorylation of carboxyl-terminal serine cluster turns off the activation. Many kinases have been implicated to be involved in the activation phosphorylation: NF- kB-inducing kinase (NIK) [8,9], mitogen-activated protein kinase/ ERK kinase kinases 1 (MEKK1) [10], MEKK2/3 [11,12], Hematopoietic progenitor kinase-1 (HPK1) [13], Mixed-lineage kinase 3 (MLK3) [14], TGF-b activated kinase 1 (TAK1) [15]. However, except TAK1, it is unclear how the upstream kinase activates the IKK complex. Second, the ubiquitin-dependent regulation of IKK activation has been studied for many years [15,16]. Recently, the regulatory subunit IKKc (or NEMO) of IKK complex has been shown to be ubiquitinated, as well as to recognize Lys63-linked polyubiquination chain on receptor- interaction protein 1 (RIP1) [17,18]. Thirdly, the regulation of IKK activation via the interacting proteins has been characterized. The best examples are heat shock proteins. For example, Cdc37 and Hsp90 have been reported to act as additional components of the IKK complex that stabilize the complex [19,20]. Hsp27 has been shown to interact with IKKb in a TNF-a-dependent manner [21]. Hsp70 also interacts with IKKc but interferes with the IKK PLoS ONE | www.plosone.org 1 March 2010 | Volume 5 | Issue 3 | e9422