Hsp60 for IKK Regulation Figure 5. Hsp60-specific antibody blocks IKK/NF-kB activation. A. Transduction of Hsp60-neutralizing antibody (Hsp60N) into the cytoplasm of HeLa cells. Mitotracker Red (Molecular Probes, USA) and DAPI indicate mitochondria and nuclei, respectively. B. The transduced Hsp60N antibody bound to endogenous Hsp60. After antibody transfection, the HeLa cell lysates were subjected to precipitation using Protein-A Sepharose beads. The precipitated proteins were immunoblotted for Hsp60. C. IKK and JNK1 activation in response to TNF-a in control IgG or Hsp60N antibody-transfected HeLa cells. The in vitro kinase activity (KA) was averaged with the values from two independent experiments, and it is represented as a fold increase of the activity versus the unstimulated and control IgG-transfected cells (lane 1). D. TNF-a-induced NF-kB transcriptional activation in antibody- transfected cells (*P,0.01 versus stimulated IgG-transfected cells). doi:10.1371/journal.pone.0009422.g005 NF-kB activation (see Fig. 6), cytosolic Hsp60 seems likely to direct cell survival in resting cancer cells. Collectively, our results suggest that the selective regulation of MnSOD and Bfl-1/A1 expression by cytosolic Hsp60 sufficiently influences cell survival via suppressing mitochondrial ROS burst. Cytosolic Hsp60 protects host cells against stressed conditions Next, the pro-survival activity of cytosolic Hsp60 was investigated in vivo. To do this, transgenic mice expressing Hsp60c were generated (Fig. 9A and 9B). The Hsp60c protein was successfully expressed in various tissues, including liver, spleen, and lung (Fig. 9C). The IKK activation was markedly enhanced in the Hsp60c-expressing transgenic mice compared to the control B6 mice when TNF-a was intravenously injected (Fig. 9D). The result indicates that the cytosolic Hsp60 enhanced the TNF-a-induced IKK activation in vivo. We then sought an animal model wherein the IKK/NF-kB-dependent cell survival is involved: the diethylnitrosamine (DEN)-induced hepatocyte death [48]. The apoptotic cell death of hepatocytes was indeed increased after DEN injection in four-week-old male mice (Fig. S3). Therefore, the DEN-induced hepatic cell death was examined in Hsp60c-expressing transgenic mice that had been primed with or without TNF-a. TUNEL staining of the liver tissue sections showed that the hepatic cell death was significantly reduced in Hsp60c-expressing transgenic mice compared to control mice (Fig. 9E and 9F). The data indicate that the cytosolic Hsp60 prevents the stress-induced cell death in vivo by promoting IKK/NF-kB activation. Discussion The present study demonstrates that the cytosolic Hsp60 interacts with the IKK complex and enhances its activation. This signaling action of cytosolic Hsp60 affects the induction of the two NF-kB target genes, Bfl-1/A-1 and MnSOD, and thus cell survival. Since Bfl-1/A-1 functions as a tBid and Bak antagonist [2,4] and MnSOD eliminates the superoxide anion inside mitochondria [49], these two genes are ultimately critical for controlling mitochondrial-derived ROS. Indeed, ablation of cytosolic Hsp60 results in the increase of cellular ROS, which in turn triggers the sustained activation of JNK/p38 via ASK-1 and finally induces the cell death, in response to TNF-a. Given that the mitochondrial ROS are causally linked to various human diseases such as cancer, vascular damage, and a variety of degenerative diseases via the accumulation of cellular damage, this new pro- survival circuitry mediated by cytosolic Hsp60 may be of particular important progression of these diseases. The transcriptional regulation of select NF-kB target genes by cytosolic Hsp60 is attractive issue but unsolved in the present study. The RNase protection assay and the microarray experiment (,24,000 RefSeq-based probe sequences per array, Illumina expression chip) were failed to demonstrate the broad effect of the cytosolic Hsp60 on expression profile of NF-kB target genes. Instead, the quantitative real-time PCR demonstrated the expres- PLoS ONE | www.plosone.org 7 March 2010 | Volume 5 | Issue 3 | e9422