Hsp60 for IKK Regulation Figure 4. Loss of cytosolic Hsp60 diminishes IKK/NF-kB activation in response to TNF-a. A. Ablation of cytosolic Hsp60 by antisense ODNs. The cytosolic and mitochondrial fractions prepared from mock or ODN-transfected HeLa cells were immunoblotted. S, sense ODN; AS-1 and AS-2, antisense ODNs. The mitochondrial fraction was loaded at a volume of one-fifth of the corresponding cytosolic fraction. In particular, Prx III, which is an antioxidant enzyme present in the mitochondrial matrix, was used as mitochondrial markers to watch the nonspecific mitochondrial rupture. B. Half-life of ectopically-expressed Hsp60c protein (HA tag) after inhibition of protein synthesis with cycloheximide. The intensity of HA band was measured and normalized by the amount of IKKa band. Data in the graph are means 6 S.D. of two independent experiments and fitted in SigmaPlot 8.0 software. C. Proteasome-dependent turnover of cytosolic Hsp60c protein. HeLa cells were pretreated with or without MG132 (5 mM) 30 min before cycloheximide treatment. D. TNF-a-induced IKK and JNK1 activation in mock or ODN-transfected 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 mock-transfected cells (lane 1). E. NF-kB transcriptional activation in mock or ODN-transfected cells. The increasing concentration of AS-ODN (100 nM or 200 nM) was tested. The relative luciferase activity was normalized to the b-galactosidase activity and data are means 6 S.D. of four independent experiments (*P,0.0001, **P,0.001 versus stimulated S-ODN-transfected cells). doi:10.1371/journal.pone.0009422.g004 indicate that the regulation of the IKK activation by cytosolic Hsp60 influences the expression of select NF-kB target genes. We next wondered whether such regulation of select target genes has an impact on cell survival. Since there is a possibility that MnSOD and Bfl-1/A1 function to suppress the mitochondrial- derived reactive oxygen species (ROS) [2,45], the level of cellular ROS was examined in ODN-transfected cells using an oxidation- sensitive fluorescence dye, CM-H2DCFDA. The AS-ODN transfection induced a marked increase of cellular ROS in response to TNF-a treatment in a time-dependent manner, compared to mock or S-ODN transfection (Fig. 8D). Since the enhanced ROS level is linked to cell death via the sustained JNK activation [46], the sustained activation of stress-activated protein kinases, JNK and p38 MAPK, was examined. Unexpectedly, the activation of both JNK and p38 MAPK were found to be clearly sustained in AS-ODN-transfected cells (Fig. 8E). The ASK-1 MAP3K is known to be responsible for the sustained activation of JNK and p38 in the ROS-mediated cell death [47]. Indeed, the ASK-1 activation was significantly induced in AS-ODN-transfect- ed cells (Fig. 8F). As a consequence of this signaling pathway including ASK-1 activation, the AS-ODN resulted in a marked induction of TNF-a-induced cell death in HeLa cells, whereas the mock or S-ODN did not at all (Fig. 8G). Likewise, the AS-ODN enhanced TNF-a-induced cell death in colon carcinoma cell lines, which showed significant level of cytosolic Hsp60 (Fig. 8H). It should be noted that the AS-ODN transfection by itself resulted in the basal activation of ROS, ASK1, and cell death. Along with the evidence that the Hsp60c overexpression induced the basal IKK/ PLoS ONE | www.plosone.org 6 March 2010 | Volume 5 | Issue 3 | e9422