Hsp60 for IKK Regulation Figure 6. Cytosol-targeted expression of Hsp60 promotes TNF-a-induced IKK/NF-kB activation. Cells were transfected with either control (CGN) or Hsp60c-encoding plasmid (HA tag) for 24 hrs and then treated with TNF-a. A. Incorporation of ectopically-expressed Hsp60c (HA tag) in IKK complex. B. TNF-a-induced IKK activation in HeLa cells. C TNF-a-induced NF-kB activation in HeLa cells (n = 4, *P,0.0001 versus unstimulated counterpart). D. TNF-a-induced NF-kB activation in the transfected IKKb2/2 3T3 cells (n = 4, *P,0.001 versus stimulated CGN-transfected cells, N.D. not detected). doi:10.1371/journal.pone.0009422.g006 sional change of the two select target genes, MnSOD and Bfl-1/A1, both of which are known to be related to the mitochondrial function. Although this odd phenomena were hardly investigated in the study, some experimental data can provide a clue. 1) Hsp60 was shown to bind preferentially to IKKa rather than to IKKb in the in vitro protein binding assay. 2) Direct interaction of Hsp60 and IKKa, but not IKKb, in the nucleus was often observed in the immunogold staining. IKKa is known to regulate NF-kB transcriptional activity in the nucleus [31,32] rather than IkB phosphorylation [50]. Supposedly, when the Hsp60-IKKa complex translocate to nucleus, the Hsp60 may directly guide IKKa to the promoter of particular gene set (e.g, MnSOD and Bfl-1/A1). This is similar to the way exemplified in the regulation of select p53 target genes by human cellular apoptosis susceptibility (hCAS) protein [51]. Otherwise, it may be possible that the Hsp60 modulates the ability of IKKa or presumably IKKb as well, to phosphorylate the other substrates such as RelA, SMRT, and histone H3 [52], which thus indirectly affects the NF-kB transcription. For eample, the IKKa-mediated phosphorylation of SMRT corepressor allows the recruitment of NF-kB to promotors [53], and IKKa-mediated phosphorylation of histone H3 affects the chromatin structure [31,32]. However, our inciting speculation needs an investigation. On the other hand, the functional significance of Hsp60-IKKa interaction was also investigated in non-canonical NF-kB signaling, RANK-induced osteoclastogenesis [54]. IKKa-mediat- ed NF-kB pathway is essential for RANK-induced osteoclastogen- esis [55,56]. As shown in Fig. S5, the reduction of cytosolic Hsp60 expression using antisense ODN (AS-1) clearly suppressed the RANK-induced osteoclast formation from the bone marrow- derived macrophage/monocytic (BMM) cells, as quantified by TRAP assay. Since TNF-a-induced osteoclast formation [57] was also markedly suppressed, this result indicates that cytosolic Hsp60 can function in the NF-kB activation pathway via interacting with non-canonical as well as canonical IKK complex. Since Hsp60 is exclusively targeted to mitochondria after translation, the cytosolic Hsp60 is presumed to be of mitochon- drial origin. In fact, the mitochondrial release of Hsp60 in staurosporine-treated Jurkat T cells has been shown [29]. We also observed the same result in staurosporine-treated HeLa cells (Fig. S4). Since the Hsp60 protein was detected in the mitochondrial intermembrane space in immunogold EM data, we believe that this pool of Hsp60 protein can be released. This is similar to the case of pro-apoptotic mitochondrial factors. For example, AIF is a mitochondrial inner membrane protein (62-kDa in mature form) and is released after proteolytic shedding to a 57-kDa form upon proapoptotic stimuli [58,59]. HtrA2/Omi (51-kDa serine protease) is released from the mitochondrial intermembrane space [60,61]. In addition, the fact that the antisense-ODN (AS-1) used in the PLoS ONE | www.plosone.org 8 March 2010 | Volume 5 | Issue 3 | e9422