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RESEARCH PAPER

Hsp70 promotes TNF-mediated apoptosis by binding IKK and impairing NF-B survival signaling

¹ Departments of Neurology, Pediatric Neurology and the Neurosciences Program, ² Department of Cell Biology, Neurobiology and Anatomy, and ³ Cincinnati Children's Hospital and Medical Center, University of Cincinnati, Cincinnati, Ohio 45267, USA; ⁴ Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02115, USA

	Abstract

Top Abstract Results Discussion Materials and methods Acknowledgments References

 
The major heat shock protein, Hsp70, can protect against cell death by directly interfering with mitochondrial apoptosis pathways. However, Hsp70 also sensitizes cells to certain apoptotic stimuli like TNF. Little is known about how Hsp70 enhances apoptosis. We demonstrate here that Hsp70 promotes TNF killing by specifically binding the coiled-coil domain of IB kinase (IKK) to inhibit IKK activity and consequently inhibit NF-B-dependent antiapoptotic gene induction. An IKK mutant, which interacts with Hsp70, competitively inhibits the Hsp70–IKK interaction and relieves heat-mediated NF-B suppression. Depletion of Hsp70 expression with RNA interference rescues TNF-mediated cell death. Although TNF may or may not be sufficient to trigger apoptosis on its own, TNF-triggered apoptosis was initiated or made worse when Hsp70 expression increased to high levels to disrupt NF-B signaling. These results provide significant novel insights into the molecular mechanism for the pro-apoptotic behavior of Hsp70 in death-receptor-mediated cell death.

[Keywords: Hsp70; NF-B; IKK; TNF; apoptosis; death receptor signaling]

Received January 21, 2004; revised version accepted March 30, 2004.

NF-B is sequestered in the cytoplasm in an inactive complex with IB proteins (Huxford et al. 1998; Malek et al. 2001). Activation of the NF-B-mediated signal is initiated through degradation of phosphorylated IB. Various stimuli activate the IB kinase (IKK) complex, which in turn phosphorylates IB, leading to NF-B activation (Chen et al. 1995; Zandi et al. 1997). NF-B is critical for survival of most cells through the induction of antiapoptotic genes (Beg et al. 1995; Beg and Baltimore 1996; Van Antwerp et al. 1996; Wang et al. 1998; Zong et al. 1999; Rudolph et al. 2000). IKK plays a central role in mediating NF-B activation. IKK is composed of two catalytic subunits, IKK and IKK, which can directly phosphorylate IB. IKK (also called NEMO) is an absolutely essential regulatory component of the IKK complex that is necessary for NF-B activation (Rothwarf et al. 1998; Yamaoka et al. 1998; Makris et al. 2002).

A number of studies have also shown that heat shock or elevated Hsp70 suppresses NF-B activity (Feinstein et al. 1997; Guzhova et al. 1997; Curry et al. 1999; Andres et al. 2002; Malhotra and Wong 2002). Although these studies imply the possibility that Hsp70 impairs NF-B signaling, the exact molecular basis of the Hsp70 and NF-B interaction is still enigmatic.

This study therefore examined the mechanism by which Hsp70 interacts with NF-B and might promote apoptosis. To do this, we studied several cell lines (Cos-1, Hela, 293 cells) that do not apparently undergo apoptosis when exposed to TNF alone. However, when these cells are exposed to TNF in combination with Hsp70 overexpression or heat shock, they die via apoptosis. Here, we demonstrate that Hsp70 associates directly and specifically with IKK and blocks the formation of the IKK complex—possibly by inhibiting oligomerization of IKK. This inhibits TNF-triggered NF-B activation and subsequently prevents NF-B-dependent anti-apoptotic gene expression. This work demonstrates how Hsp70 promotes rather than inhibits TNF-mediated cell death.

	Results

Top Abstract Results Discussion Materials and methods Acknowledgments References

 

Hsp70 inhibits NF-B activity

To determine whether Hsp70 itself specifically suppressed NF-B activity, we cotransfected Cos-1 cells with an NF-B reporter and the indicated constructs. In this transfection assay, Hsp70 and IBDN (IB dominant negative) suppressed NF-B reporter gene activity, whereas Hsp70C (C terminus of Hsp70) and Hsp70N (N terminus of Hsp70) failed to do so (Fig. 1A). HSF1 also inhibited NF-B activity. However, this inhibition was abrogated by cotransfection with Hsp70AS (antisense Hsp70), indicating that HSF1 did not directly affect NF-B activity (Fig. 1A). Hsp90 did not affect NF-B activity in resting cells but enhanced NF-B activity when cells were treated with TNF (Fig. 1A).

View larger version (102K): [in this window] [in a new window]   Figure 1. Hsp70 inhibits NF-B activation. (A) Cos-1 cells were transfected with the indicated plasmids and the NF-B reporter. Twenty-four hours after transfection, cells were treated with TNF (25 ng/mL) or vehicle for 15 min. Luciferase activity was measured 24 h later. (B) Effects of Hsp70 on Ap-1- and HSF-1-dependent transactivation. Cos-1 cells were cotransfected with an AP-1 or HSF-1 luciferase reporter along with the indicated vectors. Twenty-four hours later, cells were treated with TNF or vehicle for 15 min. After 24 h, luciferase activities were measured. (C) Hsp70- and Hsp70C-inducible cells were transiently transfected with an NF-B-dependent luciferase reporter and then treated with different concentrations of ZnCl2 for 4 h to induce His-tagged Hsp70 and His-tagged Hsp70C expression. At 16 h after ZnCl2 treatment, cells were treated with TNF (25 ng/mL) or vehicle for 15 min. NF-B activity was analyzed 24 h after TNF treatment. Hsp70 and Hsp70C expression levels were examined with an anti-His antibody. -Actin served as the lane-loading control. (D) Immunoblots of exogenous and endogenous Hsp70 expression in Hsp70 stable cells were treated with ZnCl2 (for 4 h). Sixteen hours later, cell lysates were immunoblotted with anti-Hsp70 antibody. (E) Cos-1 cells were transfected with the indicated constructs and selected cells treated with TNF (25 ng/mL) for 15 min or heated for 30 min at 45°C. Twenty-four hours later, nuclear extracts were prepared and subjected to a gel shift assay using a 32P-labeled p65 probe. (NS) Nonspecific binding. (F) Hsp70- or Hsp70C-inducible Hela cells were treated with ZnCl2 (100 µM) or vehicle for 4 h. Sixteen hours later, cells were treated with TNF (25 ng/mL) or vehicle for 15 min. Cells were stained 15 min later with an anti-p65 antibody and a TRITC-conjugated second antibody. Nuclei were visualized with Hoechst. (G) Gel shift assay with in vitro translated Hsp70, HSF1, and p65 proteins and a 32P-labeled p65 probe. (NS) Nonspecific binding.

To determine whether physiologically elevated levels of Hsp70 inhibit NF-B activity, we established Hsp70-inducible cell lines using the nonintegrating plasmid pMEP4 under the control of a Zn²⁺-regulated metallothionein promoter. Hsp70 dose-dependently inhibited NF-B activity (Fig. 1C). The modest reduction of NF-B activity in Hsp70C-expressing cells (Fig. 1C, lanes 2–4) likely occurred because of Zn²⁺-mediated induction of endogenous Hsp70 (Fig. 1C,D). Cells treated with TNF alone, in the absence of ZnCl₂, had a relative luciferase activity for NFB-luc of 100 ± 8 (see results following), which is similar to the relative luciferase activity levels for the cells stably expressing His-Hsp70C in the presence of ZnCl₂ (Fig. 1C, lanes 2–4) and for His-Hsp70 cells treated with low levels of ZnCl₂ (Fig. 1C, lane 6).

Hsp70 did not directly interfere with the function of the NF-B activation domain using a cotransfection assay with plasmid encoding Gal4 (DNA-binding domain)-p65 (activation domain) chimeric protein, a Gal-4 reporter, and Hsp70 construct (data not shown). Next, to analyze the effect of Hsp70 on NF-B DNA-binding activity, we performed a gel mobility shift assay using a p65 probe and nuclear extracts isolated from Cos-1 cells. Heat, Hsp70, IBDN, and HSF1 inhibited NF-B DNA binding (Fig. 1E, lanes 1–8). The effects of HSF1 were not a direct effect on the NF-B activity because Hsp70 antisense (Hsp70AS) blocked the effect, showing that it is HSF1 induction of Hsp70 that inhibits NF-B DNA binding (Fig. 1E, lane 8). Cellular localization data showed that Hsp70 inhibited TNF-induced translocation of p65 from the cytoplasm to the nucleus (Fig. 1F).

To test whether Hsp70 and HSF1 directly affect the NF-B DNA-binding activity, we translated p65, Hsp70, and HSF1 proteins in vitro and performed the aforementioned gel mobility shift assays. Hsp70 and HSF1 failed to directly influence the NF-B DNA binding (Fig. 1G). These negative data are shown because they help rule out the possibility that Hsp70 or HSF1 directly regulates NF-B DNA-binding activity. In order to further rule out the possibility that Hsp70 directly interacts with NF-B (p65), we constructed NLS/p65 plasmids with forced p65 nuclear localization that was independent of IB degradation. Cos-1 cells were cotransfected with NLS/p65, NF-B reporter, and Hsp70 constructs. NF-B reporter activity analysis showed that Hsp70 did not suppress NF-B activity (data not shown). This confirmed the earlier findings that Hsp70 did not directly interact with NF-B (p65).

Hsp70 inhibits TNF-induced phosphorylation of IB

Because Hsp70 inhibited NF-B activity without directly binding NF-B, this indicated that Hsp70 impaired NF-B signaling upstream of NF-B (p65). Because activation of NF-B requires phosphorylation of IB, we examined the effects of Hsp70 on the phosphorylation of IB using specific antibodies to examine phosphorylated and unphosphorylated IB proteins. The results showed that Hsp70 inhibited TNF-induced IB phosphorylation (Fig. 2A).

View larger version (33K): [in this window] [in a new window]   Figure 2. Effects of Hsp70 on IB phosphorylation. (A) Hsp70- and Hsp70C-inducible Hela cells were induced with 100 µM ZnCl2 for 4 h. Sixteen hours later, cells were treated with TNF?25 ng/mL) for 15 min. Protein levels of phospho-IB,IB, and -actin were examined 15 min after TNF treatment by Western blot. (B) Hsp70- and Hsp70C-inducible Hela cells were transiently transfected with Flag-IKK and exposed to ZnCl2 (0–125 µM) for 4 h. Sixteen hours later, the cells were treated with TNF (25 ng/mL) for 15 min. Fifteen minutes later, cells were lysed and Hsp70 and Hsp70C were immunoblotted with an anti-His antibody (top gels). An in vitro kinase assay was performed using GST–IB(1–56) as substrate (middle gels). IKK protein levels were determined with an anti-Flag antibody (lower gels).

Hsp70 directly interacts with IKK

On the basis of these observations, we considered whether Hsp70 directly interacted with one or more components of the IKK complex and inhibited IKK activity. To determine which molecule(s) of the IKK complex might be the primary target of Hsp70, we searched for a direct interaction between the three IKKs and Hsp70 using a beads pull-down assay. ³⁵S-labeled IKK was retained on Hsp70-, but not on Hsp70C-conjugated beads. IKK and IKK did not associate with Hsp70 or with Hsp70C (Fig. 3A).

View larger version (83K): [in this window] [in a new window]   Figure 3. Hsp70 interacts with IKK. (A) Hsp70 or Hsp70C was immobilized on His beads and incubated with 35S-labeled IKK, IKK, or IKK. After extensive washing, bound proteins were analyzed by SDS-PAGE. (B) Diagram of IKK and the truncation mutants used in this study. (C) His beads alone or His-Hsp70 immobilized on beads were incubated with 35S-labeled IKK and its truncation mutants. After extensive washing, the bound proteins were analyzed by SDS-PAGE. (D) Two-hybrid assays for protein–protein interactions in living cells. Cos-1 cells were transiently transfected with the Gal4-reporter together with the indicated constructs, and luciferase activity was measured 24 h later. (E) Interaction of endogenous Hsp70 and IKK. 293 cells were transfected with increasing amounts of Hsp70 siRNA (lanes 4–6) or control GFP–siRNA (lanes 1–3). After 48 h, cells were heated at 45°C for 30 min. Sixteen hours later, cells were treated for 15 min with TNF or vehicle. Cell lysates were immunoprecipitated with anti-Hsp70 antibody (IP) and then immunoblotted with anti-IKK antibody (IB). The IKK activities were detected using the whole-cell extracts with anti-phospho-IB antibody (IB, top gel). Hsp70 was detected with an anti-Hsp70 antibody (third gel from top). (F) Coimmunoprecipitation of endogenous IKK and Hsp70. 293 cells were transfected with increasing amounts of IKK siRNA (lanes 3–6). After 48 h, the cells were treated with vehicle or TNF (25 ng/mL) for 15 min. Cell lysates were immunoprecipitated with anti-IKK antibody and then immunoblotted with anti-Hsp70 antibody. The whole-cell lysates were immunoblotted with anti-IKK and anti--actin antibodies. IKK activities were detected using the whole-cell extracts with anti-phospho-IB antibody (top gel).

To further confirm that Hsp70 associated with IKK within cells, we performed a two-hybrid assay in cultured mammalian cells. Hsp70 and Hsp70C CDNAs were in frame cloned into the pM vector containing the Gal-4 DNA-binding domain. Similarly, the IKK and its mutants, and IKK, IKK, and IB CDNAs were in frame inserted into pVP16 plasmid containing the activation domain. The resulting constructs were cotransfected into Cos-1 cells with a Gal-4 reporter gene. The results confirmed the in vitro binding data showing Hsp70 interaction with IKK and IKKC1 but not with IB, IKK, or IKK (Fig. 3D). Hsp70C did not associate with any of the molecules tested (Fig. 3D).

To determine whether endogenous Hsp70 interacted with IKK at physiologically relevant levels, we exposed cells to heat and TNF, either with GFPsiRNA (control, Fig. 3E, lanes 1–3) or with Hsp70siRNA (Fig. 3E, lanes 4–6). The cells were lysed and immunoprecipitated with an anti-Hsp70 antibody. The amounts of IKK protein in the anti-Hsp70 immunoprecipitates were assessed with anti-IKK antibody (Fig. 3E). Portions of the total cell extracts were immunoblotted with -actin antibody as a control for lane loading or for siRNA specificity. The results confirmed that detectable amounts of endogenous IKK were present in the anti-Hsp70 immunoprecipitates (Fig. 3E, second gel from top) but not in the antiserum control (Fig. 3E, lane 1). In addition, IKK specifically associated with endogenous Hsp70, as the Hsp70–IKK interaction was markedly decreased in Hsp70siRNA transfectants (Fig. 3E, second gel from top, lanes 4–6). IKK activity was analyzed next using an anti-phospho-IB (P-IB) antibody. As expected, elimination of Hsp70 by RNAi rescued heat-mediated inhibition of IB phosphorylation (Fig. 3E, top gel, lanes 3–6). Conversely, using a similar approach, the endogenous Hsp70 could also be detected in the anti-IKK immunoprecipitates (Fig. 3F, second gel from top, lanes 2–4) but not in the antiserum control (Fig. 3F, lane 1). IKK activity, as assessed by P-IB levels, decreased (Fig. 3E, top gel) with decreasing levels of IKK (Fig. 3F, third gel from top).

Hsp70 inhibits the formation of the IKK complex

To define the interactions of the components of IKK and the effects of Hsp70 on the IKK complex in detail, we used the mammalian two-hybrid system to determine whether Hsp70 not only bound IKK, but also disrupted interactions between the three IKKs. IKK, IKK, and IKK cDNAs were inserted in-frame into the pM plasmid containing Gal-4 DNA-binding domain and pVP16-vector-bearing activation domains, respectively. Cos-1 cells were cotransfected with Gal-4-dependent luciferase and IKK constructs. These data showed that IKK,IKK, and IKK formed homocomplexes (IKK/, IKK/, IKK/) and heterocomplexes (IKK/, IKK/, IKK/), and these complexes were disrupted in Hsp70 overexpressing cells (Fig. 4A).

View larger version (63K): [in this window] [in a new window]   Figure 4. Hsp70 disrupts formation of the IKK complex. (A) Two-hybrid experiment. Cos-1 cells were transfected with Gal4-Luc together with the indicated constructs in the figure and in Materials and Methods. Cells were subjected to luciferase analysis 24 h later. (B) Hsp70 disrupts the IKK complex. 293 cells were transfected with His-Hsp70 or His-Hsp70C. After transfection for 48 h, the cells were treated with vehicle or with TNF (25 ng/mL) for 15 min. After immunoprecipitation (IP) with anti-IKK antibody, the IKK activity was analyzed by in vitro kinase assay using GST–IB (1–56) as substrate (top gel). The level of IKK/ present in the immunocomplexes was immunoblotted with anti-IKK/ antibody (second gel from top). Expression of Hsp70, Hsp70C, and IKK/ are shown using anti-His and IKK/ antibodies from whole-cell extracts (third and fourth gels). (C) Hsp70 or Hsp70C stable Hela cells were transiently transfected with Flag-IKK,IKK, and HA-IKK. After 48 h, cells were lysed and applied to a Superose 6 column. The fractions were subjected either to immunoblotting (top six gels) or in vitro kinase assays using the GST–IB (1–56) as substrate (lowest two gels). (D) 293 cells were transfected with the same amounts of HA-IKK (second gel) and increasing amounts of HA-IKKC1 (lanes 7–9) or IKKC2 (lanes 3–5) constructs. Twenty-four hours later, the cells were heated at 45°C for 30 min. After 16 h, the cells were treated for 15 min with TNF or vehicle. The cell lysates were immunoprecipitated with anti-Hsp70 antibody and then immunoblotted with anti-HA-IKK antibody (top gel). The total expression levels of IKK,IKKC1, IKKC2, and Hsp70 in the whole-cell lysates were immunoblotted with the appropriate antibodies. A portion of these cells was transiently transfected with an NF-B-dependent reporter and Luciferase assays (top panel), performed as described in Materials and Methods 24 h after TNF treatment.

These data suggested that Hsp70 might inhibit the assembly of the IKK complex. To test this possibility, we transiently transfected Hsp70- and Hsp70C-inducible Hela cells with Flag-IKK, Flag-IKK, and HA-IKK constructs for 48 h. Following challenge with TNF, cells were extracted and subjected to gel filtration on a Superose 6 column. Each column fraction was immunoblotted with anti-Flag (IKK/), anti-HA (IKK), and anti-His (Hsp70, Hsp70C) antibodies. IKK activity was measured using an in vitro kinase assay. Hsp70 reduced the size of the IKK complex and markedly increased the amounts of IKK in the lower molecular weight fractions (fourth row) in comparison with those from Hsp70C (third row) transfected cells (Fig. 4C). Both the HA-IKK (fourth row) and His-Hsp70 (sixth row) were detected in the same column fractions migrating at 120 kDa, a finding consistent with stable Hsp70–IKK heterocomplexes. As shown earlier, Hsp70 inhibited IKK activity (Fig. 4C, bottom two rows).

Because these results suggested that the Hsp70–IKK interaction was critically involved in regulating NF-B signaling, we confirmed whether disruption of IKK–Hsp70 interaction would be sufficient to overcome Hsp70-mediated NF-B suppression. Because Hsp70 directly bound both IKK and IKKC1, but not IKKC2 (Fig. 3C,D), we used IKKC1 to selectively disrupt the interaction between IKK and Hsp70 by competing for binding to Hsp70. Therefore, 293 cells were transfected with HA-IKK and increasing amounts of HA-IKKC1 or HA-IKKC2. Cells were exposed to TNF to activate NF-B and exposed to heat shock (45°C) for 30 min to induce endogenous Hsp70, and cell extracts were immunoprecipitated with anti-Hsp70 antibody. Appreciable amounts of HA-IKK protein could be detected with anti-HA antibody in the anti-Hsp70 immunoprecipitates (Fig. 4D, top gel, lanes 3–5) but not in the antiserum control (Fig. 4D, top gel, lane 1). Another portion of these cells was transfected with an NF-B reporter gene for NF-B activity analysis. Immunoprecipitation showed that Hsp70 interacted with IKK and that IKKC1 displaced IKK from Hsp70 immunocomplex (Fig. 4D, top gel, lanes 3–5,7–9). IKKC1-mediated IKK release from the Hsp70–IKK complex was accompanied by gradual restoration of NF-B activity (Fig. 4D, upper panel, lanes 7–9), whereas IKKC2 had no such effect (Fig. 4D, upper panel, lanes 3–5).

Hsp70 precludes IKK oligomerization, which is required for NF-B activation

These data showed that IKK formed homo-oligomers (Fig. 4A) and that the coiled-coil motif of IKK was the Hsp70-binding site (Fig. 3B,C), suggesting that the coiled-coil region serves as an effector domain to regulate the IKK complex via its oligomerization. If this conclusion is correct, the coiled-coil deletion mutant of IKK should not self-associate and should suppress NF-B signaling. We tested this by transfecting Cos-1 cells with IKKCC (the coiled-coil deletion mutant of IKK, Fig. 3B) and either Gal4- or NF-B-dependent reporters. The IKKCC failed to interact with itself or IKK and IKK, even on TNF treatment (Fig. 5A). In addition, the IKKCC, like Hsp70, inhibited TNF-mediated NF-B activation (Fig. 5B). These effects of Hsp70 and IKKCC on suppressing TNF-mediated NF-B activation are specific because our previous data showed that neither Hsp70C nor Hsp70N affected NF-B activity (Fig. 1A). These data suggested that IKK oligomerization via the coiled-coil motif was indispensable for IKK activity.

View larger version (105K): [in this window] [in a new window]   Figure 5. Hsp70 inhibits the IKK oligomerization required for NF-B activation. (A) Two-hybrid experiment for protein interactions. Cos-1 cells were transiently transfected with a Gal-4-dependent reporter and the indicated constructs and then subjected to luciferase analysis 24 h later. (B) IKKCC and Hsp70 inhibit TNF-induced (25 ng/mL) NF-B activation. Cos-1 cells were transfected with NF-B-Luc together with either IKKCC or Hsp70 and then subjected to luciferase analysis 24 h later. (C) IKK formed oligomers. 293 cells expressing HA-IKK (lanes 3–7) or HA-IKKCC (lanes 1,2) were labeled with 35S-methionine. Some of the cells were transfected with Hsp70 (lane 6) or Hsp70C (lane 7) expression vectors. Cells were treated with vehicle or TNF (25 ng/mL). Cells were lysed and the extracts were incubated with cross-linker (EGS). After cross-linking, one batch had IKK immunodepleted using IKK antibody (lane 5). IKK was immunoprecipitated with HA antibody (lanes 1–4,6,7), and the IKK immunodepleted extracts were immunoprecipitated with an anti-IKK/ antibody (lane 5). The pellets were washed and analyzed by SDS-PAGE. IKK activity (KA) in the immunoprecipitates was determined with GST–IB as substrate (bottom gel). (D) Hsp70 inhibits the IKK oligomerization in vitro. 35S-labeled IKK and IKKCC were incubated with or without purified Hsp70 and the cross-linking agent (EGS). The proteins were analyzed by SDS-PAGE. (E) IKK or IKK complexes can be visualized in vivo and are inhibited by Hsp70 or heat. GFP–IKK, GFP–IKKCC, or RFP–Hsp70 were expressed alone (panel a) or together (panels b,c) in Hela cells. Nontransfected cells were exposed either to vehicle (panel d), TNF (25 ng/mL for 15 min; panel e), or TNF (25 ng/mL for 15min) plus heat (30 min at 45°C; panel f). They were then fixed and double immunostained with anti-IKK and anti-IKK/ antibodies, and fluorescent signals were visualized by confocal microscopy.

Therefore, we next determined whether Hsp70 would block IKK oligomerization in vitro and whether IKK oligomerization was dependent on any other proteins. ³⁵S-labeled HA-IKK protein and purified Hsp70 were cross-linked with EGS followed by anti-HA antibody immunoprecipitation. Cross-linked, immunoprecipitated IKK yielded additional species with molecular weights corresponding to IKK dimers, trimers, and tetramers, with these multimers being dose-dependently inhibited by addition of Hsp70 (Fig. 5D). This occurred because Hsp70 associated with monomeric IKK to form heterodimers (Fig. 5D, lanes 3–5). These in vitro experiments support the idea that Hsp70 inhibited formation of the IKK complex by blocking oligomerization of IKK. Although these data show that Hsp70 is sufficient to prevent IKK oligomerization in vitro, future studies will be needed to determine whether Hsp70 blocks IKK oligomerization in vivo.

To further confirm the effects of Hsp70 on IKK or IKK complex in living cells, we transfected GFP/IKK (GFP, green fluorescent protein), GFP/IKKCC, and RFP/Hsp70 (RFP, red fluorescent protein) into Hela cells as reporters and examined their subcellular localization using confocal microscopy. Expression of GFP/IKK demonstrated multifocal, punctate regions of staining. They could be macromolecular foci composed of either IKK oligomeric complexes and/or complexes of IKK with other proteins. This contrasted with IKKCC expression, which showed diffuse, uniform fluorescence (Fig. 5E, panel a). These findings were observed in >95% of the cells in which GFP/IKK or GFP/IKKCC was expressed. Cotransfection of GFP/IKK and RFP/Hsp70 resulted in a dramatic redistribution of IKK from discrete macromolecular foci to uniform fluorescence (Fig. 5E, panel c)—the findings being observed in >60% of cells in which both GFP/IKK and RFP/Hsp70 were coexpressed (in some cells, the levels of Hsp70 expression may be not sufficient for the redistribution of IKK). In nontransfected cells, double labeling with antibodies to IKK and IKK/ again showed macromolecular foci in TNF-treated cells (Fig. 5E, panel e), whereas there were no IKK/IKK foci in control (no TNF treatment, Fig. 5E, panel d) or heat-shocked (Fig. 5E, panel f) cells (Fig. 5E)— the findings again being observed in >95% of cells. These data represent a clear in vivo demonstration of the ability of Hsp70 to inhibit formation of these IKK immunostained foci. The data are consistent with Hsp70 interfering with the formation of IKK oligomers (dimers, trimers, tetramers), and/or with Hsp70 interfering with the association of IKK with other proteins that make up the IKK macromolecular complex.

Hsp70 promotes TNF-mediated cell death

Because Hsp70 suppression of NF-B activity could adversely affect cell survival, we investigated the effects of Hsp70 on cell survival following treatment with several stressors. We compared the apoptotic behavior of control Hela cells to IBDN (IBDN-dominant negative used to block NF-B activation) and Hsp70 stable Hela cells. In many of the experiments, the cells were exposed to TNF for 4 h and/or heating to 45°C for 30 min. Cell death was detected by examination of the nuclear morphology using Hoechst staining. Normal cell survival was observed for cells with vector alone, vector + TNF, Hsp70C + TNF, or Hsp70, or when heated alone (Fig. 6A). Cell death, manifested by nuclei with intensely condensed and occasionally fragmented morphology, was observed with cells treated with TNF plus any of the following three treatments: overexpressing IBDN, overexpressing Hsp70, or heating (Fig. 6A).

View larger version (91K): [in this window] [in a new window]   Figure 6. Effects of Hsp70 and NF-B on cell survival. (A) Hsp70-, Hsp70C-, and IBDN-inducible Hela cells were treated with ZnCl2 (100 µM) for 4 h. Sixteen hours later, the cells were treated with vehicle or TNF (50 ng/mL) for 4 h. Nuclei were stained with Hoechst. (B) Hsp70- or Hsp70C-inducible Hela cells had Hsp70 (middle panel) or Hsp70C (right panel) induced with ZnCl2 (100 µM) for 4 h. Sixteen hours later, all cells were treated with the indicated agents. Cell viability was assessed 24 h later using MTT. (C) IBDN-, Hsp70-, or Hsp70C-inducible Hela cells, or cells with empty vector (pMEP4), were treated with increasing concentrations of ZnCl2 for 4 h. Sixteen hours later, cells were treated with vehicle or TNF (50 ng/mL). Cell viability was assessed 24 h later using MTT. (D) NF-B rescues Hsp70–TNF-induced cell apoptosis. Hsp70-inducible Hela cells were transiently transfected with an NF-B/p65 expression vector. Twenty-four hours later, the cells were treated with ZnCl2 (100 µM) for 4 h to induce Hsp70. TNF (50 ng/mL) or vehicle was then added and cell viability assessed 24 h later using MTT. (E) IKK overcomes TNF–Hsp70-induced cell apoptosis. Hsp70-inducible Hela cells were transiently transfected with IKK or IKKCC or empty vector. Twenty-four hours later, the cells were treated with vehicle or with ZnCl2 (100 µM) for 4 h to induce Hsp70. Sixteen hours later, TNF (50 ng/mL) or vehicle was added and cell viability assessed 24 h after that using MTT. (F) IBDN-, Hsp70-, or Hsp70C-inducible Hela cells were treated with ZnCl2 (100 µM) for 4 h. Sixteen hours later, cells were then treated with vehicle or TNF (50 ng/mL). Twenty-four hours later, Western blots were performed on cell extracts with antibodies to PARP, IB, and -actin. NF-B activity was assessed in cells under the same conditions using a luciferase assay (bottom panel). (G) The effects of Hsp70–IKK interactions on IKK activity and cell survival. HA-IKK or Hsp70 siRNA (lanes 8,9) or GFPsiRNA (lanes 1–4,6,7) were transiently transfected into 293 cells. After 48 h, the cells were treated either with vehicle, with TNF (50 ng/mL), and/or with heat (30 min at 45°C). Cells were lysed immediately, and the lysates immunoblotted with anti-phospho-IB (top gel), anti-Hsp70 (second gel down), anti-IKK (third gel down), and -actin antibodies. The survival of similarly treated cells was determined 24 h later using MTT. (H) Hsp70 inhibits FLIP and IAP-2 expression. 293 cells were transiently transfected with His-Hsp70- or HA-IKK-expressing constructs. Forty-eight hours later, cells were treated with vehicle or TNF, and FLIP and IAP-2 expression were assayed by immunoblots 24 h later.

In a second experiment designed to examine the effects of NF-B and dose-dependent changes of Hsp70 and Hsp70C on cell survival, treatment of control Hela cells (empty vector) with TNF plus 0.01–0.25 mM ZnCl₂ decreased cell survival from 100% to roughly 80% (Fig. 6C). In cells stably expressing IBDN, increasing induction of IBDN with increasing doses of ZnCl₂ decreased cell survival from 73% down to 22% after TNF treatment; and, in cells stably expressing Hsp70, increasing induction of Hsp70 with increasing doses of ZnCl₂ decreased cell survival from 75% down to 35% after TNF treatment. Importantly, cells stably expressing Hsp70C—which does not block NF-B activation—had near normal survival with increasing doses of ZnCl₂ from 78% up to 95%. It is notable that for both the Hela cells stably expressing the vector pMEP4 and for the cells stably expressing Hsp70C, increasing doses of ZnCl₂ actually improved cell survival by a small but significant amount in both cases. The explanation for this is not clear, because increasing ZnCl₂ doses induce endogenous Hsp70 to a modest degree (Fig. 1D). It is possible that the ZnCl₂ induces additional heat shock or other stress proteins that protect the Hela cells by a small amount in all of the experiments shown. In the case of the vector and Hsp70C-expressing cells, the small protective effects of these ZnCl₂-induced stress proteins are observed; however, in the IBDN and Hsp70-expressing cells, the combined lethal effects of increasing amounts of IBDN and Hsp70 plus TNF treatment lead to significant cell death and the small protective effects of ZnCl₂ are overwhelmed.

These results suggest that elevated Hsp70 sensitized cells to TNF killing by interfering with NF-B signaling. If this were the case, activation of NF-B would be expected to decrease apoptosis following combined exposure to Hsp70 and TNF. Hsp70 zinc-inducible cells were transiently transfected with NLS/p65 construct. Indeed, increasing levels of p65 improved cell survival following zinc induction of His-Hsp70 and TNF treatment (Fig. 6D). To provide further evidence for this, we reversed the improved cell survival obtained with increasing levels of IKK by the IKK coiled-coil deletion mutant, IKKCC (Fig. 6E).

To examine the mechanism by which Hsp70 inhibition of NF-B signaling leads to cell death, we determined whether elevated Hsp70 favored TNF-mediated caspase activation via inhibiting the NF-B pathway. The effects of Hsp70 and IBDN on caspase-3 processing were examined. The results showed that Hsp70 and IBDN, but not Hsp70C, decreased TNF-induced NF-B activity (Fig. 6F, lower panel). In addition, Hsp70 and IBDN significantly enhanced TNF-induced PARP cleavage (Fig. 6F, topmost gel).

Enforced expression of p65/NF-B or IKK did not entirely rescue Hsp70-mediated cell death (Fig. 6C,D). This might be due to the low efficiency of transient transfection in Hela cells (30%, data not shown). Additionally, Hsp70AS did not appear to block Hsp70 function efficiently (Fig. 1A,E). We therefore performed additional studies in 293 cells, where much higher rates of transfection could be obtained (90%–95% rate of transfection, data not shown). The 293 cells were transfected with IKK and Hsp70 siRNA or control GFPsiRNA for 2 d and then exposed to heat (30 min at 45°C) and/or TNF (50 ng for 15 min). IKK activity was detected using an anti-P-IB antibody. Cell survival was measured using MTT 24 h after heat and/or TNF treatment. Treatment with GFPsiRNA alone, TNF plus GFPsiRNA, or heat shock plus GFPsiRNA did not affect cell survival (Fig. 6G, lanes 1–3). The combination of TNF treatment and heat shock, however, decreased cell survival to 30% (Fig. 6G, lanes 4,7). Overexpression of HA-IKK (confirmed with HA-IKK antibody; Fig. 6G, third gel from top, lanes 5,6) enhanced IB phosphorylation (Fig. 6G, top gel, lanes 5,6) and increased cell survival to near control levels of 90% (Fig. 6G, top panel, lanes 5,6). Similarly, transfection of cells with Hsp70 siRNA almost reversed heat-shock-mediated IKK inhibition (Fig. 6G, top gel, lanes 7–9) and increased cell survival levels to near control levels of 90% despite combined heat shock and TNF treatment (Fig. 6G, top panel, lanes 7–9). Lanes 4 and 7 in Figure 6G are replicates of each experiment and are not duplicates.

Because Hsp70 inhibits NF-B transactivation and promotes caspase activation during TNF signaling (Fig. 6F), it is proposed that Hsp70 would block endogenous NF-B-dependent antiapoptotic gene expression. Indeed, the results show that expression levels of cFLIP and IAP-2, two antiapoptotic proteins (Fig. 6H, upper two gels), decreased in TNF-treated 293 cells with increasing levels of His-Hsp70 protein. This Hsp70-mediated decrease of cFLIP and IAP-2 expression was IKK dependent because increasing IKK levels significantly reversed this (Fig. 6H, last three lanes).

	Discussion

Top Abstract Results Discussion Materials and methods Acknowledgments References

 
This study demonstrates how Hsp70 negatively regulates NF-B, thus establishing the first direct, mechanistic link between Hsp70 and the NF-B signaling cascade. The Hsp70 decrease of NF-B activity tips the balance from cell survival to cell death following TNF/death receptor stimulation (Fig. 7). The ability of Hsp70 to inhibit NF-B signaling may contribute to the enhanced sensitivity of heated cells to chemotherapeutic agents and radiation treatment, and it helps explain at least some of the previous reports that demonstrate the pro-apoptotic effects of heat shock and Hsp70.

View larger version (31K): [in this window] [in a new window]   Figure 7. Proposed pro-apoptotic role of Hsp70 in TNF-mediated apoptosis. TNF simultaneously activates caspase-8 and NF-B (Micheau and Tschopp 2003). Hsp70 directly targets the coiled-coil domain of IKK and impairs TNF-triggered NF-B activation. This occurs by binding of Hsp70 to IKK, which prevents formation of the IKK complex and blocks TNF-triggered NF-B activation. Additionally, Hsp70 cannot block caspase-8-mediated cell apoptosis (Beere et al. 2000). Therefore, once Hsp70 levels are sufficient to significantly block the formation of the IKK complex, this plays an important role in shifting cells from survival to death in response to TNF challenge.

Hsp70 inhibits NF-B activation by binding IKK

Previous studies have shown that Hsp70 and heat shock modulate NF-B function, but the mechanism by which Hsp70 inhibits NF-B remained unclear (Feinstein et al. 1997; Guzhova et al. 1997; Curry et al. 1999; Andres et al. 2002; Malhotra et al. 2002). In the present study, we clarify the mechanism by which Hsp70 inhibits NF-B. Our data demonstrate that Hsp70 directly binds to IKK and this inhibits formation of the IKK complex and blocks NF-B activation.

IKK contains several distinct domains that are involved in regulating IKK activity (Rothwarf et al. 1998; May et al. 2000; Yamamoto et al. 2001). The interaction of IKK with IKK and IKK is critical for the assembly of the high-molecular-weight IKK complex that activates NF-B, and IKK appears to function as an adaptor protein to increase the interactions of key factors required for NF-B activation (Yamaoka et al. 1998). IKK is an essential component of the IKK signalsome, as demonstrated in IKK-deficient or IKK mutant cells, which are unable to trigger the NF-B response to a wide array of stimuli (Rothwarf et al. 1998; May et al. 2000; Rudolph et al. 2000; Makris et al. 2002). The large 700–900-kDa IKK complex does not form in cells lacking IKK (Yamaoka et al. 1998). A variety of proteins that interact with IKK, including RIP, A20, Tax, CIKS, vCLAP, and CYLD are involved in regulating NF-B signaling (Jin et al. 1999; Leonardi et al. 2000; Poyet et al. 2000, 2001; Zhang et al. 2000; Brummelkamp et al. 2003). We postulate and verify the possibility that Hsp70 binds IKK to hamper IKK activation. Our results show that Hsp70–IKK interaction plays a key role in NF-B signaling.

It has been well characterized that the coiled-coil domain of IKK is responsible for IKK oligomerization, which is critical for activating IKK activity (Poyet et al. 2000). The results of this study show that Hsp70 specifically binds the coiled-coil domain of IKK. These findings agree, at least in part, with those of Agou et al. (2002) who have shown that IKK (NEMO) binding via the coiled-coil domain to Hsp70 prevents incorrect interdomain pairing reactions. In the present study, we suggest that excess Hsp70 binding to IKK may prevent IKK self-association that is critical for the formation of the high-molecular-weight IKK complex. Hsp70 binding to the coiled-coil domain of IKK might lead to a chaperone-dependent change in the conformation of IKK. In this case, IKK does not form oligomers when bound to Hsp70, and it is rendered inaccessible to the IKKs and prevents assembly of the IKK complex. Because IKK activity is markedly impaired in cells that express IKKCC or Hsp70, and enforced oligomerization of IKK was able to activate NF-B (Poyet et al. 2000), these data indicate that IKK oligomerization is absolutely essential for TNF-induced NF-B activation (Tegethoff et al. 2003).

Although strong evidence is provided that Hsp70 targeting to IKK plays a negative role in NF-B signaling, the data do not rule out the possibility that Hsp70 may interact with other components of the IKK complex. The gel filtration data show that Hsp70 is detected in fractions in addition to the IKK fractions (200–500 kDa). Future studies will be required to examine the possibility that Hsp70 could have other targets in this pathway. Interestingly, we found that IKK can be directly visualized in macromolecular foci in living cells, which is in line with previous reports (Poyet et al. 2000; Heussler et al. 2002). Moreover, overexpression of Hsp70 or heat treatment significantly suppresses these macromolecular foci. These findings could suggest that Hsp70 inhibits these foci by preventing IKK oligomerization or Hsp70 suppresses these foci by inhibiting IKK binding to other proteins in the IKK complex.

Although Hsp70 did not bind directly to either IKK or IKK (Fig. 3A), Hsp70 still influenced the formation of IKK and IKK hetero- and homocomplexes (Fig. 4A). It is likely that Hsp70 indirectly regulates the assembly of the IKK complex via interacting with IKK. Because IKK is a component of the IKK and IKK hetero- and homocomplexes (Yamaoka et al. 1998; Mercurio et al. 1999), the formation of these complexes may be IKK dependent. Several reports demonstrate that IKK is required to facilitate the interactions of the IKK complex as a whole and/or that it influences individual components of the IKK complex (Mercurio et al. 1999; Poyet et al. 2000; Yamamoto et al. 2001). The exact mechanism remains to be elucidated.

The finding that transfection of Hsp70 siRNA into 293 cells restores IKK activity is strong evidence, along with the other experiments shown, that Hsp70 directly regulates IKK activity. However, it is notable that complete elimination of Hsp70 expression with Hsp70 siRNA (Fig. 3E, lane 6) did not increase IKK activity over the nonheat-shock control, where moderate amounts of Hsp70 were present (Fig. 3E, lane 2). This suggests that other heat shock proteins could also down-regulate IKK activity. Indeed, it has recently been shown that Hsp27 binds IKK and inhibits NF-B activity (Park et al. 2003). The situation is even more complex because Hsp90 can also bind the kinase domain of IKK or IKK to form part of the 900-kDa IKK complex (Chen et al. 2002). However, Hsp90 binding increased TNF-mediated NF-B activation, as shown in the present study (Fig. 1A). Therefore, although heat shock down-regulates NF-B activity (Fig. 4D), this is likely due to a complex interaction of Hsp70–IKK and Hsp27–IKK to down-regulate IKK activity, whereas Hsp90–IKK and Hsp90–IKK interactions up-regulate IKK activity. Because heat shock down-regulates NF-B activation (Fig. 4D), the effects of Hsp70 and Hsp27 on down-regulation must overwhelm the up-regulation by Hsp90. In addition, the current studies demonstrate that although low levels of Hsp70 do not appear to affect IKK activity a great deal, high-level Hsp70 expression significantly blocks IKK activity and markedly decreases NK-B activity.

Although the data show that Hsp70 specifically binds the coiled-coil motif of IKK, the regions of Hsp70 responsible for this binding are less clear. Although the ATP-binding domain of Hsp70 might be expected to bind IKK, our data show that neither the N-terminal nor the C-terminal domains of Hsp70 significantly affect NF-B activity (Fig. 1A). This result is similar to a recent study in which it was shown that full-length Hsp70 binds Apaf-1, whereas neither Hsp70C nor Hsp70N could be shown to interact with Apaf-1 (Ravagnan et al. 2001). The results of our study and that of Ravagnan et al. (2001) could suggest that full-length Hsp70 is essential for the interaction with some molecules such as IKK and Apaf-1. This is not surprising because the ATP-binding domains and the peptide-binding domains of Hsp70 are functionally coupled each other and probably essential for the complete repertoire of physiological effects of the molecule.

Hsp70 promotes apoptosis by blocking NF-B-dependent gene expression

The discovery that Hsp70 suppressed NF-B activation provides the first clear explanation for the pro-apoptotic effect of Hsp70 on cell survival. TNF applied to the three types of cells examined in this study activated NF-B but did not produce apparent apoptosis, a finding consistent with the recent report (Micheau and Tschopp 2003). TNF applied to the same three cell types overexpressing Hsp70 in this study, however, led to the failure to activate NF-B, decreased expression of NF-B antiapoptotic genes such as c-FLIP and IAP-2, and activated caspase-3-dependent cleavage of PARP. These findings are in stark contrast with the effects of Hsp70 on mitochondrial-mediated apoptosis, in which Hsp70 inhibits cell apoptosis by interfering with Apaf-1 and activation of caspase-3-mediated apoptotic pathways (Beere et al. 2000). Hsp70 appears to facilitate apoptosis that is initiated by TNF activation of its death receptors, with very high levels of Hsp70 protein being required to sensitize cells to TNF killing. These results are also generally consistent with a study that demonstrated increased cell death following inhibition of antiapoptotic genes (Goyal et al. 2000).

The death-promoting effect reported here for Hsp70 is at variance with the commonly described protective effect of Hsp70. However, some reports show that heat shock can also increase susceptibility to death, as occurs for NK or LAK cells (Jaattela 1990; Fujieda et al. 1995). In acute myeloid leukemia, apoptosis correlated with increased Hsp70 levels (Chant et al. 1996). A pro-apoptotic function of Hsp70 itself has been described after TCR/CD3 or CD95 activation in Jurkat cells overexpressing Hsp70 (Liossis et al. 1997). Hsp70 was found to accelerate the caspase-activated DNase and DNA fragmentation in TCR-stimulated T-cell apoptosis (Liu et al. 2003). It has been known for some time that heat produces radiosensitization, in which prior heat shock increases cell death in tumors produced by radiation (Dewey and Freeman 1980; Dewey 1994). The adenovirus E1A sensitizes tumor cells to lysis by macrophages through nitric oxide- and TNF-