Objectives: Acute respiratory distress syndrome is a common and highly lethal inflammatory lung syndrome. We previously have shown that an adenoviral vector expressing the heat shock protein (Hsp)70 (AdHSP) protects against experimental sepsis-induced acute respiratory distress syndrome in part by limiting neutrophil accumulation in the lung. Neutrophil accumulation and activation is modulated, in part, by the nuclear factor-[kappa]B (NF-[kappa]B) signal transduction pathway. NF-[kappa]B activation requires dissociation/degradation of a bound inhibitor, I[kappa]B[alpha]. I[kappa]B[alpha] degradation requires phosphorylation by I[kappa]B kinase, ubiquitination by the SCF[beta]^-TrCP (Skp1/Cullin1/Fbox [beta]-transducing repeat-containing protein) ubiquitin ligase, and degradation by the 26S proteasome. We tested the hypothesis that Hsp70 attenuates NF-[kappa]B activation at multiple points in the I[kappa]B[alpha] degradative pathway.

Design: Laboratory investigation.

Setting: University medical center research laboratory.

Subjects: Adolescent (200 g) Sprague-Dawley rats and murine lung epithelial-12 cells in culture.

Interventions: Lung injury was induced in rats via cecal ligation and double puncture. Thereafter, animals were treated with intratracheal injection of 1) phosphate buffer saline, 2) AdHSP, or 3) an adenovirus expressing green fluorescent protein. Murine lung epithelial-12 cells were stimulated with tumor necrosis factor-[alpha] and transfected. NF-[kappa]B was examined using molecular biological tools.

Measurements and Main Results: Intratracheal administration of AdHSP to rats with cecal ligation and double puncture limited nuclear translocation of NF-[kappa]B and attenuated phosphorylation of I[kappa]B[alpha]. AdHSP treatment reduced, but did not eliminate, phosphorylation of the [beta]-subunit of I[kappa]B kinase. In vitro kinase activity assays and gel filtration chromatography revealed that treatment of sepsis-induced lung injury with AdHSP induced fragmentation of the I[kappa]B kinase signalosome. This stabilized intermediary complexes containing I[kappa]B kinase components, I[kappa]B[alpha], and NF-[kappa]B. Cellular studies indicate that although ubiquitination of I[kappa]B[alpha] was maintained, proteasomal degradation was impaired by an indirect mechanism.

Conclusions: Treatment of sepsis-induced lung injury with AdHSP limits NF-[kappa]B activation. This results from stabilization of intermediary NF-[kappa]B/I[kappa]B[alpha]/I[kappa]B kinase complexes in a way that impairs proteasomal degradation of I[kappa]B[alpha]. This novel mechanism by which Hsp70 attenuates an intracellular process may be of therapeutic value.

Acute respiratory distress syndrome (ARDS) is a lethal lung disease and an important public health problem (1, 2). It is primarily a syndrome of excessive inflammation in which alveolar epithelial cells are damaged and ultimately may be destroyed (3, 4). Although some contributory pathophysiologic mechanisms have been identified, most are poorly understood. Increased insight into fundamental biological changes that lead to ARDS would be of scientific value and also might reveal previously unrecognized therapeutic approaches.

Normally, inflammation is a tightly regulated process. In general, proinflammatory mediator expression is balanced by activation of counter-regulatory immunosuppressive responses. This control relies, in part, on specific signaling systems that allow damaged tissues to communicate with cells and organs that may be remote from the site of injury. Both processes require coupling of extracellular signals with intracellular events, processes involving a number of specific biochemical pathways. The uncontrolled inflammation and tissue damage that occurs in ARDS likely results from a loss of this balance (1, 3–7). Impairment of an important endogenous anti-inflammatory response may underlie this problem.

Expression of the phylogenetically conserved heat shock response to noxious stimuli is part of the endogenous system that modulates inflammation (8). Impairment of this anti-inflammatory response, and in particular of the activity of the 70-kDa heat shock protein (Hsp70), might explain the uncontrolled inflammation characteristic of ARDS. Others have demonstrated that stimuli that activate the expression of heat shock proteins protect against lung injury (9, 10) via a mechanism that involves, in part, attenuation of inflammatory pathways (11). We have shown that sepsis is associated with an inappropriate failure to increase expression of Hsp70 in the lungs (5). Further, in rats with sepsis-induced ARDS, administration of an adenovirus overexpressing Hsp70 (AdHSP) prevented or limited lung injury (6). This was associated with attenuation of neutrophil accumulation, an early pathologic event in ARDS (7). Thus, enhancement of Hsp70 expression may represent an important but poorly defined therapeutic avenue. The mechanisms involved need to be investigated more thoroughly.

Activation of the nuclear transcription factor nuclear factor-[kappa]B (NF-[kappa]B) is a central signal transduction event during inflammation (12–14). The role of NF-[kappa]B in modulation of inflammatory lung injury and ARDS is well established (13). One function of the NF-[kappa]B pathway is the enhancement of the expression of genes encoding proinflammatory mediators. Important examples in lung injury include interleukin (IL)-6, which activates neutrophils, and the neutrophil chemoattractant cytokine induced neutrophil chemoattractant-1 (CINC-1) (15–19). NF-[kappa]B, in turn, is a dimeric protein most often consisting of two subunits, p50 and p65. Normally, this dimer is retained in the cytoplasm by association with the inhibitory molecule I[kappa]B[alpha] (20, 21). An essential step in NF-[kappa]B activation is I[kappa]B[alpha] degradation. This releases NF-[kappa]B for migration into the nucleus where it can initiate transcription (12, 20). The pathway leading to degradation of I[kappa]B[alpha] involves at least three sequential biochemical modifications of I[kappa]B[alpha]. These are phosphorylation, poly-ubiquitination, and proteasomal degradation. The first is catalyzed by I[kappa]B kinase (IKK), a complex molecule that contains two catalytic subunits, IKK[alpha] and IKK[beta], an essential regulatory subunit, IKK[gamma] (also called NF-[kappa]B essential modulator or NEMO) (21), and a recently identified co-modulator, the 105-kDa protein eukaryotic-like kinases (ELKS) (22–24). The dominant subunit in I[kappa]B[alpha] phosphorylation during inflammation is IKK[beta] (12). Once phosphorylated, I[kappa]B[alpha] is poly-ubiquitinated by SCF[beta]^-TrCP (Skp1/Cullin1/Fbox [beta]-transducing repeat-containing protein) ubiquitin ligase. This targets I[kappa]B[alpha] for proteolysis by the 26S proteasome (25–28).

Our previous studies suggest that AdHSP effects NF-[kappa]B activity. Here we test the hypothesis that enhancing Hsp70 expression with an adenoviral vector limits sepsis-induced acute inflammation within alveolar epithelial cells by suppressing NF-[kappa]B activation. Hsp70-associated inhibition of NF-[kappa]B is a result of its multiple effects on the I[kappa]B[alpha] degradation pathway. In accord with the observations of others, we find that Hsp70 reduces but does not abolish IKK[beta] activity (11, 29). More importantly, we have uncovered a novel mechanism of I[kappa]B[alpha] stabilization that results from an association with Hsp70. We demonstrate that Hsp70 mediates a perturbation of the protein complex that normally degrades I[kappa]B[alpha]. This complex contains phosphorylated, ubiquitinated I[kappa]B[alpha], the two subunits of active NF-[kappa]B and incomplete IKK complexes that contain ELKS, IKK[beta], and/or IKK[gamma] (NEMO). Stabilization of these intermediate protein destruction complexes attenuates proteasomal degradation of I[kappa]B[alpha], causing NF-[kappa]B retention in the cytoplasm and reduced inflammation.

All animal studies were approved by the university laboratory animal resources committees at both collaborating institutions and conformed to National Institutes of Health standards. Sepsis was induced by cecal ligation and double puncture (2CLP), as described previously (6, 7, 30). Animals were fluid resuscitated with 50 mL/kg normal saline at the time of 2CLP and every 24 hrs thereafter.

As previously described, 10¹¹ viral plaque-forming units of recombinant E1,E3-deleted adenoviral vectors expressing green fluorescent protein (AdGFP) or Hsp70 (AdHSP) (6, 7) were suspended in phosphate-buffered saline (PBS; total volume, 300 µL) and administered via a tracheal catheter. The vectors were administered immediately after 2CLP.

Cytosolic and nuclear proteins were isolated 48 hrs after 2CLP, as previously described (6, 7, 30). Total protein concentration in each lysate was determined using the Bradford assay (Bio-Rad Laboratories, Mannheim, Germany). For IL-6 and CINC-1 experiments, we used whole lung homogenates from which the nuclei had been removed. Protein loading and autoradiographic exposure were uniform throughout all experiments.

Lysates containing 30 µg of total protein were separated on 9% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Immunoblot signals were detected using enhanced chemiluminescence and quantified with scanning densitometry. NF-[kappa]B was identified with either a primary polyclonal rabbit p65 antibody (Santa-Cruz Biotechnology, Santa-Cruz, CA) or a primary polyclonal goat antibody to p50 (Santa Cruz Biotech). I[kappa]B[alpha] was identified using a rabbit I[kappa]B[alpha] polyclonal antibody (Santa Cruz Biotech). Phosphorylated I[kappa]B[alpha] was detected with an anti-mouse monoclonal antibody directed to the Ser32–36 residue (Cell Signaling Technology, Beverly, MA). IKK[beta] was identified using a rabbit polyclonal antibody (Cell Signaling Technology). Hsp70 was identified using a mouse monoclonal antibody (StressGen Biotechnologies, Canada). Ubiquitin was detected with an anti-mouse monoclonal antibody (BAbCo, Beverly, CA). [beta]-Catenin was detected with an anti-mouse monoclonal antibody (BD Transduction Laboratories, Palo Alto, CA). Rabbit antiserum against ELKS protein was a gift from Dr. Frank Mercurio, Celgene, San Diego, CA; rabbit antiserum against NEMO was a gift from Dr. Robert Weil, Pasteur Institute, Paris, France. IL-6 was identified in whole lung extracts using a polyclonal rat anti-IL-6 (PeproTech, Rocky Hill, NJ). CINC-1 was identified in whole lung extracts using a polyclonal goat anti CINC-1 (Santa Cruz Biotech). In all cases, the secondary antibodies were goat anti-rabbit immunoglobulin G or goat anti-mouse immunoglobulin G (Jackson Immunoresearch Laboratory, West Grove, PA).

Electrophoretic mobility shift analysis was performed as previously described (16, 30), with several modifications. A phosphorus-32–labeled double-stranded DNA oligonucleotide containing a consensus-[kappa]B binding site (5'-TCGAGAGATGGGGAATCCCCAGCCC-3') was used. The labeled oligonucleotide was purified on a G-25 Sephadex column. Nuclear extracts containing 5 µg of protein were incubated with binding buffer (20 mM HEPES [pH 7.9], 60 mM KCl, 2 mM EDTA, 5 mM MgCl₂, 10% glycerol, 1 mM phenylmethyl sulfonyl fluoride, 1 mM dithiothreitol, 0.1% NP-40, dIdC [1 µg/µL]) for 20 mins at room temperature. The labeled oligonucleotide was added to the reaction mixture for 20 mins. Specificity for the binding site was determined by cold competition using a 10-fold excess of unlabeled oligonucleotide and supershift analysis with either anti-P65 or anti-P50 established the identity of the bound proteins. Complexes were visualized by autoradiography.

Samples containing 500 µg of cytosolic extract were immunoprecipitated using the following antibodies: rabbit polyclonal anti-IKK[beta] (Cell Signaling Technology), rabbit I[kappa]B[alpha] polyclonal antibody (Santa Cruz Biotech), polyclonal rabbit p65 antibody (Santa Cruz Biotech), anti-mouse monoclonal [beta]-catenin antibody, anti-NEMO, and anti-ELKS. All were diluted 1:100. Samples were agitated overnight at 4°C. Protein A/G beads (Sigma, St. Louis, MO) were added, and the samples were agitated for 1 hr at 4°C and centrifuged at 14,000 rpm for 5 mins at 4°C. The resulting pellet was washed three times with lysis buffer (31). The pellet was suspended in sample buffer and boiled at 95°C for 5 mins. The resulting mixture was resuspended for use in the glutathione-S-transferase-I[kappa]B assay or for immunoblotting.

The 1–54 amino acid glutathione-S-transferase-I[kappa]B[alpha] plasmid was inserted into BL21 Escherichia coli–competent cells (31). Colonies were grown, isolated, lysed, and column purified in a standard manner. Glutathione-S-transferase-I[kappa]B[alpha] was incubated with immunoprecipitated IKK[beta] and 5 mM adenosine triphosphate for 20 mins. The components of the mixture were transferred to a membrane, and immunoblotting with mouse monoclonal anti-phospho-I[kappa]B[alpha] (Cell Signaling Technology) was performed.

Immunostaining was preformed, as previously described (12), using a 1:100 dilution of a primary rabbit antibody directed at the p65 subunit of NF-[kappa]B (Zymed, San Francisco, CA). This was followed by treatment with a secondary anti-rabbit immunoglobulin-G antibody (EnVision System Dakocytomation, DAKO, Carpinteria, CA), as previously described (32).

For gel filtration chromatography, 0.5 mL of cytosolic and nuclear extracts was loaded onto a Sephacryl S300 filtration column (Amersham Pharmacia Biotech, Uppsala, Sweden). The gel filtration buffer contained 20 mM Tris (pH 8.0), 0.1 M NaCl, and 0.02% NaN₃. Proteins were eluted from the column at a flow rate of 1 mL/min, and 1-mL fractions were collected. The column was calibrated using the following standards: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (163 kDa), bovine serum albumin (67 kDa), ovalbumin (44 kDa), and myoglobulin (17 kDa). Gel filtration chromatography fractions were loaded onto a 9% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and were transferred to nitrocellulose membranes.

Murine lung epithelial-12 cells (obtained from ATCC, Manassas, VA) were grown in Dulbecco modified Eagle medium containing 2% fetal calf serum, 100 units/mL penicillin, and 100 µg/mL streptomycin (GIBCO BRL, Grand Island, NY), infected with AdHSP or with AdGFP (5 × 10⁷ plaque-forming units/mL) for 24 hrs or left untreated. The cells were incubated with 20 µM MG-132 (Sigma) in Dulbecco modified Eagle medium for 4 hrs and treated with tumor necrosis factor (TNF)-[alpha] (20 ng/mL) (R&D Systems, Minneapolis, MN) for 20 mins or were left untreated.

The cells were washed with PBS × 1 and lysed with lysis buffer containing 50 mM Tris (pH 7.9), 150 mM NaCl, 0.1% NP-40, 1 mM dithiothreitol, 0.5 mM phenylmethyl-sulfonyl-fluoride, 1 mM Na₃-VO₄, 20 mM para-nitrophenylphosphate (Sigma), 20 mM [beta]-glycerophosphate (Sigma), and a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The lysates were incubated on ice for 10 mins, followed by centrifugation at 12,000 rpm for 30 mins at 4°C. The supernatants (cytoplasmic extracts) were collected, and the pellets were washed several times, suspended in lysis buffer, incubated for 30 mins, and centrifuged. The supernatant fractions were collected for nuclear protein detection or immunoprecipitated with rabbit polyclonal antibody directed at NEMO (diluted 1:100), as described above.

Three samples were evaluated for each experimental point. Analysis of variance with the Bonferroni correction was used to examine differences between and within groups. The significance level was set at p < .05.

We and others have demonstrated that 2CLP induces severe sepsis accompanied by acute lung inflammation in rats (5, 33). In addition, we have shown that treatment of septic rats with an adenoviral vector expressing Hsp70 limits or prevents lung inflammation and injury (6). In the studies presented here, we tested the hypothesis that this protection reflects, in part, Hsp70-mediated attenuation of the activity of the intracellular inflammatory activator NF-[kappa]B. To accomplish this, we examined the effects of treatment with an intratracheal injection of AdHSP, an adenoviral vector expressing Hsp70, on NF-[kappa]B activity 48 hrs after 2CLP. We also examined two other intratracheal injections: PBS as a control for the intratracheal injection itself and AdGFP, an adenoviral vector expressing the marker green fluorescent protein, as a control for vector effects after 2CLP.

Because NF-[kappa]B activity is defined by the nuclear accumulation and [kappa]B promoter binding of the p50/p65 heterodimer, we first performed electrophoretic mobility shift analysis (Fig. 1A) and immunoblotting (Fig. 1B). 2CLP-PBS enhanced p50/p65 heterodimer DNA binding activity and increased the amount of immunoreactive NF-[kappa]B in nuclear isolates. Treatment of septic rats with AdGFP did not alter this septic response. However, AdHSP treatment of septic rats limited both DNA binding activity and intranuclear abundance to levels that were equivalent to those observed in control animals not subjected to 2CLP. Immunostaining confirmed both the specific intranuclear staining for NF-[kappa]B after PBS treatment of sepsis and attenuation of this staining when sepsis was treated with AdHSP (Fig. 1C). These findings indicate that enhanced expression of Hsp70 suppressed or attenuated sepsis-associated accumulation of NF-[kappa]B in alveolar cell nuclei. This might, in part, explain the observed attenuation of acute lung injury.

Our previous studies demonstrated that treatment of septic rats with AdHSP dramatically attenuated neutrophil infiltration into the alveoli (6). Accumulation of neutrophils is dependent on specific chemoattractants or chemokines. The most important in rats is CINC-1 (16–19). Neutrophil activation, in turn, depends on a number of proinflammatory cytokines, especially IL-6. Expression of both CINC-1 and IL-6 is highly dependent on NF-[kappa]B activation (15–19). Our data demonstrate that, relative to nonseptic (T0) controls, expression of both CINC-1 and IL-6 was increased 48 hrs after 2CLP when treatment was limited to the administration of PBS or AdGFP (Fig. 1D). However, treatment of septic rats with AdHSP at the time of 2CLP reduced IL-6 and CINC-1 abundance to levels similar to those seen in untreated (T0) animals (Fig. 1D).

We next sought to define possible mechanisms by which Hsp70 might inhibit NF-[kappa]B activation during 2CLP sepsis. The most likely mechanism would involve failure of I[kappa]B[alpha] to disassociate from NF-[kappa]B, a process that requires degradation of this inhibitor. In accord with this, 2CLP, when treated with either PBS or AdGFP, significantly decreased I[kappa]B[alpha] abundance in the cytoplasm relative to nonseptic (T0) animals (Fig. 2A). However, intracytoplasmic I[kappa]B[alpha] abundance remained similar to that observed in nonseptic (T0) rats when septic animals were treated with intratracheal AdHSP (Fig. 2A).

Initiation of I[kappa]B[alpha] degradation requires phosphorylation of serines 32 and 36. This targets I[kappa]B[alpha] for ubiquitination and proteasomal degradation. Phosphorylation is catalyzed by the oligomeric enzyme IKK, composed of [alpha], [beta], [gamma] ([NU][EPSILON][MU][OMICRON]), and ELKS subunits. The [beta] subunit (IKK[beta]) is essential for cytoplasmic phosphorylation of I[kappa]B[alpha] in inflammation (11, 12, 20, 34). 2CLP-PBS sepsis did not alter immunoreactive IKK[beta] abundance in lung extracts relative to T0 levels (Fig. 2A). 2CLP-PBS did, however, increase the intracytoplasmic abundance of phospho-IKK[beta] in lung extracts (Fig. 2A). Although this increase was not effected by intratracheal administration of AdGFP, the change was attenuated after AdHSP treatment (Fig. 2A). Direct examination of IKK[beta] activity using an ex vivo assay demonstrated that 2CLP enhanced the ability of immunoprecipitated IKK[beta] to phosphorylate I[kappa]B[alpha] (Fig. 2B). This increase was not effected by administration of AdGFP. However, treatment of septic animals with AdHSP limited both I[kappa]B[alpha] phosphorylation (Fig. 2A) and ex vivo IKK[beta] activity (Fig. 2B).

These findings confirm that the sepsis-associated attenuation of the NF-[kappa]B pathway limits IKK[beta] phosphorylation. This explanation has been advanced by others (11, 29). However, a moderate abundance of phospho-IKK[beta] (Fig. 2A) and of residual IKK[beta] activity (Fig. 2B) remained in septic animals treated with AdHSP. Therefore, attenuation of IKK[beta] phosphorylation alone could not completely explain the effects of AdHSP on sepsis-induced activation of NF-[kappa]B. This was confirmed when we measured IKK[beta] abundance and IKK[beta] activity in TNF-stimulated murine lung epithelial-12 cells in culture (Fig. 2C). When cells were infected with AdHSP, immunoreactive IKK[beta] phosphorylation and ex vivo I[kappa]B[alpha] kinase activity were decreased but not eliminated. This led us to search for additional ways in which Hsp70 might alter NF-[kappa]B activation.

I[kappa]B[alpha] phosphorylation is catalyzed directly by the [beta] subunit of IKK (21, 22). However, kinase activity is enhanced by association of IKK[beta] with NEMO and ELKS (20, 22, 24). This forms a complex with a molecular weight of about 250–300 kDa. In addition, NEMO subunits in individual IKK complexes interact with each other to form fully active 600- to 900-kDa multimeric complexes (23, 34, 35). Based on the findings presented in Figure 2B, we hypothesized that increased Hsp70 abundance would inhibit the formation of the full IKK[beta]/IKK[alpha]/NEMO/ELKS complex or prevent the formation of complex multimers that result from NEMO-NEMO interactions (29, 34). Using gel filtration chromatography-fractionation and antibodies directed at the different components of IKK and NF-[kappa]B, we examined the abundance of each of the component proteins as a function of mass (Fig. 3A, top panels). Each of these procedures was performed on samples independently obtained from three animals.

We first examined effects in untreated animals, allowing a comparison with the different treatments of sepsis. In the whole-extract fractions, no phospho-I[kappa]B[alpha] was present in untreated (T0) animals (Fig. 3, A and B, right panels). In these samples, complexes were distributed over the entire (67–1500 kDa) molecular weight range. Experiments in which samples were co-immunoprecipitated with either NEMO or ELKS demonstrated the presence of intact multimeric IKK complexes (Fig. 3, A and B, right panels). Studies with anti-p65 demonstrated that p65 was associated with I[kappa]B[alpha] and IKK in the higher molecular weight complexes (Fig. 3B). This is consistent with a basal interaction between IKK complexes, intact NF-[kappa]B, and I[kappa]B[alpha].

After 2CLP and treatment with PBS, complexes containing NEMO were distributed throughout the entire (67–1500 kDa) molecular weight range studied, whereas IKK[beta]- and ELKS-containing complexes were detected in the higher-weight fractions (600–1500 kDa and 400–1500 kDa, correspondingly) (Fig. 3A, top left). This indicates that composition of the most functional, 900- to 1500-kDa multimers of IKK[alpha]/IKK[beta]/NEMO/ELKS was not altered by 2CLP. Some I[kappa]B[alpha] and phospho-I[kappa]B[alpha] were found in the lower molecular weight fraction, but not in the 600- to 1500-kDa fractions. This suggests that the I[kappa]B[alpha] associated with the higher molecular weight fractions had been eliminated by proteolysis. However, when septic animals were treated with AdHSP, a different molecular distribution emerged. Although NEMO- and ELKS-containing complexes remained distributed throughout the entire molecular weight range (Fig. 3A, top middle), most IKK[beta] was detected in the lower (67–440 kDa) molecular weight fractions. This implies that enhanced expression of Hsp70 in septic rats modified the complexes by limiting the association of IKK[beta] with the mature, multimeric 900- to 1500-kDa IKK complex. A significant increase in the total cytoplasmic content of I[kappa]B[alpha] and phosphorylated I[kappa]B[alpha] (Fig. 3A, top middle) indicated an alteration in I[kappa]B[alpha] degradation.

To determine where Hsp70 disrupted the IKK complex and how this contributed to the formation of immature complexes, we immunoprecipitated lung extract fractions using antibodies to different complex components. In nonseptic (T0) animals, immunoprecipitation with either NEMO or ELKS demonstrated absent phospho-I[kappa]B[alpha] and a strong association between I[kappa]B[alpha], IKK[beta], NEMO, and ELKS (Fig. 3B, right). After 2CLP and treatment with PBS and immunoprecipitation of lung extracts with anti-NEMO, all gel filtration fractions contained negligible amounts of I[kappa]B[alpha] and phospho-I[kappa]B[alpha] (Fig. 3A, left middle). In contrast, after AdHSP treatment at the time of 2CLP, I[kappa]B[alpha] and some phospho-I[kappa]B[alpha] were identified in the 67- to 440-kDa lung extract fractions. This indicates that AdHSP treatment limited the efficient, 2CLP-induced degradation of I[kappa]B[alpha], leaving both I[kappa]B[alpha] and phospho-I[kappa]B[alpha] bound to low molecular weight-NEMO-containing complexes (Fig. 3A, center middle). When protein that had been immunoprecipitated with anti-NEMO was subjected to immunoblotting with antibodies to ELKS or IKK[beta], we again found that AdHSP administration to septic rats induced a shift in the distribution of complexes from higher to lower molecular weight fractions (Fig. 3A, center middle). These cumulative data demonstrate that using AdHSP to enhance Hsp70 expression attenuated the sepsis-induced degradation of I[kappa]B[alpha], presumably by entrapping I[kappa]B[alpha] within lower molecular weight complexes containing different compositions of IKK[beta], NEMO, and ELKS.

Immunoblotting next was performed after immunoprecipitation with an antibody to ELKS. In the 2CLP PBS-treated animals, the complexes all contained NEMO (Fig. 3A, bottom). In isolates from the lungs of septic animals treated with AdHSP, enhanced expression of Hsp70 did not effect the association between ELKS and NEMO. However, the association between ELKS and I[kappa]B[alpha] was maintained in lower molecular weight fractions, but the abundance of I[kappa]B[alpha] in these fractions was markedly increased. In addition, some phospho-I[kappa]B[alpha] was detected in the 67- to 500-kDa fractions of these isolates. Finally, when samples isolated from AdHSP-treated septic animals were immunoprecipitated with an antibody directed at the p65 subunit, we observed a preferential association between Hsp70, the IKK subunits, NEMO, ELKS, p65, and most importantly, total I[kappa]B[alpha] or phospho-I[kappa]B[alpha] (Fig. 3B, middle) at low molecular weights. This indicates that treatment of septic rats with AdHSP trapped I[kappa]B[alpha] and NF-[kappa]B in a complex that also contained Hsp70, IKK[beta], NEMO, ELKS, and total I[kappa]B[alpha] or phospho-I[kappa]B[alpha]. This “entrapped” complex was of relatively low molecular weight and thus did not seem to involve formation of multimeric IKK complex.

To further test our hypothesis that Hsp70-induced modifications in the structure of IKK complexes inhibited sepsis-induced IKK[beta] activity and led to preservation of I[kappa]B[alpha], we again used the in vitro IKK[beta] activity assay (Fig. 3C). This revealed that in lung protein isolates obtained from septic rats treated with PBS, kinase activity was greatest in the higher molecular weight fractions (600–900 kDa). In homogenates isolated from lungs of septic rats administered AdHSP, IKK[beta] kinase activity was concentrated in the lower molecular weight fractions (67–200 kDa). However, AdHSP treatment of 2CLP did not eliminate IKK[beta] kinase activity in the lung; phospho-I[kappa]B[alpha] was still detected in the cytoplasm (Fig. 3C). Therefore, disruption of the IKK multimer itself could not fully explain the mechanism by which AdHSP treatment of septic rats inhibited NF-[kappa]B activity. This suggests that enhanced Hsp70 expression attenuated sepsis-induced I[kappa]B[alpha] degradation by altering something other than IKK-mediated phosphorylation.

Whereas Hsp70-associated disruption of the IKK complex attenuated but did not prevent sepsis-induced phosphorylation of I[kappa]B[alpha], NF-[kappa]B activation was blocked nearly completely. We hypothesized that Hsp70 also affected ubiquitination and proteasomal degradation. Because phosphorylated, ubiquitinated I[kappa]B[alpha] is rapidly degraded, we needed to use a proteasomal inhibitor to examine ubiquitination. This is difficult to accomplish in vivo. Therefore, we examined this process in cultured murine lung epithelial-12 cells. The proteasome was inhibited with carbobenzoxyl-leucinyl-leucinyl-leucinal, MG-132. Cytosolic extracts were subjected to immunoblotting with antibodies to Hsp70, I[kappa]B[alpha] and phospho-I[kappa]B[alpha] (Fig. 4A). I[kappa]B[alpha] was detected in untreated cells (lane 1) and in cells treated with MG-132 (lane 2). Treatment with TNF was associated with a loss of I[kappa]B[alpha] (lane 3). Addition of both MG-132 and TNF was associated with preservation of both I[kappa]B[alpha] and phospho-I[kappa]B[alpha] (lane 4). These cytoplasmic changes were accompanied by appropriate alterations in intranuclear p65 abundance (Fig. 4A, bottom). Treatment with MG-132 reduced intranuclear abundance of p65, even in TNF-stimulated cells (lanes 2 and 4). When similarly treated cells were transfected with AdHSP, some phospho-I[kappa]B[alpha] was detected in untreated samples (lane 5) and in samples treated with MG-132 (lane 6). In contrast to untransfected cells, a significant amount of I[kappa]B[alpha] and phospho-I[kappa]B[alpha] was present in transfected cells treated with TNF (lane 7) and in transfected cells treated with TNF and MG-132 (lane 8). Transfection also virtually eliminated nuclear translocation of p65 (Fig. 4A, bottom, lanes 5–8). Treatment with AdGFP did not prevent degradation of phospho-I[kappa]B[alpha] (Fig. 4A, middle). These data indicate that AdHSP-induced enhancement of Hsp70 prevented degradation of phospho-I[kappa]B[alpha] and that the effect was not a result of vector transfection.

One possible explanation for these findings is inhibition of ubiquitination. To examine this, we immunoprecipitated samples with anti-I[kappa]B[alpha] and probed with an antibody to ubiquitin (Fig. 4B, top). These findings confirmed that complexes ranging from 50 to 250 kDa were poly-ubiquitinated in AdHSP-treated cells. Unstimulated cells showed low levels of ubiquitin-containing species (lane 1). The addition of MG-132 increased this amount (lane 2). Stimulation with TNF[alpha] obliterated most of the ubiquitin-containing molecules (lane 3). Treatment with both TNF[alpha] and MG-132 substantially increased the abundance of the ubiquitinated species, presumably by blocking proteasomal degradation (lane 4). Transfection with AdHSP augmented the abundance of ubiquitinated species in all the groups (Fig. 4B, lanes 5–8). These data indicate that ubiquitinated forms are more abundant in the AdHSP-treated TNF-stimulated cells and support the hypothesis that Hsp70 does not inhibit NF-[kappa]B by blocking ubiquitination.

An alternative explanation of the findings depicted in Figure 4B is that AdHSP directly interfered with the activity of the 26S proteasome. This is consistent with our demonstration that transfection of TNF-stimulated cells with AdHSP preserved I[kappa]B[alpha] and phosphorylated, ubiquitinated I[kappa]B[alpha] (Fig. 4A, lane 7) in a manner similar to that of MG-132 (lane 4). To investigate this possibility, we examined the effect of AdHSP-induced Hsp70 expression on the TNF-stimulated ubiquitination and proteolysis of [beta]-catenin, another substrate of the E3 SCF[beta]^-TrCP ubiquitin ligase (28) (Fig. 4B, bottom). Addition of TNF to the culture medium eliminated [beta]-catenin (lane 3). Transfection with AdHSP decreased the amount of ubiquitinated [beta]-catenin immunoprecipitated from cells and did not prevent degradation of [beta]-catenin and phospho-[beta]-catenin (lane 7). We conclude that Hsp70 specifically altered phosphorylated, ubiquitinated I[kappa]B[alpha] and did not directly interfere with the proteasome (Fig. 4B, bottom).

These findings are consistent with a specific Hsp70-induced preservation of phosphorylated, ubiquitinated I[kappa]B[alpha]. Demonstration that transfecting TNF-stimulated cultured cells with AdHSP stabilized complexes that co-immunoprecipitated with antibodies to NEMO, ELKS, and phospho-I[kappa]B[alpha] or I[kappa]B[alpha] (Fig. 4C, lanes 5–8) strengthens this hypothesis.

In ARDS, uncontrolled inflammation damages lungs cells (4). Using 2CLP, a well-validated, well-accepted method to induce lung injury secondary to intra-abdominal sepsis (5–7, 9, 33), we have shown previously that administration of AdHSP, an adenoviral vector expressing the 70-kDa heat shock protein, inhibited inflammation (6). The studies presented here demonstrate that AdHSP-induced Hsp70 expression attenuates activation of the key proinflammatory transcription factor NF-[kappa]B. Our findings highlight several important aspects of the biology of both Hsp70 and NF-[kappa]B.

Among the many roles of the Hsp70-based chaperone network in the cell are the active maintenance and control of the oligomeric and active states of native proteins (36). Conversely, some investigators have proposed that Hsp70 mediates protein degradation (37–41). The data presented here suggest that the exact effect may be dependent on the Hsp70 substrate and on biological conditions. Specifically, our data indicate that AdHSP-induced Hsp70 expression in acutely injured lungs stabilized incomplete kinase-substrate complexes containing the phosphorylated, ubiquitinated I[kappa]B[alpha] and prevented proteasomal degradation. This effect of AdHSP-induced Hsp70 seems to be directed at stabilizing the I[kappa]B[alpha] protein itself because proteasomal degradation of [beta]-catenin, another SCF[beta]^-TrCP-dependent substrate, is not prevented (28). Hsp70-mediated disruption of other functional, multimeric complexes has been described in other cellular systems (24, 35, 41–44). This suggests that Hsp70 effects are likely to be diverse and not limited to a specific type of biochemical reaction or molecular substrate.

Hsp70 overexpression in acutely injured lungs prevented or disrupted formation of the mature multimeric IKK complexes required for maximal IKK[beta] activity (24, 45–47). These multimers consist of up to four complexes, each containing IKK[alpha], IKK[beta], NEMO, and ELKS (22). Our findings revealed that Hsp70 interfered with NEMO-NEMO interactions, resulting in monomerization and perhaps the dissociation of NEMO from the multimeric complex (24, 48). Such a finding would explain the decreased intensity of NEMO bands on some of the immunoblots derived from animals treated with AdHSP. This observation is important because NEMO plays a key role in the regulation of the IKK[beta] subunit (20, 24). It has been shown that dissociation of NEMO from IKK[beta] decreases cytokine- and endotoxin-stimulated phosphorylation of I[kappa]B[alpha] several-fold (46, 47). The uniform disruption of NEMO-complex interactions throughout the entire range of molecular weights supports the model proposed by Huang et al. (48), that NEMO is in a dynamic flux between its free and IKK-associated states. Although NEMO has been associated with [IOTA][kappa]B[alpha] recruitment to the activated IKK complex (46), Ducut Sigala et al. (22) demonstrated that NEMO and I[kappa]B[alpha] do not directly interact. Rather, these investigators proposed that ELKS “presents” I[kappa]B[alpha] to IKK[beta] for phosphorylation. Our data support this hypothesis, indicating an Hsp70-induced persistent association between NEMO, ELKS, and I[kappa]B[alpha].

In agreement with the observations of others, our findings demonstrate that Hsp70 interfered with I[kappa]B[alpha] phosphorylation (11, 29). However, our data indicate that this is only one of several mechanisms by which overexpressed Hsp70 attenuates NF-[kappa]B activation. The presence of significant amounts of phospho-I[kappa]B[alpha] and the preservation of some IKK[beta] activity in our samples support this contention. It seems that Hsp70-modulated stabilization of immature IKK complexes containing ELKS, NEMO, IKK[beta], p65, and phosphorylated, ubiquitinated I[kappa]B[alpha] is the key factor leading to impaired proteasomal degradation. Certainly, our results highlight the complex interactions between Hsp70 and the NF-[kappa]B activation cascade. They clearly demonstrate that, in contrast to controlled in vitro experiments, molecular activity in living organisms under pathologic conditions is complex, multifaceted, and unlikely to result from a single interaction, even in a specific pathway.

The effects of Hsp70 on NF-[kappa]B activation were similar to those of the proteasomal inhibitor MG-132 (Fig. 4A). In contrast to MG-132, however, AdHSP-maintained [beta]-catenin degradation in TNF-stimulated cells indicates that Hsp70 does not effect proteasomal degradation directly. In addition, our data confirm previous findings that proteasome inhibition by MG-132 induces expression of Hsp70. Others have shown that this protects cells from subsequent thermal injury (49, 50). These experiments, conducted using an animal model and cell culture, support our conclusion that Hsp70 prevents proteasomal degradation by stabilizing an intermediate IKK-I[kappa]B[alpha] complex. These findings invite intriguing speculation. For example, it is possible that the association of Hsp70 with the IKK-NF-[kappa]B-I[kappa]B[alpha] complex limits proteasomal access to phosphorylated, ubiquitinated I[kappa]B[alpha]. Future studies will more fully investigate the composition of this intermediate complex and its interaction with the proteasome.

Some T0 controls demonstrated mild basal NF-[kappa]B activation. This may result from exposure of the lungs to the outside environment. Although all animals were kept in a controlled environment, external stimuli likely initiate mild basal activation of the NF-[kappa]B pathway. This has been demonstrated by others (51, 52).

Our approach to the study of Hsp70 is unique. Whereas other models enhance Hsp70 abundance via a global induction of the heat shock response (9, 50, 53), we used an adenoviral vector to express only Hsp70 and to limit the response to a single tissue, the lung (5). We previously documented that AdHSP preferentially increases Hsp70 expression in pulmonary epithelial cells (6). Importantly, recent studies demonstrate that extracellular Hsp70 exerts proinflammatory effects (54, 55). An additional advantage of the gene-enhancement approach described here is that expression is confined to the intracellular compartment, circumventing potentially counterproductive extracellular effects.

Our study provides essential information regarding the basic mechanisms by which Hsp70 affects NF-[kappa]B and perhaps other multimeric proteins. The demonstration that Hsp70 induces fragmentation of the IKK signalosome and stabilizes intermediate low molecular weight complexes may explain the effects of Hsp70 on other multimeric enzymes. Alternatively, indirect prevention of proteasomal degradation may be profoundly important and could explain Hsp70-associated attenuation of the activity and kinetics of other enzymes or intracellular signal transduction pathways. This finding suggests investigation of exciting therapeutic possibilities. The ability to attenuate a key intracellular process using a naturally occurring molecule is likely to be preferable to and more specific than current approaches using unstable and toxic proteasomal inhibitors. Therefore, future exploitation of this mechanism may be of value in the treatment of inflammation, viral infection, and cancer (56–62).

We thank Eli Pikarsky, Department of Pathology, Hadassah Hebrew University School of Medicine, for help with immunohistochemical studies; Alina Maloyan, Department of Anesthesiology and Critical Care Medicine, Hadassah Hebrew University School of Medicine, for preliminary work; Mario Lebendiker, Wolfson Center for Applied Structural Biology, Institute of Life Sciences, Hebrew University, Jerusalem, Israel, for his help with gel-filtration chromatography assays; Frank Mercurio, Celgene, San Diego, CA, for providing the eukaryotic-like kinases (ELKS) antiserum; Robert Weil, Pasteur Institute, France, for providing the nuclear factor-[kappa]B essential modulator (NEMO) antiserum; Antonio DeMaio, Department of Surgery, University of California, San Diego, CA, for providing the full-length porcine Hsp70 clone and many insightful comments; Irit Alkalay, Lautenberg Center of Immunology, Hebrew University-Hadassah Medical School, Jerusalem, Israel, for providing technical assistance; and Eithan Galun, Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University School of Medicine, for his ongoing support of our research.

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*See also p. 2226. [Context Link]

Key Words: acute respiratory distress syndrome; lung injury; sepsis; heat shock proteins; proteasomal degradation; gene therapy