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The Journal of Immunology, 2005, 175: 2622-2629.
Copyright © 2005 by The American Association of Immunologists

Heat Shock Protein-70 Mediates the Cytoprotective Effect of Carbon Monoxide: Involvement of p38{beta} MAPK and Heat Shock Factor-1 1

Hong Pyo Kim*, Xue Wang*, Jinglan Zhang*, Gee Young Suh*, Ivor J. Benjamin{dagger}, Stefan W. Ryter* and Augustine M. K. Choi2,*

* Department of Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213; and {dagger} Department of Internal Medicine, Division of Cardiology, University of Utah School of Medicine, Salt Lake City, UT 84132


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Carbon monoxide (CO), a product of heme oxygenase activity, exerts antiapoptotic and anti-inflammatory effects in vitro and in vivo. The anti-inflammatory effects of CO involve the inhibition of TNF-{alpha} expression and the enhancement of IL-10 production, resulting in reduced mortality after endotoxin challenge. In this study we demonstrate for the first time that the protective effects of CO involve the increased expression of the 70-kDa inducible heat shock protein (Hsp70) in murine lung endothelial cells and fibroblasts. The p38{beta} MAPK mediated the effects of CO on cytoprotection and Hsp70 regulation. Suppression of Hsp70 expression and/or genetic deletion of heat shock factor-1, the principle transcriptional regulator of Hsp70, attenuated the cytoprotective and immunomodulatory effects of CO in mouse lung cells and in vivo. These data provide a novel mechanism for the protective effects of CO and underscore a potential application of this gaseous molecule in anti-inflammatory therapies.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Carbon monoxide (CO) 3 arises physiologically in most cell types during the oxidative catabolism of heme by the heme oxygenase (HO; E.C. 1:14:99:3) enzymes (1). Expression of the inducible isozyme HO-1 represents a protective response to injury associated with proapoptotic stimuli or inflammation (2, 3). HO-1 confers protection in several models of tissue injury, including endotoxemia, atherogenesis, ischemia/reperfusion (I/R) injury, and transplantation. The exogenous application of CO can simulate the cytoprotective effects of HO-1 in these models (3, 4, 5). To date, the physiological effects of CO have been associated with the modulation of MAPKs or the stimulation of soluble guanylyl cyclase and production of cGMP (6). In this study we demonstrate for the first time that heat shock factor (HSF)-1 acts as a novel downstream effector of the cytoprotective and anti-inflammatory potential of CO.

Exposure of biological systems to elevated temperatures induces adaptive genetic changes known as the heat shock response (HSR) that are highly conserved from prokaryotes to eukaryotes (7). The HSR involves the marked de novo synthesis of distinct heat shock proteins (HSP) in response to thermal stress against an overall attenuation of protein synthesis (8). The synthesis of HSP responds to diverse physiological stimuli (growth factors, cytokines, and hormones), pathological conditions (inflammation and autoimmune reactions), or environmental agents (heavy metals and UV radiation) (9). The HSP function as intracellular chaperones for aberrantly folded, mutated, or thermally denatured proteins (10). Induction of the HSP correlates with acquired cellular thermotolerance (11). The 70-kDa HSP (Hsp70) is the major inducible form of HSP in mammalian cells. Under physiological conditions, the expression of Hsp70 is maintained at low levels but increases dramatically after heat stress (9, 12). The transcriptional regulation of the hsp70 gene requires HSF-1, which binds to specific heat shock elements (HSE), containing the repeating consensus 5'-NGAAN-3', that occur in the 5'-regulatory regions of heat shock genes (13, 14).

In the current study we show that CO up-regulated Hsp70 and inhibited TNF-{alpha}/actinomycin D (Act D)-induced apoptosis in mouse lung endothelial cells (mLEC) and fibroblasts. Both the induction of Hsp70 as well as the cytoprotective effect afforded by CO depended on activation of p38 MAPK in mLEC and fibroblasts, as shown by chemical inhibition or gene deletion studies, respectively. Furthermore, CO protected against endotoxin shock in vivo, which is dependent on Hsp70 induction. Deletion of HSF-1 negated the cytoprotective and immunomodulatory potential of CO in vitro and in vivo. Thus, we provide a novel downstream mechanism by which CO, acting through the MAPK kinase-3/p38 MAPK pathway, exerts potent cytoprotection.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

Mice were acclimated for 1 wk with rodent chow and water ad libitum. Animals were housed according to guidelines from the American Association for Laboratory Animal Care and Research Protocols and were approved by the Animal Care and Use Committee (University of Pittsburgh School of Medicine). The p38–/– and jnk-1–/– mice were gifts from M. Su (Vertex Pharmaceuticals, Boston, MA), and R. Flavell (Yale University School of Medicine, New Haven, CT). Male C57BL/6 mice (The Jackson Laboratory) were used as a control strain for experiments using p38–/– and jnk-1–/– mice.

The hsf-1–/– mice were produced in a mixed genetic background BALB/c x 129XI/SvJ, as previously described (15). The heterozygous knockout hsf-1+/– was used as a control for the in vivo survival experiments as previously described (15). For cellular experiments, the homozygous wild-type hsf-1+/+ mice were used as a source of control cells for cytotoxicity experiments. Because the hsf-1–/– female is infertile, littermates were genotyped using tail DNA (15, 16). The primers for hsf-1–/– genotyping were: 5'-TCTCCTGTCCTGTGTGCCTAGC-3' (forward) and 5'-CAGGTCAACTGCCTACAAGACC-3' (reverse), and those for wild-type genotyping were 5'-AGGACATAGCGTTGGCTACCCGTG-3' (forward) and 5'-GCCTGCTATTGTCTTCCCAATCC-3' (reverse).

Cell culture

Primary cultures of mLEC were prepared from the lungs of wild-type mice as previously described (17) and used at confluence (passages 5–16). The mLEC were cultured in DMEM high glucose medium (Invitrogen Life Technologies) containing 15% FBS, 20 µg/ml endothelial cell growth supplements, 26.5 mmol/l HEPES, and antibiotics. Fibroblasts were cultured from the lungs of p38{beta}–/–, jnk-1–/–, and hsf-1–/– mice as previously described (18). Fibroblasts were cultured in DMEM containing 10% FCS and antibiotics. For chemical treatments, hemin (20 µmol/l), tin protoporphyrin-IX (SnPP-IX; 20 µmol/l; Frontier Scientific), 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ; 50 µmol/l), and MAPK inhibitors, PD98059 (20 µmol/l) and SB203580 (20 µmol/l; Calbiochem), were applied to culture medium from DMSO solutions at the indicated final concentrations. Inhibitors were applied with 1-h pretreatment. For cytoprotection experiments, cells were pretreated for 2 h with air or CO and treated with TNF-{alpha} (25 ng/ml)/Act D (1 µg/ml) in the absence or in the presence of CO (Fig. 2A, upper panel).



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  		FIGURE 2. Hsp70-mediated the cytoprotective effect of CO against TNF-{alpha}/Act D. Wild-type mLEC were incubated with TNF-{alpha}/Act D for 8 h in the absence or in the presence of CO (250 ppm) as depicted by the scheme, and cell viability was determined by crystal violet staining. Relative staining in untreated cells was used as a control and expressed as 100% viability (A). The cleavage of PARP or caspase-3 during TNF-{alpha}/Act D treatment in the absence or in the presence of CO was detected at the indicated times by Western blot analysis (A). The mLEC were transfected with siRNA for hsp70. The relative expressions of Hsp70 and the control proteins, HO-1, TNFR1, p38 MAPK, HSF-1, and {beta}-actin, were determined by Western immunoblot analysis (B). In mLEC transfected with Hsp 70 siRNA or control siRNA, the relative levels of TNF-{alpha} and IP-10 production were determined by ELISA (B, middle panel). Finally, the effect of caveolin-1 siRNA, an irrelevant siRNA control, on heat-inducible Hsp70 expression was also determined (B, right panel). Forty-eight hours after transfection with siRNA, the cells were treated with heat (30 min at 42°C), followed by an additional 4-h recovery period, then assayed for Hsp70 expression by Western immunoblot analysis. Control proteins were also assayed 48 h after transfection, but no heat shock was applied (B). Forty-eight hours after transfection with hsp70 siRNA or control siRNA, the mLEC were incubated with TNF-{alpha}/Act D for 8 h in the absence or in the presence of CO, and cell viability was determined (C). Data represent the mean ± SD of three independent experiments (n = 3). Student’s t test was used to generate p values for comparison of datasets as indicated. ns, not significant.

 
CO exposures

Mice or cell cultures were exposed to compressed air or CO (250 parts/million (ppm)) in modular exposure chambers essentially as previously described (3). The final CO levels in the chambers (250 ppm) were monitored using a CO analyzer (Interscan) (3).

Animal experiments

After exposure to CO for 1 h, mice were injected with LPS (1 mg/kg body weight i.p.) and exposed to CO for an additional 1 h (for TNF-{alpha} measurement) or 16 h (for IL-10 measurement) (3). The relative levels of Hsp70 and HSF-1 were assayed by Western immunoblot analysis in lung tissue 12 after LPS treatment. For survival experiments, a single dose of Escherichia coli LPS was administered (30 mg/kg body weight i.p.) to groups of heterozygous (n = 10) and knockout (n = 10) mice of both sexes (4–6 mo old), and survival was monitored for 192 h (8 days).

Cytokine analysis

Levels of serum TNF-{alpha} or IL-10 were measured with an ELISA kit (R&D Systems). Cytokine levels (TNF-{alpha} and interferon-{gamma} inducible protein-10 (IP-10) were determined in tissue culture medium using corresponding ELISA kits (R&D Systems).

Cell viability assay

Cell viability was determined by both direct cell counting and the crystal violet or MTT assay method as described previously (19). Briefly, adherent cells were stained with 0.5% crystal violet in 30% ethanol and 3% formaldehyde for 10 min at room temperature. Plates were washed six times with water. After drying, cells were lysed with 1% SDS solution, and dye uptake was measured at 550 nm with a 96-well microplate reader. Cell viability was calculated from the relative dye intensity of the mean for triplicate samples and was presented as a percentage of the control value. For all experiments, untreated cells were used as the control, and the mean dye intensity from this sample group was defined as 100% viability. The viability of cells was also evaluated with trypan blue (direct counting) and MTT assays. Because the above-described methods did not show any notable differences, representative data obtained from the crystal violet method are presented.

EMSA

Nuclear extracts were prepared by standard methods from mLEC exposed to CO or heat shock (43°C for 30 min, with 1-h recovery). For EMSA, 5 µg of nuclear protein were incubated with a 32P (5'-end)-labeled double-stranded oligonucleotide (10 fmol) containing the HSE of the mouse hsp70 promoter (–321/–350; 5'-AGACGCGAAACTGCTGGAAGATTCCTGGCC-3') (20). DNA binding reactions contained 10 mmol/l HEPES (pH 7.9), 1 mmol/l DTT, 1 mmol/l EDTA, 80 mmol/l KCl, 4% Ficoll, and 1 µg of poly(deoxyinosinic-deoxycytidylic acid) (Pharmacia Biotech). Samples were electrophoresed through a 4% polyacrylamide gel in 44.5 mmol/l Tris, 44.5 mmol/l boric acid, and 10 mmol/l EDTA (pH 8.3). The gels were dried and autoradiographed with Kodak MR film (Eastman Kodak).

Western blot analyses and immunoprecipitation

The following Abs were used for immunoblotting: rabbit anti-Hsp70 (specific for both constitutive and inducible forms); goat anti-Hsp70 (specific for inducible form); anti-lamin-B, anti-poly(ADP-ribose) polymerase (anti-PARP), anti-TNFR1, and anti-caveolin-1 (Santa Cruz Biotechnology), anti-phospho-p42/44 MAPK, anti-phospho-p38, anti-phospho-JNK, and anti-caspase-3 (Cell Signaling Technology); anti-phosphoserine (Calbiochem); and anti-HSF-1 (Neomarkers). Proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and analyzed by standard immunoblotting or immunoprecipitation procedures as previously described (21), using HRP-conjugated secondary Abs and chemiluminescence detection (Amersham Biosciences).

p38 MAPK assays

p38 MAPK activation was measured using the p38 MAPK assay kit (Cell Signaling Technology), according to the manufacturer’s instructions.

Transfection of small interference (siRNA) for hsp70

SiRNAs were targeted to the mouse hsp70 coding region, 339–359 (accession no. NM 008301; 5'-AACACCATCTTCGACGCCAAG-3') using design software from Dharmacon. The siRNA was transiently transfected into cells (48 h) with TransIT-TKO (Mirus) using the manufacturer’s protocol. A control nonspecific siRNA was provided by Dharmacon (22). Additionally, a siRNA targeting caveolin-1 (5'-TCAGCCGCGTCTACTCCAT-3') was synthesized as an irrelevant specific control.

Statistical analysis

All values were expressed as the mean ± SD from at least three independent experiments. Differences in measured variables between experimental and control groups were assessed using Student’s t test (StatView II; Abacus Concepts). Differences in mortality were assessed using the {chi}2 test for survival. A statistically significant difference was accepted at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CO increased Hsp70 expression in mLEC

Exogenous CO exposure (250 ppm) acutely induced the expression of Hsp70 in mLEC, which lasted up to 8 h and declined by 24 h (Fig. 1A). Treatment with ODQ, a specific inhibitor of soluble guanylyl cyclase activity, did not affect the induction of Hsp70 by CO treatment (Fig. 1B). To examine whether endogenous HO-derived CO can mediate the same response, mLEC were treated with hemin, a well-known inducer and substrate of HO-1. Hemin treatment induced Hsp70 expression in mLEC (Fig. 1C). Treatment with SnPP-IX, a competitive inhibitor of HO activity, decreased both basal and heme-inducible Hsp70 expression (Fig. 1C).



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  		FIGURE 1. Exogenous treatment of CO increased the expression of Hsp70 in mLEC. The mLEC were exposed to CO (250 ppm) for the indicated times, and the expression levels of Hsp70 in mLEC were examined using Western blot analyses. The ratios of Hsp70/{beta}-actin expression were calculated using Bio1D software (version 97; Vilber Lourmat). Student’s t test was used to generate p values for comparison of datasets as indicated. Data represent the mean ± SD of three independent experiments (n = 3; A). The mLEC were treated with CO for 2 h in the absence or in the presence of ODQ and assayed for Hsp70 expression (B). The mLEC were treated with hemin (16 h) in the absence and in the presence of the HO inhibitor, SnPP-IX, and assayed for Hsp70 expression (C). Data are representative of three independent experiments. {beta}-Actin was used as a loading standard (A–C).

 
CO protected against TNF-{alpha} induced apoptosis in mLEC via Hsp70 expression

To examine the potential cytoprotective effects of CO in endothelial cells, mLEC were treated with TNF-{alpha} in combination with Act D for 8 h to induce endothelial cell apoptosis. Although 4- and 16-h treatment times were also assayed (data not shown), 8 h was selected as a treatment time producing a moderate level of cytotoxicity. CO rescued wild-type mLEC from TNF-{alpha}/Act D-induced cell death (Fig. 2A, middle panel). TNF-{alpha}, Act D, or CO treatment alone did not significantly cause cell death (Fig. 2A, middle panel). The cleavage of PARP and caspase-3, two markers of cell death, observable after 8-h TNF-{alpha}/Act D treatment, were reduced by CO compared with that of air-exposed mLEC (Fig. 2A, lower panel). To confirm the role of Hsp70 in the cytoprotective effects of CO, we transfected siRNA corresponding to hsp70 into mLEC, which effectively reduced the expression of Hsp70 relative to control siRNA (Fig. 2B). Neither the control nor the Hsp70-specific siRNA affected the expression of other relevant proteins, including {beta}-actin, HO-1, TNFR1, p38 MAPK, or HSF-1 (Fig. 2B, left panel). Recent studies indicate that certain critical siRNA sequences may stimulate a pathway leading to the expression of IFNs and other immunomodulatory proteins (23). The Hsp70 siRNA used in this study, in addition to excluding any known RNA sequence reported to elicit such a pathway, had no effect on the expression of two end point targets (TNF-{alpha} and IP-10) of this pathway (Fig. 2B, middle panel). A siRNA raised against an irrelevant gene (caveolin-1) did not have any effect on heat-inducible Hsp70 expression (Fig. 2B, right panel). Suppression of hsp70 blocked the protective effect of CO against TNF-{alpha}/Act D-induced cytotoxicity (Fig. 2C).

CO induced cytoprotection and Hsp70 expression in mLEC depended on p38 MAPK

Next we determined examined the relative role(s) of p38 MAPK, which has four known isoforms ({alpha}, {beta}, {gamma}, and {delta}), in the effects of CO with respect to cytoprotection and hsp70 activation. We show that SB 203580, a specific inhibitor of p38{alpha}{beta} MAPK, abrogated the cytoprotective effect of CO against TNF-{alpha}/Act D in mLEC (Fig. 3A), whereas PD98059, a p42/44 MAPK inhibitor, had no effect. Therefore, we examined whether p38{alpha}{beta} MAPK could act as an upstream regulator of Hsp70 induction after CO exposure. Treatment of mLEC with SB 203580 also reduced the Hsp70 levels observed after TNF-{alpha}/Act D treatment and abolished the inducing effect of CO on Hsp70 expression (Fig. 3B). An increase in p38 MAPK activity was detected after 15 min of stimulation with TNF-{alpha}/Act D, which was further augmented in the presence of CO. SB 203580 completely abolished the activation of p38 MAPK by TNF-{alpha}/Act D in both the absence or the presence of CO (Fig. 3C).



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  		FIGURE 3. p38 MAPK mediated the cytoprotection and Hsp70 expression induced by CO. The mLEC were exposed to CO (250 ppm) or air for 8 h in the absence or in the presence of TNF-{alpha}/Act D and/or the p38 MAPK inhibitor SB203580 or the p42/p44 MAPK inhibitor PD98059, and cell viability was determined (A). The mLEC were treated with TNF-{alpha}/Act D for 8 h in the absence or the presence of CO and/or SB203580 and assayed for Hsp70 expression (B). {beta}-Actin served as the loading control. Data represent the mean ± SD of three independent experiments (n = 3). The mLEC were treated with TNF-{alpha}/Act D for 15 min in the absence or the presence of CO (250 ppm) and were assayed for p38 MAPK activation by monitoring the phosphorylation of a substrate (ATF-2) using IgGH as the standard (Cell Signaling Technology; C).

 
CO increased the nuclear translocation of HSF-1 in mLEC, dependent on activation of p38 MAPK

The mLEC treated with TNF-{alpha}/Act D in the presence of CO showed increased nuclear translocation of HSF-1 relative to cells treated with TNF-{alpha}/Act D in the absence of CO (Fig. 4A). We examined whether p38 MAPK acts as an upstream kinase of HSF-1 to regulate the nuclear translocation of HSF-1. Specific inhibition of p38{alpha}{beta} MAPK activation with SB 203580 diminished the translocation of HSF-1 caused by CO (Fig. 4A). Exposure to CO acutely activated HSF DNA binding activity within 10–20 min of treatment (Fig. 4B). The increased serine phosphorylation of HSF-1 was observed within 15 min of TNF-{alpha}/Act D treatment, which could be blocked by SB 203580, indicating a requirement for p38{alpha}{beta} MAPK (Fig. 4C). However, Hsp70 phosphorylation induced by TNF-{alpha}/Act D was not further modulated in the presence of CO. These data indicate that p38 MAPK is important in the pathways leading to both the phosphorylation and the nuclear translocation of Hsp70, but that only the latter process is significantly affected by CO.



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  		FIGURE 4. Role of p38 MAPK in the nuclear translocation of HSF-1. The mLEC were treated as shown in the protocol, and nuclear protein fractions were isolated and subjected to immunoblotting with anti-HSF-1 Ab. Lamin B served as a loading control. The band intensity of HSF-1 was normalized against that of lamin B. Data represent the mean ± SD of three independent experiments (n = 3; A). The mLEC were treated with CO for the indicated times, and nuclear extracts were analyzed for HSF-1 DNA binding activity by EMSA (B). Arrows indicate protein-DNA binding complexes. This figure is representative of three independent experiments. As a positive control, mLEC were exposed to heat shock for 30 min at 43°C and were allowed to recover for 1 h at 37°C. For specific competitive inhibition (CI) of the reaction, a 100-fold molar excess of unlabeled HSE oligonucleotide was added to the mixture (B). The mLEC were treated with TNF-{alpha}/Act D for 15 min in the absence or in the presence of CO (250 ppm) and/or SB203580 (20 µM). Lysates were assayed for HSF-1 phosphorylation by immunoprecipitation (IP) with anti-HSF-1, followed by immunoblotting (IB) with anti-phosphoserine. IP/IB with anti-HSF-1 served as the control (C).

 
CO-induced cytoprotection and HSP70 expression in fibroblasts requires p38MAPK

To further evaluate the involvement of p38 MAPK in CO activation of Hsp70 we re-examined the model in cells derived from p38{beta} null mice (p38{beta}–/–). Cells genetically deficient in a single p38 MAPK isoform provide a more specific tool for determination of the isoform specificity of the response because they remove the questions of selectivity associated with SB 203580, which inhibits both {alpha} and {beta} p38 MAPK isoforms. Fibroblasts were chosen because this cell type is readily harvestable from the lungs of knockout mice. The p38{beta}–/– fibroblasts displayed no detectable expression of p38{beta} (Fig. 5A, inset), relative to a detectable background in wild-type fibroblasts. CO protected against TNF-{alpha}/Act D-induced cytotoxicity in wild-type lung fibroblasts in a fashion similar to that observed in mLEC but did not afford protection in p38{beta} null fibroblasts (Fig. 5A). In contrast, the protection afforded by CO against TNF-{alpha}/Act D-induced toxicity was retained in jnk–/– fibroblasts (Fig. 5A). CO treatment augmented Hsp70 expression in wild-type cells during TNF-{alpha}/Act D treatment (4–8 h), but it failed to stimulate Hsp70 in the p38{beta}–/– cells at the same times and under the treatment conditions (Fig. 5B). Interestingly, Hsp70 levels in p38{beta}–/– fibroblasts were almost negligible at 8 h after treatment with TNF-{alpha}/Act D in the presence of CO (Fig. 5B), which was accompanied by increased cell death (Fig. 5A).



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  		FIGURE 5. p38 MAPK mediates the effects of CO on cytoprotection and Hsp70 regulation in fibroblasts. Wild-type, p38{beta}–/–, or jnk–/– fibroblasts were treated with TNF-{alpha}/Act D for 8 h in the absence or the presence of CO (250 ppm) and assayed for cell viability. Data represent the mean ± SD (n = 3–5; A). The phenotypes of p38{beta}–/– and jnk–/– were confirmed by Western immunoblotting (A, inset). Wild-type or p38{beta}–/– fibroblasts were treated with TNF-{alpha}/Act D for 0–8 h in the absence or the presence of CO (250 ppm) and were assayed for Hsp70 expression (B).

 
HSF-1 is required for the cytoprotective effect of CO

We used fibroblasts from HSF-1 null mice (hsf-1–/–) to evaluate the role of this factor in the modulation of cell survival by CO. In contrast to the protective effect observed in wild-type cells, CO did not rescue hsf-1–/– cells from TNF-{alpha}/Act D-induced cell death (Fig. 6).



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  		FIGURE 6. HSF-1 mediated the cytoprotective effects of CO in lung fibroblasts. Fibroblasts originating from wild-type (A) or hsf-1–/– knockout mice (B) were incubated with TNF-{alpha} (25 ng/ml)/Act D (1 µg/ml) for 8 h in the absence or in the presence of CO (250 ppm), and cell viability was determined. Data represent the mean ± SD (n = 3–5). Because HSF-1 (hsf-1–/–) knockout female mice are infertile, the tail of every mouse was cut to confirm the genotype (A, inset).

 
Protective effect of CO against endotoxin was lost in HSF-1 null mice

We examined the effects of CO exposure in mice for up to 72 h. CO inhalation (250 ppm) induced Hsp70 expression in the lung (Fig. 7A) as well as in other organs, including heart, liver, and skeletal muscle, but not the brain (data not shown). Increased nuclear translocation of HSF-1 was also observed in lung tissue cells after 1 h of CO exposure (Fig. 7B). LPS treatment of wild-type mice acutely induced TNF-{alpha} secretion in serum after stimulation for 1 h (Fig. 8A) and increased the production of IL-10 after 16 h (Fig. 8B). In agreement with our previous findings, CO attenuated the secretion of TNF-{alpha}, whereas CO increased the production of the anti-inflammatory cytokine, IL-10 in wild-type mice (3). As shown previously (15), the LPS-inducible production of TNF-{alpha} was increased by 1.5-fold, but that of IL-10 was not altered in hsf-1–/– mice compared with levels observed in wild-type animals (Fig. 8, A and B). Remarkably, CO did not affect TNF-{alpha} secretion or IL-10 release after systemic LPS challenge in hsf-1–/– mice.



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  		FIGURE 7. Expression of Hsp70 in vivo after CO exposure. Mice were exposed to CO for up to 3 days. At each time point, two mice were killed for lung harvest. Expression levels of Hsp70 were examined using Western blot analyses. {beta}-Actin served as a loading control for lung-derived samples (A). Animals were subjected to air or CO inhalation (250 ppm) for 1–2 h. Total lung cells were assayed for nuclear accumulation of HSF-1 as described in Fig. 4B. B, The data shown are representative of three independent experiments.

 


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  		FIGURE 8. Loss of immunomodulatory effects of CO in the absence of HSF-1. The hsf-1–/– mice (n = 4–6) or wild-type controls (hsf-1+/+) were pre-exposed to a low concentration of CO for 1 h, then treated with LPS and CO for an additional 1 h for TNF-{alpha} production (A) or for an additional 16 h to measure the release of IL-10 (B). Student’s t test was used to generate p values for comparison with values obtained for LPS-treated wild-type and LPS-treated hsf-1–/– mice. *, p < 0.05. N.D., not detected. A and B, LPS was administered to groups of heterozygous (hsf-1+/–; n = 10) and hsf-1–/– mice (n = 10), and survival rates were monitored for 8 days in the absence or in the presence of CO. In the presence of CO, a statistically significantly higher survival rate was found for hsf-1+/– relative to hsf-1–/– mice (by {chi}2 test, p < 0.05; C). Wild-type or hsf-1–/– mice were treated with LPS, followed by exposure to CO (250 ppm) or air. Lungs were removed and assayed for Hsp70 expression 12 h after initiation of treatment. The relative expression of HSF-1 in wild-type or hsf-1–/– lungs under the same conditions is shown (D).

 
To assess the protective role of CO in vivo, hsf-1–/– and heterozygous mice were administered LPS, and survival rates were monitored for 192 h. The survival rate of hsf-1–/– mice was lower than that of heterozygous controls as previously described (15). Exposure to CO (250 ppm) in wild-type mice lengthened the survival after LPS challenge to 192 h (p < 0.05 compared with wild-type mice exposed to air (Fig. 8C), which correlated with high levels of Hsp70 in lung tissue. In the absence of HSF-1, the effect of CO on the survival rate in knockout mice was significantly reduced (p < 0.05 compared with wild-type mice exposed to CO). Previously it has been shown that hsf-1–/– mice display a marked reduction of Hsp70 expression in various organs after hyperthermia treatment compared with wild-type controls (15). We have observed a reduction in inducible Hsp70 expression in the lungs of hsf-1–/– mice after i.p. injection of LPS compared with wild-type controls (Fig. 8D). CO inhalation augmented the expression of Hsp70 in wild-type mice under both control and LPS-treated conditions, but it did not stimulate or restore Hsp70 expression in the lungs of hsf1–/– mice (Fig. 8D).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Endothelial cell apoptosis is a prominent feature associated with tissue injury resulting from acute or chronic inflammation (4). Chemically induced or adenovirus-directed expression of HO-1 protects endothelial cells from apoptosis (2, 4). Recently, we have demonstrated that the antiapoptotic effect of HO-1 in endothelial cells is mediated by CO, can be mimicked by exogenous CO, and depends on the activation of p38 MAPK (4, 5). Previous results from our laboratory have revealed cytoprotective roles of CO in models of tissue injury that involve endothelial cell apoptosis (4, 5, 6). In this study we provide novel evidence that CO exerts its cytoprotective action by modulating Hsp70 expression.

In cultured mLEC, CO exposure (250 ppm) acutely induced the expression of Hsp70, which lasted up to 8 h and declined by 24 h (Fig. 1A). The cognate isoform of Hsp70 (Hsc70), is constitutively expressed in the nonstressed cell and is only slightly inducible by heat (7, 8, 9). CO treatment did not affect Hsc70 expression until 24 h of continuous exposure (our unpublished observations). HO activity represents the principal source of endogenous CO in cells and tissues. We have shown that Hsp70 responds to induction by heme, a well-known inducer of HO activity (Fig. 1C). The heme-dependent induction of Hsp70 could be blocked by an inhibitor of HO activity, suggesting that endogenous as well as exogenous CO may activate the pathway. As further proof, we have observed the elevation of Hsp70 levels in epithelial cells stably transfected with ho-1 relative to vector controls (data not shown).

Several studies have examined the protective capacity of Hsp70 against heat shock and other toxic stimuli. In rats, ischemic preconditioning of the liver strongly induced Hsp70, resulting in resistance to subsequent hepatic I/R injury (24). The down-regulation of Hsp70 can facilitate the induction of apoptosis, whereas the up-regulation of Hsp70 inhibits TNF-{alpha}-induced apoptosis (25, 26, 27). The expression of Hsp70 conferred resistance in models of cardiac I/R injury or acute respiratory distress syndrome (28, 29, 30, 31). Accumulating evidence demonstrates that the HSR can confer anti-inflammatory effects involving the inhibition of proinflammatory gene expression (32, 33, 34, 35). Hyperthermia pretreatment protected against the lethal effect of LPS or TNF-{alpha} treatment in mice, which correlated with systemic Hsp70 induction (25, 32, 33).

We demonstrate that treatment of mLEC with CO protected against TNF-{alpha}/Act D-induced toxicity (Fig. 2A). The cytoprotective effects of CO could be reversed by suppression of Hsp70 expression in mLEC (Fig. 2B) and thus strongly correlated with endogenous Hsp70 levels.

The MAPK family plays an important role in coordinating genetic responses to environmental stresses. Hsp70 expression is regulated in part by protein kinases (36, 37, 38, 39). The phosphorylation and activation of p38 MAPK and p42/44 MAPK precede the induction of Hsp70 in rat 9L cells (36). Osmotic shock induced Hsp70 expression via p38 MAPK activation in human keratinocytes and kidney cells (37, 38). HSF-1, the principal transcriptional activator of hsp70, may represent a direct target of protein kinase-dependent regulation. Several phosphorylation sites in HSF-1 contain the consensus sequence for proline-directed kinases, including p42/44 MAPK, p38 MAPK, and stress-activated protein kinase/JNK, which efficiently phosphorylate HSF-1 in vitro (36). Variation in the type or intensity of stimuli can determine the specific MAPK signaling pathways that regulate HSF-1 activation and Hsp70 induction in response to stress (36).

Four isoforms ({alpha}, {beta}, {gamma}, and {delta}) of p38 MAPK have been identified (40, 41), which can either promote or suppress apoptosis in various cell types (40, 41, 42, 43). For example, p38{alpha} induces apoptosis of cardiac myocytes, whereas p38{beta} suppresses apoptosis in the same cell type (43).

Previous studies from this laboratory have elucidated a role for p38{beta} MAPK activation in anti-inflammatory, antiapoptotic, and antiproliferative effects of CO in various models (3, 4, 5). Because the anti-inflammatory effects of CO, with respect to LPS-inducible TNF-{alpha} production in macrophages, depended on p38{beta} MAPK (3), we explored the role of this kinase as a potential upstream regulator of hsp70. Our data imply a critical role for the {beta}-isoform of p38 MAPK in mediating the effects of CO on cytoprotection and hsp70 expression because these effects were abrogated in mLEC by SB 203580, a selective inhibitor of both {alpha} and {beta} isoforms (Fig. 3, A and B), and in p38{beta} null fibroblasts (Fig. 5, A and B). The observed inhibition of Hsp70 expression by SB 203580 results from inhibition of HSF-1 activation potentially involving both phosphorylation and nuclear localization (Fig. 4). The augmentation of p38{alpha}{beta} MAPK by CO over the TNF-{alpha}/Act D-induced background led to the significant increase in HSF nuclear translocation. Activation of HSF-1 by CO was also expressed as increased HSF DNA binding activity. CO, however, did not further modulate the phosphorylation of HSF, which was also determined to require p38{alpha}{beta} MAPK activity. A possible explanation for these observations is that p38{alpha}{beta} MAPK-dependent HSF phosphorylation is saturated by TNF-{alpha}/Act D stimulation at the dose and conditions used, whereas the pathway leading to HSF nuclear translocation is not saturated by the stimulus and is sensitive to additional increases in p38{alpha}{beta} MAPK by CO treatment.

In the current study we have shown that the effects of CO on Hsp70 production do not depend on the JNK pathway. However, Morse et al. (44) demonstrated that CO signals through the JNK pathway for down-regulation of IL-6 production in LPS-treated macrophages. These experiments demonstrate variation in signaling pathways dependent on the specific downstream target modulated by CO.

Finally, we demonstrate that the anti-inflammatory properties of CO involved HSF-1. Lung fibroblasts derived from hsf-1–/– mice were not rescued from TNF-{alpha}/Act D-induced cell death as observed for their wild-type counterparts. This experiment strongly supports a role for HSF-1 in mediating the in vitro cytoprotective properties of CO. However, reconstitution experiments using HSF-1 expression vectors were not performed.

The cytoprotective action of CO was assessed in vivo in HSF-1 knockout mice (hsf-1–/–), which display an increased sensitivity to LPS-induced toxicity and lethality (15). Our data clearly showed that the protective effect of CO in mice against endotoxin shock required HSF-1 and involved decreasing the production of proinflammatory cytokines such as TNF-{alpha} (Fig. 8) and increasing the production of the anti-inflammatory cytokine, IL-10. Others have shown that the HSR strongly suppressed LPS-induced production of the proinflammatory cytokine IL-12, whereas it augmented that of IL-10 (19). In the absence of HSF-1 and the downstream expression of Hsp70, the modulatory effects of CO on cytokine production were lost. Accordingly, it has been demonstrated that overexpression of Hsp70 reduces TNF-{alpha} production in vitro or in vivo after LPS treatment (30, 32, 33, 45, 46). Inactivation of HSF-1 does not affect HSF-2 expression, yet the HSR is completely abolished in hsf-1–/– cells. In several organs of hsf-1–/– mice, HSF-2 could not compensate for HSF-1 as the main stress-inducible transactivator of the HSR (15, 16). Thus, the effects of CO reported in this study mainly depended on HSF-1 and probably exclude a role for HSF-2.

Recent studies demonstrate that components of the HSR directly target the promoters of proinflammatory genes and inhibit septic shock (47, 48). HSF-1 acts as a negative regulator of several non-heat shock genes, including IL-1{beta}, c-fos, urokinase, and TNF-{alpha} (47, 48). HSF-1 strongly binds at the 5'-untranslated region of the murine TNF-{alpha} gene and represses its transcription (47). Similarly, HSF-1 represses the transcription of the IL-1{beta} gene through physical interaction with the CCAAT/enhancer binding protein (48). These findings imply that HSF-1 is essential in mediating protection against the toxic effects of bacterial endotoxin. HSF-1 appears to carry out this function through the transcriptional repression of cytokine genes, suggesting a role for HSF-1 in antagonizing the acute phase response through redundant mechanisms.

In summary, we have shown in this study that Hsp70 and HSF-1 play vital roles in the protective effects of CO against cytokine-induced endothelial cell apoptosis and against the lethal effects of endotoxin shock. Activation of p38{beta} MAPK by CO promotes the nuclear translocation of HSF-1, which regulates the expression of cytoprotective Hsp70 in cells and tissues. Our results suggest that administration of CO may have therapeutic potential to modulate inflammatory responses under pathological conditions such as sepsis.


   Acknowledgments
 
We thank G. A. May and E. Ifedigbo for animal handling.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by an award from the American Heart Association (AHA 0335035N; to S.W.R.) and National Institutes of Health Grants R01HL60234, R01AI42365, and R01HL55330 (to A.M.K.C.). Back

2 Address correspondence and reprint requests to Dr. Augustine M. K. Choi, Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Montefiore University Hospital 628NW, 3459 Fifth Avenue, Pittsburgh, PA 15213. E-mail address: choiam{at}upmc.edu Back

3 Abbreviations used in this paper: CO, carbon monoxide; Act D, actinomycin D; HO, heme oxygenase; HSE, heat shock element; HSF, heat shock factor; HSP, heat shock protein; Hsp70, 70-kDa heat shock protein; HSR, heat shock response; IP-10, interferon (IFN)-{gamma} inducible protein-10; I/R, ischemia/reperfusion; mLEC, mouse lung endothelial cell; ODQ, 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one; PARP, poly(ADP-ribose) polymerase; ppm, parts per million; siRNA, small interference RNA; SnPPIX, tin protoporphyrin-IX. Back

Received for publication January 6, 2005. Accepted for publication May 30, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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