The Anti-inflammatory Effects of Heat Shock 72 Involve Inhibition of High-Mobility-Group Box 1 Release and Proinflammatory Function in This information is current as of September 25, 2021. Daolin Tang, Rui Kang, Weimin Xiao, Haichao Wang, Stuart K. Calderwood and Xianzhong Xiao J Immunol 2007; 179:1236-1244; ; doi: 10.4049/jimmunol.179.2.1236 http://www.jimmunol.org/content/179/2/1236 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

The Anti-Inflammatory Effects of Heat Shock Protein 72 Involve Inhibition of High-Mobility-Group Box 1 Release and Proinflammatory Function in Macrophages1

Daolin Tang,2* Rui Kang,2† Weimin Xiao,*‡ Haichao Wang,3§¶ Stuart K. Calderwood,ʈ and Xianzhong Xiao3*

High-mobility-group box 1 (HMGB1), a nuclear protein, has recently been identified as an important mediator of local and systemic inflammatory diseases when released into the extracellular milieu. Anti-inflammatory regulation by the stress response is an effective autoprotective mechanism when the host encounters harmful stimuli, but the mechanism of action remains incom- pletely delineated. In this study, we demonstrate that increases in levels of a major stress-inducible protein, heat shock protein 72

(Hsp72) by transfection attenuated LPS- or TNF-␣-induced HMGB1 cytoplasmic translocation and release. The mechanisms Downloaded from involved inhibition of the region maintenance 1 (CRM1)-dependent nuclear export pathway. Overexpression of Hsp72 inhibited CRM1 translocation and interaction between HMGB1 and CRM1 in macrophages post-LPS and TNF-␣ treat- ment. In addition, overexpression of Hsp72 strongly inhibited HMGB1-induced (TNF-␣, IL-1␤) expression and release, which correlated closely with: 1) inhibition of the MAP kinases (p38, JNK, and ERK); and 2) inhibition of the NF-␬B pathway. Taken together, these experiments suggest that the anti-inflammatory activity of Hsp72 is achieved by interfering with both the release and proinflammatory function of HMGB1. Our experimental data provide important insights into the anti-inflammatory http://www.jimmunol.org/ mechanisms of heat shock protein protection. The Journal of Immunology, 2007, 179: 1236–1244.

epsis is a systemic inflammatory syndrome that can lead flammatory cytokine that mediates the response to infection, in- to lethal organ damage. Despite advances in antibiotic jury, and inflammation (4–8). Although residing predominantly in S therapy and intensive care, the overall mortality from the nucleus of quiescent macophages/, HMGB1 can be severe exceeds 30% in the United States (1). Initiated by actively secreted in response to exogenous and endogenous in- an infection, the pathogenesis of sepsis is attributable, at least in flammatory stimuli such as endotoxin, TNF-␣, IL-1, IFN-␥, and part, to dysregulated systemic inflammatory responses charac- hydrogen peroxide (4, 9–11). In addition, HMGB1 can be pas- terized by excessive accumulation of various proinflammatory sively released by necrotic cells (6). HMGB1 has a chromosome by guest on September 25, 2021 (2, 3). region maintenance 1 (CRM1)-dependent nuclear active export Recent evidence indicates that high-mobility-group box 1 pathway (12). Once released from cells, HMGB1 can bind to cell (HMGB1),4 a ubiquitous nuclear protein, is an important proin- surface receptors including the for advanced glycation end products), TLR-2, and TLR-4 and mediate cellular responses including chemotactic cell movement and release of proinflamma- *Laboratory of Shock, Department of Pathophysiology, Xiangya School of Medicine, Changsha, People’s Republic of China; †Department of Pediatrics, Xiangya Hospital, tory cytokines (e.g., TNF-␣ and IL-1; Refs. 13–17). Anti-HMGB1 Central South University, Changsha, People’s Republic of China; ‡College of Op- Abs or specific antagonists protect mice from lethal endotoxemia tometry, University of Houston, Houston, TX 77204; §Department of Emergency Medicine, North Shore University Hospital, New York University School of Medi- or sepsis, even when the first dose are given 24 h after onset of cine, Manhasset, NY 11030; ¶Feinstein Institute for Medical Research, Manhasset, diseases (4, 18), establishing HMGB1 as an important therapeutic ʈ NY 11030; and Beth Israel Deaconess Medical Center, Harvard Medical School, target for inflammatory diseases (5, 19). Boston, MA 02215 MAPKs and the NF-␬B/I␬B pathway play essential roles in in- Received for publication April 5, 2007. Accepted for publication April 24, 2007. flammation because of the rapidity of activation and potency in The costs of publication of this article were defrayed in part by the payment of page activation of transcription of NF-␬B (20, 21). The MAPK family charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mainly includes ERK, p38 kinase, and JNK. Once activated, the 1 This work was supported by Grants 30500485 (to D.T.) and 30330280 (to X.X.) MAPKs modulate the functional responses of cells through from the National Natural Sciences Foundation of China, the Major National Basic phosphorylation of transcription factors and activation of other Research Program of China Grant G2000056908 (to X.X.), the Specialized Research kinases. NF-␬B regulates the transcription of many in- Fund for the Doctoral Program of Higher Education of China Grant 20060533009 (to X.X.), and the Innovative Program of Central South University for Post-graduate volved in immunity, inflammation, and protection from pro- Research Grant 2005-75239 (to D.T.), and in part by National Institute of General grammed cell death (22). Medical Sciences Grants R01GM063075 and R01GM070817 (to H.W.) from the National Institutes of Health. The heat shock (HS) response (HSR) is a universal stress re- sponse activated in all prokaryotes and eukaryotes and is repre- 2 D.T. and R.K. contributed equally to this paper. sented at the molecular level by the induction of heat shock protein 3 Address correspondence and reprint requests to Dr. Xianzhong Xiao, Department of Pathophysiology, Xiangya School of Medicine, Central South University, 110 (HSP) synthesis (23). The Hsp70 family is the best characterized Xiangya Road, Changsha, Hunan 410008, People’s Republic of China. E-mail ad- dress: [email protected] or Dr. Haichao Wang, Department of Emergency Medicine, North Shore University Hospital-NYU School of Medicine, 350 Commu- HSP, heat shock protein; Hsp72, heat shock protein 72; Hsp73, heat shock pro- nity Drive, NY 11030. E-mail address: [email protected] tein 73. 4 Abbreviations used in this paper: HMGB1, high-mobility group box 1; CRM1, chromosome region maintenance 1; HS, heat shock; HSR, heat shock response; Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 www.jimmunol.org The Journal of Immunology 1237 of these and include the constitutive Hsp73, and stress-inducible peroxidase-conjugated secondary Abs for1hat25°C, the signals were Hsp72. The HSPs are involved in protecting the organism from a visualized by diaminobenzidine detection (Boster Biotech) according to the variety of insults including /reperfusion and oxidative in- manufacturer’s instruction. jury (24, 25). Although the underlying mechanism of this protec- Immunocytochemical analysis tion involves their chaperone functions (e.g., preventing abnormal Cells were cultured on glass coverslips and fixed in 4% formaldehyde for protein folding or aggregation; Ref. 26), recent work also shows 30 min at room temperature before detergent extraction with 0.1% Triton that HSPs directly interferes with cell death pathways including X-100 for 10 min at 4°C. Coverslips were saturated with PBS containing and necrosis (27, 28). Induction of Hsp72 in vitro by 2%BSAfor1hatroom temperature and processed for immunofluores- heat shock or Hsp72 overexpression can reduce mortality in cence with anti-HMGB1 or anti-CRM1 Ab (BD Biosciences) followed by experimental models of septic shock, endotoxemia, and adult Cy3 (Sigma-Aldrich)- or FITC (Boster Biotech)-conjugated Ig, respec- tively. Nuclear morphology was analyzed with the fluorescent dye Hoechst respiratory distress syndrome, and can regulate expression of 33258 (Sigma-Aldrich). Between all incubation steps, cells were inflammatory genes, such as TNF-␣, IL-1, IL-12, IL-10, and washed three times for 3 min with PBS containing 0.2% BSA. Images IL-18 (29–36). were taken with a fluorescence microscope (ECLIPSE 80i; Nikon). The The mechanisms of anti-inflammatory regulation by HS or relative fluorescence intensity of HMGB1 or CRM1 in the nuclear and cytoplasmic regions of multiple representative cells (3–5 different fields Hsp72 are mostly uncharacterized. However, our previous containing ϳ50 cells) was assayed using the ImageProPlus software study showed that HS reduces LPS-induced HMGB1 active re- (Media Cybernetics). lease from cultures (37), and nuclear Hsp72 is a negative regulator of oxidative stress-induced HMGB1 cyto- Cytokine measurements plasmic translocation and release (38). Here we demonstrate Levels of TNF-␣ and IL-1␤ in the culture medium were determined by Downloaded from that Hsp72 not only inhibits HMGB1 release induced by exog- commercially obtained ELISA kits (Boster Biotech) according to the man- enous bacterial endotoxins or the endogenous proinflammatory ufacturer’s instructions. Levels of TNF-␣ and IL-1␤ mRNA were assayed by RT-PCR as previously described (36). cytokines TNF-␣ but also suppresses the proinflammatory ac- tivities of HMGB1. Immunoprecipitation analysis Cells were lysed at 4°C in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, Materials and Methods containing 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, http://www.jimmunol.org/ Cell culture and treatment 0.1% SDS, protease inhibitor mixture), and cell lysates were cleared by a brief centrifugation (12,000 ϫ g for 10 min). Concentrations of in Murine macrophage-like RAW264.7 cells were obtained from the Shang- the supernatant were determined by the BCA assay. Before immunopre- hai Type Culture Collection and cultured in RPMI 1640 (Invitrogen Life cipitation, samples containing equal amount of proteins were precleared Technologies) supplemented with 10% heat-inactivated FBS, 2 mM glu- with protein A or protein G agarose/Sepharose (Amersham Biosciences) tamine, and antibiotic-antimycotic mix in a humidified incubator with 5% (4°C, 3 h), and subsequently incubated with various irrelevant IgG or spe- CO2 and 95% air. cific Abs (5 ␮g/ml) in the presence of protein A or G agarose/Sepharose Recombinant human Hsp72 plasmid vector (pcDNA3.1-Hsp72) was in- beads for 2 h or overnight at 4°C with gently shaking. After incubation, troduced into RAW264.7 cells by liposomal delivery. After transfection, agarose/Sepharose beads were washed extensively with PBS, and proteins cells were cultured in RPMI 1640 (Invitrogen Life Technologies) supple- ϫ

were eluted by boiling in 2 SDS sample buffer before SDS-PAGE by guest on September 25, 2021 mented with geneticin G418 (Promega) for further selection of tranfected electrophoresis. cells. The surviving cells in medium containing G418 were considered to be stable cell lines that contained overexpression of Hsp72 gene, and ex- EMSA for NF-␬B activation pression of Hsp72 was confirmed by Western blotting. At 70% confluency, RAW264.7 cells were removed mechanically and Nuclear extracts were prepared, and EMSA was performed using biotin- resuspended in serum-free Opti-MEM I medium (Invitrogen Life Technol- labeled oligonucleotides to measure NF-␬B DNA binding activity (Pierce ogies). After preincubation for 2 h, RAW264.7 cells were treated with LPS Chemicals). In brief, 10 ␮g of nuclear extract were incubated with biotin- (Escherichia coli 0111:B4; Sigma-Aldrich), recombinant TNF-␣ protein labeled, double-stranded DNA fragment corresponding to the NF-␬B (PeproTech), or recombinant HMGB1 protein (Ref. 39; provided by Dr. (sense strand, 5Ј-AGTTGAGGGGACTTTCCCAGGC-3Ј) at 4°C in the Kevin J. Tracey and Dr. Huan Yang, Laboratory of Biomedical Science, presence or absence of 100ϫ unlabeled NF-␬B and subjected to electro- and Center for Immunology and Inflammation, Feinstein Institute for Med- phoresis at 110 V for4hat4°Cona5%polyacrylamide gel. The gels were ical Research, Manhasset, NY) as indicated. transferred to a nylon membrane and subjected to cross-link with a UV lamp, and the signals were detected by chemiluminescence. For supershift Preparation of cellular extracts experiments, NF-␬B p65 Ab (Santa Cruz Biotechnology) was incubated with the binding reaction mixture for 30 min at room temperature before At indicated time points after the treatment, cells were harvested and addition of the labeled oligonucleotide. washed twice with cold PBS; and nuclear and cytoplasmic extracts were prepared according to the method of Schreiber et al. (40). Statistical analysis HS treatment Significance of differences between groups was determined by two-tailed Student’s t test or Fisher’s least significance difference test, as indicated. RAW264.7 cells were sealed in screw-cap flasks containing an atmosphere Values of p Ͻ 0.05 were considered significant. of 5% CO2 and 95% air. These flasks were then immersed completely in a water bath with a measured temperature of 42.5°C. With this protocol, the medium within the flask reached 42.5°C within 5 min of immersion. After Results 1 h of immersion, cells were left at 37°C for 12 h and then treated with Hsp72 inhibits LPS- and TNF-␣-induced release and HMGB1. translocation of HMGB1 in RAW264.7 macrophages Western blotting We have previously shown that nonlethal HS inhibits LPS-induced HMGB1 release (37). To investigate whether this effect is medi- Proteins in the whole-cell lysate, subcellular fractions, or concentrated cell culture supernatants were resolved on 10% SDS-PAGE gel, and transferred ated by Hsp72, we stably transfected RAW264.7 cells with Hsp72 to a polyvinylidene fluoride membrane. After blocking, the membrane was encoding expression vector (RAW/Hsp72). Control cells were ob- incubated for2hat25°C with various primary Abs specific for HMGB1, tained by transfection of RAW264.7 cells with empty vector CRM1 (BD Biosciences), Hsp72 (Stressgen), ␤-actin, GAPDH (KangChen (RAW/Neo). Western analysis demonstrated overexpression of Biotechnology), ␤-tubulin (Sigma-Aldrich), phospho-p38 (T180/Y182; R&D Systems), phospho-ERK1/2 (Thr202/Tyr204; Upstate Biotechnology), Hsp72 in RAW/Hsp72 cells compared with RAW/Neo cells (Fig. phospho-JNK1/2 (Thr183/Tyr185; Cell Signaling Technology), or NF-␬B 1, A and B). Consistent with previous reports, LPS and TNF-␣ p65, I␬B␣ (Santa Cruz Biotechnology), respectively. After incubation with induced active HMGB1 release in macrophages, which was not 1238 EFFECT OF Hsp72 ON HMGB1 RELEASE AND CYTOKINE ACTIVITY

FIGURE 1. Effects of Hsp72 overexpression on LPS- and TNF-␣-induced HMGB1 release and translo- cation in macrophages cultures. A and B, Level of HMGB1 in the culture medium of nontransfected

RAW264.7 cells (RAW) or cells stably transfected with Downloaded from empty plasmid (RAW/Neo) or Hsp72 gene (RAW/ Hsp72) in the absence or presence of LPS (500 ng/ml) or TNF-␣ (5–20 ng/ml) for 24 h. Blots are representa- tive of three independent experiments with similar re- sults. C, Transfected RAW264.7 cells (RAW/Neo and RAW/Hsp72) incubated with LPS (500 ng/ml) or

TNF-␣ (5 ng/ml) for 12 h, and the subcellular localiza- http://www.jimmunol.org/ tion of HMGB1 was detected by immunocytochemical analysis (left). The relative fluorescence intensity in the nuclear (N) or cytoplasmic (C) regions of multiple rep- resentative cells were determined using ImageProPlus software (right) and expressed as mean Ϯ SEM (in ar- bitrary units, AU) of three independent experiments. Red, HMGB1; blue, nuclei (original magnification, p Ͻ 0.05. D, Cytosolic level of HMGB1 in ,ء .(ϫ400 the RAW/Neo (Neo) or RAW/Hsp72 (72) cells in the absence or presence of LPS (500 ng/ml) or TNF-␣ (5 by guest on September 25, 2021 ng/ml) for 12 h. ␤-Tubulin was used as a loading con- trol. Blots are representative of three independent ex- periments with similar results.

altered in control-transfected cells (RAW/Neo; Fig. 1, A and B; by 12 h after LPS or TNF stimulation, HMGB1 staining was Refs. 4, 9, and 41). However, overexpression of Hsp72 signifi- found in both the nuclear and cytoplasmic compartments of cantly inhibited LPS- and TNF-␣-induced HMGB1 release (Fig. macrophages (Fig. 1C). To confirm HMGB1 cytoplasmic trans- 1). Interestingly, although LPS did not induce Hsp72 expression location, cytoplasmic fractions were isolated and assayed for (36), TNF-␣ dose dependently induced Hsp72 expression (Fig. 1, relative content for HMGB1 or ␤-tubulin using specific Abs. A and B; Refs. 42 and 43). The levels of HMGB1 in the cytoplasmic fractions were sig- To elucidate the mechanisms underlying Hsp72-mediated nificantly increased after treatment with LPS or TNF-␣ (Fig. suppression of HMGB1 release, we determined the effects of 1D). However, overexpression Hsp72 significantly inhibited Hsp72 overexpression on LPS- or TNF-induced HMGB1 cyto- LPS- and TNF-␣-induced HMGB1 translocation (Fig. 1, C and plasmic translocation. HMGB1 was predominantly localized in D), suggesting that Hsp72 attenuates HMGB1 release partly the nucleus of unstimulated macrophages (Fig. 1C). However, through interfering with its cytoplasmic translocation. The Journal of Immunology 1239

FIGURE 2. Effects of Hsp72 overexpression on LPS- and TNF-␣-induced CRM1 translocation in mac- rophages cultures. A, Transfected RAW264.7 cells (RAW/Neo and RAW/Hsp72) were incubated with LPS (500 ng/ml) or TNF-␣ (5 ng/ml) for 12 h, and the sub- cellular localization of CRM1 was detected by immu- Downloaded from nocytochemical analysis (left). The relative fluorescence intensity in the nuclear (N) or cytoplasmic (C) regions of multiple representative cells were determined using ImageProPlus software (right) and expressed as mean Ϯ SEM (in arbitrary units, AU) of three indepen- dent experiments. Green, CRM1; blue, nuclei (original Ͻ ء ϫ magnification, 400). , p 0.05. B, Cytosolic level of http://www.jimmunol.org/ CRM1 in the RAW/Neo (Neo) or RAW/Hsp72 (72) cells in the absence or presence of LPS (500 ng/ml) or TNF-␣ (5 ng/ml) for 12 h. ␤-Actin was used as a load- ing control. Blots are representative of three indepen- dent experiments with similar results. by guest on September 25, 2021

Hsp72 inhibits LPS- and TNF-␣-induced cytoplasmic Hsp72 inhibits the interaction between HMGB1 and CRM1 by translocation of CRM1 in RAW264.7 macrophages competitively binding to HMGB1 in RAW264.7 macrophages ␣ CRM1 is a widely expressed importin involved in the maintenance after LPS and TNF- treatment of chromatin structure and export of nuclear proteins (44). Pre- Our previous study has suggested that HMGB1 may interact with viously, we and others demonstrated that CRM1 was involved CRM1 in response to oxidative stress (11). After stimulation with ␣ in LPS- or H2O2-mediated HMGB1 active export from nucleus LPS or TNF- , the amounts of CRM1 were increased in the complex to cytoplasm (11, 12). To investigate whether the inhibitory immunoprecipitated with HMGB1-specific Abs (Fig. 3A), indicating effect of Hsp72 on HMGB1 translocation is involved in the a potential enhancement of CRM1/HMGB1 interaction. However, regulation of CRM1, we examined the effects of Hsp72 in- overexpression of Hsp72 substantially attenuated this LPS- or TNF- creases on CRM1 subcellular distribution using immunocyto- ␣–induced CRM1/HMGB1 interaction (Fig. 3A). Hsp72 was not co- chemical analysis and Western blotting. In quiescent macro- immunoprecipitated with HMGB1 in resting Hsp72-transfected phages, CRM1 was primarily located in the nucleus (Fig. 2A). RAW264.7 cells (Fig. 3, B and C). After stimulation with LPS or After stimulation with LPS or TNF-␣, CRM1 was also found in TNF-␣, there was a significant increase in interaction between Hsp72 the cytoplasmic regions (Fig. 2), indicating a potential CRM1 and HMGB1 (Fig. 3, B and C). In contrast, we did not observe any cytoplasmic translocation. However, overexpression of Hsp72 interaction between Hsp72 and CRM1 even after LPS and TNF stim- in RAW/Hsp72 substantially attenuates cytoplasm translocation ulation (data not shown). It remains elusive whether Hsp72-HMGB1 of CRM1 (Fig. 2), indicating that Hsp72 inhibits cytoplasmic interaction interferes with HMGB1-CRM1 interaction, thereby sup- translocation of HMGB1 and CRM1. pressing CRM1-mediated nuclear export of HMGB1. 1240 EFFECT OF Hsp72 ON HMGB1 RELEASE AND CYTOKINE ACTIVITY Downloaded from

FIGURE 3. Interaction among Hsp72, HMGB1, and CRM1 after LPS or TNF-␣ treatment in macrophage cultures. RAW/Neo (Neo) and RAW/ Hsp72 (72) cells were treated with 500 ng/ml LPS or 5 ng/ml TNF-␣ for 6 h, and whole-cell proteins were immunoprecipitated (IP) with Abs to http://www.jimmunol.org/ Hsp72 (72) or HMGB1 (HM) as indicated. The precipitated protein com- plexes were subsequently immunoblotted (IB) with CRM1 (A)-, Hsp72 (B and C)-, or HMGB1 (A–C)-specific Abs, respectively. L, whole cell ly- sates; Sr, nonspecific rabbit serum; Sm, nonspecific mouse serum. All blots shown are representative of two experiments with similar results.

Hsp72 inhibits HMGB1-induced release of cytokines in

RAW264.7 macrophages by guest on September 25, 2021 HMGB1 can be actively secreted by activated monocytes or mac- rophages and passively released by damaged and necrotic cells, and it mediates an inflammatory response (5). HMGB1 migration to organs/tissue sites induces various inflammatory cytokines in- cluding TNF-␣, IL-1␣, IL-1␤, IL-1RA, IL-6, IL-8, MIP-1␣, and ␤ MIP-1 and thus contributes to the inflammatory cascade (45, 46). FIGURE 4. Effects of Hsp72 overexpression on HMGB1-induced cy- To evaluate the potential role of HSR and Hsp72 on the proin- tokine release in macrophages cultures. A and B, After nonlethal HS flammatory function of HMGB1, RAW264.7 cells were heat (42.5°C, 1 h), cells were allowed to recover for 12 h at 37°C and then shocked at 42.5°C for 1 h and then incubated at 37°C for 12 h. incubated with HMGB1 (1 ␮g/ml) for 4 h, and TNF-␣ (A) and IL-1␤ (B) Nonlethal HS inhibited HMGB1-induced release of cytokines levels in the culture medium were detected by ELISA. Values are mean Ϯ p Ͻ 0.05. C and D, Transfected ,ء .TNF-␣ and IL-1␤ (Fig. 4, A and B). In addition, HMGB1-induced SEM of three experiments in duplicate release of TNF-␣ and IL-1␤ was also significantly decreased in RAW264.7 cells (RAW/Neo and RAW/Hsp72) incubated with HMGB1 (1 Hsp72-overexpressing RAW/Hsp72 cells compared with RAW/ ␮g/ml) for 12 h, and TNF-␣ (C) and IL-1␤ (D) levels in the culture me- Ϯ Neo control cells (Fig. 4, C and D). These experiments suggest that dium were detected by ELISA. Values are mean SEM of three experi- .p Ͻ 0.05 ,ء .ments in duplicate the effects of Hsp72 on HMGB1 include at least two aspects: 1) inhibition of HMGB1 release; and 2) suppression of the extracel- lular proinflammatory function of HMGB1. MAPK in macrophages (Fig. 5A). However, increased expression Hsp72 inhibits HMGB1-induced activation of MAP kinases in of Hsp72 significantly inhibited HMGB1-induced phosphorylation RAW264.7 macrophages of each kinase (Fig. 5B). Activation of MAPK signaling cascades, including the ERK, p38, ␬ ␣ ␬ and JNK pathways, is an important upstream step in HMGB1- Hsp72 inhibits HMGB1-induced I B degradation, NF- B p65 ␬ induced expression and release of cytokines such as TNF-␣ and nuclear translocation, and NF- B DNA binding activity in IL-1␤ in macrophages, , and endothelial cells (14, 46, RAW264.7 macrophages 47). We investigated whether the HSR and Hsp72 increase inhibit In addition to MAPK activation, the NF-␬B HMGB1-induced cytokine release partly through interfering with pathways also involved in HMGB1-induced cellular activation, MAPK pathways. We cultured cells either recovering from HS or and NF-␬B-dependent transcriptional activity is important for cy- overexpressing Hsp72 with HMGB1 and examined the extent of tokine expression (14, 15). In quiescent cells, NF-␬B factors p50 phosphorylation of p38, JNK1/2, and ERK1/2 MAPK. HMGB1 and p65 are sequestered as inactive trimers in the cytosol through time dependently induced phosphorylation of p38, JNK, and ERK/ interaction with I␬B␣, the most important member of the inhibitors The Journal of Immunology 1241

genes in the nucleus and that such nuclear localization is effec- tively inhibited when Hsp72 is expressed to high levels. We further observed the effect of Hsp72 on HMGB1-induced NF-␬B DNA-binding activity. Indeed, HMGB1 stimulation of RAW/Neo cells led to a strong increase in NF-␬B DNA-binding activity assayed by EMSA (Fig. 6, E, lane 3 and F, lane 4). The DNA-binding activities were specific as indicated by competition with cold ␬B probe (Fig. 6, E and F, lane 6), and the presence of p65 in the NF-␬B-DNA complexes was confirmed by the substan- tial supershift in the presence of p65-specific Abs (Fig. 6, E and F, lane 7). HMGB1-inducible DNA-binding activity of NF-␬B was markedly inhibited in the cells expressing abundant Hsp72 (Fig. 6, E and F, lane 5). NF-␬B regulates the expression of proinflammatory cytokine such as TNF-␣ and IL-1␤ (21). To determine whether inhibition of NF-␬B activity influences the TNF-␣ and IL-1␤ expression, we determined cellular TNF-␣ and IL-1␤ level in RAW/Neo or RAW/ FIGURE 5. Effects of Hsp72 overexpression on HMGB1-induced Hsp72 cells at 1 h after HMGB1 stimulation. Expression of TNF-␣ Downloaded from MAPK phosphoration in macrophage cultures.Cell pretreatment with HS and IL-1␤ was increased in RAW/Neo cells after HMGB1 treat- (42.5°C, 1 h and recovery for 12 h at 37°C; A) or transfected RAW264.7 ment. In contrast, TNF-␣ and IL-1␤ expression was not signifi- cells (RAW/Neo and RAW/Hsp72; B) were incubated with HMGB1 (1 cantly increased in RAW/Hsp72 cells post-HMGB1 stimulation ␮g/ml) for 30 or 60 min, and phosphorylated p38 (P-p38), JNK1/2 (P- (Fig. 6G), implicating inhibition of NF-␬B activity by Hsp72 in JNK1/2), or ERK1/2 (P-ERK1/2) protein levels in the whole-cell lysate were detected by Western blotting. GAPDH was used as a loading control. decreasing cellular levels of HMGB1-induced proinflammatory cytokines such as TNF-␣ and IL-1␤.

All blots are representative of three experiments with similar results. http://www.jimmunol.org/ of NF-␬B(I␬B) family (21). In response to HMGB1 stimulus, Discussion rapid I␬B␣ degradation (by 15 min; Fig. 6, A and B), preceded p65 Anti-inflammatory regulation by the stress response is an effective (Fig. 6, C and D) and p50 (data not shown) translocation into the autoprotective mechanism when the host encounters harmful stim- nucleus (by 30 min) in RAW/Neo cells. However, increased in- uli. The protective roles of stress-inducible HSPs (such as Hsp72) tracellular Hsp72 (induced either by nonlethal HS or Hsp72 trans- in lethal sepsis and infection are well known, but the mechanism fection) significantly inhibited I␬B␣ degradation, and NF-␬B p65 of action remain incompletely delineated. Here we demonstrated nuclear translocation (Fig. 6). These experiments therefore show that anti-inflammatory effect of Hsp72 is involved in inhibition of by guest on September 25, 2021 that after stimulation with HMGB1, p65, the key activator of NF- HMGB1 release and its proinflammatory signaling function in ␬B-regulated transcription, becomes available to ␬B-regulated macrophages.

FIGURE 6. Effects of Hsp72 overexpression on HMGB1-induced the I␬B␣ degradation, NF-␬B p65 nuclear translocation, and NF-␬B DNA binding ac- tivation in macrophages cultures. A–D, Cell pretreat- ment with HS (42.5°C, 1 h and recovery for 12 h at 37°C) or transfected RAW264.7 cells (RAW/Neo and RAW/Hsp72) were incubated with HMGB1 (1 ␮g/ml) for indicated time, and I␬B␣ level (A and B) in cytosolic fractions, and p65 level (C and D)in cytosolic (C) or nuclear (N) fractions were detected by Western blotting. ␤-Tubulin was used as a load- ing control. E and F, Activities of transcriptional factor NF-␬B in cells pretreatment with HS (42.5°C, 1 h and recovery for 12 h at 37°C) or transfected RAW264.7 cells (N, RAW/Neo; 72, RAW/Hsp72) in the absence or presence of HMGB1 (1 ␮g/ml). At 1 h after HMGB1 stimulation, NF-␬B activities were determined by EMSA with biotin-labeled NF-␬B probe, and unlabeled (Cold) NF-␬B probe. Specificity was determined by addition of p65 Ab to the nuclear extracts. G, RAW/Neo (Neo) and RAW/ Hsp72 (72) cells were incubated with HMGB1 (1 ␮g/ml) for 1 h, and the mRNA expression levels of TNF-␣ and IL-1␤ were detected by RT-PCR. All blots are representative of three experiments with similar results. 1242 EFFECT OF Hsp72 ON HMGB1 RELEASE AND CYTOKINE ACTIVITY

FIGURE 7. Conceptual relation- ships between Hsp72 and HMGB1 release and proinflammaory func- tion in macrophages cultures. Hsp72 is an important intracellular protein to inhibition of HMGB1 release and proinflammatory function in macro- phages. On the one hand, Hsp72 at- tenuated LPS or TNF-␣ induced HMGB1 release partly through in- hibiting the CRM1-dependent nu- clear export pathway of HMGB1. On the other hand, Hsp72 inhibited

HMGB1-induced cytokine (e.g., Downloaded from TNF-␣ and IL-1␤) expression and release potentially via inhibiting MAPKs and NF-␬B activation. http://www.jimmunol.org/

HMGB1, a nuclear protein widely studied as a transcription fac- transportin, and importin-␣ (54), and interacts with nuclear loca- tor and growth factor, has recently been identified as a proinflam- tion sequence-containing proteins in the cytoplasm before their matory cytokine of lethal systemic inflammation (e.g., endotox- nuclear import (55), but the effects of Hsp72 on the function of emia and sepsis), , and local inflammation (e.g., hepatic CRM1 was previously unknown. Here, we demonstrate that ele- by guest on September 25, 2021 injury after ischemia/reperfusion and LPS-induced acute lung in- vated levels of Hsp72, such as are seen in the HSR also substan- jury; Refs. 5, 48, and 49). In vitro, HMGB1 displays delayed ki- tially attenuate the interaction between CRM1 and HMGB1 po- netics of secretion (18–24 h after stimulation), compared with the tentially by competitively binding to HMGB1. This suggests that early mediators of endotoxemia, TNF-␣ and IL-1, which are se- Hsp72 may modulate HMGB1 nucleocytoplasmic transport sys- creted within minutes of LPS stimulation (4). HMGB-1 has been tems by regulating the nuclear export of receptor protein CRM1. proven to be a successful therapeutic target in experimental models Once HMGB1 is released into the extracellular space, as a of diverse infectious and inflammatory diseases, and these findings proinflammatory cytokine, it can cause multiple organ failure and have renewed the clinical interest of specific cytokine inhibitors contribute to the pathogenesis of diverse disorders including sep- and methods (41, 50–52). sis, cardiovascular shock, rheumatoid arthritis, diabetes, and can- Previously, we found that HSR could inhibit the release and cer (19). We further observed the effects of Hsp72 on the proin- translocation of HMGB1 induced by LPS (37), which suggested flammatory function of HMGB1 in vitro. As shown previously, that HSPs may be involved in this process. Here we demonstrate HMGB1 induced the release of TNF-␣ and IL-1␤ in macrophages that overexpression of Hsp72 by gene transfection significantly (45). However, overexpression Hsp72 (induced by either nonlethal inhibits HMGB1 release in macrophages exposed to exogenous HS or Hsp72 transfection) inhibited cytokine production. This in- bacterial products (endotoxin) or endogenous proinflammatory cy- dicates that Hsp72 is an effective intracellular regulator of tokines (TNF-␣) induced active release and cytoplasmic translo- HMGB1-induced proinflammatory cytokine release. cation of HMGB1 in macrophages. The mechanisms by which We further investigated the possible mechanism of Hsp72 anti- HMGB1 is exported from the nucleus to the cytoplasm are begin- inflammatory function from HMGB1 insult in macrophages. To ning to be unraveled. When actively released after stimulation, date, three receptors are reported to mediate the extracellular func- HMGB1 is heavily acetylated and reaches the cytoplasm through tion of HMGB1. HMGB1 binding to receptor for advanced gly- the CRM1 nuclear export receptor (12, 53). In a previous study, we cation end products leads to activation of NF-␬B and ERK1/2, p38 found that H2O2-induced HMGB1 active release accompanied an and SAPK/JNK kinases (56, 57). HMGB1 also binds to TLR-2 and increase in HMGB1-CRM1 complex formation (11). In addition, TLR-4, both of which lead to a MyD88-dependent activation of others have shown that CRM1 inhibitors (such as leptomycin B) NF-␬B (15, 58). This suggests that MAPKs and NF-␬B pathways significantly block LPS-induced HMGB1 nuclear export (12). are important signal pathways involved in the proinflammatory Here, we observed that after stimulation with LPS or TNF-␣, function of HMGB1. Thus, we investigated the potential inhibitory CRM1 has a significant cytoplasmic localization, and such trans- effects of Hsp72 on HMGB1-induced activation of MAPKs and location is inhibited at elevated levels of Hsp72 (Fig. 7). By con- NF-␬B pathway. trast, Hsp73, the constitutive cytoplasmic HSP70 family member Previous studies have revealed widely divergent effects of facilitates the nuclear export of the import receptors, importin-␤, Hsp72 expression on MAPK activation in response to various The Journal of Immunology 1243 stimuli. For example, we have found that Hsp72 inhibited LPS- interleukin 1) stimulate release of high mobility group protein-1 by pituicytes. induced production of inflammatory cytokines in a MAPK-inde- Surgery 126: 389–392. 10. Rendon-Mitchell, B., M. Ochani, J. Li, J. Han, H. Wang, H. Yang, S. Susarla, pendent manner (36); whereas others have reported that Hsp72 can C. Czura, R. A. Mitchell, G. Chen, et al. 2003. IFN-␥ induces high mobility group prevent cell death via suppression of JNK (59, 60). Here we found box 1 protein release partly through a TNF-dependent mechanism. J. Immunol. 170: 3890–3897. that Hsp72 significantly inhibits HMGB1-induced phosphorylation 11. Tang, D., Y. Shi, R. Kang, T. Li, W. Xiao, H. Wang, and X. Xiao. 2007. Hy- of p38, JNK, and ERK MAPKs, supporting a potential role for drogen peroxide stimulates macrophages and monocytes to actively release Hsp72 as a negative regulator of HMGB1 signaling (Fig. 7). HMGB1. J. Leukocyte Biol. 81: 741–747. ␬ 12. Bonaldi, T., F. Talamo, P. Scaffidi, D. Ferrera, A. Porto, A. Bachi, A. Rubartelli, Transcription factors of the NF- B family play vital roles in A. Agresti, and M. E. Bianchi. 2003. Monocytic cells hyperacetylate chromatin mediating cellular responses to stress, and inflammation (22). In protein HMGB1 to redirect it towards secretion. EMBO J. 22: 5551–5560. most cells, p50 and p65 form a complex in the cytoplasm with the 13. Palumbo, R., M. Sampaolesi, F. De Marchis, R. Tonlorenzi, S. Colombetti, A. Mondino, G. Cossu, and M. E. Bianchi. 2004. Extracellular HMGB1, a signal inhibitor protein I␬B␣.I␬B␣ inactivates the p50/p65 heterodimer of tissue damage, induces mesoangioblast migration and proliferation. J. Cell by masking NF-␬B nuclear localization sequences (22). Activation Biol. 164: 441–449. of signaling pathways by extracellular signals, such as HMGB1, 14. Kokkola, R., A. Andersson, G. Mullins, T. Ostberg, C. J. Treutiger, B. Arnold, P. Nawroth, U. Andersson, R. A. Harris, and H. E. Harris. 2005. RAGE is the leads to proteolysis of I␬B␣, allowing active p50/p65 heterodimer major receptor for the proinflammatory activity of HMGB1 in rodent macro- to translocate into the nucleus, and activates ␬B containing genes phages. Scand. J. Immunol. 61: 1–9. ␣ ␤ 15. Park, J. S., D. Svetkauskaite, Q. He, J. Y. Kim, D. 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