Of HMGB1 Acetylation Release by Macrophages Through Regulation

Published November 12, 2014, doi:10.4049/jimmunol.1400359 The Journal of Immunology

PARP1 Mediates LPS-Induced HMGB1 Release by Macrophages through Regulation of HMGB1 Acetylation

Zhiyong Yang,1 Li Li,1 Lijuan Chen, Weiwei Yuan, Liming Dong, Yushun Zhang, Heshui Wu, and Chunyou Wang

The high-mobility group box protein 1 (HMGB1) is increasingly recognized as an important inflammatory mediator. In some cases, the release of HMGB1 is regulated by poly(ADP-ribose) polymerase-1 (PARP-1), but the mechanism is still unclear. In this study, we report that PARP-1 activation contributes to LPS-induced PARylation of HMGB1, but the PARylation of HMGB1 is insufficient to direct its migration from the nucleus to the cytoplasm; PARP-1 regulates the translocation of HMGB1 to the cytoplasm through upregulating the acetylation of HMGB1. In mouse bone marrow–derived macrophages, genetic and pharmacological inhibition of PARP-1 suppressed LPS-induced translocation and release of HMGB1. Increased PARylation was accompanied with the nucleus- to-cytoplasm translocation and release of HMGB1 upon LPS exposure, but PARylated HMGB1 was located at the nucleus, unlike acetylated HMGB1 localized at the cytoplasm in an import assay. PARP inhibitor and PARP-1 depletion decreased the activity ratio of histone acetyltransferases to histone deacetylases that elevated after LPS stimulation and impaired LPS-induced acetylation of HMGB1. In addition, PARylation of HMGB1 facilitates its acetylation in an in vitro enzymatic reaction. Furthermore, reactive oxygen species scavenger (N-acetyl-L-cysteine) and the ERK inhibitor (FR180204) impaired LPS-induced PARP activation and HMGB1 release. Our findings suggest that PARP-1 regulates LPS-induced acetylation of HMGB1 in two ways: PARylating HMGB1 to facilitate the latter acetylation and increasing the activity ratio of histone acetyltransferases to histone deacetylases. These studies revealed a new mechanism of PARP-1 in regulating the inflammatory response to endotoxin. The Journal of Immunology, 2014, 193: 000–000.

he high-mobility group box protein 1 (HMGB1), a mem- early phase was not newly synthesized but already existed in the ber of the HMGB superfamily, is abundant in the eu- nucleus (2). Thereafter, studies have reported that HMGB1 plays an T karyotic nucleus. HMGB1 is a highly conserved protein important role in the pathological processes of several diseases such composed of the A box, B box, and a C-terminal acidic tail (1). In as sepsis, hemorrhagic shock, acute lung injury, rheumatic arthritis, the nucleus, through nonspecific DNA binding, HMGB1 partic- and disseminated intravascular coagulation (3–6). ipates in the activities of the cell nucleus, such as DNA replica- HMGB1 cannot be secreted through a Golgi apparatus/endo- tion, cell differentiation, and regulation of gene expression (1). In plasmic reticulum–dependent secretory pathway due to the lack 1999, Wang et al. (2) first reported that after LPS stimulation, the of signal peptide. It is transported from the nucleus to the cyto- mouse macrophage cell line RAW264.7 secreted TNF-a first and plasm and then into the lysosomes and eventually released into the then HMGB1 as a delayed inflammatory mediator. Within 16 h after extracellular space through exocytosis (7). Acetylation plays an LPS and TNF-a stimulation, the expression of HMGB1 mRNA was important role in the active secretion of HMGB1 by monocytes not upregulated in macrophages, but HMGB1 protein was found to and macrophages. Acetylation of lysine residues in HMGB1 is migrate from the nucleus to the cytoplasm and was then secreted a prerequisite of its translocation to the cytoplasm (8). Stimulation into the extracellular space; it indicated that this protein released at by inflammatory mediators, such as LPS and TNF-a, is necessary for the migration of HMGB1 from the nucleus to the cytoplasm, whereas release into the extracellular space requires extracellular Pancreatic Disease Institute, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province 430022, China lysophosphatidylcholine (7). Except actively secreted by the im- mune cells, HMGB1 also can be passively released by dead cells. 1Z.Y. and L.L. contributed equally to this work. HMGB1 accumulates in the culture supernatant after repeated Received for publication February 6, 2014. Accepted for publication October 13, 2014. freezing and thawing (9). It has been reported that DNA damage This work was supported by National Natural Science Foundation of China Grants by alkylating agents activates poly(ADP-ribose) polymerase-1 30972899 and 81171840. (PARP-1), accompanied by migration of HMGB1 into the cyto- Address correspondence and reprint requests to Prof. Chunyou Wang, Pancreatic plasm and subsequent release into the extracellular space due to Disease Institute, Union Hospital, Tongji Medical College, Huazhong University of increased permeability of the cell membrane (10). This process is Science and Technology, 1277 Jiefang Avenue, Wuhan, Hubei Province 430022, China. E-mail address: [email protected] PARP-1 dependent, but the mechanism is unclear. The PARP family contains many members, of which PARP-1 was Abbreviations used in this article: AA, anacardic acid; 3-AB, 3-aminobenzamide; biotin-NAD, 6-biotin-17-NAD; BMDM, bone marrow–derived macrophage; CBP, the first to be discovered; it is also the most abundant PARP member CREB-binding protein; HAT, histone acetyltransferase; HDAC, histone deacetylase; in the cell nucleus and the one that has been most clearly charac- HMGB1, high-mobility group box protein 1; HMGB1-FL, full-length HMGB1; NAC, N-acetyl-L-cysteine; p300, E1A-associated protein p300; PARP, poly(ADP- terized (11, 12). PARP-1 participates in many physiological pro- ribose) polymerase; PCAF, E1A-associated protein p300/CREB-binding protein– cesses such as chromatin decondensation, DNA replication, DNA associated factor; ROS, reactive oxygen species; sc siRNA, scrambled small inter- repair, gene expression, cell differentiation, cell apoptosis, and gene fering RNA; TB, transport buffer. transcription (13). DNA damage has been regarded as the most Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 potent inducer of PARP-1 activation (13). PARP-1 can catalyze the

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400359 2 PARP-1 REGULATES ACETYLATION OF HMGB1 transfer of the ADP-ribosyl group in NAD+ to the carboxyl groups procedure followed the protocol of Dharmicon. Forty-eight hours after in the side chain of glutamic acid residues, resulting in O-linked transfection, the depletion of PARP-1 was confirmed by Western blotting, poly(ADP-ribose) (14). Posttranslational modification is an impor- and cells were used in subsequent experiments. tant mechanism of PARP-1 action (14). Some proteins, such as PARP activity assay PARP-1 itself, histone proteins, HMG proteins, DNA helicase, and Intracellular PARP activity was measured by cell ELISA as Bakondi et al. several transcription factors, are substrates of PARP-1 (13). It has (18) described. A total of 5 3 104 BMDMs were planted in 96-well plates. been reported that PARP-1 also participates in inflammation, be- After incubation in 100 ml PARP buffer (56 mM HEPES, 28 mM KCl, 28 m + cause the suppression of PARP-1 activity can reduce the expression mM NaCl, and 2 mM MgCl2) with 0.01% digitonin and 10 M biotin-NAD m of inflammatory cytokines in animal models of endotoxemia (15). for 30 min at 37˚C, cells were treated with 200 l 95% ethanol precooling at 220˚C for 10 min. Then, cells were washed once with PBS followed by The release of HMGB1 by LPS-challenged macrophages and blocking in 1% BSA for 30 min. After that, cells were incubated in 50 ml mouse embryonic fibroblasts is also dependent on the activation of streptavidin-HRP (1:500) at 37˚C for 30 min. Three washes with PBS were PARP-1 (16). However, the mechanism through which HMGB1 is followed by incubation in 100 ml TACS-Saphire for 15 min at room tem- released after PARP-1 activation remains unclear. In the current perature. The reaction was stopped by 1 M H2SO4. The absorbance was study, we report that LPS-induced HMGB1 release by macrophages detected at 450 nm with a microplate reader (Model 550; Bio-Rad). is mediated by the reactive oxygen species (ROS)/ERK/PARP-1 Preparation of cytoplasmic and nuclear extracts signaling pathway, and the migration of HMGB1 to the cytoplasm Cytoplasmic and nuclear extracts were prepared using the Nuclear and as well as the subsequent release is accompanied by increased Cytoplasmic Protein Extraction Kit according to the manufacturer’s PARylation of HMGB1. However, the PARylation of HMGB1 is not instructions (Beyotime Institute of Biotechnology). Briefly, cells were sufficient to direct its translocation. PARP-1 regulates the translo- scraped off, washed in ice-cold PBS, and then resuspended in 200 ml ice- cation of HMGB1 to the cytoplasm through upregulating the acet- cold cytoplasmic extraction buffer A with 1 mM PMSF, 1 mM Na4VO3, and protease inhibitor mixture. After incubation with cytoplasmic extrac- ylation of HMGB1 in two ways: PARylating HMGB1 to facilitate its tion buffer B for 1 min in an ice bath and following vortex for 5 s, cell acetylation and elevating the activity ratio of acetylases to deacety- lysates were centrifuged at 12,000 3 g for 5 min at 4˚C. Supernatants were lases, which can catalyze acetylation and deacetylation of HMGB1. aliquoted and stored at 270˚C. Nuclear pellets were resuspended in 50 ml nuclear extraction buffer. After 15 times in vortex for 15 s every 2 min at 4˚C, lysates were centrifuged at 12,000 3 g for 10 min at 4˚C. Nuclear Materials and Methods extracts were aliquoted and stored at 270˚C until use. Reagents Immunoblotting and immunoprecipitation LPS (Esherichia coli 0111:B4), SB202190, FR180204, and SP600125 was purchased from Calbiochem. Anti-HMGB1, E1A-associated protein p300 Cells were lysed in RIPA buffer supplemented with 1 mM PMSF, 1 mM (p300)/CREB-binding protein (CBP)–associated factor (PCAF), CBP, p300, Na4VO3, and protease inhibitor mixture. Lysates were sonicated and histone deacetylase (HDAC) 1, and HDAC4 Abs were obtained from centrifuged at 10,000 3 g for 10 min at 4˚C. The serum and medium were Abcam. Full-length recombinant proteins of PCAF, HDAC1, and HDAC4 first filtered with Centrifugal Filters (UFC801024; Millipore) and then were from Abcam, Cell Science, and Origene. Normal rabbit IgG, protein concentrated 30-fold with Centrifugal Filters (UFC510008; Millipore). A+G Agarose beads, HRP-conjugated secondary Abs, FITC-conjugated The concentration of proteins was measured with BCA kits. In immuno- secondary Abs, anti–PARP-1, and GAPDH Abs were from Santa Cruz precipitation, cell lysates were precleared with 1 mg normal rabbit IgG and Biotechnology. [14C]Acetyl CoA was from AppliChem. DMEM, FBS, and 20 ml protein A+G Agarose beads for 2 h at 4˚C. After centrifugation at heat-inactivated FBS were from Hyclone. DMEM/F12 and nonenzymatic 1000 3 g for 5 min, supernatants were transferred to new tubes and in- cell dissociation solution were from Cellgro. Trypsin, penicillin, and strep- cubated with 40 ml protein A+G Agarose beads and rabbit anti-HMGB1 tomycin were from Life Technologies. Centrifugal filters (UFC510008 and Ab overnight at 4˚C. Beads were collected for immunoblotting after UFC801024) were from Millipore. Rabbit anti-acetylated lysine and centrifugation and washed three times with PBS. In immunoblotting, phospho-ERK1/2 Ab were from Cell Signaling Technology. PARP-1 small samples were loaded equally for PAGE and transferred onto nitrocellulose interfering RNA (siRNA), scrambled siRNA (sc siRNA), and Dharmafector 1 membranes. The membranes were blocked with 5% nonfat milk in TBST, transfection reagent were obtained from Thermo Scientific. N-acetyl-L- probed with primary Abs for 2 h at room temperature or overnight at 4˚C, cysteine (NAC), DMSO, and digitonin were from Sigma-Aldrich. 3-Ami- and then incubated with HRP-conjugated secondary Abs at room tem- nobenzamide (3-AB) was from Alexis. HRP-conjugated anti-rabbit IgG Ab perature for 1 h. In certain experiments, HRP-conjugated Ab (TrueBlot) (TrueBlot) was from Rockland. Rabbit anti-PAR Ab, HRP-conjugated was used to recognize native rabbit IgG. The bands were determined using streptavidin, 6-biotin-17-NAD (biotin-NAD)+, and TACS-Saphire were ECL reagent (Pierce) and quantified using ImageJ Software (National from Trevigen. Histone acetyltransferase (HAT) and HDAC Activity Col- Institutes of Health) from scanned films. orimetric Assay Kits were obtained from BioVision. ELISA Isolation and culture of murine bone marrow–derived HMGB1 in culture supernatants was detected with ELISA kits (Shino test) macrophages according to the manufacturer’s instructions. Murine bone marrow–derived macrophages (BMDMs) were generated as Immunofluorescence described by Zhang et al. (17). Briefly, C57/BL6 mice (6–8 wk) were killed by cervical dislocation. Intact femurs were taken out and severed Cells plated on coverslips were fixed with 2% paraformaldehyde for 15 min proximal to each joint. Bone cavity was flushed with wash medium and washed three times with 100 mM glycine in HBSS for 10 min followed by (Dulbecco’s PBS). Wash medium was collected and then centrifuged for once with HBSS for 10 min. After permeabilized with 0.1% Triton X-100 10 min at 500 3 g at room temperature. Cell pellets were resuspended in in HBSS for 30 min, cells were incubated with anti-HMGB1 Abs (di- macrophage complete medium (DMEM/F12 with 10% FBS, 20% L-929 luted with HBSS containing 5% horse serum and 0.2% BSA in a dilution cell-conditioned medium, 10 mM L-glutamine, 100 IU/ml penicillin, and 1:500) overnight at 4˚C. Three washes with HBSS were followed by the 100 mg/ml streptomycin). A total of 4 3 105 cells was added to each incubation with FITC-conjugated secondary Ab (1:200) for 1 h. After plastic petri dish in 10 ml complete medium and cultured at 37˚C and 5% another three washes, the cells were mounted on glass slides using Prolong CO2. Another 5 ml complete medium was added to each dish on day 3. Gold antifade reagent (Molecular Probes). Images were acquired with Adherent BMDMs were harvested and used in subsequent experiments on a confocal microscope (Zeiss LSM 510 Meta; Carl Zeiss). day 7. Cells were ∼95% pure macrophages as evidenced by their expression of cell-surface markers HLA-DR, CD11b, and CD206. Expression of recombinant proteins

PARP-1 depletion The full-length HMGB1 (HMGB1-FL) was generated using forward primer 59-TGCACTGGAATTCATGGGCAAAGGAGATCC-39 and reverse A total of 2 3 106 BMDMs were planted in six-well plates and cultured primer 59-CAGTGCACTCGAGTTATTCATCATCATCATCTTC-39,whereas overnight. A pool of three target-specific 20–25 nt siRNA was used to truncated HMGB1 (HMGB1DC) was generated using reverse primer knockdown PARP-1 at a concentration of 50 nmol/l. The transfection 59-CTTCTTTTTCTTGCTTTTTTCAGCCTTG-39. The PCR products were The Journal of Immunology 3 treated with the restriction enzymes EcoR1 and XhoI, cloned in pET28a+ Statistical analysis (Novagen). For the recombinant HMGB1-EGFP protein, the gene encoding 6 human HMGB1 was cloned upstream of EGFP in pEGFP-N1 (BD Clontech), All data are expressed as mean SEM. Statistical analysis was performed , and then a SacI/NotI fragment from pHMGB1-EGFP-N1 was subcloned into using the Student t test. Differences were considered significant when p pET28a+. Proteins were expressed in E. coli BL21. His-tagged proteins were 0.05. Kaplan–Meier survival curves were compared using a log-rank test to purified on a HIS-Select HF Nickel Affinity gel (Sigma-Aldrich). The purity determine significance. of all protein preparations was confirmed by SDS-PAGE. PARylation and acetylation of proteins in vitro Results The PARylation was done using biotin-NAD instead of [32P]NAD (10). PARP-1 regulates LPS-induced HMGB1 migration from the The final reaction was performed in a 50-ml mix buffer containing 50 mM nucleus to the cytoplasm in macrophages Tris-HCl (pH 8), 25 mM MgCl2, with 5 mg PARP-activated DNA (R&D Systems), 20 mM biotin-NAD (R&D Systems), and 0.3 mg each purified PARP activity in BMDMs increased significantly, peaking at 4 h recombinant proteins. PARP (1 ml; R&D Systems) was added to each after LPS stimulation and lasting for 12 h (Fig. 1A). The HMGB1 reaction and then incubated at room temperature for 30 min. The acety- level in the medium of the BMDMs increased at 4 h after LPS m lation (19) were performed in 30 l HAT buffer including 50 mM Tris-HCl exposure (Fig. 1B), a finding that is consistent with previous (pH 8), 10% glycerol, 1 mM DTT, 1 mM PMSF, 10 mM sodium butyrate, 1mM[14C] acetyl CoA, and 0.3 mg each purified recombinant proteins. research on this topic (21). To demonstrate the roles of PARP The reaction was started by adding 1 ml recombinant PCAF, P300, and activation in LPS-induced HMGB1 release by macrophages, CBP catalytic domain (all from Enzo Life Sciences) and then incubated at we used 3-AB to suppress the activity of PARP. We found that room temperature for 30 min. For successive PARylation and acetylation, 3-AB significantly impaired the release of HMGB1 by BMDMs the probes of the PARylation were precipitated with 20% final concen- (Fig. 1B). Because PARP-1 is the main member of the PARP tration of TCA at 220˚C for 16 h. After spin at 15,000 3 g for 15 min, the pellets were washed three times with cold acetone and dissolved in 5 mlTE family (11), we knocked down PARP-1 to determine the influ- buffer (pH 8). Successive acetylation (19) were performed in 30 mlHAT ence of PARP-1 on LPS-induced HMGB1 release (Fig. 1C). We buffer including and 5 ml protein products in TE buffer. The reaction was observed a sharp decline in LPS-induced HMGB1 release after m started by adding 1 l recombinant PCAF, P300, or CBP catalytic domain PARP-1 knockdown (Fig. 1D). Therefore, we deduced that LPS- and then incubated at room temperature for 30 min. Heat-inactivated enzyme was used as control. To stop the reaction, loading buffer was added, induced HMGB1 release by macrophages is dependent on PARP- and samples were boiled for 5 min prior to loading. The samples were 1 activation. In the resting state, HMGB1 is mainly located in analyzed by SDS-PAGE, stained with Coomassie R250 (Sigma-Aldrich), the nucleus, and its migration from the nucleus to the cytoplasm then the gel was dried and exposed to Kodak XAR-5 film (Kodak), and is a prerequisite for its release into the extracellular space. Using finally quantified by Gel Pro analyzer. immunoblotting and indirect immunofluorescence labeling of Nuclear import assay HMGB1, we found that PARP-1 knockdown remarkably suppressed the LPS-induced translocation of HMGB1 to the cyto- Nuclear import assays were performed as in Cassany and Gerace (20) by minor modification. Firstly, the cytosol of BMDMs was prepared. After plasm in BMDMs (Fig. 1E, 1F). These results indicated that the washing twice in ice-cold PBS and once in washing buffer [10 mM HEPES LPS-induced migration of HMGB1 from the nucleus to the cyto- (pH 7.3), 110 mM KOAc, 2 mM Mg(OAc)2, and 2 mM DTT], BMDMs plasm, and its release was dependent on the activation of PARP-1 were homogenized with hypotonic lysis buffer [5 mM HEPES (pH 7.3), in macrophages. 10 mM KOAc, 2 mM Mg(OAc)2, 2 mM DTT, 1 mM PMSF, and 1 mg/ml 3 each leupeptin, pepstatin, and aprotinin]. After centrifugation at 1500 g The PARylation of HMGB1 companies, but not directs, its for 15 min, at 15,000 3 g for 20 min, and 100,000 3 g for 1 h at 4˚C, the final supernatants were collected and dialyzed at 4˚C against transport buffer relocalization to the cytoplasm in macrophages [TB; 20 mM HEPES (pH 7.3), 110 mM KOAc, 2 mM Mg(OAc)2,1mM PARP-1 catalyzes the PARylation of HMGB1 in vitro (10, 16). m EGTA, 2 mM DTT, 1 mM PMSF, and 1 g/ml each leupeptin, pepstatin, and To elucidate the role of PARP-1 on intracellular PARylation of aprotinin]. The cytosol was frozen in liquid nitrogen and store at 280˚C. HMGB1-GFP was PARylated or acetylated in vitro as mentioned above. HMGB1, we knocked down PARP-1 in BMDMs and found The reactions were precipitated with 20% final concentration of TCA at 220˚C that LPS-induced PARylation of HMGB1 was significantly for 16 h. After spin at 15,000 3 g for 15 min, the pellet was washed three suppressed (Fig. 2A). To further determine whether PARP-1 times with cold acetone and dissolved in TE buffer (pH 8). For the assays, regulates the relocalization of HMGB1 through PARylation of BMDMs were permeabilized for 5 min on ice in TB containing 40 mg/ml digitonin. After rinsing for 10 min with TB, cells were incubated in HMGB1, we first measured the PARylation level of HMGB1 transport mixture for 30 min at 30˚C, which contained BMDM cytosol in the nucleus, cytoplasm, and cell-culture supernatant, respec- (2 mg/ml, preincubated for 30 min at room temperature with ATP- tively. The PARylation of HMGB1 in the cytoplasm and cell- regenerating system) with 30 mM gallotannin and 30 mg/ml PARylated culture supernatant was dominantly upregulated after BMDMs m HMGB1-GFP or 10 nM trichostatin A (Sigma-Aldrich) and 30 g/ml were exposed to LPS (Fig. 2B). It indicated that the LPS-induced acetylated HMGB1-GFP. ATP-regenerating system obtained 1 mM ATP, 5 mM creatine phosphate, 20 U/ml creatine phosphokinase, and 0.5 mM PARylation of HMGB1 is catalyzed by PARP-1, and this modi- GTP. Finally, cells were fixed in 2% paraformaldehyde for 15 min and fication is accompanied by the migration of HMGB1 from the immediately examined by fluorescent microscope (Olympus BX51; nucleus to the cytoplasm of macrophages and subsequent re- Olympus). lease into the extracellular space. To further elucidate whether HDAC and HAT activity assay PARylation of HMGB1 directs its migration, HMGB1 was PARylated in an in vitro enzyme reaction and then subjected HDAC and HAT activity was measured with HDAC and HAT Activity to an import assay. As a control, GST-GFP was located in the Colorimetric Assay Kits according to the manufacturer’s instructions. The absorbance was detected at 405 or 440 nm with a microplate reader (Model cytoplasm. HMGB1-GFP and PARylated HMGB1-GFP were lo- 550; Bio-Rad). cated in the nucleus; however, acetylated HMGB1-GFP was located in the cytoplasm (Fig. 2D). In addition, anacardic acid (AA), Animal experiments an inhibitor of acetylation, showed no influence on LPS-induced C57BL/6 mice weighing 25–30 g were obtained from Experimental Ani- PARP activation (Fig. 2E) and PARylation of HMGB1 (Fig. 2F), mal Center of Tongji Medical College, Huazhong University of Science but obviously suppressed the migration of HMGB1 to the cyto- and Technology (Wuhan, China). Mice were housed under specific pathogen-free conditions with free access to water and standard mouse plasm (Fig. 2G). These data demonstrated that PARylation, not chow. All animal experiments were performed with the approval of the like acetylation, is insufficient for the translocation of HMGB1 to Institutional Animal Care and Use Committee. the cytoplasm. 4 PARP-1 REGULATES ACETYLATION OF HMGB1

FIGURE 1. PARP-1 mediates LPS-induced translocation and release of HMGB1 in macrophages. (A) LPS induced the activation of PARP in BMDMs. Cells were exposed to 100 ng/ml LPS. The activity of PARP was measured by Cell-ELISA. (B) 3-AB dampened LPS-induced release of HMGB1 in BMDMs. BMDMs pretreated with 10 mmol/l 3-AB or vehicle were exposed to 100 ng/ml LPS for indicated periods. HMGB1 of culture supernatants was detected with immunoblotting (top panel) and ELISA (bottom panel). (C) PARP-1 in BMDMs was depleted successfully using PARP-1 siRNA. BMDMs were transfected with sc siRNA or p120 siRNA. After 48 h, the cells were treated with 100 ng/ml LPS for indicated periods (D and E). (D) Depletion of PARP-1 negatively regulated LPS-induced release of HMGB1 in BMDMs. HMGB1 was measured with immunoblotting (left panel) and ELISA (right panel). (E and F) LPS-induced relocalization of HMGB1 was impaired by PARP-1 depletion in BMDMs. Intracellular HMGB1 was stained by indirect immunoﬂuorescence (E). Nuclear (Nuc) and cytoplasmic (Cyto) proteins were extracted and subjected to immunoblotting as well as concentrated medium (F). Scale bar, 100 mm. Data are mean 6 SEM of three independent experiments. *p , 0.05 compared with group without LPS treatment.

PARP-1 regulates LPS-induced HMGB1 acetylation in may regulate the nucleus-to-cytoplasm migration of HMGB1 by macrophages influencing HMGB1 acetylation. The migration of HMGB1 into the cytoplasm is dependent on the PARylation of HMGB1 facilitates its acetylation acetylation of lysine residues in monocytes or macrophages (8). PARP can catalyze the transfer of the ADP-ribosyl group from To clarify whether PARP-1 regulates the nucleus-to-cytoplasm NAD+ to the carboxyl groups of the glutamic acid residues, migration of HMGB1 by influencing HMGB1 acetylation, we resulting in O-linked PAR (14). To elucidate the relationship suppressed the activity of PARP using 3-AB and found that LPS- between PARP-1 activation and HMGB1 acetylation, we investi- induced HMGB1 acetylation was remarkably reduced (Fig. 3A). gated the influence of HMGB1 PARylation on HMGB1 acety- A similar phenomenon was observed through the knockdown of lation. HMGB1-FL and HMGB1ΔC were PARylated through PARP-1 by siRNA (Fig. 3B). These data demonstrate that PARP-1 an in vitro enzymatic reaction and subjected to acetylation by The Journal of Immunology 5

FIGURE 2. The PARylation of HMGB1 accompanies but not directs its relocalization in macrophages. (A) Cytoplasmic and extracellular HMGB1 was highly PARylated after LPS stimulation. BMDMs were exposed to LPS for 8 h. (B) Depletion of PARP-1 impaired the PARylation of HMGB1 in BMDMs. After knockdown of PARP-1, cells were exposed to 100 ng/ml LPS for indicated periods. (C) HMGB1 was successfully PARylated or acetylated in vitro. HMGB1-GFP was PARylated or acetylated as described in Materials and Methods. Reactions were analyzed by immunoblotting (top panel) or autoradiography (bottom panel). (D) PARylated HMGB1 located to the nucleus. HMGB1-GFP was PARylated or acetylated before import assay. GST-GFP was used as control. AA did not inﬂuence LPS-induced activation of PARP (E) and PARylation of HMGB1 (F). (G) AA impaired LPS-induced relocalization of HMGB1. BMDMs pretreated with 25 mmol/l AA were exposed to 100 ng/ml LPS for indicated time points (E–G). The activity of PARP was measured by Cell-ELISA (E). The culture supernatants, cytoplasmic (Cyto) and nuclear (Nuc) protein extracts (A), and cell lysates (B and F) were subjected to immunoprecipitation (IP) with anti-HMGB1 Ab. The PARylation of HMGB1 was measured by immunoblot (IB) analysis using Abs against PAR. Intracellular HMGB1 was stained by indirect immunoﬂuorescence (G). Scale bars, 50 mm(D) and 100 mm(G).

PCAF, CBP, or P300. PARylation reinforced the acetylation of PARP-1 regulates the activities of HATs and HDACs in LPS- HMGB1-FL, but not of HMGB1ΔC, by these acetylases (Fig. 4A, challenged macrophages 4B). Glutamic acid residues are abundant in the C-terminal of Acetylation is biregulated by HATs and HDACs. The acetylation of HMGB1. We found that the PARylation level of HMGB1-FL was HMGB1 is regulated by several HATs (PCAF, CBP, and P300) and higher than that of HMGB1ΔC (data not shown), a ﬁnding that is HDACs (HDAC1 and HDAC4) (8, 22). After LPS stimulation, the consistent with previous research (10). It suggests the C-tail of activity of HATs was increased, whereas that of HDACs was re- HMGB1 may be PARylated by PARP. These data indicated that the duced in macrophages, resulting in an increase in the ratio of HAT PARylation of C-tail might inﬂuence the acetylation of HMGB1. to HDAC activity (Fig. 5A, 5B). Both 3-AB and PARP-1 knockdown 6 PARP-1 REGULATES ACETYLATION OF HMGB1

PARP-1 in the enzyme system (Fig. 5D). We may infer from these data that in macrophages stimulated by LPS, PARP-1 can boost the acetylation of HMGB1 by increasing the activity ratio of HATs to HDACs that catalyze acetylation and deacetylation of HMGB1.

LPS-induced PARP-1 activation and HMGB1 release use ROS and ERK signaling pathway It was reported that HMGB1 release induced by liver ischemia was dependent on ROS production (23). ROS are recognized as the main signals leading to intracellular PARP-1 activation (24). Macrophages produce large amounts of ROS after LPS stimulation (25). Thus, we hypothesized that ROS might mediate LPS-induced PARP-1 activation and HMGB1 release. After treatment with NAC, a scavenger of ROS, PARP-1 activation in BMDMs was significantly suppressed (Fig. 6A). Moreover, the migration of HMGB1 to the cytoplasm (Fig. 6B) and the release of HMGB1 into the extracellular space FIGURE 3. PARP-1 regulates LPS-induced acetylation of HMGB1 in were remarkably reduced (Fig. 6B, 6C). It has been reported that A macrophages. ( ) 3-AB suppressed LPS-induced acetylation of HMGB1. PARP-1 can be activated by MAPKs (26). LPS can activate BMDMs pretreated with 10 mmol/l 3-AB were exposed to 100 ng/ml LPS p38MAPK, JNK, as well as the ERK signaling pathway (27); for 4 h. (B) Depletion of PARP-1 impaired LPS-induced acetylation of however, only inhibitors of the ERK pathway inhibited the acti- HMGB1. After PARP-1 knockdown, BMDMs were treated with 100 ng/ml LPS for 4 h. Cell lysates were subjected to immunoprecipitation (IP) with vation of PARP-1 (Fig. 6D). NAC inhibited the LPS-induced ac- anti-HMGB1 Ab, and then the acetylation of HMGB1 was measured using tivation of ERK (Fig. 6E). Consistently, the LPS-induced release specific Ab against acetylated-lysine (A and B). IB, immunoblot; IP, im- of HMGB1 was inhibited by FR180204, an ERK inhibitor (Fig. 6F). munoprecipitation. NAC suppressed LPS-induced activation of PARP-1 by .90% (Fig. 6A), although it just inhibited the activation of ERK by ∼50% could suppress the changes in HAT and HDAC activities in macro- at 30 min after LPS treatment (Fig. 6E). It indicated that ROS might phages after LPS stimulation and significantly reduced the ratio of use other signal pathways to mediate PARP-1 activation in this HAT to HDAC activity as compared with that in the sc siRNA group model. ROS/DNA injury was known as the classic and strong ac- (Fig. 5A, 5B). To further determine which of those HATs and HDACs tivator of PARP-1. It was reported that ERK activated PARP-1 were regulated by PARP-1, we measured the expression of PCAF, trough phosphorylation, and it was independent of DNA injury CBP, P300, HDAC1, and HDAC4 in the nucleus. We found, 4 h after (28). These data indicate that LPS-induced activation of PARP-1 LPS exposure, the expression of PCAF, CBP, and P300 was increased may use ROS/ERK pathway and ROS/DNA injury pathway. by ∼60, 300, and 280%, respectively, and the expression of HDAC1 and HDAC4 was decreased by 20 and 80%, respectively (Fig. 5C). NAC, 3-AB, and FR180204 reduces the serum HMGB1 level PARP-1 depletion inhibited LPS-induced increasing of the expression and mortality rate in a mouse model of endotoxemia of PCAF, CBP, and P300 by ∼50–70% and decreasing of HDAC4 In a mouse model of endotoxemia, the PARP-1 inhibitor 3-AB, the expression by ∼50%, but had no effect on the expression of HDAC1 ROS scavenger NAC, and the ERK inhibitor FR180204 could (Fig. 5C). We also detected whether PARP-1 PARylates those HATs reduce the HMGB1 level in the serum (Fig. 7A) as well as the andHDACsandfoundCBP,P300,andHDAC4werePARylatedby mortality rate (Fig. 7B).

FIGURE 4. PARylation of HMGB1 promotes its acetylation. (A) PARylation of HMGB1-FL promoted its acetylation in vitro. (B) PARylation of HMGB1ΔC did not inﬂuence its acetylation in vitro. HMGB1-FL (A) and HMGB1ΔC(B) were PARylated and successively acetylated as described in Materials and Methods. The reactions were analyzed by immunoblotting or autoradiography. Quantiﬁed data are shown in the bottom panels.Dataare mean 6 SEM of three independent experiments. *p , 0.05 compared with control group. The Journal of Immunology 7

FIGURE 5. PARP-1 regulates the activity of HATs and HDACs in LPS-challenged macrophages. 3-AB (A) and PARP-1 depletion (B) downregulated the activity ratio of HATs to HDACs in LPS-challenged BMDMs. BMDMs pretreated with 10 mmol/l 3-AB were exposed to 100 ng/ml LPS for indicated periods (A). After PARP-1 knockdown, BMDMs were exposed to 100 ng/ml LPS for indicated periods (B). Nucleus proteins were extracted, and colorimetric assay kits were used for measuring the activity of HATs and HDACs (A and B). The activity ratio of HATs to HDACs was calculated. (C) The effect of PARP-1 depletion on HAT and HDAC expression in the nucleus of LPS-challenged BMDMs. After PARP-1 knockdown, BMDMs were exposed to 100 ng/ml LPS for 4 h. Nucleus proteins were extracted, and the expression of PCAF, CBP, P300, HDAC1, and HDAC4 was measured by immunoblotting. Quantiﬁed data are shown in the right panel.(D) CBP, P300, and HDAC4 were PARylated by PARP-1. PCAF, CBP, P300, HDAC1, and HDAC4 were subjected to PARylation in PARP-1 catalytic system as described in Materials and Methods. Data are mean 6 SEM of three independent experiments. *p , 0.05 compared with corresponding sc siRNA group, #p , 0.05 compared with corresponding group without LPS treatment. 8 PARP-1 REGULATES ACETYLATION OF HMGB1

FIGURE 6. ROS/ERK pathway mediates LPS-induced PARP-1 activation and HMGB1 release. (A) NAC inhibited LPS-induced activation of PARP in BMDMs. (B) NAC suppressed LPS-induced release of HMGB1 by BMDMs. (C) NAC impaired LPS-induced relocalization of HMGB1 in BMDMs. Cells pretreated with 20 mM NAC were exposed to 100 ng/ml LPS for indicated periods (A–C). The activity of PARP was measured by Cell-ELISA (A). HMGB1 in culture supernatants was detected by immunoblotting (B). Intracellular HMGB1 was stained by indirect immunofluorescence (C). Scale bar, 100 mm. (D) Inhibition of ERK suppressed LPS-induced activation of PARP in BMDMs. Cells were exposed to 100 ng/ml LPS for indicated periods after pretreatment with inhibitors specific for p38 MAPK (SB202190; 5 mM), ERK (FR180204; 1 mM), and JNK (SP600125; 20 mM). The activity of PARP was measured by Cell-ELISA. (E) NAC inhibited LPS-induced phosphorylation of ERK in BMDMs. Cells were exposed to 100 ng/ml LPS for indicated periods after pretreatment with 20 mM NAC. p-ERK1/2 was detected by immunoblotting using specific Abs. Quantified data are shown in the bottom panel.(F) In- hibition of ERK impaired LPS-induced release of HMGB1 in BMDMs. Cells were exposed to 100 ng/ml LPS for indicated periods after pretreatment with 1 mM of FR180204. HMGB1 in medium supernatants was detected by immunoblotting. Data are mean 6 SEM of three independent experiments. *p , 0.05 compared with corresponding untreated group. Con, control.

Discussion extracellular space through exocytosis (2). The passive release of The active secretion of HMGB1 can be activated by inﬂammatory HMGB1 from dead cells is due to its migration from the nucleus mediators and hypoxia. This process is composed of three steps: to the cytoplasm and subsequent release into the extracellular ﬁrst, migration from the nucleus to the cytoplasm; second, enter space owing to increased cell membrane permeability (9). The into the organelles in the cytoplasm; and third, release into the translocation of HMGB1 from the nucleus to the cytoplasm, The Journal of Immunology 9

Acetylation has been regarded as a prerequisite for the LPS- induced migration of HMGB1 to the cytoplasm in monocytes and macrophages (8). In this study, PARP-1 was found to regulate the acetylation of HMGB1 in LPS-stimulated macrophages. Although PARylation was not sufficient for the migration of HMGB1 to the cytoplasm, PARylated HMGB1-FL showed higher levels of acetylation in an in vitro enzymatic reaction system, indicating that PARylation of HMGB1 can boost its acetylation. Furthermore, we found that PARylation had no influence on the acetylation of HMGB1ΔC. The C-terminal of HMGB1, which binds to HMG boxes, contains many glutamic acid residues. Further study is required to determine whether the PARylation of these glutamic acid residues would expose acetylation sites hidden by the C-terminal and facilitate the acetylation of lysine residues in HMG boxes. Under inflammatory conditions, acetylases such as CBP/p300 in monocytes and macrophages are activated, whereas the activities of HDAC1 and HDAC4 are decreased (22). This results in the acetylation of Lys27,Lys43, and Lys174–178 in HMGB1, which reduces the binding capacity of HMGB1 to DNA and lead to its subsequent migration from the nucleus to the cytoplasm, followed by secretion into the extracellular space through exocytosis (8, FIGURE 7. NAC, 3-AB, and FR180204 reduce the serum HMGB1 level 22). We found that after stimulation with LPS, the activity of and mortality rate in a mouse model of endotoxemia. Mice, 60 for survival HATs in BMDMs increased, the activity of HDACs decreased, and analysis and 30 for serum collection, were randomly divided into five groups acetylated HMGB1 increased significantly. Furthermore, 3-AB (negative control, LPS+PBS, LPS+3-AB, LPS+NAC, and LPS+FR180204). treatment and knockdown of PARP-1 suppressed the upregulation Endotoxemia model was established by i.p. injection of LPS (40 mg/kg), and PBS was used for establishing negative control. Except for negative control, of HATs activity and downregulation of HDACs activity induced mice were pretreated with PBS, 3-AB (10 mg/kg), NAC (200 mg/kg), or by LPS. It was further confirmed by measuring the expression FR180204 (2 mg/kg) for 30 min according to groups before model estab- of PCAF, CBP, P300, HDAC1, and HDAC4 in the nucleus, lishment. Sixteen hours later, mice were sacrificed, and the serum was col- which can catalyze acetylation and deacetylation of HMGB1. lected for detecting HMGB1 by immunoblotting (A). (B) Kaplan–Meier PARP-1 may regulate the activity of HATs and HDACs through survival plots for the five groups. The amount of surviving mice was PARylating those enzymes because CBP, P300, and HDAC4 were recorded every 8 h. *p , 0.05 compared with LPS+PBS group. PARylated in the PARP-1 catalytic system. Decreased ratio of HAT and HDAC activity was accompanied by a lower level of which is regulated by the modification of HMGB1 itself, is a key HMGB1 acetylation, implying that it played important roles in the step for its release into the extracellular space. The posttrans- regulation of HMGB1 acetylation. PARP-1 mediated the LPS- lational modification of HMGB1 includes methylation, acetyla- induced activity change of those acetylases and deacetylases and tion, phosphorylation, and ADP-ribosylation (8, 10, 29, 30). thus regulated the acetylation and nucleus-to-cytoplasm migration Methylation regulates the nucleus-to-cytoplasm migration of of HMGB1. HMGB1 in neutrophils (29). In monocytes and macrophages, acet- In addition, this study demonstrated that the ROS scavenger ylation and phosphorylation regulate the nucleus-to-cytoplasm NAC can remarkably inhibit the LPS-induced activation of PARP-1 migration of HMGB1 induced by LPS and TNF-a, respectively and nucleus-to-cytoplasm migration and release of HMGB1. Al- (8, 30). though it has been reported that HMGB1 release from monocytes LPS and alkylating agents have been reported to be capable of and macrophages can be induced by H2O2 through the ERK and inducing PARP-1 activation, PARylation, and final release of JNK signaling pathways (31), our data showed that among the HMGB1 (10, 16); however, the relationship between PARylation inhibitors of the p38 MAPK, JNK, and ERK signaling pathways, and the nucleus-to-cytoplasm migration of HMGB1 is not clear. only the ERK inhibitor FR180204 inhibited LPS-induced PARP-1 In this study, the translocation and final release of HMGB1 activation and HMGB1 release. NAC suppressed LPS-induced in LPS-stimulated BMDMs was found to be dependent on PARP- activation of PARP-1 by .90% despite inhibiting the activation 1 activation. PARylation of HMGB1 in LPS-challenged macro- of ERK by ∼50%. ROS might use other signal pathways to acti- phages was significantly upregulated, with higher levels of vate PARP-1, such as the classic DNA injury/PARP-1 pathway. PARylated HMGB1 in the cytoplasm and extracellular space than These results indicate that LPS-induced activation of PARP-1 may in the nucleus. This result indicates that LPS-induced nucleus- use the ROS/ERK pathway and ROS/DNA injury pathway. Data to-cytoplasm migration and release of HMGB1 is accompanied from animal experiments were consistent with the above conclu- by HMGB1 PARylation. In the nuclear import assay, we found sion, because a PARP-1 inhibitor, an ROS scavenger, and an ERK that acetylated HMGB1 is located in the cytoplasm, whereas inhibitor all reduced the serum HMGB1 level and mortality rate in PAR-modified HMGB1 is located in the nucleus, implying that a mouse model of endotoxemia. PARylation does not direct the migration of HMGB1 to the In summary, we, for the first time, to our knowledge, demon- cytoplasm solely. The acetylase inhibitor AA had no influence strated that LPS induced PARP-1 activation using the ROS/ERK on LPS-induced PARP-1 activation and HMGB1 PARylation in signaling pathway and thus elevated the activity ratio of HATs and macrophages, but still blocked the nucleus-to-cytoplasm mi- HDACs by regulating the expression of PCAF, CBP, P300, and gration and release of HMGB1, implying that when HMGB1 HDAC4 in the nucleus, resulting in the acetylation of HMGB1. In acetylation is inhibited, PARP-1 activation is insufficient for contrast, PARP-1 catalyzed PARylation of HMGB1, which facil- HMGB1 relocalization. itated acetylation of HMGB1. Increased acetylation owing to 10 PARP-1 REGULATES ACETYLATION OF HMGB1

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