Molecular Medicine

, Volume 18, Issue 3, pp 359–369 | Cite as

Poly(ADP-Ribosyl)ation of High Mobility Group Box 1 (HMGB1) Protein Enhances Inhibition of Efferocytosis

  • Kasey Davis
  • Sami Banerjee
  • Arnaud Friggeri
  • Celeste Bell
  • Edward Abraham
  • Mourad Zerfaoui
Research Article


Phagocytosis of apoptotic cells by macrophages, known as efferocytosis, is a critical process in the resolution of inflammation. High mobility group box 1 (HMGB1) protein was first described as a nuclear nonhistone DNA-binding protein, but is now known to be secreted by activated cells during inflammatory processes, where it participates in diminishing efferocytosis. Although HMGB1 is known to undergo modification when secreted, the effect of such modifications on the inhibitory actions of HMGB1 during efferocytosis have not been reported. In the present studies, we found that HMGB1 secreted by Toll-like receptor 4 (TLR4) stimulated cells is highly poly(ADP-ribosyl)ated (PARylated). Gene deletion of poly(ADP)-ribose polymerase (PARP)-1 or pharmacological inhibition of PARP-1 decreased the release of HMGB1 from the nucleus to the extracellular milieu after TLR4 engagement. Preincubation of macrophages or apoptotic cells with HMGB1 diminished efferocytosis through mechanisms involving binding of HMGB1 to phosphatidylserine on apoptotic cells and to the receptor for advanced glycation end products (RAGE) on macrophages. Preincubation of either macrophages or apoptotic cells with PARylated HMGB1 inhibited efferocytosis to a greater degree than exposure to unmodified HMGB1, and PARylated HMGB1 demonstrated higher affinity for phosphatidylserine and RAGE than unmodified HMGB1. PARylated HMGB1 had a greater inhibitory effect on Ras-related C3 botulinum toxin substrate 1 (Rac-1) activation in macrophages during the uptake of apoptotic cells than unmodified HMGB1. The present results, showing that PARylation of HMGB1 enhances its ability to inhibit efferocytosis, provide a novel mechanism by which PARP-1 may promote inflammation.



This work was supported by grant 11SDG5330014 from the American Heart Association to M Zerfaoui.

Supplementary material

10020_2012_1803359_MOESM1_ESM.pdf (501 kb)
Poly(ADP-Ribosyl)ation of High Mobility Group Box 1 (HMGB1) Protein Enhances Inhibition of Efferocytosis


  1. 1.
    Haslett C. (1999) Granulocyte apoptosis and its role in the resolution and control of lung inflammation. Am. J. Respir. Crit. Care Med. 160:S5–11.CrossRefGoogle Scholar
  2. 2.
    Voll RE, et al. (1997) Immunosuppressive effects of apoptotic cells. Nature. 390:350–1.CrossRefGoogle Scholar
  3. 3.
    Fadok VA, et al. (1998) Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF. J. Clin. Invest. 101:890–8.CrossRefGoogle Scholar
  4. 4.
    Ulloa L, Batliwalla FM, Andersson U, Gregersen PK, Tracey KJ. (2003) High mobility group box chromosomal protein 1 as a nuclear protein, cytokine, and potential therapeutic target in arthritis. Arthritis Rheum. 48:876–81.CrossRefGoogle Scholar
  5. 5.
    Czura CJ, Yang H, Amella CA, Tracey KJ. (2004) HMGB1 in the immunology of sepsis (not septic shock) and arthritis. Adv. Immunol. 84:181–200.CrossRefGoogle Scholar
  6. 6.
    Vandivier RW, Henson PM, Douglas IS. (2006) Burying the dead: the impact of failed apoptotic cell removal (efferocytosis) on chronic inflammatory lung disease. Chest. 129:1673–82.CrossRefGoogle Scholar
  7. 7.
    Vandivier RW, et al. (2009) Dysfunctional cystic fibrosis transmembrane conductance regulator inhibits phagocytosis of apoptotic cells with proinflammatory consequences. Am. J. Physiol. Lung Cell Mol. Physiol. 297:L677–86.CrossRefGoogle Scholar
  8. 8.
    Ishii Y, et al. (1998) Elimination of neutrophils by apoptosis during the resolution of acute pulmonary inflammation in rats. Lung. 176:89–98.CrossRefGoogle Scholar
  9. 9.
    Cox G, Crossley J, Xing Z. (1995) Macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary inflammation in vivo. Am. J. Respir. Cell Mol. Biol. 12:232–7.CrossRefGoogle Scholar
  10. 10.
    Ravichandran KS. (2003) “Recruitment signals” from apoptotic cells: invitation to a quiet meal. Cell. 113:817–20.CrossRefGoogle Scholar
  11. 11.
    Freeman GJ, Casasnovas JM, Umetsu DT, DeKruyff RH. TIM genes: a family of cell surface phosphatidylserine receptors that regulate innate and adaptive immunity. Immunol. Rev. 235:172–89.CrossRefGoogle Scholar
  12. 12.
    Park D, et al. (2007) BAI1 is an engulfment receptor for apoptotic cells upstream of the ELMO/ Dock180/Rac module. Nature. 450:430–4.CrossRefGoogle Scholar
  13. 13.
    Park SY, et al. (2008) Rapid cell corpse clearance by stabilin-2, a membrane phosphatidylserine receptor. Cell Death Differ. 15:192–201.CrossRefGoogle Scholar
  14. 14.
    Friggeri A, et al. (2011) Participation of the receptor for advanced glycation end products in efferocytosis. J. Immunol. 186:6191–8.CrossRefGoogle Scholar
  15. 15.
    He M, et al. (2011) Receptor for advanced glycation end products binds to phosphatidylserine and assists in the clearance of apoptotic cells. EMBO Rep. 12:358–64.CrossRefGoogle Scholar
  16. 16.
    Bianchi ME, Agresti A. (2005) HMG proteins: dynamic players in gene regulation and differentiation. Curr. Opin. Genet. Dev. 15:496–506.CrossRefGoogle Scholar
  17. 17.
    Bianchi ME. (2009) HMGB1 loves company. J. Leukoc. Biol. 86:573–6.CrossRefGoogle Scholar
  18. 18.
    Dumitriu IE, Baruah P, Manfredi AA, Bianchi ME, Rovere-Querini P. (2005) HMGB1: guiding immunity from within. Trends Immunol. 26:381–7.CrossRefGoogle Scholar
  19. 19.
    Muller S, et al. (2001) New EMBO members’ review: the double life of HMGB1 chromatin protein: architectural factor and extracellular signal. EMBO J. 20:4337–40.CrossRefGoogle Scholar
  20. 20.
    Qin YH, et al. (2009) HMGB1 enhances the proinflammatory activity of lipopolysaccharide by promoting the phosphorylation of MAPK p38 through receptor for advanced glycation end products. J. Immunol. 183:6244–50.CrossRefGoogle Scholar
  21. 21.
    Sha Y, Zmijewski J, Xu Z, Abraham E. (2008) HMGB1 develops enhanced proinflammatory activity by binding to cytokines. J. Immunol. 180:2531–7.CrossRefGoogle Scholar
  22. 22.
    Campana L, Bosurgi L, Bianchi ME, Manfredi AA, Rovere-Querini P. (2009) Requirement of HMGB1 for stromal cell-derived factor-1/ CXCL12-dependent migration of macrophages and dendritic cells. J. Leukoc. Biol. 86:609–15.CrossRefGoogle Scholar
  23. 23.
    Liu G, et al. (2008) High mobility group protein-1 inhibits phagocytosis of apoptotic neutrophils through binding to phosphatidylserine. J. Immunol. 181:4240–6.CrossRefGoogle Scholar
  24. 24.
    Rouhiainen A, Tumova S, Valmu L, Kalkkinen N, Rauvala H. (2007) Pivotal advance: analysis of proinflammatory activity of highly purified eukaryotic recombinant HMGB1 (amphoterin). J. Leukoc. Biol. 81:49–58.CrossRefGoogle Scholar
  25. 25.
    Park JS, et al. (2006) High mobility group box 1 protein interacts with multiple Toll-like receptors. Am. J. Physiol. Cell Physiol. 290:C917–24.CrossRefGoogle Scholar
  26. 26.
    Erlandsson Harris H, Andersson U. (2004) Minireview: the nuclear protein HMGB1 as a proinflammatory mediator. Eur. J. Immunol. 34:1503–12.CrossRefGoogle Scholar
  27. 27.
    Banerjee S, Friggeri A, Liu G, Abraham E. The C-terminal acidic tail is responsible for the inhibitory effects of HMGB1 on efferocytosis. J. Leukoc. Biol. 88:973–9.CrossRefGoogle Scholar
  28. 28.
    Kim MY, Zhang T, Kraus WL. (2005) Poly(ADP-ribosyl)ation by PARP-1: ‘PAR-laying’ NAD+ into a nuclear signal. Genes Dev. 19:1951–67.CrossRefGoogle Scholar
  29. 29.
    Burkle A. (2001) Physiology and pathophysiology of poly(ADP-ribosyl)ation. Bioessays. 23:795–806.CrossRefGoogle Scholar
  30. 30.
    Ditsworth D, Zong WX, Thompson CB. (2007) Activation of poly(ADP)-ribose polymerase (PARP-1) induces release of the pro-inflammatory mediator HMGB1 from the nucleus. J. Biol. Chem. 282:17845–54.CrossRefGoogle Scholar
  31. 31.
    Griffin RJ, et al. (1998) Resistance-modifying agents. 5. Synthesis and biological properties of quinazolinone inhibitors of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP). J. Med. Chem. 41:5247–56.CrossRefGoogle Scholar
  32. 32.
    Friggeri A, et al. HMGB1 inhibits macrophage activity in efferocytosis through binding to the alphavbeta3-integrin. Am. J. Physiol. Cell Physiol. 299:C1267–76.Google Scholar
  33. 33.
    Wu Y, Tibrewal N, Birge RB. (2006) Phosphatidylserine recognition by phagocytes: a view to a kill. Trends Cell Biol. 16:189–97.CrossRefGoogle Scholar
  34. 34.
    Ravichandran KS, Lorenz U. (2007) Engulfment of apoptotic cells: signals for a good meal. Nat. Rev. Immunol. 7:964–74.CrossRefGoogle Scholar
  35. 35.
    Wang H, et al. (1999) HMG-1 as a late mediator of endotoxin lethality in mice. Science. 285:248–51.CrossRefGoogle Scholar
  36. 36.
    Andersson U, Tracey KJ. (2010) HMGB1 Is a Therapeutic Target for Sterile Inflammation and Infection. Annu. Rev. Immunol. 23:139–62.Google Scholar
  37. 37.
    Wisniewski JR, Szewczuk Z, Petry I, Schwanbeck R, Renner U. (1999) Constitutive phosphorylation of the acidic tails of the high mobility group 1 proteins by casein kinase II alters their conformation, stability, and DNA binding specificity. J. Biol. Chem. 274:20116–22.CrossRefGoogle Scholar
  38. 38.
    Youn JH, Shin JS. (2006) Nucleocytoplasmic shuttling of HMGB1 is regulated by phosphorylation that redirects it toward secretion. J. Immunol. 177:7889–97.CrossRefGoogle Scholar
  39. 39.
    Ugrinova I, Mitkova E, Moskalenko C, Pashev I, Pasheva E. (2007) DNA bending versus DNA end joining activity of HMGB1 protein is modulated in vitro by acetylation. Biochemistry. 46:2111–7.CrossRefGoogle Scholar
  40. 40.
    Ugrinova I, Pasheva EA, Armengaud J, Pashev IG. (2001) In vivo acetylation of HMG1 protein enhances its binding affinity to distorted DNA structures. Biochemistry. 40:14655–60.CrossRefGoogle Scholar
  41. 41.
    Evankovich J, et al. High mobility group box 1 release from hepatocytes during ischemia and reperfusion injury is mediated by decreased histone deacetylase activity. J. Biol. Chem. 285:39888–97.CrossRefGoogle Scholar
  42. 42.
    Bonaldi T, et al. (2003) Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion. EMBO J. 22:5551–60.CrossRefGoogle Scholar
  43. 43.
    Banerjee S, Friggeri A, Liu G, Abraham E. (2010) The C-terminal acidic tail is responsible for the inhibitory effects of HMGB1 on efferocytosis. J. Leukoc. Biol. 88:973–9.CrossRefGoogle Scholar

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Authors and Affiliations

  • Kasey Davis
    • 1
  • Sami Banerjee
    • 1
  • Arnaud Friggeri
    • 1
  • Celeste Bell
    • 1
  • Edward Abraham
    • 1
  • Mourad Zerfaoui
    • 1
  1. 1.Department of MedicineUniversity of Alabama at BirminghamBirminghamUSA

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