Molecular and Cellular Biochemistry

, Volume 310, Issue 1–2, pp 77–83 | Cite as

Mono ADP-ribosylation inhibitors prevent inflammatory cytokine release in alveolar epithelial cells

  • Mariangela Del Vecchio
  • Enrico Balducci


A549, a type II alveolar epithelial cell line stimulated with LPS (10 μg/ml), released high levels of the inflammatory cytokines IL-6 and IL-8. Here, we have investigated whether ADP-ribosylation inhibitors block the LPS-triggered cytokine release in epithelial cells. When coincubating A549 with LPS and meta-iodobenzylguanidine or novobiocin, selective arginine-dependent ART-inhibitors, the release of IL-6 and IL-8 was inhibited in a concentration-dependent manner. This effect has been linked with the presence of a functionally active arginine ADP-ribosylating enzyme on the cell surface. To this aim, we amplified by RT-PCR the ART1 transcript and identified four ADP-ribosylated proteins likely substrate for ART1. The mechanism behind the cytokine inhibition in epithelial cells seems to be correlated with the presence of ART1, which behaves as an essential positive regulator of inflammatory cytokines. This novel observation indicates this enzyme as well as other novobiocin/MIBG sensitive ARTs as potential targets for the development of new therapeutic strategies.


LPS TLR-4 ADP-ribosylation inhibitors Epithelial cells Cytokines 



Toll like receptor 4






Adenosine diphosphoribose







The authors wish to thank Simona Tavarini for her excellent technical support in cytokine quantification, Giorgio Corsi for artwork, Elisabetta Soldaini for critical reading of the manuscript and Valerio Reguzzi for his assistance with statistical analysis. This work was supported by Novartis Vaccines and by Murst (fondo Ateneo ex 60%).


  1. 1.
    Ueda K, Hayaishi O (1985) ADP-ribosylation. Annu Rev Biochem 54:73–100PubMedCrossRefGoogle Scholar
  2. 2.
    Zolkiewska A, Nightingale MS, Moss J (1992) Molecular characterization of NAD: arginine ADP-ribosyltransferase from rabbit skeletal muscle. Proc Natl Acad Sci USA 89:11352–11356PubMedCrossRefGoogle Scholar
  3. 3.
    Okazaki IJ, Kim HJ, Moss J (1996) Cloning and characterization of a novel membrane-associated lymphocyte NAD:arginine ADP-ribosyltransferase. J Biol Chem 271:22052–22057PubMedCrossRefGoogle Scholar
  4. 4.
    Haag F, Koch-Nolte F, Kuhl M, Lorenzen S, Thiele HG (1994) Premature stop codons inactivate the RT6 genes of the human and chimpanzee species. J Mol Biol 243:537–546PubMedCrossRefGoogle Scholar
  5. 5.
    Allport JR, Donnelly LE, Hayes BP, Murray S, Rendell NB, Ray KP, MacDermot J (1996) Reduction by inhibitors of mono(ADP-ribosyl)transferase of chemotaxis in human neutrophil leucocytes by inhibition of the assembly of filamentous actin. Br J Pharmacol 118:1111–1118PubMedGoogle Scholar
  6. 6.
    Lodhi IJ, Clift RE, Omann GM, Sweeney JF, McMahon KK, Hinshaw DB (2001) Inhibition of mono-ADP-ribosyltransferase activity during the execution phase of apoptosis prevents apoptotic body formation. Arch Biochem Biophys 387:66–77PubMedCrossRefGoogle Scholar
  7. 7.
    Yau L, Litchie B, Thomas S, Storie B, Yurkova N, Zahradka P (2003) Endogenous mono-ADP-ribosylation mediates smooth muscle cell proliferation and migration via protein kinase N-dependent induction of c-fos expression. Eur J Biochem 270:101–110PubMedCrossRefGoogle Scholar
  8. 8.
    Yau L, Litchie B, Zahradka P (2004) MIBG, an inhibitor of arginine-dependent mono(ADP-ribosyl)ation, prevents differentiation of L6 skeletal myoblasts by inhibiting expression of myogenin and p21(cip1). Exp Cell Res 301:320–330PubMedCrossRefGoogle Scholar
  9. 9.
    Takizawa H (1998) Airway epithelial cells as regulators of airway inflammation (Review). Int J Mol Med 1:367–378PubMedGoogle Scholar
  10. 10.
    Nakamura H, Yoshimura K, Jaffe HA, Crystal RG (1991) Interleukin-8 gene expression in human bronchial epithelial cells. J Biol Chem 266:19611–19617PubMedGoogle Scholar
  11. 11.
    Richman-Eisenstat JB, Jorens PG, Hebert CA, Ueki I, Nadel JA (1993) Interleukin-8: an important chemoattractant in sputum of patients with chronic inflammatory airway diseases. Am J Physiol 264:L413–L418PubMedGoogle Scholar
  12. 12.
    Le Page C, Wietzerbin J (2003) Modulation of the activation of extracellular signal-regulated kinase (ERK) and the production of inflammatory mediators by ADP-ribosylation inhibitors. Biol Chem 384:1509–1513PubMedCrossRefGoogle Scholar
  13. 13.
    Heine H, Ulmer AJ, Flad HD, Hauschildt S (1995) Lipopolysaccharide-induced change of phosphorylation of two cytosolic proteins in human monocytes is prevented by inhibitors of ADP-ribosylation. J Immunol 155:4899–4908PubMedGoogle Scholar
  14. 14.
    Lieber M, Smith B, Szakal A, Nelson-Rees W, Todaro G (1976) A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells. Int J Cancer 17:62–70PubMedCrossRefGoogle Scholar
  15. 15.
    Balducci E (2005) A filter plate-based assay for mono adenosine 5′-diphosphate-ribosyltransferases. Anal Biochem 344:278–280PubMedCrossRefGoogle Scholar
  16. 16.
    Guillot L, Medjane S, Le-Barillec K, Balloy V, Danel C, Chignard M, Si-Tahar M (2004) Response of human pulmonary epithelial cells to lipopolysaccharide involves Toll-like receptor 4 (TLR4)-dependent signaling pathways: evidence for an intracellular compartmentalization of TLR4. J Biol Chem 279:2712–2718PubMedCrossRefGoogle Scholar
  17. 17.
    Banasik M, Komura H, Shimoyama M, Ueda K (1992) Specific inhibitors of poly(ADP-ribose) synthetase and mono(ADP-ribosyl)transferase. J Biol Chem 267:1569–1575PubMedGoogle Scholar
  18. 18.
    Smets LA, Loesberg C, Janssen M, Van Rooij H (1990) Intracellular inhibition of mono(ADP-ribosylation) by meta-iodobenzylguanidine: specificity, intracellular concentration and effects on glucocorticoid-mediated cell lysis. Biochim Biophys Acta 1054:49–55PubMedCrossRefGoogle Scholar
  19. 19.
    Rankin PW, Jacobson EL, Benjamin RC, Moss J, Jacobson MK (1989) Quantitative studies of inhibitors of ADP-ribosylation in vitro and in vivo. J Biol Chem 264:4312–4317PubMedGoogle Scholar
  20. 20.
    Ungerstedt JS, Blomback M, Soderstrom T (2003) Nicotinamide is a potent inhibitor of proinflammatory cytokines. Clin Exp Immunol 131:48–52PubMedCrossRefGoogle Scholar
  21. 21.
    Yau L, Elliot T, Lalonde C, Zahradka P (1998) Repression of phosphoenolpyruvate carboxykinase gene activity by insulin is blocked by 3–aminobenzamide but not by PD128763, a selective inhibitor of poly(ADP-ribose) polymerase. Eur J Biochem 253:91–100PubMedCrossRefGoogle Scholar
  22. 22.
    Freissmuth M, Gilman AG (1989) Mutations of GS alpha designed to alter the reactivity of the protein with bacterial toxins. Substitutions at ARG187 result in loss of GTPase activity. J Biol Chem 264:21907–21914PubMedGoogle Scholar
  23. 23.
    Balducci E, Micossi LG (2002) NAD-dependent inhibition of the NAD-glycohydrolase activity in A549 cells. Mol Cell Biochem 233:127–132PubMedCrossRefGoogle Scholar
  24. 24.
    Nemoto E, Yu Y, Dennert G (1996) Cell surface ADP-ribosyltransferase regulates lymphocyte function-associated molecule-1 (LFA-1) function in T cells. J Immunol 157:3341–3349PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2007

Authors and Affiliations

  1. 1.Novartis Vaccines and DiagnosticsSienaItaly
  2. 2.Department of Comparative Morphology and BiochemistryUniversity of CamerinoCamerinoItaly

Personalised recommendations