Cellular and Molecular Life Sciences

, Volume 67, Issue 10, pp 1687–1697 | Cite as

Ordered transcriptional factor recruitment and epigenetic regulation of tnf-α in necrotizing acute pancreatitis

  • J. Sandoval
  • J. Pereda
  • J. L. Rodriguez
  • J. Escobar
  • J. Hidalgo
  • L. A. B. Joosten
  • L. Franco
  • J. Sastre
  • G. López-RodasEmail author
Research Article


Τhe expression of the critical initiator cytokine TNF-α was strongly upregulated in vivo in acute necrotic pancreatitis (AP) in rodents and in vitro in TNF-α activated acinar AR42J cells. Upregulation of tnf-α, inos, icam-1 and il-6 occurred both in TNF-α receptor 1 and 2 knock-out mice, but not in TNF-α knock-out mice, in cerulein-induced acute pancreatitis. Chromatin immunoprecipitation analysis showed that transcriptional factors (ELK-1, SP1, NF-κB and EGR-1) and chromatin modification complexes (HDAC1, HDAC2, GCN5, PCAF and CBP) were recruited and/or released from the promoter in a strictly ordered mechanism. Activation of tnf-α gene was also accompanied by an ordered increased level of histone H3K9, H3K14 and H3K18-acetylation and H3K4 methylation, as well as H4K5 acetylation. A better knowledge of the molecular mechanisms that control tnf-α gene regulation will provide deeper understanding of the initiation and development of the inflammatory processes occurring in acute pancreatitis triggered by TNF-α cytokine.


Acute necrotic pancreatitis Chromatin immunoprecipitation ChIP Epigenetic Tumor necrosis factor alpha 



This work was supported by research grants from the Ministerio de Ciencia y Tecnología (BFU2007-63120, CSD2006-49 to G. López-Rodas and SAF2006-06963, SAF2009-09500, CSD2007-00020 to J. Sastre) and from the European Commission FP6 Integrated Project Exogenesis (LSHM-CT-2004-005272 to J.Hidalgo).

Supplementary material

18_2010_272_MOESM1_ESM.tif (909 kb)
Suplementary Figure S1. Effect of pentoxyfilline in tnf-α expression and in transcriptional factor recruitment in tnf-α promoter. (A) Quantitative RT-PCR analysis of tnf-α gene expression. β-Actin gene was used as negative control of taurocholate-induced expression. (B) ChIP assay of tnf-α promoter transcriptional factor occupation. α-Actin gene was used as a negative control of the ChIP assay. (C) ImageJ analysis of PCR signals. P: pentoxyfilline treatment; T: taurocholate treatment; P+T: pentoxyfilline plus taurocholate treatment. The statistical significance is indicated as follows: **P < 0.01 vs. control or time 0; ##P < 0.01 vs. taurocholate (TIFF 909 kb)


  1. 1.
    Pandol SJ (2006) Acute pancreatitis. Curr Opin Gastroenterol 22:481–486CrossRefPubMedGoogle Scholar
  2. 2.
    Pereda J, Sabater L, Aparisi L, Escobar J, Sandoval J, Vina J, Lopez-Rodas G, Sastre J (2006) Interaction between cytokines and oxidative stress in acute pancreatitis. Curr Med Chem 13:2775–2787CrossRefPubMedGoogle Scholar
  3. 3.
    Cosen-Binker LI, Gaisano HY (2007) Recent insights into the cellular mechanisms of acute pancreatitis. Can J Gastroenterol 21:19–24PubMedGoogle Scholar
  4. 4.
    Chan YC, Leung PS (2007) Acute pancreatitis: animal models and recent advances in basic research. Pancreas 34:1–14CrossRefPubMedGoogle Scholar
  5. 5.
    Pandol SJ, Saluja AK, Imrie CW, Banks PA (2007) Acute pancreatitis: bench to the bedside. Gastroenterology 132:1127–1151CrossRefPubMedGoogle Scholar
  6. 6.
    Kusske AM, Rongione AJ, Reberl HA (1996) Cytokines and acute pancreatitis. Gastroenterology 110:639–642CrossRefPubMedGoogle Scholar
  7. 7.
    Bhatia M, Brady M, Shokuhi S, Christmas S, Neoptolemos JP, Slavin J (2000) Inflammatory mediators in acute pancreatitis. J Pathol 190:117–125CrossRefPubMedGoogle Scholar
  8. 8.
    Norton ID, Clain JE (2001) Optimising outcomes in acute pancreatitis. Drugs 61:1581–1591CrossRefPubMedGoogle Scholar
  9. 9.
    Norman J (1998) The role of cytokines in the pathogenesis of acute pancreatitis. Am J Surg 175:76–83CrossRefPubMedGoogle Scholar
  10. 10.
    Sakorafas GH, Tsiotou AG (2000) Etiology and pathogenesis of acute pancreatitis: current concepts. J Clin Gastroenterol 30:343–356CrossRefPubMedGoogle Scholar
  11. 11.
    Libermann TA, Baltimore D (1990) Activation of interleukin-6 gene expression through the NF-kappa B transcription factor. Mol Cell Biol 10:2327–2334PubMedGoogle Scholar
  12. 12.
    Shimada M, Andoh A, Hata K, Tasaki K, Araki Y, Fujiyama Y, Bamba T (2002) IL-6 secretion by human pancreatic periacinar myofibroblasts in response to inflammatory mediators. J Immunol 168:861–868PubMedGoogle Scholar
  13. 13.
    Zhou A, Scoggin S, Gaynor RB, Williams NS (2003) Identification of NF-kappa B-regulated genes induced by TNFalpha utilizing expression profiling and RNA interference. Oncogene 22:2054–2064CrossRefPubMedGoogle Scholar
  14. 14.
    Hou J, Baichwal V, Cao Z (1994) Regulatory elements and transcription factors controlling basal and cytokine-induced expression of the gene encoding intercellular adhesion molecule 1. Proc Natl Acad Sci USA 91:11641–11645CrossRefPubMedGoogle Scholar
  15. 15.
    Roebuck KA, Rahman A, Lakshminarayanan V, Janakidevi K, Malik AB (1995) H2O2 and tumor necrosis factor-alpha activate intercellular adhesion molecule 1 (ICAM-1) gene transcription through distinct cis-regulatory elements within the ICAM-1 promoter. J Biol Chem 270:18966–18974CrossRefPubMedGoogle Scholar
  16. 16.
    Baker SJ, Reddy EP (1996) Transducers of life and death: TNF receptor superfamily and associated proteins. Oncogene 12:1–9PubMedGoogle Scholar
  17. 17.
    Brockhaus M, Schoenfeld HJ, Schlaeger EJ, Hunziker W, Lesslauer W, Loetscher H (1990) Identification of two types of tumor necrosis factor receptors on human cell lines by monoclonal antibodies. Proc Natl Acad Sci USA 87:3127–3131CrossRefPubMedGoogle Scholar
  18. 18.
    Lewis M, Tartaglia LA, Lee A, Bennett GL, Rice GC, Wong GH, Chen EY, Goeddel DV (1991) Cloning and expression of cDNAs for two distinct murine tumor necrosis factor receptors demonstrate one receptor is species specific. Proc Natl Acad Sci USA 88:2830–2834CrossRefPubMedGoogle Scholar
  19. 19.
    Pastor CM, Frossard JL (2001) Are genetically modified mice useful for the understanding of acute pancreatitis? FASEB J 15:893–897CrossRefPubMedGoogle Scholar
  20. 20.
    Pastor CM, Matthay MA, Frossard JL (2003) Pancreatitis-associated acute lung injury: new insights. Chest 124:2341–2351CrossRefPubMedGoogle Scholar
  21. 21.
    Tsai EY, Falvo JV, Tsytsykova AV, Barczak AK, Reimold AM, Glimcher LH, Fenton MJ, Gordon DC, Dunn IF, Goldfeld AE (2000) A lipopolysaccharide-specific enhancer complex involving Ets, Elk-1, Sp1, and CREB binding protein and p300 is recruited to the tumor necrosis factor alpha promoter in vivo. Mol Cell Biol 20:6084–6094CrossRefPubMedGoogle Scholar
  22. 22.
    Goldfeld AE, Maniatis T (1989) Coordinate viral induction of tumor necrosis factor alpha and interferon beta in human B cells and monocytes. Proc Natl Acad Sci USA 86:1490–1494CrossRefPubMedGoogle Scholar
  23. 23.
    Falvo JV, Uglialoro AM, Brinkman BM, Merika M, Parekh BS, Tsai EY, King HC, Morielli AD, Peralta EG, Maniatis T, Thanos D, Goldfeld AE (2000) Stimulus-specific assembly of enhancer complexes on the tumor necrosis factor alpha gene promoter. Mol Cell Biol 20:2239–2247CrossRefPubMedGoogle Scholar
  24. 24.
    Falvo JV, Brinkman BM, Tsytsykova AV, Tsai EY, Yao TP, Kung AL, Goldfeld AE (2000) A stimulus-specific role for CREB-binding protein (CBP) in T cell receptor-activated tumor necrosis factor alpha gene expression. Proc Natl Acad Sci USA 97:3925–3929CrossRefPubMedGoogle Scholar
  25. 25.
    Niederau C, Ferrell LD, Grendell JH (1985) Caerulein-induced acute necrotizing pancreatitis in mice: protective effects of proglumide, benzotript, and secretin. Gastroenterology 88:1192–1204PubMedGoogle Scholar
  26. 26.
    Frossard JL, Pastor CM (2002) Experimental acute pancreatitis: new insights into the pathophysiology. Front Biosci 7:d275–d287CrossRefPubMedGoogle Scholar
  27. 27.
    Sandoval J, Rodriguez JL, Tur G, Serviddio G, Pereda J, Boukaba A, Sastre J, Torres L, Franco L, López-Rodas G (2004) RNAPol-ChIP: a novel application of chromatin immunoprecipitation to the analysis of real-time gene transcription. Nucleic Acids Res 32:e88CrossRefPubMedGoogle Scholar
  28. 28.
    Gukovskaya AS, Gukovsky I, Zaninovic V, Song M, Sandoval D, Gukovsky S, Pandol SJ (1997) Pancreatic acinar cells produce, release, and respond to tumor necrosis factor-alpha. Role in regulating cell death and pancreatitis. J Clin Invest 100:1853–1862CrossRefPubMedGoogle Scholar
  29. 29.
    Duell EJ, Casella DP, Burk RD, Kelsey KT, Holly EA (2006) Inflammation, genetic polymorphisms in proinflammatory genes TNF-A, RANTES, and CCR5, and risk of pancreatic adenocarcinoma. Cancer Epidemiol Biomarkers Prev 15:726–731CrossRefPubMedGoogle Scholar
  30. 30.
    Baumann B, Wagner M, Aleksic T, von Wichert G, Weber CK, Adler G, Wirth T (2007) Constitutive IKK2 activation in acinar cells is sufficient to induce pancreatitis in vivo. J Clin Invest 117:1502–1513CrossRefPubMedGoogle Scholar
  31. 31.
    Rodriguez JL, Sandoval J, Serviddio G, Sastre J, Morante M, Perrelli MG, Martinez-Chantar ML, Vina J, Vina JR, Mato JM, Avila MA, Franco L, López-Rodas G, Torres L (2006) Id2 leaves the chromatin of the E2F4–p130-controlled c-myc promoter during hepatocyte priming for liver regeneration. Biochem J 398:431–437CrossRefPubMedGoogle Scholar
  32. 32.
    Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159CrossRefPubMedGoogle Scholar
  33. 33.
    Grewal HP, Mohey el Din A, Gaber L, Kotb M, Gaber AO (1994) Amelioration of the physiologic and biochemical changes of acute pancreatitis using an anti-TNF-alpha polyclonal antibody. Am J Surg 167:214–218 discussion 218–9CrossRefPubMedGoogle Scholar
  34. 34.
    Hughes CB, Gaber LW, Mohey el-Din AB, Grewal HP, Kotb M, Mann L, Gaber AO (1996) Inhibition of TNF alpha improves survival in an experimental model of acute pancreatitis. Am Surg 62:8–13PubMedGoogle Scholar
  35. 35.
    Denham W, Yang J, Fink G, Denham D, Carter G, Ward K, Norman J (1997) Gene targeting demonstrates additive detrimental effects of interleukin 1 and tumor necrosis factor during pancreatitis. Gastroenterology 113:1741–1746CrossRefPubMedGoogle Scholar
  36. 36.
    Bazzoni F, Beutler B (1996) The tumor necrosis factor ligand and receptor families. N Engl J Med 334:1717–1725CrossRefPubMedGoogle Scholar
  37. 37.
    Quintana A, Giralt M, Rojas S, Penkowa M, Campbell IL, Hidalgo J, Molinero A (2005) Differential role of tumor necrosis factor receptors in mouse brain inflammatory responses in cryolesion brain injury. J Neurosci Res 82:701–716CrossRefPubMedGoogle Scholar
  38. 38.
    Coimbra R, Melbostad H, Hoyt DB (2004) Effects of phosphodiesterase inhibition on the inflammatory response after shock: role of pentoxifylline. J Trauma 56:442–449CrossRefPubMedGoogle Scholar
  39. 39.
    Pereda J, Sabater L, Cassinello N, Gomez-Cambronero L, Closa D, Folch-Puy E, Aparisi L, Calvete J, Cerda M, Lledo S, Vina J, Sastre J (2004) Effect of simultaneous inhibition of TNF-alpha production and xanthine oxidase in experimental acute pancreatitis: the role of mitogen activated protein kinases. Ann Surg 240:108–116CrossRefPubMedGoogle Scholar
  40. 40.
    Gomez-Cambronero L, Camps B, De La Asuncion JG, Cerda M, Pellin A, Pallardo FV, Calvete J, Sweiry JH, Mann GE, Vina J, Sastre J (2000) Pentoxifylline ameliorates cerulein-induced pancreatitis in rats: role of glutathione and nitric oxide. J Pharmacol Exp Ther 293:670–676PubMedGoogle Scholar
  41. 41.
    Yang C, Shapiro LH, Rivera M, Kumar A, Brindle PK (1998) A role for CREB binding protein and p300 transcriptional coactivators in Ets-1 transactivation functions. Mol Cell Biol 18:2218–2229PubMedGoogle Scholar
  42. 42.
    Merika M, Williams AJ, Chen G, Collins T, Thanos D (1998) Recruitment of CBP/p300 by the IFN beta enhanceosome is required for synergistic activation of transcription. Mol Cell 1:277–287CrossRefPubMedGoogle Scholar
  43. 43.
    Li QJ, Yang SH, Maeda Y, Sladek FM, Sharrocks AD, Martins-Green M (2003) MAP kinase phosphorylation-dependent activation of Elk-1 leads to activation of the co-activator p300. EMBO J 22:281–291CrossRefPubMedGoogle Scholar
  44. 44.
    Deng WG, Zhu Y, Wu KK (2003) Up-regulation of p300 binding and p50 acetylation in tumor necrosis factor-alpha-induced cyclooxygenase-2 promoter activation. J Biol Chem 278:4770–4777CrossRefPubMedGoogle Scholar
  45. 45.
    Hiroi M, Ohmori Y (2003) The transcriptional coactivator CREB-binding protein cooperates with STAT1 and NF-kappa B for synergistic transcriptional activation of the CXC ligand 9/monokine induced by interferon-gamma gene. J Biol Chem 278:651–660CrossRefPubMedGoogle Scholar
  46. 46.
    Wiper-Bergeron N, Salem HA, Tomlinson JJ, Wu D, Hache RJ (2007) Glucocorticoid-stimulated preadipocyte differentiation is mediated through acetylation of C/EBPbeta by GCN5. Proc Natl Acad Sci USA 104:2703–2708CrossRefPubMedGoogle Scholar
  47. 47.
    Rahman I, Marwick J, Kirkham P (2004) Redox modulation of chromatin remodelling: impact on histone acetylation and deacetylation, NF-kappaB and pro-inflammatory gene expression. Biochem Pharmacol 68:1255–1267CrossRefPubMedGoogle Scholar
  48. 48.
    Panne D (2008) The enhanceosome. Curr Opin Struct Biol 18:236–242PubMedGoogle Scholar
  49. 49.
    Revilla Y, Granja AG (2009) Viral mechanisms involved in the transcriptional CBP/p300 regulation of inflammatory and immune responses. Crit Rev Immunol 29:131–154PubMedGoogle Scholar
  50. 50.
    Merika M, Thanos D (2001) Enhanceosomes. Curr Opin Genet Dev 11:205–208CrossRefPubMedGoogle Scholar
  51. 51.
    Alvarez M, Rhodes SJ, Bidwell JP (2003) Context-dependent transcription: all politics is local. Gene 313:43–57CrossRefPubMedGoogle Scholar
  52. 52.
    Vanden Berghe W, Ndlovu MN, Hoya-Arias R, Dijsselbloem N, Gerlo S, Haegeman G (2006) Keeping up NF-kappaB appearances: epigenetic control of immunity or inflammation-triggered epigenetics. Biochem Pharmacol 72:1114–1131CrossRefGoogle Scholar
  53. 53.
    Engdahl R, Monroy MA, Daly JM (2007) 15-Deoxy-Delta12, 14-prostaglandin J2 (15d-PGJ2) mediates repression of TNF-alpha by decreasing levels of acetylated histone H3 and H4 at its promoter. Biochem Biophys Res Commun 359:88–93CrossRefPubMedGoogle Scholar
  54. 54.
    Miao F, Gonzalo IG, Lanting L, Natarajan R (2004) In vivo chromatin remodeling events leading to inflammatory gene transcription under diabetic conditions. J Biol Chem 279:18091–18097CrossRefPubMedGoogle Scholar

Copyright information

© Springer Basel AG 2010

Authors and Affiliations

  • J. Sandoval
    • 1
  • J. Pereda
    • 2
  • J. L. Rodriguez
    • 1
  • J. Escobar
    • 2
  • J. Hidalgo
    • 3
  • L. A. B. Joosten
    • 4
    • 5
  • L. Franco
    • 1
  • J. Sastre
    • 2
  • G. López-Rodas
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of ValenciaValenciaSpain
  2. 2.Department of Physiology, School of PharmacyUniversity of ValenciaValenciaSpain
  3. 3.Department of PhysiologyAutonomous University of BarcelonaBarcelonaSpain
  4. 4.Department of MedicineRadboud University Nijmegen Medical CentreNijmegenThe Netherlands
  5. 5.Rheumatology Research and Advanced TherapeuticsRadboud University Nijmegen Medical CentreNijmegenThe Netherlands

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