Article | Published:

RIPK1 and death receptor signaling drive biliary damage and early liver tumorigenesis in mice with chronic hepatobiliary injury


Hepatocyte apoptosis is intrinsically linked to chronic liver disease and hepatocarcinogenesis. Conversely, necroptosis of hepatocytes and other liver cell types and its relevance for liver disease is debated. Using liver parenchymal cell (LPC)-specific TGF-beta-activated kinase 1 (TAK1)-deficient (TAK1LPC-KO) mice, which exhibit spontaneous hepatocellular and biliary damage, hepatitis, and early hepatocarcinogenesis, we have investigated the contribution of apoptosis and necroptosis in hepatocyte and cholangiocyte death and their impact on liver disease progression. Here, we provide in vivo evidence showing that TAK1-deficient cholangiocytes undergo spontaneous necroptosis induced primarily by TNFR1 and dependent on RIPK1 kinase activity, RIPK3, and NEMO. In contrast, TAK1-deficient hepatocytes die by FADD-dependent apoptosis, which is not significantly inhibited by LPC-specific RIPK1 deficiency, inhibition of RIPK1 kinase activity, RIPK3 deficiency or combined LPC-specific deletion of TNFR1, TRAILR, and Fas. Accordingly, normal mouse cholangiocytes can undergo necroptosis, while primary hepatocytes are resistant to it and die exclusively by apoptosis upon treatment with cell death-inducing stimuli in vitro, likely due to the differential expression of RIPK3. Interestingly, the genetic modifications that conferred protection from biliary damage also prevented the spontaneous lethality that was often observed in TAK1LPC-KO mice. In the presence of chronic hepatocyte apoptosis, preventing biliary damage delayed but did not avert hepatocarcinogenesis. On the contrary, inhibition of hepatocyte apoptosis fully prevented liver tumorigenesis even in mice with extensive biliary damage. Altogether, our results suggest that using RIPK1 kinase activity inhibitors could be therapeutically useful for cholestatic liver disease patients.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Guicciardi ME, Malhi H, Mott JL, Gores GJ. Apoptosis and necrosis in the liver. Compr Physiol. 2013;3:977–1010.

  2. 2.

    Schwabe RF, Luedde T. Apoptosis and necroptosis in the liver: a matter of life and death. Nat Rev Gastroenterol Hepatol. 2018;15:738–52.

  3. 3.

    Kondylis V, Pasparakis M. RIP kinases in liver cell death, inflammation and cancer. Trends Mol Med. 2019;25:47–63.

  4. 4.

    Luedde T, Beraza N, Kotsikoris V, van Loo G, Nenci A, De Vos R, et al. Deletion of NEMO/IKKgamma in liver parenchymal cells causes steatohepatitis and hepatocellular carcinoma. Cancer Cell. 2007;11:119–32.

  5. 5.

    Liedtke C, Bangen JM, Freimuth J, Beraza N, Lambertz D, Cubero FJ, et al. Loss of caspase-8 protects mice against inflammation-related hepatocarcinogenesis but induces non-apoptotic liver injury. Gastroenterology. 2011;141:2176–87.

  6. 6.

    Ehlken H, Krishna-Subramanian S, Ochoa-Callejero L, Kondylis V, Nadi NE, Straub BK, et al. Death receptor-independent FADD signalling triggers hepatitis and hepatocellular carcinoma in mice with liver parenchymal cell-specific NEMO knockout. Cell Death Differ. 2014;21:1721–32.

  7. 7.

    Kondylis V, Polykratis A, Ehlken H, Ochoa-Callejero L, Straub BK, Krishna-Subramanian S, et al. NEMO prevents steatohepatitis and hepatocellular carcinoma by inhibiting RIPK1 kinase activity-mediated hepatocyte apoptosis. Cancer Cell. 2015;28:582–98.

  8. 8.

    Vucur M, Reisinger F, Gautheron J, Janssen J, Roderburg C, Cardenas DV, et al. RIP3 inhibits inflammatory hepatocarcinogenesis but promotes cholestasis by controlling caspase-8- and JNK-dependent compensatory cell proliferation. Cell Rep. 2013;4:776–90.

  9. 9.

    Koppe C, Verheugd P, Gautheron J, Reisinger F, Kreggenwinkel K, Roderburg C, et al. I kappa B kinase alpha/beta control biliary homeostasis and hepatocarcinogenesis in mice by phosphorylating the cell-death mediator receptor-interacting protein kinase 1. Hepatology . 2016;64:1217–31.

  10. 10.

    Schneider AT, Gautheron J, Feoktistova M, Roderburg C, Loosen SH, Roy S, et al. RIPK1 suppresses a TRAF2-dependent pathway to liver cancer. Cancer Cell. 2017;31:94–109.

  11. 11.

    Boege Y, Malehmir M, Healy ME, Bettermann K, Lorentzen A, Vucur M, et al. A dual role of caspase-8 in triggering and sensing proliferation-associated DNA damage, a key determinant of liver cancer development. Cancer Cell. 2017;32:342–59 e10.

  12. 12.

    Dara L, Liu ZX, Kaplowitz N. Questions and controversies: the role of necroptosis in liver disease. Cell Death Discov. 2016;2:16089.

  13. 13.

    Ting AT, Bertrand MJ. More to life than NF-kappaB in TNFR1 signaling. Trends Immunol. 2016;37:535–45.

  14. 14.

    Annibaldi A, Meier P. Checkpoints in TNF-induced cell death: implications in inflammation and cancer. Trends Mol Med. 2018;24:49–65.

  15. 15.

    Dondelinger Y, Jouan-Lanhouet S, Divert T, Theatre E, Bertin J, Gough PJ, et al. NF-kappa B-independent role of IKK alpha/IKK beta in preventing RIPK1 kinase-dependent apoptotic and necroptotic cell death during TNF signaling. Mol Cell. 2015;60:63–76.

  16. 16.

    Lafont E, Draber P, Rieser E, Reichert M, Kupka S, de Miguel D, et al. TBK1 and IKKepsilon prevent TNF-induced cell death by RIPK1 phosphorylation. Nat Cell Biol. 2018;20:1389–99.

  17. 17.

    Xu D, Jin T, Zhu H, Chen H, Ofengeim D, Zou C, et al. TBK1 suppresses RIPK1-driven apoptosis and inflammation during development and in aging. Cell . 2018;174:1477–91 e19.

  18. 18.

    Bettermann K, Vucur M, Haybaeck J, Koppe C, Janssen J, Heymann F, et al. TAK1 suppresses a NEMO-dependent but NF-kappa B-independent pathway to liver cancer. Cancer Cell. 2010;17:481–96.

  19. 19.

    Kellendonk C, Opherk C, Anlag K, Schutz G, Tronche F. Hepatocyte-specific expression of Cre recombinase. Genesis . 2000;26:151–3.

  20. 20.

    Inokuchi S, Aoyama T, Miura K, Osterreicher CH, Kodama Y, Miyai K, et al. Disruption of TAK1 in hepatocytes causes hepatic injury, inflammation, fibrosis, and carcinogenesis. Proc Natl Acad Sci USA. 2010;107:844–9.

  21. 21.

    Gautheron J, Vucur M, Reisinger F, Cardenas DV, Roderburg C, Koppe C, et al. A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis. Embo Mol Med. 2014;6:1062–74.

  22. 22.

    Roychowdhury S, McMullen MR, Pisano SG, Liu X, Nagy LE. Absence of receptor interacting protein kinase 3 prevents ethanol-induced liver injury. Hepatology. 2013;57:1773–83.

  23. 23.

    Wang L, Du F, Wang X. TNF-alpha induces two distinct caspase-8 activation pathways. Cell . 2008;133:693–703.

  24. 24.

    Dara L, Johnson H, Suda J, Win S, Gaarde W, Han D, et al. Receptor interacting protein kinase 1 mediates murine acetaminophen toxicity independent of the necrosome and not through necroptosis. Hepatology. 2015;62:1847–57.

  25. 25.

    Suda J, Dara L, Yang L, Aghajan M, Song Y, Kaplowitz N, et al. Knockdown of RIPK1 markedly exacerbates murine immune-mediated liver injury through massive apoptosis of hepatocytes, independent of necroptosis and inhibition of NF-kappaB. J Immunol. 2016;197:3120–9.

  26. 26.

    Van TM, Polykratis A, Straub BK, Kondylis V, Papadopoulou N, Pasparakis M. Kinase-independent functions of RIPK1 regulate hepatocyte survival and liver carcinogenesis. J Clin Invest. 2017;127:2662–77.

  27. 27.

    Afonso MB, Rodrigues PM, Simao AL, Ofengeim D, Carvalho T, Amaral JD, et al. Activation of necroptosis in human and experimental cholestasis. Cell Death Dis. 2016;7:e2390.

  28. 28.

    Gunther C, He GW, Kremer AE, Murphy JM, Petrie EJ, Amann K, et al. The pseudokinase MLKL mediates programmed hepatocellular necrosis independently of RIPK3 during hepatitis. J Clin Invest. 2016;126:4346–60.

  29. 29.

    Pasparakis M, Vandenabeele P. Necroptosis and its role in inflammation. Nature. 2015;517:311–20.

  30. 30.

    Anderton H, Bandala-Sanchez E, Simpson DS, Rickard JA, Ng AP, Di Rago L, et al. RIPK1 prevents TRADD-driven, but TNFR1 independent, apoptosis during development. Cell Death Differ. 2019;26:877–89.

  31. 31.

    Yang L, Inokuchi S, Roh YS, Song J, Loomba R, Park EJ, et al. Transforming growth factor-beta signaling in hepatocytes promotes hepatic fibrosis and carcinogenesis in mice with hepatocyte-specific deletion of TAK1. Gastroenterology. 2013;144:1042–54 e4.

  32. 32.

    Mihaly SR, Ninomiya-Tsuji J, Morioka S. TAK1 control of cell death. Cell Death Differ. 2014;21:1667–76.

  33. 33.

    Inokuchi-Shimizu S, Park EJ, Roh YS, Yang L, Zhang B, Song J, et al. TAK1-mediated autophagy and fatty acid oxidation prevent hepatosteatosis and tumorigenesis. J Clin Invest. 2014;124:3566–78.

  34. 34.

    Pescatore A, Esposito E, Draber P, Walczak H, Ursini MV. NEMO regulates a cell death switch in TNF signaling by inhibiting recruitment of RIPK3 to the cell death-inducing complex II. Cell Death Dis. 2016;7:e2346.

  35. 35.

    Irrinki KM, Mallilankaraman K, Thapa RJ, Chandramoorthy HC, Smith FJ, Jog NR, et al. Requirement of FADD, NEMO, and BAX/BAK for aberrant mitochondrial function in tumor necrosis factor alpha-induced necrosis. Mol Cell Biol. 2011;31:3745–58.

  36. 36.

    Shimizu Y, Peltzer N, Sevko A, Lafont E, Sarr A, Draberova H, et al. The linear ubiquitin chain assembly complex acts as a liver tumor suppressor and inhibits hepatocyte apoptosis and hepatitis. Hepatology. 2017;65:1963–78.

  37. 37.

    Filliol A, Piquet-Pellorce C, Le Seyec J, Farooq M, Genet V, Lucas-Clerc C, et al. RIPK1 protects from TNF-alpha-mediated liver damage during hepatitis. Cell Death Dis. 2016;7:e2462.

  38. 38.

    Filliol A, Piquet-Pellorce C, Raguenes-Nicol C, Dion S, Farooq M, Lucas-Clerc C, et al. RIPK1 protects hepatocytes from Kupffer cells-mediated TNF-induced apoptosis in mouse models of PAMP-induced hepatitis. J Hepatol. 2017;66:1205–13.

  39. 39.

    Seehawer M, Heinzmann F, D’Artista L, Harbig J, Roux PF, Hoenicke L, et al. Necroptosis microenvironment directs lineage commitment in liver cancer. Nature. 2018;562:69–75.

  40. 40.

    Yanger K, Zong Y, Maggs LR, Shapira SN, Maddipati R, Aiello NM, et al. Robust cellular reprogramming occurs spontaneously during liver regeneration. Genes Dev. 2013;27:719–24.

  41. 41.

    Eftychi C, Karagianni N, Alexiou M, Apostolaki M, Kollias G. Myeloid TAKI [corrected] acts as a negative regulator of the LPS response and mediates resistance to endotoxemia. PLoS ONE. 2012;7:e31550.

  42. 42.

    Schmidt-Supprian M, Bloch W, Courtois G, Addicks K, Israel A, Rajewsky K, et al. NEMO/IKK gamma-deficient mice model incontinentia pigmenti. Mol Cell. 2000;5:981–92.

  43. 43.

    Mc Guire C, Volckaert T, Wolke U, Sze M, de Rycke R, Waisman A, et al. Oligodendrocyte-specific FADD deletion protects mice from autoimmune-mediated demyelination. J Immunol. 2010;185:7646–53.

  44. 44.

    Dannappel M, Vlantis K, Kumari S, Polykratis A, Kim C, Wachsmuth L, et al. RIPK1 maintains epithelial homeostasis by inhibiting apoptosis and necroptosis. Nature . 2014;513:90–4.

  45. 45.

    Polykratis A, Hermance N, Zelic M, Roderick J, Kim C, Van TM, et al. Cutting edge: RIPK1 Kinase inactive mice are viable and protected from TNF-induced necroptosis in vivo. J Immunol. 2014;193:1539–43.

  46. 46.

    Newton K, Sun X, Dixit VM. Kinase RIP3 is dispensable for normal NF-kappa Bs, signaling by the B-cell and T-cell receptors, tumor necrosis factor receptor 1, and Toll-like receptors 2 and 4. Mol Cell Biol. 2004;24:1464–9.

  47. 47.

    Van Hauwermeiren F, Armaka M, Karagianni N, Kranidioti K, Vandenbroucke RE, Loges S, et al. Safe TNF-based antitumor therapy following p55TNFR reduction in intestinal epithelium. J Clin Invest. 2013;123:2590–603.

  48. 48.

    Hao Z, Hampel B, Yagita H, Rajewsky K. T cell-specific ablation of Fas leads to Fas ligand-mediated lymphocyte depletion and inflammatory pulmonary fibrosis. J Exp Med. 2004;199:1355–65.

  49. 49.

    Grosse-Wilde A, Voloshanenko O, Bailey SL, Longton GM, Schaefer U, Csernok AI, et al. TRAIL-R deficiency in mice enhances lymph node metastasis without affecting primary tumor development. J Clin Invest. 2008;118:100–10.

  50. 50.

    Uriarte I, Banales JM, Saez E, Arenas F, Oude Elferink RP, Prieto J, et al. Bicarbonate secretion of mouse cholangiocytes involves Na(+)-HCO(3)(−) cotransport in addition to Na(+)-independent Cl(−)/HCO(3)(−) exchange. Hepatology. 2010;51:891–902.

Download references


We thank V. Dixit and Genentech for anti-RIPK3 antibody and Ripk3−/− mice. We thank J. Kuth, C. Uthoff-Hachenberg, E. Gareus, B. Kühnel and E. Stade for technical assistance. This work was supported by grants from Worldwide Cancer Research (award no. 15–0228) and the European Research Council (grant agreement no. 323040 to MP). VK was supported by a Marie Curie Career Development Fellowship (FP7-PEOPLE-2010-IEF; proposal no. 275767).

Author information

SK-S performed genetic crosses, tissue sampling, IHC, immunoblotting, qRT-PCR, and data interpretation and drafted the manuscript. VK conducted genetic crosses, tissue sampling, IHC analysis, in vitro experiments in NMCs and hepatocytes and data interpretation. SS, KH, and PS performed histopathological analysis of mouse livers. MA and GK provided Tak1FL and HW TrailrFL mice. JMB provided NMCs. VK and MP coordinated the project and wrote the manuscript.

Correspondence to Vangelis Kondylis.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Edited by G. Melino

Supplementary information

Legends for Supplementary Figures

Figure S1

Figure S2

Figure S3

Figure S4

Figure S5

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8