Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Targeting CASP8 and FADD-like apoptosis regulator ameliorates nonalcoholic steatohepatitis in mice and nonhuman primates

A Corrigendum to this article was published on 01 October 2017

This article has been updated

Abstract

Nonalcoholic steatohepatitis (NASH) is a progressive disease that is often accompanied by metabolic syndrome and poses a high risk of severe liver damage. However, no effective pharmacological treatment is currently available for NASH. Here we report that CASP8 and FADD-like apoptosis regulator (CFLAR) is a key suppressor of steatohepatitis and its metabolic disorders. We provide mechanistic evidence that CFLAR directly targets the kinase MAP3K5 (also known as ASK1) and interrupts its N-terminus-mediated dimerization, thereby blocking signaling involving ASK1 and the kinase MAPK8 (also known as JNK1). Furthermore, we identified a small peptide segment in CFLAR that effectively attenuates the progression of steatohepatitis and metabolic disorders in both mice and monkeys by disrupting the N-terminus-mediated dimerization of ASK1 when the peptide is expressed from an injected adenovirus-associated virus 8–based vector. Taken together, these findings establish CFLAR as a key suppressor of steatohepatitis and indicate that the development of CFLAR-peptide-mimicking drugs and the screening of small-molecular inhibitors that specifically block ASK1 dimerization are new and feasible approaches for NASH treatment.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Cflar is ubiquitinated, and Cflar expression is downregulated in fatty liver.
Figure 2: Cflar protects against hepatic steatosis and inflammation.
Figure 3: Hepatocyte-specific Cflar deletion exacerbates insulin resistance and glucose metabolic disorder.
Figure 4: Inactivation of Ask1–Jnk1 signaling is essential for Cflar function.
Figure 5: CFLAR directly interacts with ASK1 to interrupt its N-terminus-mediated dimerization.
Figure 6: Therapeutic effect of CFLAR(S1) on steatohepatitis and metabolic disorders in monkeys.

Similar content being viewed by others

Change history

  • 26 July 2017

    In the version of this article initially published, the authors inadvertently left out information in the Online Methods section regarding a second injection of AAV8-CFLAR(S1) 7 weeks after the first injection in the monkey experiments to ensure stable expression of CFLAR(S1) in the livers of the monkeys that received the injections. This correction does not change any results or conclusions of the paper. The error has been corrected in the HTML and PDF versions of the article.

References

  1. Michelotti, G.A., Machado, M.V. & Diehl, A.M. NAFLD, NASH and liver cancer. Nat. Rev. Gastroenterol. Hepatol. 10, 656–665 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Byrne, C.D. & Targher, G. NAFLD: a multisystem disease. J. Hepatol. 62 (Suppl. 1), S47–S64 (2015).

    Article  PubMed  Google Scholar 

  3. Mittal, S. et al. Hepatocellular carcinoma in the absence of cirrhosis in United States veterans is associated with nonalcoholic fatty liver disease. Clin Gastroenterol Hepatol. 14, 124–131 e1 (2016).

    Article  CAS  PubMed  Google Scholar 

  4. Zhang, H.J. et al. Effects of moderate and vigorous exercise on nonalcoholic fatty liver disease: a randomized clinical trial. JAMA Intern. Med. 176, 1074–1082 (2016).

    Article  PubMed  Google Scholar 

  5. Nascimbeni, F. et al. From NAFLD in clinical practice to answers from guidelines. J. Hepatol. 59, 859–871 (2013).

    Article  PubMed  Google Scholar 

  6. LaBrecque, D.R. et al. World Gastroenterology Organization global guidelines: nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. J. Clin. Gastroenterol. 48, 467–473 (2014).

    Article  PubMed  Google Scholar 

  7. Yki-Järvinen, H. Nonalcoholic fatty liver disease as a cause and a consequence of metabolic syndrome. Lancet Diabetes Endocrinol. 2, 901–910 (2014).

    Article  CAS  PubMed  Google Scholar 

  8. Czech, M.P. Obesity notches up fatty liver. Nat. Med. 19, 969–971 (2013).

    Article  CAS  PubMed  Google Scholar 

  9. Wang, W., Zhang, Y., Yang, L. & Li, H. The innate immune signaling in cancer and cardiometabolic diseases: friends or foes? Cancer Lett. 387, 46–60 (2017).

    Article  CAS  PubMed  Google Scholar 

  10. Zhang, X.J., Zhang, P. & Li, H. Interferon regulatory factor signaling in cardiometabolic diseases. Hypertension 66, 222–247 (2015).

    Article  CAS  PubMed  Google Scholar 

  11. The Institute of Model Animals of Wuhan University. China. Eur. Heart J. 37, 3257–3259 (2016).

  12. Gehrke, N. et al. Acute organ failure following the loss of anti-apoptotic cellular FLICE-inhibitory protein involves activation of innate immune receptors. Cell Death Differ. 22, 826–837 (2015).

    Article  CAS  PubMed  Google Scholar 

  13. Xiao, J. et al. Cellular FLICE-inhibitory protein protects against cardiac remodeling after myocardial infarction. Basic Res. Cardiol. 107, 239 (2012).

    Article  PubMed  Google Scholar 

  14. Li, H. et al. Cellular FLICE-inhibitory protein protects against cardiac remodeling induced by angiotensin II in mice. Hypertension 56, 1109–1117 (2010).

    Article  CAS  PubMed  Google Scholar 

  15. Cha, S.I. et al. Compartmentalized expression of c-FLIP in lung tissues of patients with idiopathic pulmonary fibrosis. Am. J. Respir. Cell Mol. Biol. 42, 140–148 (2010).

    Article  CAS  PubMed  Google Scholar 

  16. Monteleone, I. et al. A functional role of flip in conferring resistance of Crohn's disease lamina propria lymphocytes to FAS-mediated apoptosis. Gastroenterology 130, 389–397 (2006).

    Article  CAS  PubMed  Google Scholar 

  17. Esmailzadeh, S., Huang, Y., Su, M.W., Zhou, Y. & Jiang, X. BIN1 tumor suppressor regulates Fas–Fas ligand-mediated apoptosis through c-FLIP in cutaneous T cell lymphoma. Leukemia 29, 1402–1413 (2015).

    Article  CAS  PubMed  Google Scholar 

  18. Schattenberg, J.M. et al. Ablation of c-FLIP in hepatocytes enhances death-receptor-mediated apoptosis and toxic liver injury in vivo. J. Hepatol. 55, 1272–1280 (2011).

    Article  CAS  PubMed  Google Scholar 

  19. Kohl, T. et al. Diabetic liver injury from streptozotocin is regulated through the caspase-8 homolog cFLIP, involving activation of JNK2 and intrahepatic immunocompetent cells. Cell Death Dis. 4, e712 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Schattenberg, J.M. et al. Increased hepatic fibrosis and JNK2-dependent liver injury in mice exhibiting hepatocyte-specific deletion of cFLIP. Am. J. Physiol. Gastrointest. Liver Physiol. 303, G498–G506 (2012).

    Article  CAS  PubMed  Google Scholar 

  21. Browning, J.D. & Horton, J.D. Molecular mediators of hepatic steatosis and liver injury. J. Clin. Invest. 114, 147–152 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Scorletti, E. & Byrne, C.D. Omega-3 fatty acids, hepatic lipid metabolism and nonalcoholic fatty liver disease. Annu. Rev. Nutr. 33, 231–248 (2013).

    Article  CAS  PubMed  Google Scholar 

  23. Houstis, N., Rosen, E.D. & Lander, E.S. Reactive oxygen species have a causal role in multiple forms of insulin resistance. Nature 440, 944–948 (2006).

    Article  CAS  PubMed  Google Scholar 

  24. Gao, D. et al. The effects of palmitate on hepatic insulin resistance are mediated by NADPH oxidase 3–derived reactive oxygen species through JNK and p38 MAPK pathways. J. Biol. Chem. 285, 29965–29973 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Li, M. et al. MicroRNA-223 ameliorates alcoholic liver injury by inhibiting the IL-6–p47phox oxidative stress pathway in neutrophils. Gut. http://dx.doi.org/10.1136/gutjnl-2016-311861 [Epub ahead of print] (2016).

  26. Taylor, P.R. et al. Activation of neutrophils by autocrine IL-17A–IL-17RC interactions during fungal infection is regulated by IL-6, IL-23, ROR-γt and dectin-2. Nat. Immunol. 15, 143–151 (2014).

    Article  CAS  PubMed  Google Scholar 

  27. Kleiger, G. & Mayor, T. Perilous journey: a tour of the ubiquitin–proteasome system. Trends Cell Biol. 24, 352–359 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Chang, L. et al. The E3 ubiquitin ligase itch couples JNK activation to TNF-α-induced cell death by inducing c-FLIP(L) turnover. Cell 124, 601–613 (2006).

    Article  CAS  PubMed  Google Scholar 

  29. Kamata, H. et al. Reactive oxygen species promote TNF-α-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases. Cell 120, 649–661 (2005).

    Article  CAS  PubMed  Google Scholar 

  30. Tilg, H. & Moschen, A.R. Evolution of inflammation in nonalcoholic fatty liver disease: the multiple parallel hits hypothesis. Hepatology 52, 1836–1846 (2010).

    Article  CAS  PubMed  Google Scholar 

  31. Tilg, H. & Moschen, A.R. Insulin resistance, inflammation and nonalcoholic fatty liver disease. Trends Endocrinol. Metab. 19, 371–379 (2008).

    Article  CAS  PubMed  Google Scholar 

  32. Arthur, J.S. & Ley, S.C. Mitogen-activated protein kinases in innate immunity. Nat. Rev. Immunol. 13, 679–692 (2013).

    Article  CAS  PubMed  Google Scholar 

  33. Kyriakis, J.M. & Avruch, J. Mammalian MAPK signal transduction pathways activated by stress and inflammation: a 10-year update. Physiol. Rev. 92, 689–737 (2012).

    Article  CAS  PubMed  Google Scholar 

  34. Fujino, G. et al. Thioredoxin and TRAF family proteins regulate reactive-oxygen-species-dependent activation of ASK1 through reciprocal modulation of the N-terminal homophilic interaction of ASK1. Mol. Cell. Biol. 27, 8152–8163 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ji, Y.X. et al. The ubiquitin E3 ligase TRAF6 exacerbates pathological cardiac hypertrophy via TAK1-dependent signaling. Nat. Commun. 7, 11267 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Nam, H.J. et al. Structure of adeno-associated virus serotype 8, a gene therapy vector. J. Virol. 81, 12260–12271 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Neuschwander-Tetri, B.A. et al. Farnesoid X nuclear receptor ligand obeticholic acid for noncirrhotic, nonalcoholic steatohepatitis (FLINT): a multicenter, randomized, placebo-controlled trial. Lancet 385, 956–965 (2015).

    Article  CAS  PubMed  Google Scholar 

  38. Sanyal, A.J. et al. Pioglitazone, vitamin E or placebo for nonalcoholic steatohepatitis. N. Engl. J. Med. 362, 1675–1685 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Lavine, J.E. et al. Effect of vitamin E or metformin for treatment of nonalcoholic fatty liver disease in children and adolescents: the TONIC randomized controlled trial. J. Am. Med. Assoc. 305, 1659–1668 (2011).

    Article  CAS  Google Scholar 

  40. Nobili, V. et al. Effect of vitamin E on aminotransferase levels and insulin resistance in children with nonalcoholic fatty liver disease. Aliment. Pharmacol. Ther. 24, 1553–1561 (2006).

    Article  CAS  PubMed  Google Scholar 

  41. Neuschwander-Tetri, B.A. Future treatments of NASH. Curr. Hepatol. Rep. 15, 125–133 (2016).

    Article  Google Scholar 

  42. Musso, G., Cassader, M. & Gambino, R. Nonalcoholic steatohepatitis: emerging molecular targets and therapeutic strategies. Nat. Rev. Drug Discov. 15, 249–274 (2016).

    Article  CAS  PubMed  Google Scholar 

  43. Xiang, M. et al. Targeting hepatic TRAF1–ASK1 signaling to improve inflammation, insulin resistance and hepatic steatosis. J. Hepatol. 64, 1365–1377 (2016).

    Article  CAS  PubMed  Google Scholar 

  44. Xie, L. et al. DKK3 expression in hepatocytes defines susceptibility to liver steatosis and obesity. J. Hepatol. 65, 113–124 (2016).

    Article  CAS  PubMed  Google Scholar 

  45. Lin, J.H., Zhang, J.J., Lin, S.L. & Chertow, G.M. Design of a phase 2 clinical trial of an ASK1 inhibitor, GS-4997, in patients with diabetic kidney disease. Nephron 129, 29–33 (2015).

    Article  CAS  PubMed  Google Scholar 

  46. Liu, Y., Yin, G., Surapisitchat, J., Berk, B.C. & Min, W. Laminar flow inhibits TNF-induced ASK1 activation by preventing dissociation of ASK1 from its inhibitor 14-3-3. J. Clin. Invest. 107, 917–923 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Davis, R.J. Signal transduction by the JNK group of MAP kinases. Cell 103, 239–252 (2000).

    Article  CAS  PubMed  Google Scholar 

  48. Vernia, S. et al. The PPAR-α–FGF21 hormone axis contributes to metabolic regulation by the hepatic JNK signaling pathway. Cell Metab. 20, 512–525 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Aguirre, V. et al. Phosphorylation of Ser307 in insulin receptor substrate 1 blocks interactions with the insulin receptor and inhibits insulin action. J. Biol. Chem. 277, 1531–1537 (2002).

    Article  CAS  PubMed  Google Scholar 

  50. Schultze, S.M., Hemmings, B.A., Niessen, M. & Tschopp, O. PI3K–AKT, MAPK and AMPK signaling: protein kinases in glucose homeostasis. Expert Rev. Mol. Med. 14, e1 (2012).

    Article  CAS  PubMed  Google Scholar 

  51. Kodama, Y. & Brenner, D.A. c-Jun N-terminal kinase signaling in the pathogenesis of nonalcoholic fatty liver disease: multiple roles in multiple steps. Hepatology 49, 6–8 (2009).

    Article  CAS  PubMed  Google Scholar 

  52. Cazanave, S.C. et al. JNK1-dependent PUMA expression contributes to hepatocyte lipo-apoptosis. J. Biol. Chem. 284, 26591–26602 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Ratziu, V., Goodman, Z. & Sanyal, A. Current efforts and trends in the treatment of NASH. J. Hepatol. 62 (Suppl. 1), S65–S75 (2015).

    Article  CAS  PubMed  Google Scholar 

  54. Negash, A.A. & Gale, M. Jr. Hepatitis regulation by the inflammasome signaling pathway. Immunol. Rev. 265, 143–155 (2015).

    Article  CAS  PubMed  Google Scholar 

  55. Rotman, Y. & Sanyal, A.J. Current and upcoming pharmacotherapy for nonalcoholic fatty liver disease. Gut 66, 180–190 (2017).

    Article  CAS  PubMed  Google Scholar 

  56. Wree, A., Broderick, L., Canbay, A., Hoffman, H.M. & Feldstein, A.E. From NAFLD to NASH to cirrhosis—new insights into disease mechanisms. Nat. Rev. Gastroenterol. Hepatol. 10, 627–636 (2013).

    Article  CAS  PubMed  Google Scholar 

  57. Zhang, X. et al. Rhesus macaques develop metabolic syndrome with reversible vascular dysfunction responsive to pioglitazone. Circulation 124, 77–86 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Kleiner, D.E. et al. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 41, 1313–1321 (2005).

    Article  PubMed  Google Scholar 

  59. Wang, P.X. et al. Hepatocyte TRAF3 promotes liver steatosis and systemic insulin resistance through targeting TAK1-dependent signaling. Nat. Commun. 7, 10592 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Deng, K.Q. et al. Suppressor of IKK is an essential negative regulator of pathological cardiac hypertrophy. Nat. Commun. 7, 11432 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Kraus, D. et al. Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity. Nature 508, 258–262 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Oh, D.Y. et al. A Gpr120-selective agonist improves insulin resistance and chronic inflammation in obese mice. Nat. Med. 20, 942–947 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Wang, X.A. et al. Interferon regulatory factor 9 protects against hepatic insulin resistance and steatosis in male mice. Hepatology 58, 603–616 (2013).

    Article  CAS  PubMed  Google Scholar 

  64. Wang, X.A. et al. Interferon regulatory factor 3 constrains IKKβ–NF-κB signaling to alleviate hepatic steatosis and insulin resistance. Hepatology 59, 870–885 (2014).

    Article  CAS  PubMed  Google Scholar 

  65. Sridhar, S. et al. Cellular immune correlates of protection against symptomatic pandemic influenza. Nat. Med. 19, 1305–1312 (2013).

    Article  CAS  PubMed  Google Scholar 

  66. Straussman, R. et al. Tumor micro-environment elicits innate resistance to RAF inhibitors through HGF secretion. Nature 487, 500–504 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Rudin, C.M. et al. Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer. Nat. Genet. 44, 1111–1116 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank L. Ma (Wuhan University), a biostatistical expert, for his help with the statistical analyses in our present study and L. Zhang (Chinese Academy of Medical Sciences) for the Jnk1-KO (B6.129S1-Mapk8) and Jnk2-KO (B6.129S2-Mapk9) mice. This work was supported by grants from the National Science Fund for Distinguished Young Scholars (no. 81425005; H.L.), the Key Project of the National Natural Science Foundation (no. 81330005 and 81630011; H.L.), the National Science and Technology Support Project (no. 2014BAI02B01 and 2015BAI08B01; H.L.), the National Key Research and Development Program (no. 2013YQ030923-05 (H.L.) and 2016YFF0101504 (Z.-G.S.)).

Author information

Authors and Affiliations

Authors

Contributions

P.-X.W., Y.-X.J. and X.-J.Z. contributed equally to this work, and designed and performed experiments, analyzed data and wrote the manuscript; L.-P.Z., Z.-Z.Y., P.Z., L.-J.S. and X.Y. performed experiments, analyzed data and provided useful advice on the manuscript; J.F., S.T. and Y.W. performed the experiments with monkeys; X.-Y.Z. performed western blot experiments; J.G. and X.Z. constructed the genetically engineered mice used in this study; Q.-F.W. established the mouse models of NASH; J.L., L.W. and Q.X. performed the PET experiments; Z.-G.S., Z.W. and Z.H. helped design the project and edited the manuscript; and H.L. supervised the study, designed experiments and wrote the manuscript.

Corresponding author

Correspondence to Hongliang Li.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–22 and Supplementary Tables 1–5 (PDF 13718 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, PX., Ji, YX., Zhang, XJ. et al. Targeting CASP8 and FADD-like apoptosis regulator ameliorates nonalcoholic steatohepatitis in mice and nonhuman primates. Nat Med 23, 439–449 (2017). https://doi.org/10.1038/nm.4290

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.4290

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing