Free fatty acids sensitise hepatocytes to TRAIL mediated cytotoxicity

Correspondence to: Dr Gregory J Gores
Miles and Shirley Fiterman Center for Digestive Diseases, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA; gores.gregory@mayo.edu

Harmeet Malhi, Fernando J Barreyro, Hajime Isomoto, Steven F Bronk, Gregory J Gores, Miles and Shirley Fiterman Center for Digestive Diseases, Mayo Clinic College of Medicine, Rochester, MN, USACorrespondence to: Dr Gregory J Gores
Miles and Shirley Fiterman Center for Digestive Diseases, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA; gores.gregory@mayo.edu

Revised 2007 Mar 12; Accepted 2007 Apr 10.

Abstract

Background

Elevated circulating free fatty acids (FFA) contribute to the development of hepatic steatosis and promote hepatocyte apoptosis by incompletely defined mechanisms. Although the death ligand TRAIL has been implicated in a variety of pathological liver diseases, the role of TRAIL in mediating apoptosis of FFA induced steatotic hepatocytes is unknown.

Aim

We examined TRAIL cytotoxicity in an in vitro model of hepatocyte steatosis induced by FFA.

Methods

Hepatocytes (Huh 7 cells, HepG2 cells, and primary rat hepatocytes) were rendered steatotic by incubation with oleic acid. Apoptosis was assessed morphologically and biochemically by caspase activity. TRAIL receptor regulation was examined using immunoblot analysis and siRNA for targeted knockdown. c‐jun N‐terminal kinase (JNK) inhibition was attained with SP600125.

Results

Oleic acid sensitised the cells to TRAIL but not TNF‐α cytotoxicity. FFA sensitisation to TRAIL occurred at much lower concentrations than required for FFA mediated sensitisation to Fas, or FFA induced lipoapoptosis. Oleic acid treatment led to upregulation of the cognate TRAIL receptor death receptor 5 (DR5) but not death receptor 4 (DR4). The upregulation of DR5 was JNK dependent. siRNA targeted knockdown of either DR5 or DR4 demonstrated that DR5 was responsible for FFA sensitisation to TRAIL killing. DR5 expression was enhanced in steatotic human liver samples.

Conclusion

Our results suggest that FFA induced hepatocyte steatosis sensitises to TRAIL by a DR5 mediated JNK dependent mechanism.

Keywords: cellular steatosis, death receptor, SP600125

Abstract

Steatosis is a common histological feature of several liver disorders including non‐alcoholic steatohepatitis (NASH), alcohol induced liver disease (ALD), hepatitis C, and drug mediated liver injuries.¹²³⁴ Multiple metabolic derangements lead to the development of hepatocyte steatosis.⁵ Insulin resistance and elevated circulating free fatty acids (FFA) have been implicated in the development of steatosis in NASH and other forms of hepatic steatosis.⁶ FFA are injurious to many cells by inducing apoptosis, so called lipoapoptosis.⁷⁸ Indeed, hepatocyte apoptosis is a prominent feature of steatotic liver diseases and serum biomarkers of apoptosis are elevated in patients with NASH.⁹ Hepatocyte apoptosis is commonly mediated by the death receptors tumour necrosis factor alfa (TNF‐α), tumour necrosis factor related apoptosis inducing ligand (TRAIL), and Fas ligand (FasL). Although TNF‐α and FasL are both implicated in the pathogenesis of NASH,³⁶ the role of TRAIL in the pathogenesis of steatotic liver injury is unexplored.

Of five known TRAIL receptors, death receptor 4 (DR4) and death receptor 5 (DR5) are classic death receptors, expressed in fetal and adult liver.¹⁰ Enhanced expression of these death receptors is one mechanism by which cells can be sensitised to TRAIL. DR4 and DR5 expression can be upregulated by p53 dependent and p53 independent pathways.¹¹¹²¹³¹⁴ In particular, DR5 is also regulated by a c‐jun N‐terminal kinase (JNK) dependent manner.¹⁵ On ligand binding, DR5 and DR4 are able to activate canonical death signalling pathways leading to activation of caspases culminating in the classic morphological changes of apoptosis.¹⁰ Based on these concepts, the objective of this study was to ascertain if steatotic hepatocytes are sensitised to TRAIL cytotoxicity.

Acknowledgements

The authors would like to acknowledge the excellent secretarial support of Ms Erin Nystuen‐Bungum.

Acknowledgements

Abbreviations

ALD - alcohol induced liver disease

DAPI - 4′,6‐diamidino‐2‐phenylindole

DISC - death inducing signalling complex

DR - death receptor

EPA - eicosapentaenoic acid

FFA - free fatty acid

HCV - hepatitis C virus

JNK - C‐jun N‐terminal kinase

MAPK - mitogen activated protein kinase

NAFLD - non‐alcoholic fatty liver disease

NASH - non‐alcoholic steatohepatitis

OA - oleic acid

PCR - polymerase chain reaction

PVDF - poly(vinylidene) fluoride

siRNA - small interfering RNA

TNF‐α - tumour necrosis factor alfa

TRAIL - tumour necrosis factor related apoptosis inducing ligand

Abbreviations

Footnotes

Competing interests: None.

This work was supported by NIH grant DK 41876 and the Mayo and Palumbo.

Footnotes

References

1. Chou A H, Tsai H F, Wu Y Y. et al Hepatitis C virus core protein modulates TRAIL‐mediated apoptosis by enhancing Bid cleavage and activation of mitochondria apoptosis signaling pathway. J Immunol 20051742160–2166. [[PubMed]
2. Feldstein A E, Canbay A, Angulo P. et al Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis. Gastroenterology 2003125437–443. [[PubMed]
3. Ribeiro P S, Cortez‐Pinto H, Sola S. et al Hepatocyte apoptosis, expression of death receptors, and activation of NF‐kappaB in the liver of nonalcoholic and alcoholic steatohepatitis patients. Am J Gastroenterol 2004991708–1717. [[PubMed]
4. Wu J, Danielsson A, Zern M AToxicity of hepatotoxins: new insights into mechanisms and therapy. Expert Opin Invest Drugs 19998585–607. [[PubMed][Google Scholar]
5. Sanyal A JMechanisms of disease: pathogenesis of nonalcoholic fatty liver disease. Nat Clin Pract Gastroenterol Hepatol 2005246–53. [[PubMed][Google Scholar]
6. Feldstein A E, Canbay A, Guicciardi M E. et al Diet associated hepatic steatosis sensitizes to Fas mediated liver injury in mice. J Hepatol 200339978–983. [[PubMed]
7. Unger R HMinireview: weapons of lean body mass destruction: the role of ectopic lipids in the metabolic syndrome. Endocrinology 20031445159–5165. [[PubMed][Google Scholar]
8. Listenberger L L, Schaffer J EMechanisms of lipoapoptosis: implications for human heart disease. Trends Cardiovasc Med 200212134–138. [[PubMed][Google Scholar]
9. Wieckowska A, Zein N N, Yerian L M. et al In vivo assessment of liver cell apoptosis as a novel biomarker of disease severity in nonalcoholic fatty liver disease. Hepatology 20064427–33. [[PubMed]
10. Kimberley F C, Screaton G RFollowing a TRAIL: update on a ligand and its five receptors. Cell Res 200414359–372. [[PubMed][Google Scholar]
11. Wu G S, Burns T F, McDonald E R., 3rd et al Killer/DR5 is a DNA damage‐inducible p53‐regulated death receptor gene. Nat Genet 199717141–143. [[PubMed]
12. Sheikh M S, Burns T F, Huang Y. et al p53‐dependent and ‐independent regulation of the death receptor KILLER/DR5 gene expression in response to genotoxic stress and tumor necrosis factor alpha. Cancer Res 1998581593–1598. [[PubMed]
13. El‐Deiry W SRegulation of p53 downstream genes. Semin Cancer Biol 19988345–357. [[PubMed][Google Scholar]
14. Takimoto R, El‐Deiry W SWild‐type p53 transactivates the killer/DR5 gene through an intronic sequence‐specific DNA‐binding site. Oncogene 2000191735–1743. [[PubMed][Google Scholar]
15. Higuchi H, Grambihler A, Canbay A. et al Bile acids up‐regulate death receptor 5/TRAIL‐receptor 2 expression via a c‐Jun N‐terminal kinase‐dependent pathway involving Sp1. J Biol Chem 200427951–60. [[PubMed]
16. Spivey J R, Bronk S F, Gores G J. Glycochenodeoxycholate‐induced lethal hepatocellular injury in rat hepatocytes. Role of ATP depletion and cytosolic free calcium. J Clin Invest 19939217–24.
17. Malhi H, Bronk S F, Werneburg N W. et al Free fatty acids induce JNK‐dependent hepatocyte lipoapoptosis. J Biol Chem 200628112093–12101. [[PubMed]
18. Ren Y G, Wagner K W, Knee D A. et al Differential regulation of the TRAIL death receptors DR4 and DR5 by the signal recognition particle. Mol Biol Cell 2004155064–5074.
19. Zou W, Liu X, Yue P. et al c‐Jun NH2‐terminal kinase‐mediated up‐regulation of death receptor 5 contributes to induction of apoptosis by the novel synthetic triterpenoid methyl‐2‐cyano‐3,12‐dioxooleana‐1, 9‐dien‐28‐oate in human lung cancer cells. Cancer Res 2004647570–7578. [[PubMed]
20. Harris C A, Johnson E M., Jr BH3‐only Bcl‐2 family members are coordinately regulated by the JNK pathway and require Bax to induce apoptosis in neurons. J Biol Chem 200127637754–37760. [[PubMed]
21. Kharbanda S, Saxena S, Yoshida K. et al Translocation of SAPK/JNK to mitochondria and interaction with Bcl‐x(L) in response to DNA damage. J Biol Chem 2000275322–327. [[PubMed]
22. Deng X, Xiao L, Lang W. et al Novel role for JNK as a stress‐activated Bcl2 kinase. J Biol Chem 200127623681–23688. [[PubMed]
23. Chang L, Kamata H, Solinas G. et al The E3 ubiquitin ligase itch couples JNK activation to TNFalpha‐induced cell death by inducing c‐FLIP(L) turnover. Cell 2006124601–613. [[PubMed]
24. Kim B J, Ryu S W, Song B JJNK‐ and p38 kinase‐mediated phosphorylation of Bax leads to its activation and mitochondrial translocation and to apoptosis of human hepatoma HepG2 cells. J Biol Chem 200628121256–21265. [[PubMed][Google Scholar]
25. Nehra V, Angulo P, Buchman A L. et al Nutritional and metabolic considerations in the etiology of nonalcoholic steatohepatitis. Dig Dis Sci 2001462347–2352. [[PubMed]
26. Ashkenazi A, Pai R C, Fong S. et al Safety and antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest 1999104155–162.
27. Han L H, Sun W S, Ma C H. et al Detection of soluble TRAIL in HBV infected patients and its clinical implications. World J Gastroenterol 200281077–1080.
28. Janssen H L, Higuchi H, Abdulkarim A. et al Hepatitis B virus enhances tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) cytotoxicity by increasing TRAIL‐R1/death receptor 4 expression. J Hepatol 200339414–420. [[PubMed]
29. Mundt B, Kuhnel F, Zender L. et al Involvement of TRAIL and its receptors in viral hepatitis. Faseb J 20031794–96. [[PubMed]
30. Zheng S J, Wang P, Tsabary G. et al Critical roles of TRAIL in hepatic cell death and hepatic inflammation. J Clin Invest 200411358–64.
31. Mundt B, Wirth T, Zender L. et al Tumour necrosis factor related apoptosis inducing ligand (TRAIL) induces hepatic steatosis in viral hepatitis and after alcohol intake. Gut 2005541590–1596.
32. Qiao L, Han S I, Fang Y. et al Bile acid regulation of C/EBPbeta, CREB, and c‐Jun function, via the extracellular signal‐regulated kinase and c‐Jun NH2‐terminal kinase pathways, modulates the apoptotic response of hepatocytes. Mol Cell Biol 2003233052–3066.
33. Wang Y, Schattenberg J M, Rigoli R M. et al Hepatocyte resistance to oxidative stress is dependent on protein kinase C‐mediated down‐regulation of c‐Jun/AP‐1. J Biol Chem 200427931089–31097. [[PubMed]
34. Bruckner S R, Estus SJNK3 contributes to c‐jun induction and apoptosis in 4‐hydroxynonenal‐treated sympathetic neurons. J Neurosci Res 200270665–670. [[PubMed][Google Scholar]
35. Hirosumi J, Tuncman G, Chang L. et al A central role for JNK in obesity and insulin resistance. Nature 2002420333–336. [[PubMed]
36. Schattenberg J M, Singh R, Wang Y. et al JNK1 but not JNK2 promotes the development of steatohepatitis in mice. Hepatology 200643163–172. [[PubMed]
37. Gunawan B K, Liu Z X, Han D. et al c‐Jun N‐terminal kinase plays a major role in murine acetaminophen hepatotoxicity. Gastroenterology 2006131165–178. [[PubMed]
38. Wang Y, Singh R, Lefkowitch J H. et al Tumor necrosis factor‐induced toxic liver injury results from JNK2‐dependent activation of caspase‐8 and the mitochondrial death pathway. J Biol Chem 200628115258–15267.
39. Pan G, O'Rourke K, Chinnaiyan A M. et al The receptor for the cytotoxic ligand TRAIL. Science 1997276111–113. [[PubMed]
40. Sheridan J P, Marsters S A, Pitti R M. et al Control of TRAIL‐induced apoptosis by a family of signaling and decoy receptors. Science 1997277818–821. [[PubMed]