The cirrhotic hepatocyte: navigating between Scylla and Charybdis

Abstract

In the chronically injured and cholestatic liver, the various hepatic cell types are exposed to a vicious cocktail of toxic and potentially lethal ingredients, such as cytokines, reactive oxygen species (ROS), bile acids, endotoxin and Fas-Ligand expressed on immune cells. Some liver cells thrive in this hostile environment, e.g. stellate cells, which start to proliferate and produce excessive amounts of extracellular matrix and inflammatory and immune cells, which infiltrate the liver and produce cytokines. Others, e.g. hepatocytes, perish in this environment. It is still a matter of debate what the predominant mechanism of hepatocyte cell death is in chronic liver injury: necrosis or apoptosis. This debate is not trivial, because any intervention aimed to protect hepatocytes in the injured liver must be based on a correct understanding of the mechanism of cell death, e.g. apoptosis is a highly regulated process involving numerous steps and components with ample opportunity to intervene and inhibit apoptosis. Prevention of necrosis requires a fundamentally different approach aimed at reducing the necrosis-causing agent. In the chronically injured liver, liver enzymes in serum are elevated. Although increased AST and ALT levels are considered as markers of necrotic cell death, it may result from necrosis secondary to apoptosis (necro-apoptosis) [[1]Jaeschke H Lemasters J.J Apoptosis versus oncotic necrosis in hepatic ischemia/reperfusion injury.Gastroenterology. 2003; 125: 1246-1257Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar]. With the introduction of methods to detect apoptosis, e.g the TUNEL assay, apoptosis was proposed as the major mechanism of cell death in chronic cholestatic liver injury [[2]Miyoshi H Rust C Roberts P.J Burgart L.J Gores G.J Hepatocyte apoptosis after bile duct ligation in the mouse involves Fas.Gastroenterology. 1999; 117: 669-677Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar]. However, the TUNEL assay appeared to be prone to artifacts. Therefore, more specific methods have been applied, e.g. active caspase-3 staining and morphological criteria. Based on these latter methods, apoptosis as the predominant mode of cell death was questioned and necrosis has been proposed as the major mode of cell death in the cirrhotic liver [3Oh S.-H Yun K.-J Nan J.-X Sohn D.-H Lee B.-H Changes in the expression and immunolocalization of protein associated with toxic bile salts-induced apoptosis in rat hepatocytes.Arch Toxicol. 2003; 77: 110-115Crossref PubMed Scopus (25) Google Scholar, 4Schoemaker M.H Gommans W.M Conde de la Rosa L Homan M Klok P Trautwein C et al.Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation.J Hepatol. 2003; 39: 153-161Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 5Gujral J.S Farhood A Jaeschke H Apoptosis and oncotic necrosis during acute liver injury after bile duct ligation.Hepatology. 2003; 38 (abstract #175): 240ACrossref Google Scholar]. Bile acids, Fas-Ligand, reactive oxygen species (ROS) and cytokines like TNFα are all potent inducers of apoptotic cell death in normal primary cultures of hepatocytes. This raises the question why in vivo in the chronically injured or cirrhotic liver, apoptosis is not the prevailing mode of hepatocyte cell death. It appears that the hepatocyte in the chronically injured liver is somehow resistant against apoptotic cell death. Several mechanisms have been proposed which contribute to this resistant phenotype [4Schoemaker M.H Gommans W.M Conde de la Rosa L Homan M Klok P Trautwein C et al.Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation.J Hepatol. 2003; 39: 153-161Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 6Trauner M Boyer J.L Bile salt transporters: molecular characterization, function and regulation.Physiol Rev. 2003; 83: 633-671PubMed Google Scholar, 7Schoemaker M.H Conde de la Rosa L Homan M Vrenken T.E Havinga R Poelstra K et al.TUDCA protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways.Hepatology. 2004; (in press)PubMed Google Scholar, 8Lieser M.J Park J Natori S Jones B.A Bronk S.F Gores G.J Cholestasis confers resistance to the rat liver mitochondrial permeability transition.Gastroenterology. 1998; 115: 693-701Abstract Full Text Full Text PDF PubMed Google Scholar, 9Miyoshi H Rust C Guicciardi M.E Gores G.J NF-κB is activated in cholestasis and functions to reduce liver injury.Am J Pathol. 2001; 158: 967-975Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar]. First of all, the expression of the bile acid importer NTCP (Na+-dependent taurocholate cotransporting polypeptide) is downregulated in chronic liver diseases, in particular cholestatic liver diseases [[6]Trauner M Boyer J.L Bile salt transporters: molecular characterization, function and regulation.Physiol Rev. 2003; 83: 633-671PubMed Google Scholar]. Since bile acid-induced apoptosis is absolutely dependent on bile acid uptake [[7]Schoemaker M.H Conde de la Rosa L Homan M Vrenken T.E Havinga R Poelstra K et al.TUDCA protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways.Hepatology. 2004; (in press)PubMed Google Scholar], reduced NTCP expression contributes to the protection against bile acid induced apoptosis. Furthermore, in cholestasis hepatocytes are exposed to a mixture of many different bile acids, including pro-, anti- and non-apoptotic bile acids. The actual apoptotic potential of this bile acid mixture remains to be determined. Recently, another mechanism of increased resistance against apoptosis in the cholestatic liver has been described: activation of the transcription factor NF-κB [4Schoemaker M.H Gommans W.M Conde de la Rosa L Homan M Klok P Trautwein C et al.Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation.J Hepatol. 2003; 39: 153-161Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 9Miyoshi H Rust C Guicciardi M.E Gores G.J NF-κB is activated in cholestasis and functions to reduce liver injury.Am J Pathol. 2001; 158: 967-975Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar]. NF-κB regulates the transcription of numerous inflammation-associated genes, including many anti-apoptotic genes [[10]Schoemaker M.H Ros J.E Homan M Trautwein C Liston P Poelstra K et al.Cytokine regulation of pro- and anti-apoptotic genes in rat hepatocytes: NF-κB-regulated inhibitor of apoptosis protein-2 (cIAP2) prevents apoptosis.J Hepatol. 2002; 36: 742-750Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar]. Indeed, inhibition of NF-κB increases hepatocyte apoptosis in chronic cholestasis [[9]Miyoshi H Rust C Guicciardi M.E Gores G.J NF-κB is activated in cholestasis and functions to reduce liver injury.Am J Pathol. 2001; 158: 967-975Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar]. Activation of NF-κB prior to exposure to bile acids protects against bile acid-induced apoptosis, although bile acids themselves do not activate NF-κB [4Schoemaker M.H Gommans W.M Conde de la Rosa L Homan M Klok P Trautwein C et al.Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation.J Hepatol. 2003; 39: 153-161Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 7Schoemaker M.H Conde de la Rosa L Homan M Vrenken T.E Havinga R Poelstra K et al.TUDCA protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways.Hepatology. 2004; (in press)PubMed Google Scholar]. In vivo, NF-κB in hepatocytes is activated by cytokines derived from inflammatory cells including Kupffer cells and increased expression of the NF-κB-activating cytokines TNFα and interleukin-1 has been reported in chronic cholestasis [4Schoemaker M.H Gommans W.M Conde de la Rosa L Homan M Klok P Trautwein C et al.Resistance of rat hepatocytes against bile acid-induced apoptosis in cholestatic liver injury is due to nuclear factor-kappa B activation.J Hepatol. 2003; 39: 153-161Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 11Bemelmans M.H Gouma D.J Greve J.W Buurman W.A Cytokines tumor necrosis factor and interleukin-6 in experimental biliary obstruction in mice.Hepatology. 1992; 15: 1132-1136Crossref PubMed Scopus (195) Google Scholar]. Bile acids activate additional survival pathways, such as PI-3/Akt-kinase and the p38 and ERK MAP kinases, in various liver cell types, including hepatocytes, cholangiocytes and stellate cells [7Schoemaker M.H Conde de la Rosa L Homan M Vrenken T.E Havinga R Poelstra K et al.TUDCA protects rat hepatocytes from bile acid-induced apoptosis via activation of survival pathways.Hepatology. 2004; (in press)PubMed Google Scholar, 12Rao Y.-P Studer E.J Stravitz R.T Gupta S Qiao L Dent P Hylemon P.B Activation of the Raf-1/MEK/ERK cascade by bile acids occurs via the epidermal growth factor receptor in primary rat hepatocytes.Hepatology. 2002; 35: 307-314Crossref PubMed Scopus (87) Google Scholar, 13Werneburg N.W Yoon J.-H Higuchi H Gores G.J Bile acids activate the EGF receptor via a TGF-α-dependent mechanism in human cholangiocyte cell lines.Am J Physiol. 2003; 285: G31-G36Crossref Scopus (21) Google Scholar, 14Svegliati-Baroni G Ridolfi F Schoemaker M Saccomanno S Homan M Jansen P.L.M et al.Bile acids induce survival signals in hepatic stellate cells through a PKC- and ERK-dependent mechanism.Hepatology. 2002; 36 (abstract #611): 316AGoogle Scholar, 15Qiao L Yacoub A Studer E Gupta S Pei X.Y Grant S Hylemon P.B Dent P Inhibition of the MAPK and PI3K pathways enhances UDCA-induced apoptosis in primary rodent hepatocytes.Hepatology. 2002; 35: 779-789Crossref PubMed Scopus (130) Google Scholar]. Bile acid-mediated activation of the EGF-receptor and subsequent activation of ERK appears to be a common mechanism in these cell types [12Rao Y.-P Studer E.J Stravitz R.T Gupta S Qiao L Dent P Hylemon P.B Activation of the Raf-1/MEK/ERK cascade by bile acids occurs via the epidermal growth factor receptor in primary rat hepatocytes.Hepatology. 2002; 35: 307-314Crossref PubMed Scopus (87) Google Scholar, 13Werneburg N.W Yoon J.-H Higuchi H Gores G.J Bile acids activate the EGF receptor via a TGF-α-dependent mechanism in human cholangiocyte cell lines.Am J Physiol. 2003; 285: G31-G36Crossref Scopus (21) Google Scholar, 14Svegliati-Baroni G Ridolfi F Schoemaker M Saccomanno S Homan M Jansen P.L.M et al.Bile acids induce survival signals in hepatic stellate cells through a PKC- and ERK-dependent mechanism.Hepatology. 2002; 36 (abstract #611): 316AGoogle Scholar]. In this issue of the Journal, Black and colleagues deepen our understanding of the resistance phenotype of hepatocytes in the chronically injured and cirrhotic liver [[16]Black D Bird M.A Samson C.M Lyman S Lange P.A Schrum L.W et al.Primary cirrhotic hepatocytes resist TGFβ-induced apoptosis through a ROS-dependent mechanism.J Hepatol. 2004; 40: 942-951Abstract Full Text Full Text PDF PubMed Google Scholar]. Black et al. observed increased resistance against TGF-β-induced apoptosis in hepatocytes isolated from cirrhotic livers. Interestingly, TGF-β-induced apoptosis in normal hepatocytes is mediated via a ROS-dependent mechanism: inhibition of ROS exposure prevented TGF-β-induced apoptosis in normal hepatocytes. In contrast, basal ROS production was increased in hepatocytes isolated from cirrhotic livers and inhibition of ROS exposure in cirrhotic hepatocytes increased TGF-β-induced apoptosis. Subsequent investigations demonstrated that the initiation of the caspase-cascade via TGF-β-induced caspase-8 activation was less effective in cirrhotic hepatocytes due to decreased pro-caspase-8 protein levels in cirrhotic hepatocytes. This is a seminal study since it describes a completely novel mechanism of resistance against apoptosis and because it provides directions how to exploit this knowledge in future therapeutic applications. TGF-β is a pro-apoptotic cytokine for hepatocytes and is produced by non-parenchymal cells, in particular activated stellate cells, in chronic liver diseases. Therefore, hepatocyte exposure to increased concentrations of TGF-β is common in most chronic liver diseases, regardless of their etiology. The most intriguing observation in the study of Black is the “conditioning” of hepatocytes, converting increased ROS exposure from a pro-apoptotic factor into an anti-apoptotic factor. Considering that bile acids increase intracellular ROS generation in hepatocytes, “conditioning” of hepatocytes could also occur via chronic exposure to bile acids [17Sokol R.J Straka M.S Dahl R Devereaux M.W Yerushalmi B Gumpricht E et al.Role of oxidant stress in the permeability transition induced in rat hepatic mitochondria by hydrophobic bile acids.Pediatr Res. 2001; 49: 519-531Crossref PubMed Scopus (121) Google Scholar, 18Sokol R.J Winklhofer-Roob B.M Devereaux M.W McKim Jr, J.M Generation of hydroperoxides in isolated rat hepatocytes and hepatic mitochondria exposed to hydrophobic bile acids.Gastroenterology. 1995; 109: 1249-1256Abstract Full Text PDF PubMed Scopus (241) Google Scholar]. The findings of Black et al. are also highly relevant for any attempt to treat chronic liver diseases with anti-oxidants. Indeed, the disappointing results of anti-oxidants in the treatment of chronic liver diseases could be explained by the fact that any beneficial effects of anti-oxidants, e.g. lowering the exposure to inflammatory cell derived extracellular ROS and lipid peroxidation, are offset by the collapse of the anti-apoptotic phenotype in hepatocytes. Obviously, we need to know the mechanism(s) of the beneficial effect of increased basal ROS production in cirrhotic hepatocytes. Although Black and colleagues do not address this issue, there are some clues. First of all is the involvement of the JNK pathway. Reactive oxygen species activate JNK and activation of JNK in hepatocytes is considered to be pro-apoptotic event [19Czaja M Liu H Wang Y Oxidant-induced hepatocyte injury from menadione is regulated by ERK and AP-1 signaling.Hepatology. 2003; 37: 1405-1413Crossref PubMed Scopus (119) Google Scholar, 20Qiao L Han S.I Fang Y Park J.S Gupta S Gilfor D 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. 2003; 23: 3052-3066Crossref PubMed Scopus (133) Google Scholar, 21Marderstein E.L Bucher B Guo Z Feng X Reid K Geller D.A Protection of rat hepatocytes from apoptosis by inhibition of c-Jun N-terminal kinase.Surgery. 2003; 134: 280-284Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar]. In cirrhotic hepatocytes chronically exposed to ROS-generating factors continuous JNK-activation could be prevented via some kind of de-sensitization mechanism, contributing to an anti-apoptotic phenotype. It remains to be explained why inhibition of ROS exposure then leads to a sudden increase in apoptosis. Another explanation could be that chronic ROS exposure leads to induction of the gene heme-oxygenase-1 (HO-1). HO-1 is an oxidative stress-induced gene and it is well known that expression of HO-1 protects against oxidative stress-induced cell death [[22]Katori M Busuttil R.W Kupiec-Weglinski J.W Heme oxygenase-1 system in organ transplantation.Transplantation. 2002; 74: 905-912Crossref PubMed Scopus (192) Google Scholar]. Indeed, HO-1 expression contributes to the phenomenon of protective pre-conditioning in organ transplantation [[22]Katori M Busuttil R.W Kupiec-Weglinski J.W Heme oxygenase-1 system in organ transplantation.Transplantation. 2002; 74: 905-912Crossref PubMed Scopus (192) Google Scholar]. Both hypotheses can be tested experimentally and additional possibilities may exist. Another important observation in the study of Black et al. is the reduced content of pro-caspase-8 protein in cirrhotic hepatocytes. Since pro-caspase-8 mRNA levels were not investigated, we do not know whether this regulation is at the transcriptional or post-transcriptional level. Not much is known about the regulation of the caspase-8 promoter and mRNA expression, although gene-silencing and downregulation of caspase-8 mRNA has been observed in several tumors [23Harada K Toyooka S Shivapurkar N Maitra A Reddy J.L Matta H et al.Deregulation of caspase 8 and 10 expression in pediatric tumors and cell lines.Cancer Res. 2002; 62: 5897-5901PubMed Google Scholar, 24Shivapurkar N Toyooka S Eby M.T Huang C.X Sathyanarayana U.G Cunningham H.T et al.Differential inactivation of caspase-8 in lung cancers.Cancer Biol Ther. 2002; 1: 65-69Crossref PubMed Scopus (8) Google Scholar, 25Liedtke C Groger N Manns M.P Trautwein C The human caspase-8 promoter sustains basal activity through SP1 and ETS-like transcription factors and can be up-regulated by a p53-dependent mechanism.J Biol Chem. 2003; 278: 27593-27604Crossref PubMed Scopus (71) Google Scholar]. This is extremely relevant for the present study since one of the hypotheses of Black et al. is that the increased resistance of cirrhotic hepatocytes to TGF-β-induced apoptosis facilitates inappropriate hepatocyte proliferation and predisposes to hepatocellular carcinoma. Reduction of caspase-8 activity to preserve hepatocytes in chronic liver injury could then turn out to be a wolf in sheep's clothing because it may facilitate aberrant hepatocyte proliferation and tumor development and it may be futile since necrotic rather than apoptotic hepatocyte death prevails in chronic liver injury. In contrast, transient reduction of caspase-8 function may be a promising therapy for acute liver injury characterised by massive apoptosis of hepatocytes as recently reported by Zender et al. [[26]Zender L Hutker S Liedtke C Tillmann H.L Zender S Mundt B et al.Caspase 8 small interfering RNA prevents acute liver failure in mice.Proc Natl Acad Sci USA. 2003; 100: 7797-7802Crossref PubMed Scopus (358) Google Scholar]. One potential pitfall of the study of Black et al. is the preservation of the anti-apoptotic phenotype of cirrhotic hepatocytes after isolation and in culture. Indeed, although there is strong evidence that NF-κB is activated in cholestatic liver injury in vivo, Black et al. did not demonstrate any involvement of NF-κB in the resistance against TGF-β induced apoptosis in cultured cirrhotic hepatocytes. The preservation of anti-apoptotic mechanisms in cultured cirrhotic hepatocytes, in particular NF-κB activation, but also other mechanisms involving activity of kinases, remains to be addressed and is of considerable importance to obtain a full comprehension of the anti-apoptotic phenotype of cirrhotic hepatocytes. Finally, we should not forget that in chronic liver injury hepatocytes die and eventually liver failure will develop. Strengthening the anti-apoptotic phenotype of already apoptosis-resistant hepatocytes may be a futile approach or even undesirable because of the risk for cancer development. Much more benefit can be expected from preventing necrotic cell death. In fact, the anti-apoptotic phenotype of cirrhotic hepatocytes may be the cause of necrotic cell death, since inhibition of apoptosis often shifts the balance to necrotic cell death [27Samali A Nordgren H Zhivotovsky B Peterson E Orrenius S A comparative study of apoptosis and necrosis in HepG2 cells: oxidant-induced caspase inactivation leads to necrosis.Biochem Biophys Res Commun. 1999; 255: 6-11Crossref PubMed Scopus (177) Google Scholar, 28Kalai M Van Loo G Vanden Berghe T Meeus A Burm W Saelens X Vandenabeele P Tipping the balance between necrosis and apoptosis in human and murine cells treated with interferon and dsRNA.Cell Death Differ. 2002; 9: 981-994Crossref PubMed Scopus (113) Google Scholar, 29Van den Berghe T Kalai M van Loo G Declercq W Vandenabeele P Disruption of HSP90 function reverts tumor necrosis factor-induced necrosis to apoptosis.J Biol Chem. 2003; 278: 5622-5629Crossref PubMed Scopus (142) Google Scholar, 30Conde de la Rosa L Schoemaker M.H Homan M Jansen P.L.M Moshage H Reactive oxygen species induce hepatocyte cell death via different mechanisms: a balance between apoptosis and necrosis.Hepatology. 2003; 38 (abstract #866): 577APubMed Google Scholar]. All in all, there are many mechanisms which protect cholestatic or cirrhotic hepatocytes against apoptosis: MAP kinases like ERK and p38, PI-3/Akt kinase, transcriptional events such as downregulation of bile acid importers and NF-κB-mediated induction of anti-apoptotic genes. Black et al. have now reported a novel mechanism: reduction of caspase-8 function. Involvement of other pathways like JNK and HO-1 remain to be investigated. All these pathways suggest numerous novel intervention targets aimed at modulating apoptosis in the chronically injured or cirrhotic liver. The question is: should we inhibit or promote apoptosis? Inhibition of apoptosis will strengthen the resistant phenotype and may promote proliferation of malignant hepatocytes and development of hepatocellular carcinoma. On the other hand, reverting the resistant phenotype may lead to excess hepatocyte apoptosis and liver failure. The true challenge will be to avoid both Scylla and Charybdis. In the end, reducing necrotic cell death, e.g. by reducing inflammation, may be the answer to this challenge [[31]Gujral J.S Farhood A Bajt M.L Jaeschke H Neutrophils aggravate acute liver injury during obstructive cholestasis in bile duct-ligated mice.Hepatology. 2003; 38: 355-363Crossref PubMed Scopus (261) Google Scholar].