Activation and function of hepatocyte NF-κB in postischemic liver injury


  • Potential conflict of interest: Nothing to report.


The inhibitor of NF-κB (I-κB) kinase (IKK) complex consists of 3 subunits, IKK1, IKK2, and NF-κB essential modulator (NEMO), and is involved in the activation of NF-κB by various stimuli. IKK2 or NEMO constitutive knockout mice die during embryogenesis as a result of massive hepatic apoptosis. Therefore, we examined the role of IKK2 in TNF-induced apoptosis and ischemia/reperfusion (I/R) injury in the liver by using conditional knockout mice. Hepatocyte-specific ablation of IKK2 did not lead to impaired activation of NF-κB or increased apoptosis after TNF-alpha stimulation whereas conditional NEMO knockout resulted in complete block of NF-κB activation and massive hepatocyte apoptosis. In a model of partial hepatic I/R injury, mice lacking IKK2 in hepatocytes displayed significantly reduced liver necrosis and inflammation than wild-type mice. AS602868, a novel chemical inhibitor of IKK2, protected mice from liver injury due to I/R without sensitizing them toward TNF-induced apoptosis and could therefore emerge as a new pharmacological therapy for liver resection, hemorrhagic shock, or transplantation surgery.

Luedde T, Assmus U, Wustefeld T, Meyer Zu Vilsendorf A, Roskams T, Schmidt-Supprian M, Rajewsky K, Brenner DA, Manns MP, Pasparakis M, Trautwein C. Deletion of IKK2 in hepatocytes does not sensitize these cells to TNF-induced apoptosis but protects from ischemia/reperfusion injury. J Clin Invest 2005;115:849–859. (Reprinted with permission.)


Warm hepatic ischemia/reperfusion (I/R) injury is often a component of liver surgery, trauma, and hemorrhagic shock. Consequences of I/R injury include liver failure and, in more severe cases, remote organ failure, both of which have significant rates of morbidity and mortality. Using experimental animal models, substantial progress has been made toward elucidating the mechanisms of tissue injury resulting from hepatic I/R. These models have characterized the liver injury induced by I/R as dependent upon an intense acute inflammatory response involving activation of the transcription factor, nuclear factor κB (NF-κB), leading to the production of proinflammatory cytokines and chemokines and upregulation of vascular cell adhesion molecules.1–3 These events cause substantial recruitment of activated neutrophils into the hepatic parenchyma, leading to hepatocellular injury through neutrophil release of oxidants and proteases. A greater understanding of the mechanisms through which this inflammatory response is initiated and propagated could lead to the generation of new therapeutics modalities.

The involvement of NF-κB in the hepatic inflammatory response has been the subject of many studies, and in most cases it has been assumed that NF-κB is the primary transcriptional regulator of inflammatory genes after injury. The NF-κB system was clearly designed by nature to be immediately responsive to cellular stress. The term NF-κB actually applies to a number of dimeric combinations of proteins of the Rel family, which possess transcriptional activating properties.4 The classical form of NF-κB, which is also the most relevant to liver pathologies, consists of a heterodimer of p50 and p65 (RelA) proteins.3, 4 This complex is retained in the cytoplasm by proteins of the inhibitory κB (IκB) family. To date, at least seven IκB proteins have been identified in vertebrates.4 Of these, IκBα has been found to be the most physiologically relevant.

Two divergent mechanisms for the activation of NF-κB in liver after I/R have been proposed (Fig. 1). The first, which is the classical pathway of activation, entails activation of the IκB kinase (IKK) complex, which is comprised of two kinase subunits, IKK1 (or IKKα) and IKK2 (or IKKβ), and a regulatory subunit, NF-κB essential modifier (NEMO; also known as IKKγ).4 Activation of this kinase complex results in phosphorylation of IκBα on serine residues 32 and 36 by the IKK2 subunit, leading to polyubiquitination and subsequent degradation by the 26S proteosome.4 The second mechanism involves activation of a Src family kinase resulting in phosphorylation of the tyrosine residue at position 42 of IκBα.5, 6In vitro studies have suggested that hypoxia is the primary physiological effector of IκBα tyrosine phosphorylation.5, 7 Both mechanisms appear to be operant during warm I/R injury in vivo, most likely reflecting the complexity of the insult, which includes oxidant stress, proinflammatory cytokine production, and hypoxia..

Figure 1.

Pathways to NF-κB activation in hepatocytes following I/R. I/R, ischemia/reperfusion; IKK, inhibitory κB kinase.

One important aspect of NF-κB activation during liver I/R injury that has remained elusive is the cell-specific function of this transcriptional activator. Activation of NF-κB in Kupffer cells is presumed to be largely responsible for the wave of proinflammatory cytokines released shortly after reperfusion, although this contention has never been rigorously tested. The function of hepatocyte NF-κB in postischemic injury also has not been well defined, but several studies have documented the critical role of NF-κB in preventing hepatocyte apoptosis in response to tumor necrosis factor α (TNF-α).8, 9 The study by Luedde et al.10 sheds new light on the regulation and function of hepatocyte NF-κB during postischemic injury. The authors use a conditional, hepatocyte-specific knockout of IKK2 to address this issue. This approach is powerful, because constitutive IKK2 knockout mice die during embryogenesis.11–13 Using this system, the authors show that hepatocyte IKK2, and subsequent NF-κB activation in hepatocytes, plays an active role in the development of the inflammatory response induced by I/R. With deletion of hepatocyte IKK2, liver NF-κB activation after reperfusion was ablated. This was accompanied by greatly diminished expression of the proinflammatory mediators TNF-α and inducible nitric oxide synthase, reduced hepatic recruitment of neutrophils, and substantial protection from necrosis. Similar results were also observed using oral administration of the IKK2 inhibitor, AS602868. These findings provide strong evidence that activation of proinflammatory genes in hepatocytes plays a major role in the propagation of inflammatory injury.

The study, however, does not identify the precise manner in which disruption of the NF-κB pathway by IKK2 deletion blunts the hepatic inflammatory response. TNFα, a central mediator in this response,14 was examined only by immunohistochemistry, which does not provide quantitative information about its gene expression. Moreover, hepatocytes are not thought to be a primary source of TNFα during I/R injury. Hepatocytes are known to be a source of chemokines,15 and although these mediators were not investigated, their gene expression is regulated by NF-κB. As such, disruption of hepatocyte NF-κB might be expected to reduce production of CXC chemokines, which are required for the recruitment of neutrophils.2

Previous work in other laboratories has suggested that hepatic NF-κB activation during I/R injury may occur predominantly via tyrosine phosphorylation of IκBα.3, 16 However, these studies differ in the degree of IκBα degradation (indicative of serine phosphorylation of IκBα) observed in the liver after I/R and do not directly evaluate the function of candidate tyrosine kinases for NF-κB activation. The data presented by Luedde et al.10 sharply contrast the notion of tyrosine phosphorylation of IκBα as a major pathway for activation of NF-κB by definitively demonstrating that lack of IKK2 prevents activation of NF-κB in the liver after I/R.

Another interesting question raised by the Luedde et al. study is whether inhibition or gene deletion of hepatocyte IKK2 has any effect on liver recovery or regeneration after I/R. There are few data on the mechanisms involved in postischemic liver recovery, but given the antiapoptotic, pro-proliferative effects of NF-κB in liver regeneration, NF-κB is likely to play an important role in this process. Thus it would be interesting to know whether disruption of NF-κB signaling in hepatocytes, which Luedde et al. show is protective against acute injury, is detrimental to more long-term liver recovery. Nonetheless, the new information provided by this study advances our understanding of the transcriptional mechanisms contributing to liver I/R injury. Furthermore, the successful use of an orally active inhibitor of IKK2 to limit liver injury suggests that pharmacological manipulation of this system is possible, which may have great benefit to a number of liver diseases involving NF-κB.