The cytokine tumor necrosis factor alpha (TNF-α; TNF) plays a critical role early in liver regeneration following partial hepatectomy (PH). TNF stimulates at least three different pathways leading to nuclear factor kappa B (NF-κB) activation, apoptosis signaling by way of caspase-8 (Casp8), and activation of cJun N-terminal kinases (JNK). The present study aimed to better define the role of Casp8 during liver regeneration. We performed PH in mice lacking Casp8 specifically in hepatocytes (Casp8Δhepa) and determined their liver regeneration capacity by measuring liver mass restoration and kinetics of cell cycle progression. Casp8Δhepa mice showed an accelerated onset of DNA synthesis after PH, delayed hepatocyte mitosis, but overall normal liver mass restoration. Analysis of immediate TNF-dependent signaling pathways revealed that loss of Casp8 prevents proteolytic cleavage of the receptor-interacting protein 1 (RIP1) in hepatocytes and subsequently triggers premature activation of NF-κB and JNK/cJun related signals. In order to define the role of NF-κB in this setting we blocked NF-κB activation in Casp8Δhepa mice by concomitant inactivation of the NF-κB essential modulator (NEMO) in hepatocytes. Lack of NEMO largely reverted aberrant DNA synthesis in Casp8Δhepa mice but resulted in incomplete termination of the regeneration process and hepatomegaly. Conclusion: Casp8 comprises a nonapoptotic function during liver regeneration by balancing RIP1, NF-κB, and JNK activation. While loss of Casp8 triggers NF-κB activation and thus improves liver regeneration, combined loss of Casp8 and NEMO impairs a controlled regenerative response and drives hepatomegaly. (Hepatology 2013;58:1779–1789)
The cytokine tumor necrosis factor alpha (TNFα; TNF) mediates pleiotropic effects by triggering inflammation and cell proliferation by way of nuclear factor kappa B (NF-κB), apoptosis through caspase-8 (Casp8), or activation of cJun N-terminal kinases (JNK). It has been identified as a crucial mediator for the priming phase of liver regeneration. Genetic inactivation of TNF-receptor 1 (TNF-R1) results in decreased NF-κB and JNK signaling leading to impaired hepatocyte proliferation after 70% partial hepatectomy (PH).
In the adult liver, hepatocytes are long-lived and rarely undergo proliferation, yet they retain a remarkable ability to proliferate. This allows the liver to restore its original mass within 7 to 10 days after PH. The regenerative response is initiated by a series of signaling events that allow the quiescent hepatocytes to reenter the cell cycle and undergo several rounds of proliferation until the original liver mass is restored.
Binding of TNF to TNF-R1 rapidly initiates assembly of a plasma membrane bound complex-I, composed of TNF-R1, the tumor necrosis factor receptor type 1-associated death domain protein (TRADD), the protein kinase RIP1, and the TNF receptor-associated factor 2 (TRAF2). Complex-I induces immediate downstream activation of both the JNK and NF-κB signaling pathways and prevents apoptosis in part by inducing antiapoptotic proteins such as FLIPL. Upon inhibition of NF-κB signaling, a competing complex (complex-II) is formed immediately after TNF ligation. Complex-II includes the adapter proteins TRADD, FADD (Fas-associated protein with death domain), and the proapoptotic protease pro-caspase-8, which eventually initiates the apoptotic signal cascade. Constitutive targeted disruption of Casp8 results in embryonic lethality presumably due to an abundance of developmental defects. More recent studies revealed that Casp8 plays also an essential role for prevention of an alternative mode of programmed cell death, termed necroptosis. We recently reported that loss of Casp8 in hepatocytes protects from acute Fas and lipopolysaccharide (LPS)-induced liver injury but also triggers increased nonapoptotic cell death in mice lacking the NF-κB essential modulator (NEMO) involving enhanced RIP1 kinase activity and necroptosis.
The aim of the present study was to investigate the consequences of genetic Casp8 inactivation in hepatocytes for liver regeneration following PH. We demonstrate that loss of Casp8 leads to an accelerated onset of hepatocyte priming and DNA synthesis following PH without affecting proper termination of liver growth. We provide evidence that this protective effect is due to early NF-κB activation associated with premature expression of the upstream RIP1 kinase. Our findings may have an impact for the evaluation of human therapies using low-molecular caspase-inhibitors.
Casp8 is the most apical caspase during extrinsic apoptosis mediated by death-receptors. In addition, Casp8 may have nonapoptotic functions, e.g., by regulating NF-κB transcriptional activity. In the present study we characterized the consequences of hepatocyte-specific Casp8 deletion for liver regeneration in mice. We tested the hypothesis that loss of Casp8 would either prevent proper termination of liver growth or affect the early events of TNF-dependent signaling after PH.
The present study revealed that termination of liver regeneration is independent of Casp8. However, loss of Casp8 resulted in deregulation of all interphase cyclins and in accelerated onset of DNA synthesis after PH. These unexpected effects could be linked to premature RIP1 kinase activation affecting the downstream NF-κB and JNK/cJun pathways, respectively. Concomitant NEMO deletion restored normal onset of hepatocyte proliferation in Casp8-deficient livers but eventually induced hepatomegaly.
Previous work from Ben Moshe et al. revealed completely opposite results compared to our own study, including high mortality postsurgery, impaired early liver regeneration, and delayed expression of interphase cyclins. However, that study used a different experimental setting (33% hepatectomy) and a different Casp8 knockout allele (ΔExon 1-2). We thus conclude that the effects of Casp8 depletion on liver regeneration could be allele-specific and may depend on the strength of the regeneration stimulus.
Casp8Δhepa mice showed normal liver regeneration after PH despite excessive DNA synthesis. This was unexpected, as aberrant DNA replication could result in enhanced cell division and in augmented liver growth. However, even 6 months after PH we did not detect any signs of hepatomegaly or precarcinogenic lesions in Casp8Δhepa mice (data not shown). These results suggest that termination of liver regeneration is predominantly controlled by nonapoptotic, Casp8-independent mechanisms. We further conclude that Casp8-deficient hepatocytes undergo delayed G1/M transition and slow progression through mitosis, as evidenced by impaired induction, phosphorylation, and nuclear translocation of cyclin B (indicative of late M-phase transition) and poor phosphorylation of histone H3 demonstrating low prophase activity. Thus, accelerated DNA synthesis is most likely compensated by delayed mitosis progression eventually resulting in normal liver mass restoration.
Importantly, accelerated onset of DNA synthesis in Casp8Δhepa mice was also associated with earlier induction of cyclin D gene expression. Several experimental data demonstrated that the cyclin D gene promoter is regulated by NF-κB and by way of cJun and cFos in a JNK-dependent manner.[22-25] Thus, our data suggest that the early start of DNA synthesis in Casp8Δhepa liver is best explained by premature NF-κB or JNK/cJun activation.
However, our experiments using Casp8ΔhepaNEMOΔhepa double-deficient mice clearly demonstrated that accelerated onset of DNA replication in Casp8Δhepa livers is dependent on the NEMO/NF-κB axis and not due to aberrant JNK/cJun activation. Additional ablation of NEMO in Casp8Δhepa mice completely rescued the kinetics of liver regeneration, although it resulted in constitutive cJun activation.
In addition, Casp8ΔhepaNEMOΔhepa mice revealed improved survival after PH (75% total survival, 90% survival in type I) in comparison to single NEMOΔhepa mice, which showed 50% mortality due to excessive liver apoptosis and strong oxidative stress. Interestingly, liver resection even improved the spontaneous necrotic liver injury in Casp8ΔhepaNEMOΔhepa mice. Therefore, loss of Casp8—and thus accumulation of RIP1—seems to predispose to liver necrosis in a purely inflammatory setting, while it appears highly protective in the setting of surgical liver injury. Additionally, our data demonstrate that NEMO and Casp8 expression are of major relevance to tightly balance the precise timing of liver regeneration by synergistically controlling NF-κB and cJun activation and thus cyclin D expression.
Ultimately, our data indicate that all observations in Casp8Δhepa mice can be attributed to increased sensitivity towards exocrine TNF and accelerated induction of RIP1 in Casp8-deficient hepatocytes. RIP1 is proteolytically degraded by Casp8 and we provided direct evidence that loss of Casp8 prevented RIP1 cleavage in primary hepatocytes. Instead, even low doses of external TNF enabled accelerated RIP1 induction in Casp8-deficient cells. We recently demonstrated that elevated expression of RIP1 in Casp8Δhepa mice can result in RIP1/RIP3 complex formation and nonapoptotic liver injury resembling features of necroptosis in the Concanavalin A model of acute hepatitis. However, our present data strongly suggest that after PH premature RIP1 induction is rather protective. Following PH, we could not detect pronecrotic RIP1-RIP3 colocalization in Casp8Δhepa liver tissue, but identified excessive RIP1 in hepatocyte nuclei. In line with our findings, a recent study demonstrated that RIP1 is directly involved in TNF gene transcription under certain conditions. Thus, it is tempting to speculate that improved RIP1 stability in Casp8-deficient cells triggers autocrine TNF gene expression in hepatocytes, which would also explain elevated TNF gene expression in Casp8Δhepa mice. However, our data from primary hepatocytes using different dosages of TNF indicate that increased sensitivity of Casp8-deficient hepatocytes towards low-dose TNF is of greater relevance to explain our findings, as this was sufficient to trigger enhanced activation of all downstream signals including RIP1, NF-κB, JNK1, and JNK2, which pushes these cells towards cell cycle entry.
Upon TNF stimulation, RIP1 is recruited to the TNF receptor complex and contributes to activation of NF-κB by way of binding to NEMO, which is the regulatory subunit of the IKK complex. Previous data demonstrated that phosphorylation of p65 at Ser536, which was constitutively found in Casp8-deficient hepatocytes, is performed by IKK kinase, further highlighting the importance of the RIP1-NEMO-NF-κB axis for accelerated onset of liver regeneration in Casp8Δhepa mice. In addition, overexpression of RIP1 also induces JNK activation. However, by analyzing Casp8ΔhepaNEMOΔhepa mice we provided indirect evidence that enhanced JNK/cJun activation is not involved in premature cyclin D induction after PH. Thus, hepatoprotection and accelerated liver regeneration in Casp8Δhepa mice is best explained by aberrant high RIP1 expression and improved NF-κB activation. Our conclusions are illustrated in Supporting Fig. 4.
In summary, our study demonstrates that loss of Casp8 is protective in the priming phase of liver regeneration in a nonapoptotic manner as it triggers the RIP1/NF-κB axis. These findings could be clinically and potentially therapeutically relevant in patients undergoing extended surgical liver resection.