A direct in vivo RNAi screen identifies MKK4 as a key regulator of liver regeneration. Cell 2013;153:389-401 (Reproduced with permission.), , , , , , et al.
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The liver harbors a distinct capacity for endogenous regeneration; however, liver regeneration is often impaired in disease and therefore insufficient to compensate for the loss of hepatocytes and organ function. Here we describe a functional genetic approach for the identification of gene targets that can be exploited to increase the regenerative capacity of hepatocytes. Pools of small hairpin RNAs (shRNAs) were directly and stably delivered into mouse livers to screen for genes modulating liver regeneration. Our studies identify the dual-specific kinase MKK4 as a master regulator of liver regeneration. MKK4 silencing robustly increased the regenerative capacity of hepatocytes in mouse models of liver regeneration and acute and chronic liver failure. Mechanistically, induction of MKK7 and a JNK1-dependent activation of the AP1 transcription factor ATF2 and the Ets factor ELK1 are crucial for increased regeneration of hepatocytes with MKK4 silencing.
The unique ability of the liver to regenerate was appreciated by the ancient Greeks, who believed that Prometheus was punished for stealing fire from the gods by having his liver eaten by an eagle each night, only to have it regrow by the next day. Today, we still appreciate the liver's amazing regenerative potential, and know this potential is compromised when the liver is diseased. The dismal prognosis for patients with liver failure is, in part, due to impairment of the regenerative capacity of damaged hepatocytes combined with the dearth of donor livers. Additionally, patients undergoing hepatic resection of malignancies, living related donors, and recipients of small-for-size livers would all benefit if healthy hepatocytes could be coaxed to regenerate better. A recent study published in Cell by Wuestefeld et al. made the surprising finding that suppressing the MAP kinase family member, Mkk4, in hepatocytes improves their regenerative potential. This provides a new and much-needed avenue for pursuing therapeutic approaches to improve liver regeneration in clinical settings.
Acute and chronic liver injury has been studied in mice using genetic models (such as the fumarylacetoacetate hydrolase [Fah] knockout) and toxin-mediated hepatic injury (by carbon tetrachloride [CCl4] exposure or FAS activation). In these cases, most hepatocytes are rendered dysfunctional and it is a challenge for these damaged hepatocytes to regenerate. In contrast, when an entire liver lobe is resected, as in living related liver donors or in rodents following 70% partial hepatectomy, the hepatocytes that remain are normal and functional, and restore the liver mass by undergoing proliferation and hypertrophy. Using partial hepatectomy, researchers have uncovered a number of genes required for liver regeneration, such as c-Met or c-Jun. Here, the authors use an unbiased in vivo screen to identify a pathway that promotes the ability of hepatocytes to repopulate a damaged liver. They are among the first to show that suppression of a gene, Mkk4, enhances hepatocyte regeneration. However, an interesting twist is that loss of Mkk4 does not suppress its downstream targets, but instead activates them by inducing its partner kinase, Mkk7 (Fig. 1). Thus, the Mkk4/Mkk7 pathway promotes cell cycle activation and is required for the proliferative response to a mitogenic stimulus that arises either from cell injury or from loss of liver mass.
The simple hypothesis underlying this study was that the same genes that are deleted in liver cancers may be those that suppress hepatocyte proliferation under noncancerous conditions. Genes they previously showed to be deleted in human hepatocellular carcinomas were knocked down in hepatocytes using a pool of 631 short hairpin RNAs (shRNAs). Next, they tested whether any of these shRNAs improved the ability of hepatocytes to repopulate a damaged liver in the Fah knockout or CCl4 treated mice. Mkk4 emerged as the most effective target in both. These findings were substantiated by experiments where FAS-mediated hepatocyte apoptosis was reduced and mouse survival following FAS-mediated liver injury was enhanced when Mkk4 was silenced.
Replacing damaged livers with healthy hepatocytes is an unrealized goal of regenerative therapy. As a step towards this end, the authors report that transplanting primary hepatocytes treated with shMkk4 into Fah-deficient mice improves survival by over 50%. This suggests that manipulating Mkk4 signaling primary hepatocytes from humans either in vivo or prior to transplantation may sustain patients with acute liver failure.
Interestingly, shRNAs targeting two other genes (Edem1 and Mapk4) were also identified as beneficial in the FAH model and one (Fam43a) provided a benefit in response to CCl4. These targets were not pursued in the current study but will be interesting to evaluate in the future.
Chronic liver injury can lead to fibrosis and cirrhosis by way of activation of hepatic stellate cells; and by knocking down Mkk4 in hepatocytes, fibrosis after chronic CCl4 treatment was reduced. This points to a nonautonomous effect of hepatocytes on stellate cells. The authors conclude that the increased proliferation and decreased apoptosis of hepatocytes restrains the fibrogenic signal which induces stellate cell activation. These data illustrate that loss of Mkk4 promotes liver regeneration, recovery from injury and resection, and reduces fibrosis.
MAPK signaling pathways are involved in both promoting proliferation, largely mediated by the ERKs, and in blocking proliferation and promoting apoptosis, mediated by JNK and p38 family members. Previous studies demonstrate that loss of Mkk4 prevents hepatocyte differentiation and proliferation during liver development,[6-8] and a large body of literature demonstrates that Jnk activation blocks regeneration and promotes liver injury. As shown in Fig. 1, Mkk4 functions with Mkk7 to phosphorylate Jnk. To identify downstream Mkk4 targets responsible for its antiregenerative functions, the authors performed a kinase array on Mkk4-deficient livers. This surprisingly uncovered increased phosphorylation of Jnk and its downstream targets, the Atf2 and Elk1 transcription factors. This is counterintuitive, as loss of Mkk4 should reduce Jnk phosphorylation. Their finding that increased Mkk7 phosphorylation occured induced in Mkk4-deficient livers provides a potential explanation for this surprising result.
Next, the functional relevance of these targets was tested by delivering a vector with one shRNA targeting Mkk4 and another targeting Mkk7, Jnk, Atf2, or Elk1 to the liver. Remarkably, removing any one of these other genes blocks the regenerative advantage conferred by Mkk4 knockdown in the FAH model. Furthermore, Elk1 knockout mice fail to restore liver mass following partial hepatectomy. Together, these data suggest the model in Fig. 1 whereby Mkk4 loss promotes activation of Mkk7, Jnk, Atf2, and Elk1, promoting hepatocyte cell cycle reentry and liver regeneration.
A plausible physiological role for this pathway is to prevent inappropriate or unscheduled proliferation to reduce carcinogenesis or to limit the replication of damaged cells. As such, the potential for hepatocyte hyperproliferation and transformation is the flip-side to enhancing regeneration. Indeed, Mkk4 has been implicated as a tumor suppressor in some cancers,[11, 12] but not in hepatocellular carcinoma. While the current study does not find an increase in liver tumors in mice within a year of stable Mkk4 knockdown, the possibility that long-term Mkk4 suppression may lead to tumorigenesis cannot be excluded. This is an important caveat, as patients suffering from endstage liver disease are prone to liver cancer and thus they may be more susceptible to the tumorigenic effects of Mkk4 suppression. Another caveat is that this study applied liver-damaging agents to mice only after Mkk4 knockdown, whereas patients would only presumably be treated after injury has occurred. Thus, it is important to determine whether loss of Mkk4 is equally effective if it occurs in an already damaged liver.
In summary, these studies use a novel, unbiased approach to undertake gene discovery for new regulators that could be exploited clinically to improve liver repopulation and survival in patients with hepatic injury. An interesting but unexplored finding was that Irs1, the insulin signaling pathway component and Jnk target, was induced in Mkk4-deficient livers. This might suggest the exciting possibility that modulating Mkk4 and Jnk signaling could mediate insulin sensitivity in other settings, such as type II diabetes. Pursuing the proregenerative function of the Mkk4-Mkk7-Jnk pathway, which has primarily been investigated for its role in promoting liver injury, may facilitate clinical studies with Mkk4 inhibitors in patients who could benefit from enhanced liver regeneration.
Vinitha Jacob, B.S.1,3
Kirsten C. Sadler, Ph.D.,1,2,3
1Department of Medicine Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, NY
2Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, NY
3Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY