Receptor tyrosine kinases MET and EGFR are critically involved in initiation of liver regeneration. Other cytokines and signaling molecules also participate in the early part of the process. Regeneration employs effective redundancy schemes to compensate for the missing signals. Elimination of any single extracellular signaling pathway only delays but does not abolish the process. Our present study, however, shows that combined systemic elimination of MET and EGFR signaling (METKO+EGFRi mice) abolishes liver regeneration, prevents restoration of liver mass and leads to liver decompensation. METKO or simply EGFRi mice had distinct and signaling-specific alterations in Ser/Thr phosphorylation of mTOR, AKT, ERK1/2, PTEN, AMPKα etc. In the combined MET and EGFR signaling elimination of METKO+EGFRi mice, however, alterations dependent on either MET or EGFR combined to create shutdown of many programs vital to hepatocytes. These included decrease in expression of enzymes related to fatty acid metabolism, urea cycle, cell replication, and mitochondrial functions and increase in expression of glycolysis enzymes. There was however increase in expression of genes of plasma proteins. Hepatocyte average volume decreased to 35% of control with proportional decrease in dimensions of the hepatic lobules. Mice died at 15–18 days after hepatectomy with ascites, increased plasma ammonia and very small livers. Conclusion The study shows that MET and EGFR separately control many non-overlapping signaling endpoints, allowing for compensation when only one of the signals is blocked. The combined elimination of the signals however is not tolerated. The results provide critical new information on interactive MET and EGFR signaling and the contribution of their combined absence to regeneration arrest and liver decompensation.

Taken together, these data indicate that hepatocyte to BE transdifferentiation is regulated by HGF and EGF receptors and that PI3 kinase-mediated signaling independent of AKT is a crucial component of the transdifferentiation process.

Exogenous interleukin 6 (IL-6), synthesized at the initiation of the acute phase response, is considered responsible for signaling hepatocytes to produce acute phase proteins. It is widely posited that IL-6 is either delivered to the liver in an endocrine fashion from immune cells at the site of injury, or alternatively, in a paracrine manner by hepatic immune cells within the liver. A recent publication showed there was a muted IL-6 response in lipopolysaccharide (LPS)-injured mice when nuclear NFκB was specifically inactivated in the hepatocytes. This indicates hepatocellular signaling is also involved in regulating the acute phase production of IL-6. Herein, we present extensive in vitro and in vivo evidence that normal hepatocytes are directly induced to synthesize IL-6 mRNAs and protein by challenge with LPS, a bacterial hepatotoxin, and by HGF, an important regulator of hepatic homeostasis. As the IL-6 receptor is found on the hepatocyte, these results reveal that induction of the acute phase response can be regulated in an autocrine as well as endocrine/paracrine fashion. Further, herein we provide data indicating that following partial hepatectomy (PHx), HGF differentially regulates IL-6 production in hepatocytes (induces) versus immune cells (suppresses), signifying disparate regulation of the cell sources involved in IL-6 production is a biologically relevant mechanism that has previously been overlooked. These findings have wide ranging ramifications regarding how we currently interpret a variety of in vivo and in vitro biological models involving elements of IL-6 signaling and the hepatic acute phase response.

Liver regeneration after a two-thirds partial hepatectomy (PHx) is a complex process requiring interaction and cooperation of many growth factors and cytokines and cross talk between multiple pathways. Along with hepatocyte growth factor and its receptor MET (HGF-MET), the epidermal growth factor receptor (EGFR) signaling pathway is activated within 60 minutes after PHx. To investigate the role of EGFR in liver regeneration, we used two EGFR-specific short hairpin silencing RNAs to inhibit EGFR expression in regenerating normal rat liver. Suppression of EGFR mRNA and protein was evident in treated rats. There was also a demonstrable decrease but not complete elimination of bromo-deoxyuridine incorporation and mitoses at 24 hours after PHx. In addition, we observed up-regulation of MET and Src as well as activation of the ErbB-3-ErbB-2-PI3K-Akt pathway and down-regulation of STAT 3, cyclin D1, cyclin E1, p21, and C/EBP ␤. The decrease in the ratio of C/EBP ␣ to C/EBP ␤ known to occur after PHx was offset in shEGFR-treated rats. Despite suppression of hepatocyte proliferation lasting into day 3 after PHx, liver weight restoration occurred. Interestingly, hepatocytes in shEGFR-treated rats were considerably larger when compared with ScrRNA-treated controls. The data indicate that although the MET and EGFR pathways are similar, the contributions made by MET and EGFR are unique and are not compensated by each other or other cytokines. Partial hepatectomy (PHx), in which two thirds of the rat liver is surgically removed, has been extensively used to study the highly complex phenomenon of liver regeneration. Although hepatocytes in normal adult liver are quiescent and rarely divide, they do retain an astounding ability to reenter the cell cycle and regenerate on surgical insult or injury. PHx in rats/mice results in rapid induction of more than 100 genes that are not expressed in the normal resting liver.1 A rapid up-regulation of genes encoding transcriptional factors like AP1 breakdown of extracellular matrix by uPA and release of pre-existing stores of HGF is observed within 60 minutes of a PHx. 2The hepatocytes leave the quiescent G0 phase and enter the cell cycle. Methods to identify extrahepatic signals leading to liver regeneration have included mitogenic effects on hepatocyte cultures, stimulation of DNA synthesis in the liver of normal (unoperated) animals, and decrease in regeneration-related events in animals genetically or pharmacologically depleted of the agent under study. Of the various agents implicated in liver regeneration, HGF and ligands of the epidermal growth factor receptor (EGFR) are the only ones that stimulate DNA synthesis in hepatocyte cultures maintained in chemically defined media.3 They are also the only ones that stimulate DNA synthesis in the liver of normal mice and rats. 4 -6 HGF and EGF signaling pathways are activated within 60 minutes after a PHx, 7,8 as evidenced by tyrosine phosphorylation of MET and EGFR within 30 to 60 minutes after PHx. There is evidence of cross talk and c...

Hepatocyte growth factor (HGF) has been shown to be indispensable for liver regeneration because it serves as a main mitogenic stimulus driving hepatocytes toward proliferation. We hypothesized that ablating HGF in adult mice would have a negative effect on the ability of hepatocytes to regenerate. Deletion of the HGF gene was achieved by inducing systemic recombination in mice lacking exon 5 of HGF and carrying the Mx1-cre or Cre-ERT transgene. Analysis of liver genomic DNA from animals 10 days after treatment showed that a majority (70–80%) of alleles underwent cre-induced genetic recombination. Intriguingly, however, analysis by RT-PCR showed the continued presence of both unrecombined and recombined forms of HGF mRNA after treatment. Separation of liver cell populations into hepatocytes and non-parenchymal cells showed equal recombination of genomic HGF in both cell types. The presence of the unrecombined form of HGF mRNA persisted in the liver in significant amounts even after partial hepatectomy (PH), which correlated with insignificant changes in HGF protein and hepatocyte proliferation. The amount of HGF produced by stellate cells in culture was indirectly proportional to the concentration of HGF, suggesting that a decrease in HGF may induce de novo synthesis of HGF from cells with residual unrecombined alleles. Carbon tetrachloride (CCl4)-induced regeneration resulted in a substantial decrease in preexisting HGF mRNA and protein, and subsequent PH led to a delayed regenerative response. Thus, HGF mRNA persists in the liver even after genetic recombination affecting most cells; however, PH subsequent to CCl4 treatment is associated with a decrease in both HGF mRNA and protein and results in compromised liver regeneration, validating an important role of this mitogen in hepatic growth.

Epidermal growth factor receptor (EGFR) is a critical regulator of hepatocyte proliferation and liver regeneration. Our recent work indicated that EGFR can also regulate lipid metabolism during liver regeneration after partial hepatectomy. Based on these findings, we investigated the role of EGFR in a mouse model of nonalcoholic fatty liver disease (NAFLD) using a pharmacological inhibition strategy. C57BL6/J mice were fed a chow diet or a fast-food diet (FFD) with or without EGFR inhibitor (canertinib) for 2 months. EGFR inhibition completely prevented development of steatosis and liver injury in this model. In order to study if EGFR inhibition can reverse NAFLD progression, mice were fed the FFD for 5 months, with or without canertinib treatment for the last 5 weeks of the study. EGFR inhibition remarkably decreased steatosis, liver injury, and fibrosis and improved glucose tolerance. Microarray analysis revealed that ~40% of genes altered by the FFD were differentially expressed after EGFR inhibition and, thus, are potentially regulated by EGFR. Several genes and enzymes related to lipid metabolism (particularly fatty acid synthesis and lipolysis), which were disrupted by the FFD, were found to be modulated by EGFR. Several crucial transcription factors that play a central role in regulating these lipid metabolism genes during NAFLD, including peroxisome proliferator-activated receptor gamma (PPARγ), sterol regulatory element-binding transcription factor 1 (SREBF1), carbohydrate-responsive element-binding protein, and hepatocyte nuclear factor 4 alpha, were also found to be modulated by EGFR. In fact, chromatin immunoprecipitation analysis revealed that PPARγ binding to several crucial lipid metabolism genes (fatty acid synthase, stearoylcoenzyme A desaturase 1, and perilipin 2) was drastically reduced by EGFR inhibition. Further upstream, EGFR inhibition suppressed AKT signaling, which is known to control these transcription factors, including PPARγ and SREBF1, in NAFLD models. Lastly, the effect of EGFR in FFD-induced fatty-liver phenotype was not shared by receptor tyrosine kinase MET, investigated using MET knockout mice. Conclusion: Our study revealed a role of EGFR in NAFLD and the potential of EGFR inhibition as a treatment strategy for NAFLD. Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.30696/suppinfo. For the 2-month study, male C57BL6/J mice, 6-8 weeks old, were fed ad libitum (1) chow diet, (2) FFD (Western diet, high saturated fats [21% by weight; 42% Supported by the Cleveland Foundation and the Menten Endowment Foundation of the University of Pittsburgh.

Particularly interesting new cysteine-histidine-rich protein (PINCH) protein is part of the ternary complex known as the IPP (integrin linked kinase (ILK)-PINCH-Parvin-α) complex. PINCH itself binds to ILK and to another protein known as Rsu-1 (Ras suppressor 1). We generated PINCH 1 and PINCH 2 Double knockout mice (referred as PINCH DKO mice). PINCH2 elimination was systemic whereas PINCH1 elimination was targeted to hepatocytes. The genetically modified mice were born normal. The mice were sacrificed at different ages after birth. Soon after birth, they developed abnormal hepatic histology characterized by disorderly hepatic plates, increased proliferation of hepatocytes and biliary cells and increased deposition of extracellular matrix. After a sustained and prolonged proliferation of all epithelial components, proliferation subsided and final liver weight by the end of 30 weeks in livers with PINCH DKO deficient hepatocytes was 40% larger than the control mice. The livers of the PINCH DKO mice were also very stiff due to increased ECM deposition throughout the liver, with no observed nodularity. Mice developed liver cancer by one year. These mice regenerated normally when subjected to 70% partial hepatectomy and did not show any termination defect. Ras suppressor 1 (Rsu-1) protein, the binding partner of PINCH is frequently deleted in human liver cancers. Rsu-1 expression is dramatically decreased in PINCH DKO mouse livers. Increased expression of Rsu-1 suppressed cell proliferation and migration in HCC cell lines. These changes were brought about not by affecting activation of Ras (as its name suggests) but by suppression of Ras downstream signaling via RhoGTPase proteins. In conclusion, our studies suggest that removal of PINCH results in enlargement of liver and tumorigenesis. Decreased levels of Rsu-1, a partner for PINCH and a protein often deleted in human liver cancer, may play an important role in the development of the observed phenotype.

Summary Liver regeneration under normal circumstances proceeds through proliferation of all cellular elements of the liver. Studies with rodent models have shown that when proliferation of hepatocytes is inhibited, progenitor cells arising from the biliary compartment transdifferentiate into “oval/progenitor” cells, which proceed to differentiate into hepatocytes. Recent studies have shown that the same pathways may operate in human liver failure. The growth factor receptors (HGF [hepatocyte growth factor] receptor) and epidermal growth factor receptor are key mitogenic receptors for both hepatocytes and progenitor cells. Our current study used the biliary and progenitor marker EpCAM (epithelial cell adhesion molecule) to detect “regenerative clusters” of mixed cholangiocyte-hepatocyte differentiation. We determined that expression of metabolic equivalent and epidermal growth factor receptor occurs in biliary cells, progenitor cells, and hepatocytes, whereas activation of metabolic equivalent and epidermal growth factor receptor is limited to regenerative cluster hepatocytes. These histologic events are associated with expression of apoptosis-inducing FAS and mitoinhibitory protein glypican 3. Cell proliferation was overall suppressed in regenerative clusters. Transdifferentiation of biliary and progenitor cells appears to be regulated by a complex interaction of signals promoting and arresting growth.