Combined systemic elimination of MET and epidermal growth factor receptor signaling completely abolishes liver regeneration and leads to liver decompensation

Abstract

Receptor tyrosine kinases MET and epidermal growth factor receptor (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 (MET knockout + EGFR-inhibited mice) abolishes liver regeneration, prevents restoration of liver mass, and leads to liver decompensation. MET knockout or simply EGFR-inhibited mice had distinct and signaling-specific alterations in Ser/Thr phosphorylation of mammalian target of rapamycin, AKT, extracellular signal-regulated kinases 1/2, phosphatase and tensin homolog, adenosine monophosphate-activated protein kinase α, etc. In the combined MET and EGFR signaling elimination of MET knockout + EGFR-inhibited 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, increased expression of genes of plasma proteins. Hepatocyte average volume decreased to 35% of control, with a proportional decrease in the dimensions of the hepatic lobules. Mice died at 15-18 days after hepatectomy with ascites, increased plasma ammonia, and very small livers.

Conclusion: MET and EGFR separately control many nonoverlapping signaling endpoints, allowing for compensation when only one of the signals is blocked, though the combined elimination of the signals 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. (Hepatology 2016;64:1711-1724).

Figure 1

Suppression of cell proliferation and arrest of liver regeneration in METKO+EGFRi mice. A. Percent of hepatocytes in DNA synthesis (Ki67 positive nuclei) following PHx of mice in the four different treatment categories. The values of the bars reflect the mean and standard error of minimally three separate mice in each treatment group. (The numbers over the bars indicate the absolute values of the mean). The Day 14 value of hepatocyte proliferation in MET-KO+EGFRi mice was 0% and is placed under an arbitrary horizontal bar. B. Comparison of “liver-to-body weight” (LBWR) ratio at days 7 and 14 after PHx with that of normal mice prior to and immediately after PHx. The values of the bars reflect the mean and standard error of minimally three separate mice in each treatment group. (The numbers over the bars indicate the absolute values of the mean). C:. Glutamine synthetase immunohistochemistry of post-PHx Day 14 of mouse liver in control (upper photo) and MET-KO+EGFRi mice (lower photo) (Magnification: 100X). (D): The mean average volume of hepatocytes in the different treatment groups at Day 14 after PHx. (Data derived from E below) E: A hematoxylin stain photomicrograph of control (left) and MET-KO+EGFRi mice (right) at Day 14 after PHx (Magnification 200X). F: Immunohistochemistry for CDKN1A (p21) in the nuclei of hepatocytes in Control (upper) and METKO+EGFRi mice (lower) at Day 4 after PHx. (Magnification: 400X). Most of the nuclei in the METKO+EGFRi liver section are positive for p21 (black arrowheads).

Figure 2. Western immunoblot analysis of activation status of MET, EGFR and downstream effects on activation of key signaling pathways. For details and interpretation, see Results

A. Western blot analysis of total and activated Met, EGFR ERBB2 and ERBB3. Beta actin was used as loading control. B. Western Blot analysis of mTOR-AKT-PTEN Pathway. C. Western Blot analysis of Cyclin D1, phospho-Erk1/2, AMPK alpha, Phospho-AMPKα (Thr172), Acetyl-CoA carboxylase and Phospho-Acetyl-CoA Carboxylase (Ser79).

Figure 3

A: Gene Cluster Analysis of microarray data. Heatmap of gene expression in livers of the different categories at day 14 after PHx and GO analysis of the genes in Cluster 3 (indicated by arrow) in which gene expression decreases only when both MET and EGFR are suppressed. B: The complexity of the functions and processes controlled Gene Cluster 3 is illustrated by the adjacent three pie charts illustrating Gene Ontogeny results for the functions performed by the genes which are enriched in that cluster. C: The sum of the expression of each gene present in Cluster 3 was obtained by adding the expression values of each of the four categories of treatment. That sum was normalized to 100%. The top 25 genes expressed in the control group of Cluster 3 were analyzed and shown in Fig. 3C. Each treatment category is shown with a specific color. Results show the percent expression of the top 25 most expressed genes in Control treatment at Day 14 after PHx, relative to the other three treatment categories.

Figure 4. Dysregulation of oxidative stress pathways in METKO+EGFRi mice

RT-qPCR analysis of genes associated with the oxidative stress response in METKO+EGFRi mice at Days 0, 2, 7, 14 post hepatectomy. Y-axes indicate fold-change compared to the geometric mean of the six reference genes of the array plate (see Methods). Significant downregulation was observed in genes that protect against oxidative stress, such as Cat, Gcs, Gclc, GCLM, GstP1. Genes that promote oxidative stress, such as Txnip were upregulated, suggestive of a compromised response against oxidative stress. (For function details and references, see Supplemental Table S3).

Figure 5

Altered gene expression of multiple fundamental enzymes and proteins associated with hepatocyte metabolism and differentiation. A. Gene expression of enzymes associated with different aspects of fatty acid synthesis. Expression data at Day 14 for each treatment group are compared to the expression of Day 0 Each expression point is shown by colored-tagged bars and the absolute expression values are shown above the corresponding bar for Control Day 0 (prior to hepatectomy) and Day 14 of regeneration for the METKO+EGFR inhibited mice. All values are suppressed at Day 14 of METKO+EGFR inhibited mice as compared to any other category. Decreased expression of the two mitochondria-localized urea cycle enzymes OTC (B) and CPS1 (C). Values are compared between Days 0, 2, 7 and 14 after PHx. A schematic (D) demonstrates the location of the different enzymes of the urea cycle and their associated products, illustrating the location of both enzymes in the mitochondria. E. Log ratio of RNA expression of the ratio of [(expression in METKO+EGFRi)/(Average expression of the other categories)] for genes associated with mitochondrial functions, Krebs cycle and glycolysis. There is a general shift for down-regulation of expression of mitochondrial and Krebs cycle genes and upregulation of genes associated with glycolysis at Day 14 in the METKO+EGFRi group. F. Enhanced expression of plasma proteins produced by hepatocytes at Day 14 in METKO+EGFRi mice (red line bars). For all data in Figure 4F, each bar demonstrates gene expression results obtained by pooling of equal weights of samples from at least four separate mice. The expression value of each bar shown was statistically significant within the array at a p-value of less than 0.05%. For more details and associated references, see text. There is general upregulation of expression of most of the plasma proteins secreted by hepatocytes of METKO+EGFRi group (shown in red), with the exception of 17 genes in the EGFRi group (shown in purple) which surpass the expression of the METKO+EGFRi group.

Figure 6. A schematic diagram of the study and main findings

In standard liver regeneration after 2/3 partial hepatectomy, liver mass is mostly restored within 14–20 days. In the combined absence of MET and EGFR signaling, however, liver regeneration is completely arrested with ensuing liver failure and animal death.