The adaptations and changes in the livers of ILK-Liver-KO- mice have been previously described (8
). Partial hepatectomies were performed in 30 week old mice with hepatocyte-targeted ILK removal, using Cre expressing mice of the same age as control. Changes in liver weights and proliferation kinetics are shown in , and . In , the data show the liver weights (in grams) prior to and after partial hepatectomy performed in control and ILK-KO-Liver mice. The same data are plotted in as percent of liver weight regained after the operation. Both and show that whereas in control mice liver weight returns to the original size prior to hepatectomy, in ILK-KO-Liver mice liver weight surpasses (at Day 7) that of the pre-hepatectomy size and it continues to grow beyond that. At day 14 after PHx, a time in which according to all literature liver regeneration has terminated, the liver weight of the ILK-KO-Liver mice surpasses that of the pre-hepatectomy level by 58%-58%. The control mice exhibited the typical pattern of termination of liver regeneration with liver weights returning to their pre-PHx weights (1.36±0.2 grams) by 14 days after PHx.
Liver weights of Control and ILK-KO-Liver mice at partial hepatectomy (PHx) and different times thereafter.
Figure 1 The data show the percentile of liver weight regained after partial hepatectomy in the control and ILK-KO-Liver mice. Following partial hepatectomy, the liver weights of Control and ILK-KO-Liver mice drop down to 54% and 53% of the original, respectively. (more ...)
Fig. 2 A. Percent of PCNA positive cells in the livers of ILK-KO-Liver and Control mice at various time points after PHx. ILK-KO-Liver mice have sustained excess cell proliferation at 5, 7 and 14 days post-PHx. The data show the mean and standard error from (more ...)
The detail proliferation kinetics of hepatocytes at different times after PHx are shown in . In both types of mice, there is a typical initial increase (2 and 3 days post PHx) and later decrease (5, 7 and 14 days post-PHx) in cell proliferation, as assessed by proliferating cell nuclear antigen (PCNA) immunohistochemistry. Compared to Control mice, the ILK-KO-Liver mice exhibited a slightly slower increase in initial cell proliferation at 2 days post-PHx, but had sustained cell proliferation at 3, 5, 7 and 14 days post-PHx (). The main difference in liver histology between the two types of mice was the persistent hepatocyte proliferation of hepatocytes in the ILK-KO-Liver mice at 5, 7 and 14 days after PHx ().
A detailed microarray analysis (Affymetrix) was performed to investigate mechanisms involved in the termination defect in the ILK-KO-Liver mice. No major difference in gene expression of potential extracellular signals (2
) was seen with the exception of hepatocyte growth factor (HGF). HGF gene expression was 5-fold higher before PHx in the ILK-KO-Liver mice and remained higher at 7 and 14 days post-PHx (). There was increased protein for both HGF and its receptor Met for Days 7 and 10 in the ILK-KO-Liver mice, whereas a decline for the same time points was seen in the Control mice (). We have shown in previous studies (13
) that beta catenin migrates to hepatocyte nuclei from the earliest stages after PHx. The data in show that there is increased presence of beta catenin in nuclei in the ILK-KO-Liver mice. We also investigated AKT/Protein Kinase B, a signaling kinase linked to both ILK and HGF (15
). ILK-KO-Liver mice had substantially higher total as well as phosphorylated AKT protein especially at Days 7-14 (). We further investigated ERK1/2 (p42/p44 MAPK) and p38 MAPK, the two main downstream mitogen-activated kinase pathways activated by HGF. Previous studies have reported the role of p42/p44 MAPK in activation and p38 MAPK in inhibition of cell proliferation (16
). There was no difference in the total protein of p42/p44 MAPK (data not shown) or p38MAPK. Differences were noticed however in phospho-p38 MAPK (). We observed an increase in phosphorylation (activation) of p38 MAPK in the ILK-KO-Liver liver at 2, 3 and 5 days after PHx, which decreased at 7 and 14 days post PHx. This is consistent with the lower cell proliferation observed in the ILK-KO-Liver mice at 2 days post-PHx and later sustained proliferation at 7 and 14 days after PHx. Previously, it has been demonstrated that P38 MAPK can inhibit cell cycle progression during liver regeneration (18
Figure 3 Changes in HGF and its receptor cMet and levels of total and nuclear beta catenin after partial hepatectomy. In A, data indicated expression values of HGF RNA in pooled samples of three animals per point. (CO: Control mice). B and C show representative (more ...)
Figure 4 Changes in cell cycle related proteins in ILK-KO-Liver mice after PHx, compared to Control. A and B: Western blots of total and phospho- AKT and total and phospho-p38 MAPK at different times after PHx. (C) Western blot analysis of c-myc and p-27 showing (more ...)
To further analyze signaling pathways during the time of prolonged proliferation in the KO mice (days 7-14 after PHx), we investigated levels of cell cycle genes. We observed overall increased and sustained expression of c-Myc in the ILK-KO-Liver mice over the 14 day post- PHx time course. Additionally, expression of the CDK2 inhibitor p27, a cell cycle inhibitor, was consistently lower in ILK-KO-Liver mice at 5, 7 and 14 days post-PHx. (). TGFβ is another molecule implicated in termination of liver regeneration due to its anti-proliferative activity (19
). However, no significant changes in TGFβ1 protein levels were seen between the two groups ().
Recently the role of Hippo Kinase pathway in regulation of organ size in Drosophila as well as of liver in mammals has been reported (20
). The mammalian Hippo Kinase pathway converges on yes-associated protein (YAP), which plays a role in liver size regulation and cancer development (20
). We investigated whether ILK loss causes changes in YAP expression during liver regeneration. Western blot analysis for YAP indicated substantially higher YAP protein in ILK-KO-Liver livers at 5, 7 and 14 days after PHx (). Phosphorylation of YAP results in its nuclear export and degradation, which leads to decrease in cell proliferation. The ILK-KO-Liver mice had overall lower phosphorylated YAP protein at all time points following PHx as compared to the Control mice. Furthermore, immunohistochemistry for YAP indicated higher cytoplasmic and nuclear YAP in ILK-KO-Liver mice with loss of the restriction of positive immunohistochemistry to periportal regions observed in the Control mice (). These data indicate that ILK signaling contributes to control of the Hippo kinase pathway, and particularly YAP activation in the liver.
To understand the whether the termination defect in ILK-KO-Liver mice is coupled with defective hepatic differentiation, a comprehensive microarray analysis was conducted using liver mRNA collected at 0, 2, 3, 5, 7 and 14 days after PHx. The microarray study revealed that in addition to excessive liver weigh, ILK-KO-Liver mice also have problems in regulating the differentiation status of the liver at the end of regeneration. We compared the expression of the 120 most expressed genes at time 0 (day of hepatectomy) in each of the two categories of mice and followed their expression levels by Affymetrix gene arrays at different days after PHx (). In Control mice, levels of expression of the top most expressed genes returned mostly to pre-PHx values at Day 14 after PHx. In ILK-KO-Liver mice on the other hand, there was a wide increase in the expression of many of the same top 120 genes and several of them remained elevated at Day 14. Selenoprotein P was the most elevated gene for the ILK-KO-Liver mice at Day 14 after PHx. Major Urinary Protein 1 became elevated in Control mice at Day 5 but returned to normal levels by Day 14. It did remain elevated in the ILK-KO-Liver mice. These data indicate that targeted ablation of ILK and the associated secondary changes result in defective control of hepatocyte differentiation and block return of gene expression to pre-PHx levels, in addition to inappropriate termination of growth and excess liver weight accumulation.
Figure 5 The top 120 expressed genes in time 0 (pre-PHx) in ILK-KO mice were identified by ranking of expression values in the Affymetrix gene array results. (for details, please supplement of reference (8)). The figure shows the expression of these genes at different (more ...)