Liver-specific ablation of integrin-linked kinase in mice results in abnormal histology, enhanced cell proliferation, and hepatomegaly

Abstract

Hepatocyte differentiation and proliferation are greatly affected by extracellular matrix (ECM). Primary hepatocytes cultured without matrix dedifferentiate over time, but matrix overlay quickly restores differentiation. ECM also is critical in liver regeneration where ECM degradation and reconstitution are steps in the regenerative process. Integrin-linked kinase (ILK) is a cell-ECM-adhesion component implicated in cell-ECM signaling by means of integrins. We investigated the role of ILK in whole liver by using the LoxP/Cre model system. ILK was eliminated from the liver by mating homozygous ILK-floxed animals with mice expressing Cre-recombinase under control of the alpha fetoprotein enhancer and albumin promoter. After ablation of ILK, animals are born normal. Soon after birth, however, they develop histologic abnormalities characterized by disorderly hepatic plates, increased proliferation of hepatocytes and biliary cells, and increased deposition of extracellular matrix. Cell proliferation is accompanied by increased cytoplasmic and nuclear stabilization of beta-catenin. After this transient proliferation of all epithelial components, proliferation subsides and final liver to body weight ratio in livers with ILK deficient hepatocytes is two times that of wild type. Microarray analysis of gene expression during the stage of cell proliferation shows up-regulation of integrin and matrix-related genes and a concurrent down-regulation of differentiation-related genes. After the proliferative stage, however, the previous trends are reversed resulting in a super-differentiated phenotype in the ILK-deficient livers.

Conclusion: Our results show for the first time in vivo the significance of ILK and hepatic ECM-signaling for regulation of hepatocyte proliferation and differentiation.

Fig. 1

ILK expression level is decreased in the liver of the ILK-KO livers and the reduction is due to elimination of the ILK from hepatocytes. (A) Western blotting analysis with an anti-ILK monoclonal antibody carried out on total cell lysates extracted from whole livers from the ILK-KO mice. β-Actin is used as loading control. The last lane corresponds to a wild-type animal (Control). (B) Bar graph showing the ILK expression level in whole liver, isolated nonparenchymal cells (solid white bars), and isolated hepatocytes (solid black bars) from the control and AFP/ALB KO animals.

Fig. 2

Histologic changes in ILK-KO mice. Representative photomicrographs of hematoxylin-eosin-stained liver sections from ILK-KO mice at (A) 2 weeks, (C) 6 weeks, (E) 10 weeks, and (G) 30 weeks of age. (B,D,F,H) Histology of Control mice at the same age points. All photographs were taken at 100× magnification. There are no significant histologic changes at 2 weeks between KO and Control mice. After 6 weeks of age, hepatic histology in the ILK-KO mice also showed tracts composed of proliferating biliary cells and stellate cells shown by arrows (Fig. 3). Hepatocytes with unusually large nuclei were observed (D,E, arrowheads). These changes decreased at 30 weeks of age.

Fig. 3

Deposition of extracellular matrix, proliferation of biliary epithelial cells and stellate cells, hepatocyte proliferation, and apoptosis in ILK-KO mice. Extensive deposition of extracellular matrix was observed in ILK-KO mice as shown by reticulin staining at (A) 10 weeks compared to (B) control mice shows accumulation of extracellular matrix in the livers of Liver-ILK-KO mice. (C) Proliferation of biliary ductules was observed in ILK deficient mice. Expression of the biliary specific transcription factor HNF1 β is shown in the nuclei of portal ductules (black arrow) as well as in the biliary cells proliferating and forming extra portal tracts (yellow arrow). (D) Desmin positive stellate cells (black arrows) are also proliferating in tandem with the biliary cells. (C,D) Consecutive sections. (A-D) Magnification: 100×. (E) Hepatocytes in mitosis (star mark), apoptosis (black arrow), and some with atypical nuclei (yellow arrow) (magnification: 400×).

Fig. 4

Quantitative assessment of hepatocyte proliferation and apoptosis and liver enlargement in ILK-KO mice. Hepatocyte proliferation measured by (A) PCNA analysis and (B) by mitotic index and apoptosis measured by (C) TUNEL assay was estimated in wild-type and ILK-KO mice at various ages. Each data point is the mean and SE of three independent measurements. Asterisks indicate data statistically significant at a P value < 0.05 as determined by Student’s nonpaired t test. (D) Liver weight to body weight ratios of wild-type and ILK-KO mice at 30 weeks of age. Each data point is the mean ± SE from more than three measurements per point. (E,F) PCNA-positive nuclei (shown by arrow) in Liver-ILK-KO (E) and control mice. (G,H) TUNEL-positive apoptotic cells (arrow) shown in (G) (Liver-ILK-KO mice). No such cells were seen in (H) (control mice). (E-H) Magnification: 200×. (I) Representative livers of control and Liver-ILK-KO mice at 30 weeks of age indicating the difference in liver size between the two groups. Arrows indicate the gallbladder.

Fig. 5

Expression of different integrin chains in ILK-KO or WT mice at different age points. Each lane was generated from material extracted from pooled liver homogenates of three mice.

Fig. 6

(A) Differences in gene expression between ILK-KO and Control mice at different ages. The 85 most-expressed genes in Liver-ILK-KO mice over Control mice at 30 weeks of age are listed in Table 1. The differences in expression of these genes between Liver-ILK-KO and Control mice in ages from 2 weeks to 30 weeks is shown in the y axis of this figure. The x axis indicates the age of the mice. The red line indicates zero difference in expression for the specific gene between the two groups. Genes markedly over-expressed in the KO animals at 30 weeks of age (most of them hepatocyte-specific genes) were underexpressed in the same animals at 2 weeks of age, with progressive increase thereafter. (B) Expression of C/EBP α and β in Liver-ILK-KO and Control mice at different times after birth. The y axis indicates expression values as determined by the oligo-DNA microarrays (see Materials and Methods). The x-axis indicates age of the mice.