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

Authors

  • Vasiliki Gkretsi,

    1. Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA
    Current affiliation:
    1. Institute of Immunology, Biomedical Sciences Research Centre “Alexander Fleming”, Vari, Greece
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  • Udayan Apte,

    1. Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA
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  • Wendy M. Mars,

    1. Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA
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  • William C. Bowen,

    1. Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA
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  • Jian-Hua Luo,

    1. Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA
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  • Yu Yang,

    1. Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA
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  • Yan P. Yu,

    1. Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA
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  • Ann Orr,

    1. Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA
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  • René St.-Arnaud,

    1. Shriners Hospital and McGill University, Montréal, Québec, Canada
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  • Shoukat Dedhar,

    1. British Columbia Cancer Agency and Vancouver Hospital, Jack Bell Research Center, Vancouver, British Columbia, Canada
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  • Klaus H. Kaestner,

    1. Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA
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  • Chuanyue Wu,

    1. Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA
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  • George K. Michalopoulos

    Corresponding author
    1. Division of Cellular and Molecular Pathology, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA
    • Department of Pathology, University of Pittsburgh School of Medicine, S-410 Biomedical Science Tower, Pittsburgh, PA 15261
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    • fax: 412-648-9846


  • Potential conflict of interest: Nothing to report.

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 α 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 β-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. (HEPATOLOGY 2008;48:1932-1941.)

Liver supports multiple homeostatic functions of the body and maintains an appropriate size for the needs of the organism by combining (as needed) cell proliferation (to restore or increase weight) and apoptosis (to decrease weight, or in response to injury). Liver consists of hepatocytes, the main functional cells of the liver, as well as endothelial cells, stellate cells, Kupffer cells, and biliary cells. For all the hepatic cells to function properly, the interaction with extracellular matrix (ECM) is of utmost importance.1 Previous studies have shown that liver undergoes matrix breakdown and remodeling at the early stages of regeneration after partial hepatectomy.1–5 Hepatocytes depend on ECM for their differentiation in culture and they rapidly lose hepatocyte specific gene expression when maintained in the absence of matrix. Differentiation and normal hepatocyte cyto-architecture is quickly restored, however, on addition of complex matrix environments (e.g., Matrigel or collagen gels).1, 6–8 Restoration of differentiation is accompanied with decrease in hepatocyte proliferation and loss of responsiveness to hepatocyte growth factor (HGF) and epidermal growth factor (EGF).9

Communication between cells and the ECM is achieved through integrins and the associated integrin-proximal adhesion molecules.10 Through multiple protein–protein interactions and signaling events, these molecules transmit signals from the ECM to the interior of the cell and regulate many fundamental cellular processes. Integrin-linked kinase (ILK) is a β1- and β3-integrin-interacting cell matrix adhesion protein that has been shown to be crucial for a number of cellular processes such as survival, differentiation, proliferation, migration, and angiogenesis.11–15

Recent studies have shown that ILK plays a key role in the activation of hepatic stellate cells and thus in the development of liver fibrosis16 as well as during liver wound healing.17, 18 We showed recently that removal of ILK from hepatocytes in culture results in loss of differentiation,19 whereas acute elimination of ILK by injection of adenovirus expressing Cre recombinase in the tail vein of ILKflox/flox mice led to massive hepatocyte apoptosis.20 In our current study, we used the mouse model of targeted ablation of a gene in a specific organ by the LoxP-Cre system and eliminated ILK from mouse liver by expression of Cre recombinase in ILKflox/flox mouse livers through promoters whose expression is specific to hepatocytes.21

Abbreviations

AFP, α fetoprotein; AFP/ALB ILK-KO, ILK eliminated by Cre controlled by AFP enhancer driven albumin promoter; CO, control mice; ECM, extracellular matrix; EGF, epidermal growth factor; FAK, focal adhesion kinase; HGF, hepatocyte growth factor; ILK, integrin-linked kinase; KO, knock-out; PHx, partial hepatectomy; PCNA, proliferating cell nuclear antigen; PINCH, particularly interesting Cys-His-rich protein; TUNEL, terminal deoxynucleotidyl transferase-mediated nick-end labeling; WT, wild type.

Materials and Methods

Antibodies.

The following primary antibodies were used in this study: mouse monoclonal (mAb) anti-ILK antibody (clone 69) (Santa Cruz Biotechnology, Santa Cruz, CA), PCNA (Dako, Carpinteria, CA), and anti-β-actin mAb (Chemicon, Temecula, CA). Goat-anti-mouse secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). All integrin antibodies were purchased from Cell Signaling Technologies (Danvers, MA) except Integrin-β5 and Integrin-α1, which were purchased from Santa Cruz Biotechnology. Integrin-α7 antibodies were generated in the laboratory of Dr. Jian-Hua Luo as described.22

Animals.

The ILK-floxed animals used in this study were generated as described23 and were cross-bred with mice expressing Cre-recombinase under the control of hepatocyte-specific AFP-enhancer-albumin promoter. The AFP-enhancer-albumin promoter Cre-expressing mouse strains were provided by Dr. Klaus Kaestner (University of Pennsylvania). The Cre-expressing animals were maintained in a heterozygous state to avoid any Cre-induced toxicity. “Control” animals were taken from Cre positive mice that retained wild-type ILK alleles or mice that were Cre-positive but had only one copy of the floxed ILK gene and one of the WT (ILKloxP/+). All animals were housed in the animal facility of the University of Pittsburgh and all experiments were carried out under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pittsburgh. Animals were treated humanely according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health.

Liver-Specific Inactivation of the ILK Gene.

The ILK-floxed mice were mated with mice expressing Cre-recombinase under the control of α fetoprotein (AFP) enhancer-albumin promoter.21 Genotyping was carried out by polymerase chain reaction from DNA isolated from the mouse tails using ILK primer sequences published previously. For the genotyping of the Cre transgenic animals we used the following set of primers: 5′-GCG GTC TGG CAG TAA AAA CTA TC-3′ (forward) and 5′-GTG AAA CAG CAT TGC TGT CAC TT-3′.

Liver Collagenase Perfusion and Separation of Hepatocytes from Nonparenchymal Cells.

Mouse hepatocytes from the ILK-knockout (ILK-KO) animals under the three different liver-specific promoters, as well as their respective controls, were isolated by an adaptation of the calcium two-step collagenase perfusion technique, as described.24 After the liver perfusion, hepatocytes were separated from the nonparenchymal cells of the liver by several centrifugation steps. Briefly, the cell pellet obtained from the liver perfusion was centrifuged at 1,000 g for 5 minutes and the supernatant was saved. The pellet was washed subsequently with Hank's buffered salt solution and centrifuged at 1,000 g for 5 minutes, and the supernatant was saved for later use while the pellet was kept as the fraction that corresponds to hepatocytes. The supernatants from the last two centrifugations were combined and centrifuged at 1,200 g for 5 minutes. The resulting supernatants were then centrifuged at 1,000 g for 5 minutes and the pellet was saved and washed once again with Hank's buffered salt solution. This pellet contained all the nonparenchymal cells of the liver (stellate cells, endothelial cells, Kupffer cells, biliary cells). The controls used for each ILK-KO animal were their Cre negative ILK-floxed siblings.

Results

Mice with ILK KO Targeted to the Liver Are Born Normal.

To study the effects of ablation of ILK in vivo, we mated homozygous ILK-floxed animals with heterozygous ILK-floxed mice expressing Cre-recombinase under control of the AFP-enhancer-albumin promoter. Mice were sacrificed and their livers harvested at day 1 after birth, and at 14 days, 5 weeks, 8-10 weeks, 16 weeks, and 30-35 weeks of age. Mice with ILK liver-targeted elimination were born normal. The livers of the ILK-liver-KO mice had apparently normal histology and size, indistinguishable between the KO and the control mice (data not shown). It is not clear whether ILK is not required for hepatic development or whether ILK is not completely removed during that stage. Although this has not been described for systems using AFP/Alb Cre, it is seen with Cre expressed under control only by the albumin promoter.25

ILK Is Removed from the Liver of the Liver-Targeted KO Animals.

To determine whether the ILK gene was indeed deleted in the Cre expressing mice, we carried out western blot analysis with anti-ILK antibody in whole-cell lysates from liver of 30-week-old animals. As shown in Fig. 1A, ILK was knocked down efficiently in AFP-albumin-ILK-KO animals. However, there was still a certain amount of ILK remaining in the KO animals (Fig. 1A), most likely reflecting expression of ILK in nonparenchymal cells. Stellate cells are known to contaminate hepatocyte cultures by variable percentages (up to 5%).26

Figure 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.

ILK Is Removed from the Hepatocytes and Not from the Other Hepatic Cell Types.

We next sought to determine in which population of liver cells the ILK was ablated. We perfused livers with collagenase to isolate specific hepatic cell subpopulations and subsequently separated the hepatocytes from the nonparenchymal cells.26 As shown in Fig. 1B, ILK expression in the nonparenchymal cells is almost the same between the control and the liver-targeted KOs, whereas it is reduced dramatically in the hepatocyte fraction of the same mice (the residual ILK presence in hepatocytes is probably due to the well-documented minor contamination of the hepatocyte fraction by stellate cells).26 This suggests that ILK is indeed removed from the hepatocytes but not the nonparenchymal cells of the liver (endothelial cells, Kupffer cells, stellate cells, and biliary cells), thus explaining why a significant amount of ILK protein is still present in whole cell lysates from the whole liver tissue of the liver-targeted KO animals (Fig. 1A,B). This cellular profile of ablation of ILK is consistent with the expression of Cre only in hepatocytes in the AFP-Cre livers. These findings are also consistent with a recent publication in which strong expression of ILK was found in the stellate cells.16

Histologic Abnormalities of the Livers of ILK-KO Animals with Progressing Age of the Animals.

As mentioned above, the livers of 1-day-old ILK liver-KOs were grossly and histologically normal. The histology appeared similar also at 2 weeks of age (Fig. 2A,B). As mice progressed in age, however, their livers exhibited abnormalities as compared to the control animals. The hepatocyte plates were disorganized in ILK-KO mice with an increased number of hepatocytes in proliferation and in apoptosis. These changes were even more evident between 5 and 10 weeks, with both mitoses and apoptoses subsiding by 30 weeks. Histologic changes in comparison between ILK liver-KO and Control mice for the ages of 2 to 30 weeks are shown in Fig. 2. Extensive proliferation of biliary epithelial cells was observed in ILK-KO, shown in lower magnification in Fig. 2C,E,G and at higher magnification in Fig. 3C (see below).

Figure 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.

Figure 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×).

Figure 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.

Enhanced Matrix Deposition and Biliary Cell Proliferation in ILK-KO Mice.

There was progressive deposition of fine connective tissue material in the ILK-KO livers (staining positive with silver stain for reticulin, Fig. 3A,B). The fibrils were few in number at the early age (2 weeks, data not shown), but increase to the point that most hepatocytes are surrounded by individual reticulin fibers by 10 weeks of age (Fig. 3A). There was an overall decrease from 16 to 30 weeks however. This accumulation of ECM was self-limited. The surface of the livers was smooth and there was no cirrhosis. At 30 weeks of age, although the rest of the organs appeared normal, the livers from the ILK liver-targeted KO were paler, larger, and their texture was more rigid than their control counterparts.

From 2 to 16 weeks, in addition to the increase in hepatocyte proliferation, there was also proliferation and expansion of biliary epithelial cells (Fig. 3C). These cells were characterized as biliary by virtue of their morphology, their tendency to organize into incomplete duct structures, and the fact that their nuclei were staining positive for the transcription factor HNFβ1. The tracts containing biliary epithelial cells also contained desmin-positive stellate cells (Fig. 3D). Proliferation of biliary cells started at 5 weeks, culminated at 16 weeks, and subsided by 30 weeks. The mixed cell populations and the histologic expansion along oblong tracts emanating from the portal triads strongly resembled proliferation of oval cells described in suppressed regeneration models.27, 28 None of these changes were seen in livers of Control (Cre-positive mice that retained wild-type ILK alleles or mice that were Cre-positive but had only one copy of the floxed ILK gene and one of the WT [ILKloxP/+]) (data not shown).

Increase in Cell Proliferation and Liver Size in ILK-KO Livers.

We monitored the cell kinetics in Control and ILKloxP/loxP AFP Cre mice at 2, 5, 10, and 30 weeks of age. The data in Fig. 4A-C shows the percent of hepatocytes in the cell cycle (PCNA-positive nuclei; Fig. 3E,F), and numbers of cells in mitosis and apoptosis (TUNEL-positive cells; Fig. 4G,H) as a function of age of the mice. In normal livers, the percent of PCNA-positive hepatocytes, cells in mitosis, and TUNEL-positive hepatocytes declined with age. In livers with targeted elimination of ILK, the percent of PCNA-positive cells remained elevated with eventual decline by 30 weeks. The number of cells in mitosis did decline but was at all times higher than in control mice. The numbers of hepatocytes in apoptosis increased from 2 to 5 weeks and declined thereafter, but remained at all times about three-fold higher than in control livers. The sustained cell proliferation in the ILK-KO livers resulted in increase in liver weight and size (Fig. 4D,I) at the end of 30 weeks, where ILK-KO livers were approximately twice the size of control livers.

Sequential Changes in Integrin Expression.

The histologic changes described above prompted us to examine the expression levels of different integrins as a function of the age of the mice. ILK normally links with integrins containing chains β1 and β3. We investigated the possibility that in the absence of integrin signaling through ILK, hepatocytes alter their integrin patterns to provide alternative signaling mechanisms as part of their adaptation to the absence of ILK. The expression of several integrins was assessed by western blot analysis as a function of age of the mice (Fig. 5). There were transient changes occurring between 2 and 30 weeks of age. Many changes were seen at 5 weeks, corresponding to the peak of ploidy-related changes in hepatocytes.29 At 30 weeks, however, when most of the biliary and hepatocyte proliferation subsides, integrin chain β1 was absent from the ILK-KO mice whereas expression of integrin chain β5 and αV and α7 was enhanced.

Figure 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.

Gene Expression Alterations in the ILK-KO Livers.

Matrix signaling is very important for maintenance of hepatocyte gene expression and control of proliferation. In view of likely changes in matrix signaling given the absence of ILK and the alterations in expression of integrin β chains, we investigated the gene expression patterns in the livers of ILK-KO mice at 2, 5, 10, and 30 weeks age, using GeneChip Microarray analysis (Affymetrix). We concentrated first on numeric value differences in gene expression (as opposed to fold change in gene expression) under the rationale that large differences in gene expression values are likely to be seen in genes expressed abundantly in the liver, as opposed to large fold changes in minimally-expressed genes. The top 85 most overexpressed genes in the KO mice over control at 30 weeks of age are shown in Table 1. Most of the genes are well recognized as hepatocyte-specific, including several apolipoproteins, albumin, hemopexin, vitronectin, transferrin, transthyretin, fibrinogen, carbamoyl-phosphate synthetase, CYP family members 2e1, 3a11, 2a5, and 2c70, etc. When the expression of these 85 genes is assessed over all the ages of the mice, the results were surprising in that the genes most overexpressed in ILK-KO over the Control mice at age 30 weeks were highly under-expressed in the previous time points (Fig. 6A). The surprising overexpression of many hepatocyte-associated genes at 30 weeks of age suggested that livers had adapted to a different set of ECM and integrin mediated signals required to maintain hepatocyte differentiation. Detail data on gene expression related to Fig. 6A are shown in Table 1 and in the Supporting Table 1. There was a decline in focal adhesion kinase, another enzyme associated with integrin signaling. At 2, 5, 10, and 30 weeks, expression of FAK in ILKloxP/loxP AFP Cre mice over Control was +171, −369, −35, and −808. The accumulation of matrix during the period from 2 to 30 weeks of age was also reflected in gene expression of ECM-related proteins, as shown in Supporting Figure 1. Several ECM-related proteins had a transient increase at 10 weeks followed by an overall decline at 30 weeks. The dramatic enhancement in expression of hepatocyte-associated genes at 30 weeks, however, did correlate with enhanced expression of some hepatocyte-associated transcription factors (Fig. 6B). With increasing age of the mice there were changes in expression of C/EBPα and C/EBPβ. At 30 weeks, C/EBPα and C/EBPβ both remained elevated in AFP/ALB ILK-KO compared to Control. The ratio of C/EBPα to C/EBPβ, normally being high in the resting liver of Control mice, was reversed in the AFP/ALB ILK-KO mice, mimicking changes seen after partial hepatectomy.30

Table 1. Difference in Expression Units of Liver Genes in ILK-KO (Liver) and Control Mice
Liver Genes
  1. Numbers to the left of the gene name indicate the (KO-Control) difference in gene expression. The genes below are the top 85 in terms of overexpression in the ILK-KO (liver) over the Control mice at 30 weeks of age.

54423 Apolipoprotein A130531 CYP 2a5
53940 Apolipoprotein A230495 Fructose biphosphatase 1
53566 Apolipoprotein E30421 Glutathione peroxidase
48880 Serine protease inhibitor 1-330292 Thyroid hormone responsive SPOT14 homolog
46690 Serine protease inhibitor 1-230147 Ferritin light chain
46419 Transferrin29906 RNAase A-4
44396 Transthyretin29875 Kininogen
44001 Hemopexin29871 beta-2 microglobulin
43478 Hepcidin29718 S-adenosylhomocysteine hydrolase
43174 Albumin29501 Plasminogen
42475 CYP2e129149 Peroxiredoxin
41721 Fatty acid binding protein 128835 Ubiquitin C
41535 Serine protease inhibitor 1-128546 Carbonic anhydrase 3
40615 alpha-2-HS-glycoprotein28157 Selenophosphate synthetase 2
40072 Fibrinogen alpha27628 Diazepam binding inhibitor
39740 Fibrinogen beta27599 Isocitrate dehydrogenase 2
38940 Glutathione S-transferase 127440 Aldolase 2, B isoform
38758 Ceruloplasmin27156 Phenylalanine hydroxylase
38707 Complement C327130 Ferritn Heavy Chain
38663 Apolipoprotein H27084 Aldo-keto reductase family 1, C6
37797 IGF BP426189 alpha-trypsin inhibitor, heavy chain 4
37676 alpha-2 macroglobulin25911 Adenosine kinase
36622 Haptoglobin25630 Methionine adenosyltransferase I
36256 Fibrinogen gamma25353 Glutathione-S-transferase, mu 1
35552 Apolipoprotein C-III25163Aldehyde dehydrogenase 2
35513 CYP3a11 Steroid Inducible25030 Aldehyde dehydrogenase 1 family, member L1
35482 4-hydroxyphenylpyruvic acid dioxygenase24750 Cytochrome b-5
35368 Apoliprotein C124493 Sterol carrier protein 2, liver
35311 Antithrombin 124486 Solute carrier family 38
35104 Alpha 1 microglobulin/bikunin24337 Interferon induced transmembrane protein 3
34632 Vitronectin24204 Acetyl-Coenzyme A acyltransferase 1B
34466 Selenoprotein P, plasma, 124155 Progesterone receptor membrane component 1
34284 Apolipoprotein A-V24068Regucalcin
34020 Esterase 124036 Polymeric immunoglobulin receptor
34019 Phenol UDP-glucuronosyltransferase23934 Paraoxonase 1
33241 Retinol binding protein23482 L-3-hydroxyacyl-Coenzyme A dehydrogenase
32662 Murinoglobulin 123236 Ubiquitin B
31966 Alcohol dehydrogenase 123182 CYP 2c70
31743 Carbamoyl-phosphate synthetase 123178 Apoliprotein C IV
31532 Glycine N-methyltransferase 
31335 Betaine-homocysteine methyltransferase 
31291 Clusterin 
31110 Arginosuccinate synthetase 1 
31048 Urate Oxidase 
30761 Actin, beta 
Figure 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 under-expressed 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.

Discussion

The removal of ILK from hepatocytes interrupts an important part of the signaling cascade initiated by components of ECM and mediated through integrins. Cell-ECM adhesion proteins such as FAK and ILK are major mediators of integrin signaling, thus affecting many cellular processes. Although they both have domains suggesting a kinase function, there is considerable debate whether the functions mediated by ILK proceed through such kinase activity or through its association with PINCH and Parvin and the formation of a stable ternary complex at the cell-ECM adhesion sites. The preponderance of the evidence suggests that both aspects of ILK are involved and that ILK constitutes an important mediator of integrin signaling.11, 31-37 Thus, elimination of ILK is likely to cause disruption of integrin signaling and to interfere with the effects of extracellular matrix on hepatocytes. The findings of this study, and also our recent work with acute removal of ILK from hepatocytes in culture19 and whole animals20 by means of adenoviral vectors, raise important questions on the role of ECM signaling on hepatocyte proliferation, differentiation, and apoptosis. There are two findings in relation to ILK removal and hepatocytes that seem contradictory:

  • 1Previous work has shown that addition of ECM components to primary hepatocyte cultures inhibits hepatocyte proliferation.6 It is not surprising that elimination of the matrix signaling through elimination of ILK leads to increased hepatocyte proliferation and increased number of mitoses.
  • 2Previous studies from our laboratory, however, have also shown that acute elimination of ILK from the ILK-floxed hepatocytes in vitro and in vivo by adenoviral delivery of Cre-recombinase gives rise to massive apoptosis in cultured cells and fulminant hepatitis in the whole animal.20 We have interpreted those findings as consistent with the type of apoptosis known as “anoikis,” occurring in epithelial cells after acute removal of ECM from their basement membrane. It is known that ILK protects other cell types from anoikis.35 Because we do not observe massive apoptosis in the livers that ILK was removed by transgenic Cre expression, we have to assume that the slower pace of removal by genetic means allows for some form of adaptation that prevents massive apoptosis, as manifested by altered expression of integrins and matrix proteins in the ILK-KO livers at 30 weeks. Previous studies have shown that integrin β1 is a key integrator of growth and differentiation related signals and its loss between 16 and 30 weeks is bound to have profound effects on hepatocyte growth and differentiation.38

The wave of proliferation and apoptosis in hepatocytes and biliary cells seen between 2-30 weeks of age raises the issue of whether hepatocyte proliferation or apoptosis is the primary event. Does slow removal of ILK cause hepatocyte apoptosis as a primary event (with compensatory proliferation to maintain liver mass as a result), or hepatocyte proliferation as a primary event, caused by removal of the mito-inhibitory ECM signaling (with apoptosis being a compensatory event to prevent excessive liver weight)? Because these possibilities are not mutually exclusive, it is possible, that both phenomena may be occurring at the same time. We believe that the predominant effect of removal of ILK by genetic elimination is the enhanced proliferation of hepatocytes (and biliary epithelial cells), occurring because of the removal of the proliferation-inhibitory effects of ECM on hepatocytes. Our interpretation is based on two findings. First, at 30 weeks there is enlargement of the ILK-KO livers to twice the size of the wild-type mice. This would be unlikely if hepatocyte apoptosis was the primary driving force for the histologic changes seen. The second finding is the simultaneous proliferation of both hepatocytes and biliary cells. It has been shown repeatedly that when liver regeneration is stimulated and hepatocyte proliferation is inhibited, biliary cells become activated into the transiently amplifying population of progenitor cells known as “oval cells” (that in turn become hepatocytes).28, 39 If hepatocytes can proliferate normally and thus carry out liver regeneration (as in most circumstances), proliferation of biliary/oval cells does not occur. In our study, however, both hepatocytes and biliary epithelium are intensely proliferating at the same time (in the absence of chemical injury, bile duct ligation, etc.). We believe our data are most compatible with the interpretation that that hepatocytes are proliferating because of the removal of the mito-inhibitory signal of ECM. Because hepatocytes seem to be proliferating rather intensely, and because there is no decrease in liver mass to trigger activation of the oval cell compartment, the proliferation of the biliary epithelium is enigmatic. Biliary cells derive from embryonic hepatoblasts. This differentiation occurs at day 16 to 17 of mouse embryonic development. Activation of the AfpCre however presumably occurs earlier, at days 10 to 11.21 Thus, it is likely that many, if not most, of the biliary epithelial cells in the apparently normal biliary ductules are missing ILK and thus enter into the same “hyperproliferation” mode as the hepatocytes.

Evidence for mito-inhibitory effects of integrin signaling also is now accumulating from studies with neoplastic cells. Disruption of integrin signaling is associated with enhancement of the malignant phenotype including enhanced proliferation and tendency to metastasize. This has been shown with hepatocellular carcinoma, prostate adenocarcinoma, and leiomyosarcomas.22

Microarray analysis (see Supporting Information for more details) provided us with more insight into the details of changes in hepatocyte differentiation and the observed changes after elimination of ILK. The results in Fig. 6 show that after elimination of ILK, and thus decrease in signaling from ECM required to maintain hepatocyte differentiation, there is a decrease in hepatocyte-associated gene expression at 2 weeks of age. This is also associated with the initiation of enhanced proliferation at the same time and it is very similar to what is seen in hepatocyte primary cultures.24 In the latter, HGF and EGF stimulate hepatocyte proliferation along with a concomitant drop in hepatocyte gene expression. Multiple studies have shown that when matrix is reintroduced into the cultures, hepatocyte differentiation is restored and proliferation stops.24 Our studies provide the first evidence that interference with matrix signaling in whole liver has the same effect as with hepatocytes in culture. The results in Figs. 5 and 6, however, also show that in the absence of ILK, there is an “adaptation” process that includes altered expression of transcription factors, integrins, and (Supporting Figure 1) synthesis of ECM with a different composition (notable increase of syndecans 1, 2, and 4, increase in collagen type III and decrease in collagens IV and VI). The finally adapted liver with substituted alternatives to ILK/ECM signaling of control animals seems to be in an over-compensated state in terms of hepatocyte differentiation, with dramatic changes in expression of hepatocyte associated genes and some key hepatocyte-associated transcription factors (C/EBPα and C/EBPβ).

In conclusion, the significant changes seen with genetic elimination of ILK show the importance of matrix signaling in regulation of hepatocyte proliferation and differentiation.

Acknowledgements

We are grateful to Ben Cieply for carrying out some of the genotyping for the mice, to Dr. Satdarshan P. Monga for helpful suggestions, and to Dennis P. Michalopoulos for help with analysis of the microarray results.

Ancillary

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