Journal of Hepatology
Volume 42, Issue 1 , Pages 87-93, January 2005

RGD peptides confer survival to hepatocytes via the β1-integrin-ILK-pAkt pathway

Department of Pathology, Leiden University Medical Center, Leiden, Building 1, L1-Q, P.O. Box 9600, 2300 RC Leiden, The Netherlands

Received 14 June 2004; received in revised form 17 August 2004; accepted 21 September 2004. published online 11 October 2004.

Article Outline

Background/Aims

Allogeneic cell transplantation is characterized by a lack of sustained survival of the transplanted cells in the recipient. Activation of the appropriate integrin-linked signaling pathways in cells can promote cell survival. The purpose of this study was to determine how presence or absence of anti-β1 integrin chain antibodies or RGD peptides affects the survival of hepatocytes.

Methods

Hepatocytes of BN rats were isolated. Hepatocyte survival was tested after the hepatocytes had been cultured in the presence of anti-β1 integrin antibodies or RGD peptides. Hepatocytes that had been given a different treatment were stained for caspase 3 (apoptosis marker) and phospho-Akt Ser 473 (survival marker) and were measured for their integrin-linked kinase (ILK) activity.

Results

Ligation of integrins using antibodies against the β1 integrin chain or RGD peptides protected isolated hepatocytes from apoptosis and resulted in an increased ILK activity and persistent phosphorylation of protein kinase B/Akt at serine 473.

Conclusions

Integrin activation in isolated hepatocytes contributes to the activation of ILK, phosphorylation of Akt on serine residue 473, and inhibition of apoptosis. Integrin signaling through the ILK-phospho Akt pathway protects isolated hepatocytes from apoptosis. This notion may potentially be applied to render the transplantation of hepatocytes more effective.

Keywords: Apoptosis, Rat, Extracellular matrix, Caspase 3, Akt, ILK

 

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1. Introduction 

Survival of anchorage-dependent cells requires integrin-mediated adhesion to extracellular matrix (ECM) components, which include fibronectin, collagens, and laminins [1]. Integrins act as a bridge between ECM components, the cytoskeleton, and other proteins, and regulate cell survival, proliferation, differentiation and migration [2]. Many of the integrins, like α5β1, αvβ1, and the αvβ3 integrins, recognize peptide sequences of ECM components that include the tripeptide arg-gly-asp (RGD), a sequence that is shared by several extracellular matrix proteins [3], [4]. Numerous studies have shown that RGD interacts with integrins to activate several tyrosine kinases and protein kinase signal transduction pathways [5]. Cell attachment mediated by the α5β1 and the αvβ3 integrin promotes cell survival by upregulation of bcl-2 [6], [7]. Integrins are believed to mediate signaling effects by binding and activating different intracellular proteins. One of these proteins is integrin-linked kinase (ILK), which can phosphorylate β1 and β3 integrins [8]. ILK is reported to function as the effector of phoshatidylinositol 3-kinase (PI3-K) signaling. As a result of this signal, protein kinase B (PKB)/Akt on serine residue 473 is directly phosphorylated [9], [10], and apoptosis is suppressed [11], [12], [13]. When integrin-ECM adhesion is lost, cells die via detachment-induced apoptosis, which is called anoikis [14], [15]. Prior to hepatocyte transplantation, hepatocytes are removed from their natural environment, causing anoikis [16], [17]. Furthermore, it will take approximately 18h for donor hepatocytes to join adjacent host hepatocytes after hepatocyte infusion in the portal vein [18]. During these 18h, hepatocytes will not be attached to any ECM protein.

Allogeneic cell transplantation is characterized by a lack of sustained survival of the transplanted cells in the recipient. We have shown that isolated hepatocytes that were treated with antibodies directly against α1- or β1-integrin chains significantly improve their intraparenchymal survival upon transplantation [19]. Nevertheless, the mechanisms by which application of antibodies against the β1-integrins or RGD peptides affects survival of hepatocytes are unclear. It is important to establish which integrins are responsible for protective signaling in hepatocytes, if one intends to manipulate integrin activation as a preventive or therapeutic tool in cell transplantations. Our findings imply that treatment with anti-β1 antibodies or RGD peptides confers survival to isolated hepatocytes via the β1-integrin-ILK-pAkt pathway.

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2. Materials and methods 

2.1. Hepatocyte isolation 

Inbred female Brown Norway (BN) rats (n=15) weighing 150–200g were obtained from Harlan (Horst, The Netherlands). The hepatocytes were isolated with a modification of the Seglen collagenase digestion technique [20]. In brief, after cannulation of the portal vein, the liver was perfused with a calcium free Hepes-based buffer (pH 7.4) at 37°C at a flow rate of 30ml/min. Recirculation was established for 9min with Hepes buffered at pH 7.4 containing 0.5mg/ml collagenase type IV (Sigma Chemicals Co, St Louis, USA). The cells were dispersed in Ca2+-free Hepes buffer and filtered through a 40μm pore diameter sieve. Viability was determined by 0.02% Trypan blue dye exclusion and amounted to at least 90%.

2.2. Culture of hepatocytes 

Hepatocyte suspensions of 1×106 viable BN cells per ml were incubated with hamster anti-β1 integrin chain (IgM) monoclonal antibodies (Becton Dickenson, Alphen aan den Rijn, The Netherlands) in concentrations of 2.5, 10, 20 or 40μg/ml for 10min at 37°C or with 0.01, 0.1, 1.0 or 2.0mM RGD (H-Arg-Gly-Asp-OH) peptides, 0.1mM cyclic RGD (both from Brunswig Chemie, Amsterdam, The Netherlands), or 0.1mM proNectin F (Protein Polymer Technologies, San Diego, CA, USA), a repetitive RGD polymer [21], for 30min at 37°C. The control peptide with a scrambled amino acid sequence Arg-Gly-Glu-Ser was purchased from Sigma-Aldrich Chemicals (Zwijndrecht, The Netherlands) and used at a final concentration of 0.1mM. Isolated hamster IgM from hamster serum (Jackson Immunoresearch laboratories, West Grove, USA) was used as non-relevant IgM (40μg/ml). Hepatocytes were suspended in RPMI-1640 medium, which contained 100μg/ml penicillin, 100μg/ml streptomycin, 10ng/ml insulin and 10% fetal calf serum, and the hepatocytes were plated at a density of 0.8×105 cells per cm2. These 24-well culture plates were precoated with polyhydroxyethylmethacrylate (polyHEMA) (Sigma-Aldrich Chemicals, Zwijndrecht, The Netherlands) to prevent the cells from becoming attached to the plates [22]. The cells were cultured at 37°C in a humidified atmosphere of 5% CO2 and 95% oxygen.

2.3. DNA fragmentation analysis by gel electrophoresis 

Hepatocytes were either cultured with or without anti-β1 integrin MoAbs or RGD peptides for 5h. Hepatocytes were also cultured with an irrelevant normal hamster IgM as control for the anti-β1 MoAbs. The cells were centrifuged and washed twice with ice-cold PBS. The cells were resuspended in a lysis buffer (10mM Tris–HCl, 1mM EDTA, pH 8.0, 0.5% Triton X-100). The samples were treated with the lyses buffer containing 10μg/ml of RNAse at 37°C. After 1h of incubation, 0.6% SDS and 0.4mg/ml proteinase K were added. This was followed by incubation for 1h at 37°C. DNA was precipitated with 5M NaCl and ethanol. A 100bp DNA ladder (GibcoBRL, Breda, The Netherlands) was used as a marker. Electrophoresis was performed at 50V for 60min on a 1% agarose gel. DNA was stained with SYBRgreen (Molecular Probes, Leiden, The Netherlands) and visualized under ultraviolet light. As a positive control for the DNA fragmentation, hepatocytes were treated with anti-Fas antibodies (JO-2, Pharmingen, San Diego, USA) and cultured on collagen IV for 5h.

2.4. Detection of apoptosis by active caspase-3 and survival by phospho-Akt Ser 473 immunostaining 

Hepatocytes were cultured for 18h, fixed in 4% paraformaldehyde overnight, and dehydrated in a graded ethanol series. The cells were embedded in paraffin and cut into 3μm sections. Active caspase 3 was detected with diluted (1:3000) rabbit polyclonal antibody CM1 (Becton Dickenson, Alphen aan den Rijn, The Netherlands) [23]. The diluted (1:50) rabbit anti-phospho-Akt Ser 473 antibody (Westburg bv, Leusden, The Netherlands) was used as a survival marker. The antigen was retrieved by boiling the deparaffinized sections for 10min in 0.01M citrate buffer at pH 6.0. Overnight incubation with the primary antibody was followed, after washing, by 1h of incubation with diluted (1:100) biotinylated swine anti-rabbit IgG antibody (Dako, Glostrup, Denmark). Finally, the sections were treated with peroxidase-labeled SABC complex (Dako, Glostrup, Denmark) and developed with H2O2 and 3,3′-diamebenzidinetetrahydrochloride (DAB) as a chromogen. The percentage of caspase 3 positive or phospho-Akt 473 positive cells was determined by calculating the number of caspase 3 positive cells or phospho-Akt 473 positive hepatocytes per 100 hepatocytes (three fields per section were measured at a magnification of 400×). All values are given as means±SD.

2.5. Integrin‐linked kinase immunoblot and activity assay 

Hepatocytes were either cultured with or without anti-β1 integrin MoAbs at 40μg/ml or with 0.1mM RGD peptides, or with 0.1mM scrambled peptides on polyHEMA for 0, 30, 60min, or 18h. Cells were lysed on ice for 10min in a buffer containing 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 5mM EDTA, 0.1M Tris–HCl (pH 7.4), 1mM Na3VO4, 2mM PMSF, 1μg/ml leupeptin and 1μg/ml aprotinin. For immunoblot analysis, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on to PVDF membranes by wet transfer (45min at 70V). Membranes were blotted with the rabbit anti-ILK antibody (Upstate biotechnology, New York, USA) at a concentration of 2μg/ml in Tris-buffered saline (50mM Tris, 0.138M NaCl, 2.7mM KCl, pH 7.6) containing 0.1% (v/v) Tween-20 and 5% (w/v) BSA. Blots were incubated with swine anti-rabbit horseradish-peroxidase-labeled secondary antibody, visualized by means of enhanced chemiluminescence, and the images were captured with a cooled CCD camera system (Fuji LAS1000).

ILK kinase activity was determined in cell extracts with in vitro immunoprecipitation kinase assays that have been previously described [8]. Myelin basic protein (MBP) (Sigma-Aldrich Chemicals, Zwijndrecht, The Netherlands) was used as a substrate for ILK in separate experiments, and phosphorylated proteins were subjected to electrophoresis on 15% SDS/PAGE gels. [32P] ATP was used as the phosphate donor in the kinase assay, and [32P]MBP was detected by autoradiography or phosphorimaging analysis of the gels.

2.6. Detection of cross-linking of β1-integrins 

Hepatocytes were incubated in the presence or absence of anti-β1 integrin MoAbs at 40μg/ml or with 0.1mM RGD peptides for 60min at 37°C. Cells were washed in the presence of 0.01% sodium azide. This was followed by 30min of incubation with diluted (1:25) mouse anti-hamster IgM antibody (Pharmingen, Palo Alto, CA, USA), and visualized by 30min of incubation with anti-mouse IgG/FITC. Cells were photographed with a fluorescence microscope (Zeiss, Sliedrecht, The Netherlands).

2.7. Statistical analysis 

Differences in hepatocyte apoptosis and survival after different hepatocyte treatments were compared with Student's t test. P<0.05 was considered to be statistically significant. All data are expressed as means±SD.

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3. Results 

3.1. Both anti-β1 integrin antibodies and RGD peptides prevent anoikis-induced DNA laddering 

Induction of hepatocyte apoptosis is caused by disruption of cell–matrix interactions. We studied the effects of anti-β1 integrin antibodies and RGD peptides on freshly isolated hepatocytes. The hepatocytes were cultured on plastic dishes that had been precoated with a non-toxic film of polyHEMA to prevent their attachment. The presence of apoptosis of the hepatocytes was assessed by showing DNA-fragmentation. Hepatocytes cultured on polyHEMA showed less DNA fragmentation when they had been treated with anti-β1 integrin antibodies before culture. When hepatocytes were treated with RGD-peptides and cultured on polyHEMA, DNA fragmentation was completely prevented after 5h of culture. Hepatocytes that had been pretreated with the anti-Fas antibody, and which were cultured on collagen IV, served as the positive control for apoptosis and showed abundant DNA laddering (Fig. 1).

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  • Fig. 1. 

    Inhibition of DNA fragmentation of isolated hepatocytes treated with anti-β1 antibodies, or RGD peptides. Freshly isolated hepatocytes were treated with normal hamster IgM (B), anti-β1 antibodies (C), or RGD peptides (E) and cultured on polyHEMA for 5h. Anti-Fas antibody-treated hepatocytes, which were cultured on collagen IV, served as a positive control (D). Lane A shows untreated hepatocytes. DNA fragmentation was analyzed by agarose gel electrophoresis and SYBRgreen staining.

3.2. RGD concentration-dependent prevention of caspase 3 activity 

We determined the amount of caspase 3 positive hepatocytes by immunostaining in order to investigate the influence of the presence of the anti-β1 integrin antibodies and RGD peptides on the caspase 3 activity. In control cultures 39.8±3.9% of hepatocytes stained positively, which indicates that apoptosis took place. Whereas 20.1±3.4% of the anti-β1 integrin antibodies (Fig. 2(c)) and 8.6±2.9% of the RGD (Fig. 2(d)) pretreated hepatocytes were positive for caspase 3. This represents a decrease by a factor 2 (P<0.02) and factor 4 (P<0.001), respectively, in the level of apoptosis. Hepatocytes that underwent apoptosis after cyclic RGD and repeating RGD peptide motifs (pronectin F) treatment amounted to 17.6±4.5 and 18.5±1.4%, respectively, (Fig. 2(e)).

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  • Fig. 2. 

    Percentage of caspase 3 positive hepatocytes. Caspase 3 immunostaining of untreated (a) and RGD pretreated (b) hepatocytes. Freshly isolated hepatocytes were treated with different concentrations of (c) anti-β1 antibodies, (d) RGD peptides or with (e) cyclic RGD, pronectin F or scrambled peptides and cultured on polyHEMA for 18h. The percentage of caspase 3 positive hepatocytes was determined by calculating the number of caspase 3 positive hepatocytes per 100 hepatocytes. Data are expressed as means±SD of 5 independent experiments. *P<0.005 compared to the untreated hepatocytes. †P<0.001 compared to the untreated hepatocytes. [This figure appears in colour on the web.]

3.3. Integrin ligation increases phosphorylation of Akt on Ser 473 

Because integrin receptor activation can lead to ILK-mediated phosphorylation of Akt on serine residue 473, we determined if anti-β1 integrin antibodies or RGD peptides could induce Akt phosphorylation in hepatocytes. We determined the amount of phospho-Akt Ser 473-positive hepatocytes by immunostaining. Only 20% of the untreated hepatocytes were positive for phospho-Akt Ser 473, whereas 80% of the anti-β1 integrin antibodies (Fig. 3(a)) and the RGD (Fig. 3(b)) pretreated hepatocytes were positive for phospho Akt Ser 473 (P<0.005). This represents an increase by a factor 4 in the number of phospho Akt Ser 473 positive hepatocytes. We concluded that phosphorylation of Akt Ser 473 increased through the ligation of integrins by either anti-β1 integrin antibodies or RGD peptides.

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  • Fig. 3. 

    Percentage of phospho-Akt positive hepatocytes. Freshly isolated hepatocytes were treated with different concentrations of (a) anti-β1 antibodies or (b) RGD peptides, and cultured on polyHEMA for 18h. The hepatocytes were stained for phospho-Akt Ser 473. Data are expressed as means±SD of 5 independent experiments. *P<0.005 compared to the untreated hepatocytes. †P<0.001 compared to the untreated hepatocytes.

3.4. Integrin ligation results in increased integrin‐linked kinase activity 

Integrin-linked kinase (ILK) is a serine/threonine kinase that is known to associate with the cytoplasmic tail of the β1 integrin. ILK has been reported to increase phosphorylation of Akt on serine residue 473. Western blot with anti-ILK shows equivalent amounts of the protein in each extract (Fig. 4(a)). ILK activity was increased within 30min of exposure of hepatocytes to RGD peptides (Fig. 4(b)) and remained at an increased level after 18h of culture, in comparison to the ILK activity of untreated hepatocytes (Fig. 4(c)). The activation of ILK in response to stimulation of hepatocytes was blocked by scrambled peptides (Fig. 4(c)). ILK activity increased within 30min of exposure of hepatocytes to anti-β1 integrin antibodies. The increase of ILK activity was transient with activity returning towards basal levels within 60min (Fig. 4(c)).

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  • Fig. 4. 

    Integrin-linked kinase (ILK) is upregulated following integrin ligation in hepatocytes. (a) Western blot with anti-ILK shows equivalent amounts of the protein in each extract. (b) ILK assay using MBP as a substrate. The phosphorylation of the substrate by ILK is detected with [32P]ATP. The ILK assay was performed with immunoprecipitation of cell extracts of hepatocytes that were either not treated (−) or treated with anti-beta 1 antibodies or RGD peptides for 30min. (c) Ratio ILK activity: ILK activity of hepatocytes, which were either not treated or pretreated with anti-β1 antibodies, RGD peptides, or scrambled (scr.) peptides for 30, 60min or 18h versus the ILK activity of freshly isolated hepatocytes.

3.5. Clustering of β1-integrins on the surface of hepatocytes 

We hypothesized that ligand-induced clustering of the β1 integrins on the cell surface of the hepatocytes might result in survival. To characterize the process of integrin activation-induced cell survival, we compared the distribution of β1-integrins before and after activation. Fluorescein-labeled secondary antibodies were used to quantify attachment of the antibodies to the β1 integrins. After activation with anti-β1 integrin monoclonal antibody, β1 integrins were predominantly clustered, or capped, which could be observed as a patchy fluorescence pattern on the cell surface (Fig. 5(b) and (c)). Capping of the integrins on hepatocytes was still detectable after 18h of culture (data not shown). Hepatocytes treated with RGD peptides also induced clustering of β1-integrins (Fig. 5(d)). On non-activated hepatocytes, β1-integrins remained diffusely distributed along the surfaces of cells (Fig. 5(e)).

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  • Fig. 5. 

    Clustering of beta-1 integrins. (a) The presence of β1 integrins on hepatocytes in the liver. Binding of anti-β1-integrin moAbs (b and c) or RGD peptides (d) results in rapid redistribution of β1-integrins into clusters on the hepatocytes. Note the uniform distribution of β1 integrins on untreated cells (e).

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4. Discussion 

In the present study, we show that anti-β1 integrin antibodies are able to mimic a β1 integrin-mediated signal for the phosphorylation of Akt on serine 473 and thus inhibit the activation of caspases in isolated hepatocytes. Previous studies on apoptosis in mammary epithelial cells demonstrated that caspases are activated in the absence of a β1-integrin mediated signal, which leads to apoptosis. The β1-mediated signal is normally delivered by ECM proteins [24]. Integrin-linked kinase (ILK) is a serine/threonine kinase that has been known to associate with the cytoplasmic tail of the β1 integrin. ILK has been reported to increase phosphorylation of Akt on Ser 473 [8], [9], [10], [25], [26]. Observations that the application of antibodies against the fibronectin receptor (α5β1, α3β1 integrins) and the vitronectin receptor (αvβ3, αvβ5 integrins) leads to activation of ILK in mammary epithelial cells suggest that ILK may also interact with the β3 and β5 integrin subunits [8]. These integrin subunits bind to their ligands by recognizing the RGD sequence [3]. ILK showed an increased and longer activation when hepatocytes were treated with RGD peptides than with anti-β1 integrin antibodies, suggesting that ligation of β1 integrins by RGD peptides results in a higher ILK activation than after ligation with anti-beta 1 integrin antibodies. In this study we show that RGD-containing peptides are able to induce dose-dependent survival in hepatocytes and that treatment with RGD peptides results in a 1.38-fold increase in cell numbers after 18h of culture as compared to untreated hepatocytes (data not shown). The activation of ILK in hepatocytes by RGD peptides shown in this study is in keeping with data that show RGD peptides can activate ILK in human glomerular mesangial cells, which results in an increased secretion of transforming growth factor β1 [27]. In accordance with our current findings, activation of integrins that use a peptide containing the amino acid sequence EIKLLIS derived from the α chain of laminin [28] protects hippocampal neurons from apoptosis [29]. The α3β1 integrin activation by EIKLLIS peptides led to activation of ILK and phospho-Akt [29]. Our results and those of others demonstrate that specific integrin receptor-binding peptides activate integrin-receptors and protect cells from apoptosis. Susceptibility to cell death in adhesion-prevented epithelial cells are also regulated by signalling pathways involving phosphatidylinositol 3-kinase (PI3K), janus kinase 2 (JAK2), focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) [1], [30], [31]. Whether these kinases will be activated after hepatocytes that are treated with RGD peptides or anti-β1 antibodies is an interesting question for future studies. Furthermore, it is important to take into account that apoptosis in isolated hepatocytes is not only caused by disruption of cell–matrix interactions, but also by other factors as hypoxia, free radicals etc [16]. In this study, caspase 3 is a marker for the level of apoptosis. It is known that caspase 3 is a downstream caspase, causing degrading of cytoplasmic and nuclear proteins. Earlier markers for apoptosis as annexin V binding, or activation of caspases like caspase 8 and 9 are not measured in this study, indicating that the total number of cells going into apoptosis might be higher.

Several studies have shown that small peptides containing RGD motifs can activate cytosolic caspase 3 by direct interaction with the RGD recognition site of caspase 3 [32], [33]. The RGD peptides enter the cell by a passive, non-integrin-dependent pathway. Apoptosis that is induced by RGD peptides has most frequently been described in lymphocytes and immortalized cell lines. To confirm these data, we have also treated lymphocytes with RGD peptides and found induction of apoptosis (data not shown). There are several possible explanations for the apparent discrepancy between our current findings, which either showed decreased caspase 3 activity in hepatocytes that had been treated with RGD peptides or increased caspase 3 activity in lymphocytes and fibroblasts that had been treated with RGD peptides [33], [34], [35]. Lymphocytes exhibit anchorage-independent growth and appear not to require integrin-mediated signals to survive. Buckley et al. reported induction of apoptosis in anchorage-dependent fibroblasts by treatment with RGD peptides [33]. These fibroblasts were cultured on type I collagen, where cell attachment is RGD-independent [36]. Since lymphocytes and fibroblasts do not require integrins for attachment to RGD-regions within the ECM, exogenous RGD peptides enter these cells instead of binding to their outer membranes. These results indicate that activation of integrins by integrin-binding peptides or antibodies can suppress apoptosis in anchorage-dependent cells. Since these RGD peptides directly activate apoptosis in lymphocytes and cause a survival signal in anchorage-dependent cells, they may be helpful in allogeneic cell transplantations in various manners.

Attempts to cluster β1 integrins by mAbs resulted in an increase in hepatocyte viability. Since the used anti-β1 integrin antibodies are IgM mAbs and contain multiple antigen-binding sites for β1 integrins, it is indeed conceivable that cross-linking of the integrins on the hepatocytes' cell surface caused the observed enhancement of survival. Monovalent RGD peptides induced integrin clustering as effectively as intact antibodies did, which indicates that ligand-induced integrin clustering was not caused by antibody cross-linking. This indicates that cross-linking of integrins by an exogenous ligand is not required for survival signaling.

We propose that integrin activation in isolated hepatocytes contributes to the activation of ILK, phosphorylation of Akt on serine residue 473, and inhibition of apoptosis. Integrin signaling through the ILK-phospho Akt pathway protects isolated hepatocytes from apoptosis. This notion may be applied to render transplantation of hepatocytes more effective.

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Acknowledgements 

The authors wish to thank Margriet Ouwens and Dennis Hoogervorst for excellent technical assistance. Part of this study was performed with financial support of BTS grant 98161 from Senter, Ministry of Economic Affairs, The Netherlands.

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PII: S0168-8278(04)00432-5

doi:10.1016/j.jhep.2004.09.010

Journal of Hepatology
Volume 42, Issue 1 , Pages 87-93, January 2005

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