Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation Research. 2006;99:354-361
Published online before print July 13, 2006, doi: 10.1161/01.RES.0000237022.72726.01
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
99/4/354    most recent
01.RES.0000237022.72726.01v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Esfandiarei, M.
Right arrow Articles by McManus, B. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Esfandiarei, M.
Right arrow Articles by McManus, B. M.
Related Collections
Right arrow Infectious endocarditis
Right arrow Apoptosis
Right arrow Cell signalling/signal transduction
Right arrow Heart failure - basic studies
Right arrowRelated Article
(Circulation Research. 2006;99:354.)
© 2006 American Heart Association, Inc.

Molecular Medicine

Novel Role for Integrin-Linked Kinase in Modulation of Coxsackievirus B3 Replication and Virus-Induced Cardiomyocyte Injury

Mitra Esfandiarei, Agripina Suarez, Ansel Amaral, Xiaoning Si, Maziar Rahmani, Shoukat Dedhar, Bruce M. McManus

From The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research/Providence Health Care Research Institute (M.E., A.S., A.A., X.S., M.R., B.M.M.), Department of Pathology and Laboratory Medicine; and Department of Biochemistry and Molecular Biology (S.D.), Department of Cancer Genetics, British Columbia Cancer Agency, University of British Columbia, Vancouver, Canada.

Correspondence to Dr Bruce M. McManus, The James Hogg iCAPTURE Centre/Providence Health Care Research Institute, UBC-St. Paul’s Hospital, Rm 388 Burrard West, 1081 Burrard St, Vancouver, British Columbia V6Z 1Y6, Canada. E-mail bmcmanus{at}mrl.ubc.ca


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Viral myocarditis is a major cause of sudden cardiac death in children and young adults. Among viruses, coxsackievirus B3 (CVB3) is the most common agent for myocarditis. Recently, more consideration has been given to the role of signaling pathways in pathogenesis of enteroviral myocarditis, providing new platform for identifying a new potential therapeutic target for this, so far, incurable disease. Previously, we reported on the role of the protein kinase-B/Akt in CVB3 replication and virus-induced cell injury. Here, we report on regulation of virus-induced Akt activation by the integrin-linked kinase in infected mouse cardiomyocytes and HeLa cells. This study also presents the first observation that inhibition of ILK in CVB3-infected cells significantly improves the viability of infected cells, while blocking viral replication and virus release. Complementary experiments using a constitutively active form of Akt1 revealed that the observed protective effect of ILK inhibition is dependent on the associated downregulation of virus-induced Akt activation. To our knowledge, this is the first report of such beneficial effects of ILK inhibition in a viral infection model and conveys new insights in our efforts to characterize a novel therapeutic target for treatment of enteroviral myocarditis.


Key Words: viral myocarditis • CVB3 • integrin-linked kinase • cell death • PKB/Akt


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Myocarditis, an inflammatory disease of heart muscle, is a major cause of unexpected and sudden cardiac death in people under 40 years of age.1 More than 20 viruses have been associated with myocarditis, causing mild to severe injury in the myocardium with ultimate manifestation of end-stage dilated cardiomyopathy and heart failure.1,2 Among them, coxsackievirus B3 (CVB3), a small nonenveloped single-stranded RNA enterovirus in Picornaviridae family, has been implicated in 25% to 40% cases of acute myocarditis and dilated cardiomyopathy in infants and young adolescents.3–5

CVB3-induced myocarditis is known historically as an immune-mediated disease.6–8 However, direct CVB3-induced injury during acute phase of disease and before target-organ immune cell infiltration has been shown to be a very important determinant of disease progression and prognosis.9–11 The fate of infected cells and severity of disease are related to the balance between multiple and contemporaneous proapoptotic and antiapoptotic processes, both viral and host cell in origin.

In both cell cultures and experimental animal models, CVB3 infection leads to the release of mitochondrial cytochrome c and subsequent cleavage of executioner caspases in the cytoplasm of infected cells.12–15 These events result in morphological features of apoptotic cell death and virus-induced cytopathic effects (CPE). Host cell signaling may rebalance cellular homeostasis, block apoptotic cell death, and diminish viral progeny release. Of the host cell signals, the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway has been implicated in survival, metabolism, proliferation, and apoptosis.16,17

Previously, we reported on activation of Akt during CVB3 infection through a PI3K-dependent pathway.18 However, much remains to be uncovered about the actual mechanisms underlying virus-induced Akt activation and its significance in virus-induced cardiac cell death.

Full Akt activation requires phosphorylation of Thr308 residue on the catalytic domain and Ser473 residue on its C-terminal hydrophobic domain.19 The 3-phosphoinositide–dependent kinase 1 (PDK-1), the kinase responsible for Akt phosphorylation on Thr308, has been identified and thoroughly investigated.20,21 Several studies in PDK-1 knockout cells have emphasized the existence of a distinct Ser473 kinase.22 Cumulative evidence suggests that integrin-linked kinase (ILK), a serine-threonine protein kinase containing 4 ankyrin-like repeats at the N terminus, a central pleckstrin homology (PH)-like domain, and a catalytic domain at C terminus,23–27 is the upstream kinase responsible for Akt phosphorylation on Ser473 in vitro.28–32 Conversely, genetic studies in mouse fibroblasts, Caenorhabditis elegans, and Drosophila revealed that ILK kinase activity may not be required for complete Akt activation.33–36

In the present study, we investigated whether ILK is an upstream kinase mediating virus-induced Akt activation in our viral infection models. We also studied the effect of ILK inhibition on virus replication and progeny release as well as virus-induced CPE, all of which are important events in the pathogenesis of viral myocarditis.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Cell Culture and Virus Infection
HeLa cells (HeLa S3) were obtained from the American Type Culture Collection (ATCC). Mouse atrial cardiomyocyte cell line (HL-1) was established and provided by Dr William Claycomb (Louisiana State University Health Sciences Center, New Orleans). CVB3 (Nancy strain) was a kind gift from Dr Reinhard Kandolf (University Hospital Benjamin Franklin, Berlin, Germany). (See the online data supplement, available at http://circres.ahajournals.org.)

Constructs
Adenoviral constructs encoding constitutively active form of murine Akt1 tagged with the HA epitope (Ad-Myr-Akt1) and control green fluorescence protein (GFP) (Ad-GFP) were kindly provided by Dr Kenneth Walsh (Whitaker Cardiovascular Institute, Boston University School of Medicine, Mass) and Dr Jason Dyck (University of Alberta, Edmonton, Canada) and described previously.37 ILK cDNAs including His-V5–tagged kinase-dead ILK (S343A) and His-V5–tagged kinase-deficient ILK (E359K) have been explained elsewhere31,32 (see the online data supplement).

ILK Inhibition
HeLa and HL-1 cells were pretreated with various doses of specific ILK inhibitors KP392 and QLT0267 (QLT Inc, Vancouver, British Columbia, Canada) for 2 hours before infection. Inhibitor-containing medium was removed and replaced by serum-free medium during virus incubation to avoid any possible interference with virus binding. For ILK RNA inhibition, a 21-base pair double-stranded small interfering RNA (siRNA) molecule targeting the PH domain of ILK or a control nonspecific siRNA were used as previously described.30

Western Blot and Kinase Assay
For Western blot, 40 to 80 µg of extracted protein was fractionated by 9% to 10% sodium dodecyl sulfate-polyacrylamide gels, and protein expression, phosphorylation, and cleavage were measured. Alternatively, 250 µg of protein was used for immunoprecipitation and kinase assay as described previously31,32 (see the online data supplement for more details).

Viral RNA Synthesis and Viral Release
In situ hybridization technique and agar overlay assay were used to measure virus RNA replication and viral progeny release, respectively. (See the online data supplement for a detailed description.)

Statistical Analysis
Two-way analysis of variance with multiple comparisons, and paired Student t tests were performed. Values shown are the mean±SD. A probability value of <0.005 was considered significant.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
CVB3 Infection Enhances ILK Kinase Activity and Akt Phosphorylation in HL-1 Mouse Cardiomyocytes
To characterize the activation dynamics of Akt, mouse cardiomyocytes were grown in Claycomb medium. Confluent HL-1 cells represent the contractile phenotype of adult cardiomyocytes (see Movie in the online data supplement). Cells were infected with CVB3 and cellular extracts were collected at various time points post-infection. CVB3 infection enhanced ILK kinase activity (Figure 1B) and increased Akt phosphorylation on Ser473 and Thr308, with a phosphorylation peak around 4 hours postinfection that coincided with the expression of viral protein (VP1) in infected cells (Figure 1A).


Figure 1
View larger version (36K):
[in this window]
[in a new window]
  		Figure 1. CVB3 infection enhances ILK kinase activity and Akt phosphorylation in HL-1 cells. A, CVB3 enhanced Akt phosphorylation on both Ser473 and Thr308 sites. ß-Actin protein was measured to ensure equal protein loading. Note that Akt phosphorylation coincided with VP1 expression. B, CVB3 infection increased ILK kinase activity. The activity of immuno-precipitated ILK in samples was measured using GSK3ß protein as a substrate. C, ILK inhibition in HL-1 cells blocked virus-induced Akt phosphorylation on Ser473. Cells were pretreated with ILK inhibitors KP392 and QLT0267 for 2 hours. D, ILK inhibition had no effect on virus-induced Akt phosphorylation on Thr308 site.

Virus-Induced Akt Phosphorylation on Ser473 Is ILK Dependent
To examine whether ILK is an upstream kinase regulator of Akt-Ser473 phosphorylation, cells were pretreated with increasing doses of specific small-molecule ILK inhibitors KP392 and QLT0267, and Akt phosphorylation on both sites was assessed. ILK Inhibition significantly blocked Ser473 phosphorylation, while having no effect on Thr308 phosphorylation (Figure 1C and 1D). Similarly, transient overexpression of both kinase-dead (S343A) and kinase-deficient (E359K) mutants of ILK in HeLa cells resulted in considerable inhibition of virus-induced Ser473 phosphorylation (supplemental Figure I).

Inhibition of ILK Significantly Blocks CVB3 Replication
As previously reported, blocking Akt phosphorylation leads to a significant decrease in virus replication in vitro.18 Here, we have assessed the effect of ILK inhibition on various phases of CVB3 replication. ILK inhibitors KP392 and QLT0267 markedly diminished viral protein expression and progeny release in a dose-dependent manner (Figure 2A and 2B). ILK Inhibition by siRNA (100 nmol/L for 96 hours) also reduced viral replication (Figure 2C).


Figure 2
View larger version (25K):
[in this window]
[in a new window]
  		Figure 2. ILK inhibition blocks viral VP1 synthesis and viral release in HL-1 cells. A, ILK inhibitors blocked VP1 synthesis in a dose-dependent manner. Infected HL-1 cells were treated with either DMSO or increasing doses of ILK inhibitors. B, ILK inhibitor markedly decreased virus release. Supernatants from infected cardiomyocyte cultures were collected and assayed for viral release using the agar overlay plaque assay. The data represent 3 independent experiments (*P<0.005). C, ILK siRNA significantly blocked ILK expression and viral VP1 synthesis in mouse cardiomyocytes. HL-1 cells were transfected with increasing amounts of ILK siRNA before virus infection.

Similarly, in infected HeLa cells, ILK inhibition caused a substantial decline in VP1 expression and viral RNA synthesis (Figure 3A and 3B). Furthermore, HeLa cells were also transfected with 2 kinase-inactive mutants of ILK (S343A and E359K). Overexpression of ILK inactive forms substantially blocked virus replication (Figure 3C), which was associated with apparent downregulation of virus-induced Akt phosphorylation (supplemental Figure I).


Figure 3
View larger version (41K):
[in this window]
[in a new window]
  		Figure 3. ILK inhibition significantly blocks viral replication in infected HeLa cells. A, ILK inhibitors blocked viral protein synthesis in a dose-dependent manner. Virus-infected HeLa cells were pretreated with either DMSO or increasing doses of ILK inhibitors. B, ILK inhibitor blocked viral RNA transcription (black arrows) and replication in infected HeLa cells. In situ hybridization of CVB3-infected HeLa cells was performed using a viral strand-specific riboprobe (original magnification, x200). C, Overexpression of the kinase-dead or -deficient mutant of ILK also decreased viral protein (VP1) synthesis.

ILK Inhibition Suppresses Virus-Induced CPE and Enhances Host Cell Viability
In virus-infected cells, excessive viral replication is associated with the activation of apoptotic pathways. We have previously shown that the late virus-induced apoptosis/CPE and the secondary necrosis facilitate viral progeny release, a process necessary for disease progression.13,15 On the other hand, in a variety of experimental models, ILK inhibition has been shown to promote cell cycle arrest and disrupts cell adhesion and migration leading to cell death.28,38

Taking into account that ILK inhibition markedly reduced virus replication, it was important to determine whether inhibition of ILK in virus-infected cells would also induce cardiomyocyte cell death. To examine this hypothesis, we investigated the effect of ILK inhibition on virus-induced CPE and host cell viability. ILK inhibition significantly decreased virus-induced CPE but enhanced the viability of infected cardiomyocytes in a dose-dependent manner, as determined by morphological features and CellTiter 96 AQueous Assay (MTS) (Figure 4A and 4B). Furthermore, fluorescent staining of infected HL-1 cells using the Double Live/Dead staining method confirmed that ILK inhibition resulted in significant decrease in virus-induced cell death (Figure 4C).


Figure 4
View larger version (29K):
[in this window]
[in a new window]
  		Figure 4. Specific ILK inhibitor QLT0267 markedly decreases CVB3-induced cytopathic effects. A, ILK inhibition led to a significant decline in virus-induced CPE. Photomicrographs of HL-1 cells treated with either ILK inhibitor QLT0267 (5 µmol/L) or DMSO before and after virus infection (original magnification, x200). B, ILK inhibition also increased viability of virus-infected cells as measured by MTS assay. C, The number of dead cells (red cells) was reduced in cell culture treated with ILK inhibitor. Virus-infected HL-1 cells, treated with either ILK inhibitor or DMSO, were stained with Cyto-dye and propidium iodide (PI) for 30 minutes. Dead cells were stained with PI (red), whereas live cells were stained with Cyto-dye (green). Original magnification, x200.

Additionally, experiments in HeLa cells using ILK inhibitors also corroborated our findings in mouse cardiomyocytes, suggesting that ILK inhibition was beneficial to virus-infected host cells by overturning virus-induced CPE (supplemental Figure IIA). Notably, inhibition of ILK had no significant effect on virus-induced caspase-3 cleavage in infected HeLa cells, while significantly diminishing cellular cytopathic features (supplemental Figure IIB and IIC), giving more credibility to the previously proposed hypothesis that a caspase-independent pathway is also involved in the process of destructive morphological changes caused by CVB3.13,15

Constitutively Active Form of Akt1 Subverts the Protective Effects of ILK Inhibition in Infected Cardiomyocytes
Previously, we provided evidence that Akt activation is required for a full productive virus replication.18 Here, to investigate the potential causal relationship between the loss of ILK activation, the resulting Akt inhibition, and subsequent suppression of virus-induced CPE, mouse cardiomyocytes were transfected with either a constitutively active form of Akt1 (Ad-Myr-Akt1) or the GFP (Ad-GFP) construct. Transfection efficiency was evaluated by careful screening of Akt phosphorylation, glycogen synthase kinase 3-ß (GSK3-ß) phosphorylation (indication for Akt activity), and HA-tagged protein expression in a serum-starved condition (Figure 5A). To exclude any potential cytotoxic effect caused by adenoviral infection, and to evaluate GFP expression (transfection efficiency), transfected cells were observed at 48 hours posttransfection, using bright-field and fluorescent microscopy techniques (Figure 5B).


Figure 5
View larger version (39K):
[in this window]
[in a new window]
  		Figure 5. Adenoviral transfection of HL-1 mouse cardiomyocytes with a constitutively active form of Akt1. A, Subconfluent cells were infected with adenoviral vector expressing either a constitutively active form of Akt1 (Ad-Myr-Akt1) or GFP (Ad-GFP) at the multiplicity of infection of 100. To ensure transfection efficiency, transfected cells were serum starved for 24 hours, cell lysates were collected, and Akt expression and phosphorylation, GSK3-ß phosphorylation (as indicator of Akt activity), and HA-tagged protein expression were assessed using Western blot analysis. B, Overexpression of the Ad-Myr-Akt1 or GFP protein had no cytotoxic effects on HL-1 cells. Photomicrograph of HL-1 cells showing the morphology of Ad-myr-Akt1–transfected cells at 48 hours following transfection. Note the high expression of GFP in Ad-GFP–transfected HL-1 cells. Original magnification, x200.

In this part of the study, to increase the sensitivity of the assessment, cardiomyocytes infected with either Ad-Myr-Akt1 or Ad-GFP were treated with a low dose of ILK inhibitor QLT0267 (1.5 µmol/L). As shown before, at the above concentration, virus replication is slightly, but not completely, blocked (Figures 2A and 3UpA), providing a condition in which even a slight change in virus replication or cytopathic effects would be detectible. Although overexpression of an active form of Akt1, to some extent increased viral protein expression (Figure 6A), the effect on virus release and cardiomyocytes viability was particularly significant. It was evident that overexpression of Myr-Akt1 overcame the protective effect of ILK inhibition; augmented CVB3 release (Figure 6B), and reduced cellular viability (Figure 6C), indicating an elevated rate of cardiomyocyte death (Figure 6D). Remarkably, Overexpression of active Akt1 had no measurable effect on virus-induced caspase-3 cleavage in CVB3/QLT0267-treated cardiomyocytes (supplemental Figure III). Similar observations were made when Ad-Myr-Akt1 transfected HeLa cells were treated with ILK inhibitor QLT0267 (supplemental Figure IVA and IVB).


Figure 6
View larger version (26K):
[in this window]
[in a new window]
  		Figure 6. Constitutively active form of Akt1 increases viral protein synthesis and virus progeny release and enhances cell death in infected cardiomyocytes. A, In QLT0267-treated HL-1 cells, overexpression of an active form of Akt1 increased viral protein synthesis. HL-1 cells were transfected with either Ad-Myr-Akt1 or Ad-GFP for 48 hours and then treated with ILK inhibitors before CVB3 infection. B, The active form of Akt1 significantly enhanced CVB3 release from QLT0267-treated cells. C, Constitutively active form of Akt1, augmented virus-induced cell death. The viability of QLT-treated HL-1 in the presence or absence of an active form of Akt1 was measured using a standard MTS assay. D, Virus-infected HL-1 cells were stained with Cyto-dye and propidium iodide (PI) for 30 minutes at 16 hours post-infection (dead cells, red; live cells, green). Original magnification, x200.

Inhibition of {alpha}Vß1 and {alpha}Vß3 Integrins With RGD Peptides Does Not Inhibit CVB3 Infection
ILK has been shown to interact with the cytoplasmic domain of the ß1 and ß3 integrins linking extracellular matrix components to cytoplasmic signaling and structural networks.39,40 Because our results characterized a regulatory role for ILK during CVB3 infection, we sought to determine whether integrin subunits also played a role in CVB3 infection. Hence, mouse cardiomyocytes were treated with increasing doses of {alpha}Vß1 cyclic blocking peptide GRGDNP (H-Gly-Arg-Gly-Asp-Asn-Pro-OH) and {alpha}Vß3 cyclic binding peptide XJ735 (Cyclo[-Ala-Arg-Gly-Asp-3-aminomethylbenzoyl]) before infection as well as during virus incubation. Following the infection period, cells were replenished with fresh serum-free medium.

At desired time points postinfection, viral protein expression (indication of virus entry) and virus-induced morphological changes (indication of virus replication) were evaluated. Blocking ß1 and ß3 integrin function with RGD peptides had no effect on virus replication and virus-induced cytopathic effects in infected cardiomyocytes (Figure 7).


Figure 7
View larger version (117K):
[in this window]
[in a new window]
  		Figure 7. Inhibition of {alpha}Vß1 and {alpha}Vß3 integrins with RGD peptides does not inhibit CVB3 infection. A, {alpha}{nu}ß1 and {alpha}{nu}ß3 blocking peptides had no measurable effects on CVB3 replication. HL-1 Cardiomyocytes were treated with increasing doses of GRGDNP ({alpha}{nu}ß1 blocker) and XJ735 ({alpha}{nu}ß3 blocker) cyclic peptides for 1 hour before and during virus incubation. B, Virus-induced cytopathic features in infected cardiomyocytes. Original magnification, x400.

To ensure the effectiveness of RGD peptide treatment, in a separate experiment, cardiomyocytes were treated with both blocking peptides for 24 hours and morphological changes were monitored using bright-field microscopy. The disruption of extracellular matrix/integrin interaction and cellular detachment were apparent in HL-1 culture (supplemental Figure V). Similar results were obtained in HeLa treated with increasing doses of monoclonal blocking antibody against {alpha}Vß3 integrin (supplemental Figure VI).


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
Successful exploration for an alternative therapeutic approach for viral myocarditis necessitates an understanding of virus-host cell interaction and cellular events during viral infection. It is well known that CVB3 directly injures cardiac myocytes before infiltrative inflammatory responses and generally through the activation and subsequent cleavage of the components of apoptotic pathways including caspases.9–11,14,15 During the course of viral infections, viruses release proteins that modify normal cellular function and structure. In response to the assault, host cells may activate prosurvival defenses aimed to preserve cell viability, confront virus-induced cytopathic effect, and delay or avert virus release. How viruses evolve strategies to highjack or delay such defense mechanisms and/or manipulate them to augment their own replication is currently the subject of intense research.

In recent years, extensive efforts have been targeted toward understanding the interplay between pro- and antiapoptotic signaling pathways activated by either virus or the host cell. Activation of the PI3K/Akt, extracellular signal-regulated kinase (Erk) 1/2 mitogen-activated protein kinase (MAPK), and p38 MAPK pathways in CVB3 infection have been mapped and thoroughly studied.9,16,18,41–44 Among them, Akt activation particularly has been shown to be beneficial for virus replication, wherein inhibition of Akt significantly blocks virus replication, apparently through a caspase-independent pathway.18

Akt acts as a key protein mediator for a myriad of distinct cellular responses associated with cell survival, proliferation, differentiation, migration, and apoptosis.16,17,45 Complete Akt activation requires phosphorylation of both Thr308 and Ser437 residues.19 To date, several kinases have been shown to phosphorylate Akt on Ser473. Among them, are the MAPK-activated protein kinase-2 (MK2), p38 MAP kinase, protein kinase C (PKC), and the mammalian target of rapamycin (mTOR). Recently, through several in vitro studies, ILK has also been proposed as a potential upstream kinase responsible for Akt phosphorylation on Ser473.28–32 However, other genetic studies suggest that ILK kinase activity may not be essential for full Akt activation (at least in vertebrates) and that the observed dependency is cell and agonist specific.33–36,39

Increased level of ILK expression has been reported in various cancers, making it a potential and attractive therapeutic target for cancer treatment.39,40,46 However, not much information is available on the potential role of ILK during the episode of viral infections, in general, and enteroviral myocarditis, in particular. Recently, the cDNA microarray and Northern blot analyses of extracellular matrix gene expression in myocardium of mouse infected with CVB3 have shown a 2.4-fold increase in ILK mRNA expression as compared with control group, pointing toward a potential role for ILK in disease progression.47 However, the role and consequence of ILK upregulation in viral myocarditis remains elusive.

Here, we suggest a crucial role for ILK in regulation of CVB3-induced Akt activation in early phase of infection. Our findings in mouse cardiomyocytes (HL-1) and human epithelial cells (HeLa) showed that inhibition of ILK activity and expression by various means significantly blocked virus-triggered Akt phosphorylation on Ser473 without having any effect on Thr308 phosphorylation. ILK inhibition had a major effect on CVB3 life cycle leading to a significant decline in viral RNA transcription, viral protein synthesis, and virus progeny release. All these events eventually rescued cells from virus-induced CPE and considerably improve the viability of infected cells. Overexpression of an active form of Akt1 dramatically reversed the protective effect of QLT0267 in infected cells, indicating that (1) protective effects of ILK inhibition was through down regulation of Akt activation and (2) Akt activation during CVB3 infection was detrimental and disadvantageous to the host cell but was beneficial to virus replication. These findings suggest that the outcome of Akt activation may be highly dependent on cell environment and the type of agonist.

Following its activation, ILK interacts with several components of the focal adhesion complex through its catalytic domain.23,39 This interaction is necessary to establish a strong association between ILK and actin cytoskeleton, leading to cell proliferation and spreading. Inhibition of Rho GTPases such as RhoA, Rac, and Cdc42 as well as of F-actin assembly can block CVB3 replication in Caco-2 cell line because of the inhibition of virus movement toward the tight junction and subsequent movement through the cytosol of infected cells.48 ILK is a crucial regulator of actin rearrangements by activating Rho GTPases and by phosphorylating components of the focal adhesion complex including {alpha}- and ß-Parvin and PINCH.30,49,50 It is therefore possible that ILK may regulate CVB3 movement within the infected cell by modulating focal adhesions and actin cytoskeleton rearrangements, which is crucial for virus movement and replication. Currently, research is ongoing in our laboratory to investigate this hypothesis.

ILK has also been shown to anchor to cytoplasmic tails of integrin ß1 and ß3 regulating cell–cell and/or cell–extracellular matrix interaction in response to various stimuli. Cumulative evidence indicates that integrin subunits may play a role in several viral infections by facilitating virus entry.51–57 There are reports of colocalization of human coxsackievirus-adenovirus receptor (CAR) with integrins {alpha}Vß3 and {alpha}Vß5 in the heart of patients diagnosed with end-stage dilated cardiomyopathy.58 Agrez et al59 have studied the role of integrin subunits in CVB1 infection and shown that overexpression of integrin {alpha}Vß5 enhances CVB1 lytic infection in human colon cancer cells.

Our findings demonstrated that inhibiting ß1 and ß3 integrin function with RGD peptides had no effects on CVB3 entry and replication, as well as virus-induced CPE. However, the data do not rule out the potential involvement of other motifs on ß1 and ß3 integrins as well as a potential role for other integrin subunits in CVB3 entry and replication.

To our knowledge, this is the first report of a potential regulatory role for ILK in a viral infection model. Here, we have provided evidence that ILK plays a critical role in CVB3 pathogenesis, by modulating virus replication and virus-induced cellular injury through an Akt-dependent mechanism. Further in vivo studies using ILK inhibitors and knockout mouse models will also provide valuable information on efficacy of ILK inhibition and should provide a foundation to establish and develop an effective therapeutic approach to treat enteroviral myocarditis.


*    Acknowledgments
 
Sources of Funding

This work was supported by research grants from the Heart and Stroke Foundation of British Columbia and the Yukon (to B.M.M.), the Canadian Institutes of Health Research (to B.M.M. and S.D.), and the National Cancer Institute of Canada (to S.D.). M.E. is a recipient of studentships from the Michael Smith Foundation for Health Research, the Heart and Stroke Foundation of Canada, and the Canadian Institutes for Health Research.

Disclosure

None.


*    Footnotes
 
Original received March 1, 2006; revision received June 2, 2006; accepted June 28, 2006.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Woodruff JF. Viral myocarditis. A review. Am J Pathol. 1980; 101: 425–484.[Medline] [Order article via Infotrieve]
  2. Hosenpud JD, Bennett LE, Keck BM, Boucek MM, Novick RJ. The Registry of the International Society for Heart and Lung Transplantation: eighteenth Official Report-2001. J Heart Lung Transplant. 2001; 20: 805–815.[CrossRef][Medline] [Order article via Infotrieve]
  3. Leonard EG. Viral myocarditis. Pediatr Infect Dis J. 2004; 23: 665–666.[Medline] [Order article via Infotrieve]
  4. Feldman AM, McNamara D. Myocarditis. N Engl J Med. 2000; 343: 1388–1398.[Free Full Text]
  5. Kandolf R, Ameis D, Kirschner P, Canu A, Hofschneider PH. In situ detection of enteroviral genomes in myocardial cells by nucleic acid hybridization: an approach to the diagnosis of viral heart disease. Proc Natl Acad Sci U S A. 1987; 84: 6272–6276.[Abstract/Free Full Text]
  6. Mason JW, O’Connell JB, Herskowitz A, Rose NR, McManus BM, Billingham ME, Moon TE. A clinical trial of immunosuppressive therapy for myocarditis. The Myocarditis Treatment Trial Investigators. N Engl J Med. 1995; 333: 269–275.[Abstract/Free Full Text]
  7. Rose NR, Wolfgram LJ, Herskowitz A, Beisel KW. Postinfectious autoimmunity: two distinct phases of coxsackievirus B3-induced myocarditis. Ann N Y Acad Sci. 1986; 475: 146–156.[Medline] [Order article via Infotrieve]
  8. Huber SA, Lodge PA. Coxsackievirus B-3 myocarditis in Balb/c mice. Evidence for autoimmunity to myocyte antigens. Am J Pathol. 1984; 116: 21–29.[Abstract]
  9. Lim BK, Nam JH, Gil CO, Yun SH, Choi JH, Kim DK, Jeon ES. Coxsackievirus B3 replication is related to activation of the late extracellular signal-regulated kinase (ERK) signal. Virus Res. 2005; 113: 153–157.[CrossRef][Medline] [Order article via Infotrieve]
  10. McManus BM, Chow LH, Wilson JE, Anderson DR, Gulizia JM, Gauntt CJ, Klingel KE, Beisel KW, Kandolf R. Direct myocardial injury by enterovirus: a central role in the evolution of murine myocarditis. Clin Immunol Immunopathol. 1993; 68: 159–169.[CrossRef][Medline] [Order article via Infotrieve]
  11. Chow LH, Beisel KW, McManus BM. Enteroviral infection of mice with severe combined immunodeficiency. Evidence for direct viral pathogenesis of myocardial injury. Lab Invest. 1992; 66: 24–31.[Medline] [Order article via Infotrieve]
  12. Kyto V, Saraste A, Saukko P, Henn V, Pulkki K, Vuorinen T, Voipio-Pulkki LM. Apoptotic cardiomyocyte death in fatal myocarditis. Am J Cardiol. 2004; 94: 746–750.[CrossRef][Medline] [Order article via Infotrieve]
  13. Carthy CM, Yanagawa B, Luo H, Granville DJ, Yang D, Cheung P, Cheung C, Esfandiarei M, Rudin CM, Thompson CB, Hunt DW, McManus BM. Bcl-2 and Bcl-xL overexpression inhibits cytochrome c release, activation of multiple caspases, and virus release following coxsackievirus B3 infection. Virology. 2003; 313: 147–157.[CrossRef][Medline] [Order article via Infotrieve]
  14. Saraste A, Arola A, Vuorinen T, Kyto V, Kallajoki M, Pulkki K, Voipio-Pulkki LM, Hyypia T. Cardiomyocyte apoptosis in experimental coxsackievirus B3 myocarditis. Cardiovasc Pathol. 2003; 12: 255–262.[CrossRef][Medline] [Order article via Infotrieve]
  15. Carthy CM, Granville DJ, Watson KA, Anderson DR, Wilson JE, Yang D, Hunt DW, McManus BM. Caspase activation and specific cleavage of substrates after coxsackievirus B3-induced cytopathic effect in HeLa cells. J Virol. 1998; 72: 7669–7675.[Abstract/Free Full Text]
  16. Song G, Ouyang G, Bao S. The activation of Akt/PKB signaling pathway and cell survival. J Cell Mol Med. 2005; 9: 59–71.[Medline] [Order article via Infotrieve]
  17. Woodgett JR. Recent advances in the protein kinase B signaling pathway. Curr Opin Cell Biol. 2005; 17: 150–157.[CrossRef][Medline] [Order article via Infotrieve]
  18. Esfandiarei M, Luo H, Yanagawa B, Suarez A, Dabiri D, Zhang J, McManus BM. Protein kinase B/Akt regulates coxsackievirus B3 replication through a mechanism which is not caspase dependent. J Virol. 2004; 78: 4289–4298.[Abstract/Free Full Text]
  19. Kobayashi T, Cohen P. Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3-phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2. Biochem J. 1999; 339 (pt 2): 319–328.[CrossRef][Medline] [Order article via Infotrieve]
  20. Lawlor MA, Alessi DR. PKB/Akt: a key mediator of cell proliferation, survival and insulin responses? J Cell Sci. 2001; 114: 2903–2910.[Medline] [Order article via Infotrieve]
  21. Alessi DR, Deak M, Casamayor A, Caudwell FB, Morrice N, Norman DG, Gaffney P, Reese CB, MacDougall CN, Harbison D, Ashworth A, Bownes M. 3-Phosphoinositide-dependent protein kinase-1 (PDK1): structural and functional homology with the Drosophila DSTPK61 kinase. Curr Biol. 1997; 7: 776–789.[CrossRef][Medline] [Order article via Infotrieve]
  22. Williams MR, Arthur JS, Balendran A, van der Kaay J, Poli V, Cohen P, Alessi DR. The role of 3-phosphoinositide-dependent protein kinase 1 in activating AGC kinases defined in embryonic stem cells. Curr Biol. 2000; 10: 439–448.[CrossRef][Medline] [Order article via Infotrieve]
  23. Hannigan GE, Leung-Hagesteijn C, Fitz-Gibbon L, Coppolino MG, Radeva G, Filmus J, Bell JC, Dedhar S. Regulation of cell adhesion and anchorage-dependent growth by a new beta 1-integrin-linked protein kinase. Nature. 1996; 379: 91–96.[CrossRef][Medline] [Order article via Infotrieve]
  24. Brakebusch C, Fassler R. The integrin-actin connection, an eternal love affair. EMBO J. 2003; 22: 2324–2333.[CrossRef][Medline] [Order article via Infotrieve]
  25. Wu C, Dedhar S. Integrin-linked kinase (ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes. J Cell Biol. 2001; 155: 505–510.[Abstract/Free Full Text]
  26. Persad S, Attwell S, Gray V, Delcommenne M, Troussard A, Sanghera J, Dedhar S. Inhibition of integrin-linked kinase (ILK) suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer cells. Proc Natl Acad Sci U S A. 2000; 97: 3207–3212.[Abstract/Free Full Text]
  27. D’Amico M, Hulit J, Amanatullah DF, Zafonte BT, Albanese C, Bouzahzah B, Fu M, Augenlicht LH, Donehower LA, Takemaru K, Moon RT, Davis R, Lisanti MP, Shtutman M, Zhurinsky J, Ben-Ze’ev A, Troussard AA, Dedhar S, Pestell RG. The integrin-linked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP-responsive element-binding protein-dependent pathways. J Biol Chem. 2000; 275: 32649–32657.[Abstract/Free Full Text]
  28. Edwards LA, Thiessen B, Dragowska WH, Daynard T, Bally MB, Dedhar S. Inhibition of ILK in PTEN-mutant human glioblastomas inhibits PKB/Akt activation, induces apoptosis, and delays tumor growth. Oncogene. 2005; 24: 3596–3605.[CrossRef][Medline] [Order article via Infotrieve]
  29. Nho RS, Xia H, Kahm J, Kleidon J, Diebold D, Henke CA. Role of integrin-linked kinase in regulating phosphorylation of Akt and fibroblast survival in type I collagen matrices through a beta1 integrin viability signaling pathway. J Biol Chem. 2005; 280: 26630–26639.[Abstract/Free Full Text]
  30. Attwell S, Mills J, Troussard A, Wu C, Dedhar S. Integration of cell attachment, cytoskeletal localization, and signaling by integrin-linked kinase (ILK), CH-ILKBP, and the tumor suppressor PTEN. Mol Biol Cell. 2003; 14: 4813–4825.[Abstract/Free Full Text]
  31. Persad S, Attwell S, Gray V, Mawji N, Deng JT, Leung D, Yan J, Sanghera J, Walsh MP, Dedhar S. Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343. J Biol Chem. 2001; 276: 27462–27469.[Abstract/Free Full Text]
  32. Delcommenne M, Tan C, Gray V, Rue L, Woodgett J, Dedhar S. Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase. Proc Natl Acad Sci U S A. 1998; 95: 11211–11216.[Abstract/Free Full Text]
  33. Sakai T, Li S, Docheva D, Grashoff C, Sakai K, Kostka G, Braun A, Pfeifer A, Yurchenco PD, Fassler R. Integrin-linked kinase (ILK) is required for polarizing the epiblast, cell adhesion, and controlling actin accumulation. Genes Dev. 2003; 17: 926–940.[Abstract/Free Full Text]
  34. Hill MM, Feng J, Hemmings BA. Identification of a plasma membrane Raft-associated PKB Ser473 kinase activity that is distinct from ILK and PDK1. Curr Biol. 2002; 12: 1251–1255.[CrossRef][Medline] [Order article via Infotrieve]
  35. Mackinnon AC, Qadota H, Norman KR, Moerman DG, Williams BD. C. elegans PAT-4/ILK functions as an adaptor protein within integrin adhesion complexes. Curr Biol. 2002; 12: 787–797.[CrossRef][Medline] [Order article via Infotrieve]
  36. Zervas CG, Gregory SL, Brown NH. Drosophila integrin-linked kinase is required at sites of integrin adhesion to link the cytoskeleton to the plasma membrane. J Cell Biol. 2001; 152: 1007–1018.[Abstract/Free Full Text]
  37. Kovacic S, Soltys CL, Barr AJ, Shiojima I, Walsh K, Dyck JR. Akt activity negatively regulates phosphorylation of AMP-activated protein kinase in the heart. J Biol Chem. 2003; 278: 39422–39427.[Abstract/Free Full Text]
  38. Obara S, Nakata M, Takeshima H, Katagiri H, Asano T, Oka Y, Maruyama I, Kuratsu J. Integrin-linked kinase (ILK) regulation of the cell viability in PTEN mutant glioblastoma and in vitro inhibition by the specific COX-2 inhibitor NS-398. Cancer Lett. 2004; 208: 115–122.[CrossRef][Medline] [Order article via Infotrieve]
  39. Hannigan G, Troussard AA, Dedhar S. Integrin-linked kinase: a cancer therapeutic target unique among its ILK. Nat Rev Cancer. 2005; 5: 51–63.[CrossRef][Medline] [Order article via Infotrieve]
  40. Edwards LA, Shabbits JA, Bally M, Dedhar S. Integrin-linked kinase (ILK) in combination molecular targeting. Cancer Treat Res. 2004; 119: 59–75.[Medline] [Order article via Infotrieve]
  41. Si X, Luo H, Morgan A, Zhang J, Wong J, Yuan J, Esfandiarei M, Gao G, Cheung C, McManus BM. Stress-activated protein kinases are involved in coxsackievirus B3 viral progeny release. J Virol. 2005; 79: 13875–13881.[Abstract/Free Full Text]
  42. Cunningham KA, Chapman NM, Carson SD. Caspase-3 activation and ERK phosphorylation during CVB3 infection of cells: influence of the coxsackievirus and adenovirus receptor and engineered variants. Virus Res. 2003; 92: 179–186.[CrossRef][Medline] [Order article via Infotrieve]
  43. Luo H, Yanagawa B, Zhang J, Luo Z, Zhang M, Esfandiarei M, Carthy C, Wilson JE, Yang D, McManus BM. Coxsackievirus B3 replication is reduced by inhibition of the extracellular signal-regulated kinase (ERK) signaling pathway. J Virol. 2002; 76: 3365–3373.[Abstract/Free Full Text]
  44. Opavsky MA, Martino T, Rabinovitch M, Penninger J, Richardson C, Petric M, Trinidad C, Butcher L, Chan J, Liu PP. Enhanced ERK-1/2 activation in mice susceptible to coxsackievirus-induced myocarditis. J Clin Invest. 2002; 109: 1561–1569.[CrossRef][Medline] [Order article via Infotrieve]
  45. Haslam RJ, Koide HB, Hemmings BA. Pleckstrin domain homology. Nature. 1993; 363: 309–310.[Medline] [Order article via Infotrieve]
  46. Yoganathan TN, Costello P, Chen X, Jabali M, Yan J, Leung D, Zhang Z, Yee A, Dedhar S, Sanghera J. Integrin-linked kinase (ILK): a "hot" therapeutic target. Biochem Pharmacol. 2000; 60: 1115–1119.[CrossRef][Medline] [Order article via Infotrieve]
  47. Zhang ZC, Li SJ, Yang YZ, Chen RZ, Ge JB, Chen HZ. Microarray analysis of extracellular matrix genes expression in myocardium of mouse with Coxsackie virus B3 myocarditis. Chin Med J (Engl). 2004; 117: 1228–1231.[Medline] [Order article via Infotrieve]
  48. Coyne CB, Bergelson JM. Virus-induced Abl and Fyn kinase signals permit coxsackievirus entry through epithelial tight junctions. Cell. 2006; 124: 119–131.[CrossRef][Medline] [Order article via Infotrieve]
  49. Guo L, Wu C. Regulation of fibronectin matrix deposition and cell proliferation by the PINCH-ILK-CH-ILKBP complex. FASEB J. 2002; 16: 1298–1300.[Abstract/Free Full Text]
  50. Zhang Y, Guo L, Chen K, Wu C. A critical role of the PINCH-integrin-linked kinase interaction in the regulation of cell shape change and migration. J Biol Chem. 2002; 277: 318–326.[Abstract/Free Full Text]
  51. Monaghan P, Gold S, Simpson J, Zhang Z, Weinreb PH, Violette SM, Alexandersen S, Jackson T. The {alpha}v{beta}6 integrin receptor for Foot-and-mouth disease virus is expressed constitutively on the epithelial cells targeted in cattle. J Gen Virol. 2005; 86: 2769–2780.[Abstract/Free Full Text]
  52. Wang X, Huang DY, Huong SM, Huang ES. Integrin alphavbeta3 is a coreceptor for human cytomegalovirus. Nat Med. 2005; 11: 515–521.[CrossRef][Medline] [Order article via Infotrieve]
  53. Parry C, Bell S, Minson T, Browne H. Herpes simplex virus type 1 glycoprotein H binds to alphavbeta3 integrins. J Gen Virol. 2005; 86: 7–10.[Abstract/Free Full Text]
  54. Weigel-Kelley KA, Yoder MC, Srivastava A. Alpha5beta1 integrin as a cellular coreceptor for human parvovirus B19: requirement of functional activation of beta1 integrin for viral entry. Blood. 2003; 102: 3927–3933.[Abstract/Free Full Text]
  55. Ciarlet M, Crawford SE, Cheng E, Blutt SE, Rice DA, Bergelson JM, Estes MK. VLA-2 (alpha2beta1) integrin promotes rotavirus entry into cells but is not necessary for rotavirus attachment. J Virol. 2002; 76: 1109–1123.[Abstract/Free Full Text]
  56. Triantafilou M, Triantafilou K, Wilson KM, Takada Y, Fernandez N, Stanway G. Involvement of beta2-microglobulin and integrin alphavbeta3 molecules in the coxsackievirus A9 infectious cycle. J Gen Virol. 1999; 80 (pt 10): 2591–2600.[Abstract/Free Full Text]
  57. Summerford C, Bartlett JS, Samulski RJ. AlphaVbeta5 integrin: a co-receptor for adeno-associated virus type 2 infection. Nat Med. 1999; 5: 78–82.[CrossRef][Medline] [Order article via Infotrieve]
  58. Noutsias M, Fechner H, de Jonge H, Wang X, Dekkers D, Houtsmuller AB, Pauschinger M, Bergelson J, Warraich R, Yacoub M, Hetzer R, Lamers J, Schultheiss HP, Poller W. Human coxsackie-adenovirus receptor is colocalized with integrins alpha(v)beta(3) and alpha(v)beta(5) on the cardiomyocyte sarcolemma and upregulated in dilated cardiomyopathy: implications for cardiotropic viral infections. Circulation. 2001; 104: 275–280.[Abstract/Free Full Text]
  59. Agrez MV, Shafren DR, Gu X, Cox K, Sheppard D, Barry RD. Integrin alpha v beta 6 enhances coxsackievirus B1 lytic infection of human colon cancer cells. Virology. 1997; 239: 71–77.[CrossRef][Medline] [Order article via Infotrieve]

Related Article:

Integrin-Linked Kinase Plays a Key Role in Coxsackievirus Replication
Charles J. Lowenstein
Circ. Res. 2006 99: 346-347. [Full Text] [PDF]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
F. Acconcia, C. J. Barnes, R. R. Singh, A. H. Talukder, and R. Kumar
Phosphorylation-dependent regulation of nuclear localization and functions of integrin-linked kinase
PNAS, April 17, 2007; 104(16): 6782 - 6787.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
C. J. Lowenstein
Integrin-Linked Kinase Plays a Key Role in Coxsackievirus Replication
Circ. Res., August 18, 2006; 99(4): 346 - 347.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow All Versions of this Article:
99/4/354    most recent
01.RES.0000237022.72726.01v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Esfandiarei, M.
Right arrow Articles by McManus, B. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Esfandiarei, M.
Right arrow Articles by McManus, B. M.
Related Collections
Right arrow Infectious endocarditis
Right arrow Apoptosis