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Cell Biology International (2007) 31, 1207–1213 (Printed in Great Britain)
Coxsackievirus B3 affects endothelial tight junctions: Possible relationship to ZO-1 and F-actin, as well as p38 MAPK activity
Yuanrong Jua1, Tao Wangb1, Yang Lic, Wei Xine, Shuyun Wangd** and Jianfeng Lie*
aIntensive Care Unit, Shandong Provincial Hospital, Jinan, PR China
bShandong Provincial Medical Imaging Institute, Jinan, PR China
cDepartment of Medicine, Binzhou Medical College, Yantai, PR China
dDepartment of Pathophysiology, Medical School of Shandong University, Jinan, PR China
eCentral Lab of Shandong Provincial Hospital, Jinan, PR China


Tight junction (TJ) plays a pivotal role in preventing the invasion of pathogens from the blood to extracellular environment. However, the mechanisms by which Group B coxsackievirus 3 (CVB3) can get through TJ from the apical surface still remain obscure. In the present study, the human umbilical vein endothelial cell (HUVEC) was utilized to investigate the alterations in F-actin and ZO-1 status, permeability as well as p38 mitogen-activated protein kinase (MAPK) activity in response to CVB3 by means of fluorescence labeling, flow cytometry, and macromolecule permeability assay. We found that CVB3 was able to induce reorganization of F-actin and redistribution of ZO-1, increase the level of F-actin, and elevate the permeability of FITC-albumin. Moreover, CVB3-mediated the above effects involve in P38 MAPK activation. Our preliminary study indicates that CVB3-induced alteration in permeability may be attributed to disruption of F-actin and ZO-1 organizations and that SB203580, a specific P38 MAPK inhibitor, can reverse these effects. The precise mechanisms underlying the CVB3-mediated effects on HUVECs need to be studied further.

Keywords: Coxsackievirus, HUVEC, F-actin, ZO-1, p38 MAPK.

1Both these authors contributed equally to this work.

*Correspondence to: Central Lab of Shandong Provincial Hospital, Rd 324 Jingwu, 250021 Jinan, PR China. Tel.: +86 531 8518 6912.

**Corresponding author.

1 Introduction

Group B coxsackievirus (CVB) consists of six serotypes, of which CVB3 in particular is considered to be the most frequent cause of human viral heart disease as well as other related disorders (Tracy et al., 1990; Baboonian et al., 1997; Pallansch, 1997). CVB3 infections are characterized by primary viral replication at the portal of entry, such as the nasopharynx and intestine, and then viruses invade the bloodstream leading to viremia (Beck et al., 1990). Subsequently, viruses must either pass through the vascular endothelium by transcytosis or infection or be carried past the endothelial barrier by infected circulating cells, which can finally migrate into the target tissues (Carson et al., 1999). Obviously, CVB3 must induce endothelial barrier dysfunction during the process to cross the endothelium.

Tight junction (TJ), which is formed by endothelial cell at the apical side of endothelia and epithelia, constitutes as a intercellular barrier and intramembrane fence to separate the apical space from the basaolateral membrane and to restrict the diffusion of ions and small molecules (Ohtake et al., 2003; Martin et al., 2004; Fischbarg et al., 2006). The components of TJ are extremely complex that comprise integral membrane proteins, junctional adhesion molecule, and intracellular junction related proteins (Madara, 1998; Nitz et al., 2003; Romero et al., 2003). The integral proteins in combination with membrane-associated proteins constitute the TJ complexes that are anchored to cytoskeleton (Mark and Davis, 2002). The maintenance of TJ depends on the equilibrium of competing contractile and tethering forces created by the cytoskeletal and adhesive proteins (Birukova et al., 2004).

Of these TJ components, zonula occludens-1 (ZO-1) and F-actin, in particular, are recognized to be key elements in the structural organization of TJ complexes. ZO-1 belongs to the family of multidomain proteins pertinent to membrane and cytoskeletal elements at specialized sites of cell-cell contacts (Fanning et al., 1998; Grisendi et al., 1998). Moreover, ZO-1 can directly interact with F-actin, suggesting that ZO-1 may serve as a link between the TJ proteins and the actin cytoskeleton to mediate the anchoring or assembly of actin filaments at TJ (Fanning et al., 1998). Accumulating evidence suggests that disrupting the organization of TJ components, including ZO-1 and F-actin, can affect TJ integrity, which in turn weakens the barrier integrity and consequently, creates a route that favors certain pathogen invasion (Yan et al., 1996; Huber et al., 2001).

To date, however, the precise mechanisms by which CVB3 can get through TJ from the apical surface still remain obscure (Coyne and Bergelson, 2006). Therefore, the present study was designed to investigate the alterations in F-actin and ZO-1 status as well as permeability of HUVECs in response to CVB3, with special attention given to whether or not p38 MAPK signaling pathway participated in modulation of the CVB3-mediated process because p38 activation regulates a variety of fundamental cellular functions.

2 Materials and methods

2.1 Materials

CVB3 (Nancy strain) were originally obtained from the American Type Culture Collection (ATCC). HUVEC was purchased from Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. DMEM was from GIBCO (USA) and fetal calf serum was from Biosource (USA). Rhodamine-phalloidin, which can bind specifically to F-actin due to stabilizing it by binding secondary sequences, or adjacent monomers, was bought from Molecular Probes (Eugene, OR, USA). Rabbit anti-ZO-1-FITC, rabbit-anti-mouse-FITC IgG were purchased form Zymed Laboratories (San Francisco, CA, USA). SB203580, a specific inhibitor of p38, was from Calbiochem (Germany). TRITC-albumin, MTT, HEPES, Triton X-100, and DMSO were purchased from Sigma (St. Louis, MO, USA).

2.2 Cell culture

HUVEC was utilized because the cells can maintain endothelial morphology and characteristics and not differentiate into other type cells. HUVECs were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 100U/ml penicillin, and 100μg streptomycin at 37°C in a humidified atmosphere composed of 95% air and 5% CO2. Cells were regularly subcultured with 0.25% trypsin and seeded into Petri dish or 96-well plate or Transwell Cell Culture Chamber System (diameter, 6.5mm; pore size, 0.4μm, Costar, Cambridge, MA, USA) for different experiments.

2.3 Determination of F-actin and ZO-1 distribution

To determine the distribution patterns of F-actin and ZO-1 in HUVECs in response to CVB3, double immunofluorescent staining were performed and viewed with laser confocal microscope (Cohen et al., 2001). Briefly, cells at a density of 105cells/ml were seeded in Petri dish, allowed to reach confluence. For CVB3 infection, cells were exposed to virus (100 TCID50) for 1h, washed, and then incubated with fresh medium without CVB3. After 12h, 24h, 48h incubation at 37°C, cells were washed twice with PBS, fixed with 3.7% paraformaldehyde in PBS for 20min, and then rinsed with PBS containing 0.3% Triton X-100 for 20min, respectively. Subsequently, 5μl mouse monoclonal antibody to ZO-1 (1:100 dilution) was added, and kept for 1h at 4°C. The specimens were incubated with FITC-goat anti-mouse immunoglobulin (IgG, 1:100 dilution) as well as rhodamine-phalloidin probe for 1h under dark condition. The labeled cells were observed with a laser confocal microscope (Leica TCS SP2, Leica, Germany).

2.4 Measurement of F-actin and ZO-1 levels

To measure the expression level of ZO-1 in HUVECs, the cells were immunostained with monoclonal antibody against the human ZO-1. Briefly, cells were cultured in flasks, treated with CVB3 (100 TCID50) for 1h, and then exposed to fresh medium without CVB3. After 12, 24, 48h incubation at 37°C, cells were harvested, washed twice with PBS, and fixed with 3.7% paraformaldehyde in PBS for 20min, respectively. Subsequently, the samples were rinsed with PBS containing 0.3% Triton X-100, 5μl mouse monoclonal antibody to ZO-1 was added, and kept for 1h at 4°C. Then, the cells (105) were washed with PBS and incubated with FITC-goat anti-mouse immunoglobulin (IgG) for 45min at 4°C and analyzed by flow cytometry. As for the determination of F-actin content, Rhodamine-phalloidin probe was applied to label cells for direct immunofluorescence analysis, and then samples were subjected to flow cytometry.

2.5 Detection of cell permeability

To detect the permeability across the HUVEC monolayer, the cells were cultured in Tanswell plates. When the cells fully reached confluence, experiments were performed. Briefly, cells were either left untreated or stimulated with CVB3 (100 TCID50) alone for 1h or pretreated with SB203580 followed by 1h CVB3 intervention, and then incubated in fresh medium without CVB3 for 12, 24 and 48h, respectively. At indicated time points, the passage of FITC-albumin across the HUVEC monolayer was employed in the present study. In each determination, 100μl (1g/L) FITC-albumin was added to upper compartment of Transwell (the lumenal side) and incubated at 37°C in a 5% CO2 atmosphere for 45min. Subsequently, 100μl sample was taken from upper as well as lower chamber of Transwell (the ablumenal side) respectively, put into dark 96-well microtitre plates, and then determined using a HTS 700 (Japan) plate reader at 485nm with reference at 595nm. The readings were converted with the use of a standard curve to albumin concentration. Meanwhile, the volume in lower chamber of Transwell was calculated. FITC-albumin in samples was quantified in a permeability coefficient (Pa) as expressed in the following equation as previously described (Tinsley et al., 2000):where [A] is the abluminal albumin concentration, t is the time in seconds, A is the area of the membrane in cm2, V is the volume of abluminal side, and [L] is luminal concentration.

2.6 Investigation of P38 MAPK activity

To investigate the role of p38 MAPK in CVB3-mediated alterations on HUBEC, SB203580, a specific p38 MAPK inhibitor, was applied as previously described (Kiemer et al., 2002). Briefly, cells were either left untreated or pretreatment with serial concentrations of SB203580 (0.25μmol/L, 2.5μmol/L, 25μmol/L, 50μmol/L) for 30min followed by CVB3 (100 TCID50) stimulation. The effects of SB203580 on F-actin and ZO-1 distributions, F-actin and ZO-1 levels and cell permeability were explored following the procedures as described above.

2.7 Statistics

Where appropriate, results were expressed as means±SD and were examined for statistical significance by means of Student's t-tests and ANOVA. Value of P<0.05 were considered to be statistically significant. Each experiment was performed at least in triplicate unless stated otherwise.

3 Results

3.1 CVB3 induces reorganization of F-actin and redistribution of ZO-1

Fig. 1 (top panel) displays the normal distribution of intracellular F-actin and ZO-1 in untreated confluent HUVECs. The F-actin and ZO-1 staining in normal control appeared to be smooth continuous staining at intercellular borders of adjacent endothelial cells, which can support our following studies. As also shown in Fig. 1, CVB3 at 100 TCID 50 significantly affected the F-actin and ZO-1distribution in a time dependent manner. When endothelial cells exposed to CVB3 for 12h, the F-actin and ZO-1 patterns displayed local discontinuities. With the increase in incubation time, the changes became more significant. At 24h and 48h time points, cells showed an increase and thickening of F-actin bundles as well as the formation of stress fibers. Labeled ZO-1 showed numerous sawtooth-shaped structures or short spurs, which were often associated with the formation of intercellular gaps. This suggests that the effect of CVB3 on the distribution of intracellular F-actin and ZO-1 was apparent.

Fig. 1

Effects of CVB3 on the redistribution of intracellular F-actin and ZO-1 in HUVECs for indicated time points (from top to bottom). Top panel for normal control, Second panel for 12h, Third panel for 24h, and Bottom panel for 48h. a: F-actin; b: ZO-1; and c: Merged image of a and b.

3.2 CVB3 increases the level of F-actin, but not influences the content of ZO-1

In this study, the contents of F-actin and ZO-1 were expressed as mean fluorescent intensity (MFI). As illustrated in Fig. 2, CVB3 stimulation resulted in a significant increase in F-actin content compared with untreated cells. However, the amount of ZO-1 was not significantly different in cells treated with CVB3 compared with untreated cells.

Fig. 2

Effects of CVB3 on the expression of F-actin and ZO-1 proteins in HUVECs. MFI represented the relative levels of endothelial F-actin and ZO-1 proteins at 12h, 24h, and 48h. *P<0.05 vs 0h (normal control).

3.3 CVB3 increases the permeability of FITC-albumin across HUVEC monolayer

To further explore the effect of CVB3 on monolayer integrity, we measured the clearance of album across the HUVEC monolayer. Fig. 3 showed that there was a significant increase in permeability of FITC-albumin in the CVB3-treated group compared with control monolayer, thereby confirming that CVB3 did affect the HUVEC monolayer integrity.

Fig. 3

Effect of CVB3 on permeability of HUVEC monolayer. Pa represented the relative value of albumin permeability at 12h, 24h, and 48h. *P<0.05 vs 0h (normal control).

3.4 CVB3-mediated the above effects involve in P38 MAPK activation

P38, a key member of MAPKs, has been shown to be activated by various stimuli. Upon activation, it is able to phosphorylate different transcription factors to exert the pivotal roles in signaling transduction pathways involved in the regulation of a diverse range of pathophysiological processes. In order to determine whether P38 MAPK participated in CVB3-mediated effects on the distribution and expression of F-actin and ZO-1 as well as permeability, SB203580, a specific P38 MAPK inhibitor, was employed in this study. In the absence of any other agents, SB203580 alone failed to alter the distribution of F-actin and ZO-1, the basal contents of F-actin and ZO-1, and the permeability of normal endothelial cells (data not shown, n=6).

However, administration of serial concentrations of SB203580 to pretreat HUVECs followed by CVB3 insult reversed the disruption of F-actin and ZO-1 distribution in a concentration-dependent manner. In detail, 0.25μmol/L SB203580 had no effect on attenuating CVB3-induced actin polymerization and ZO-1 redistribution, 2.5μmol/L SB203580 could partly reduce CVB3-induced formation of stress fiber and ZO-1 reorganization, while 25μmol/L and 50μmol/L SB203580 could prevent CVB3-mediated changes of F-actin and ZO-1 (Fig. 4). Moreover, SB203580 was able to attenuate the increased content of F-actin induced by CVB3 in a dose-dependent manner, whereas SB203580 could not affect the amount of ZO-1 protein (Fig. 5A,B). In addition, we found that introduced CVB3 first followed by SB203580 (25μmol/L) could also reverse the changes in F-actin and ZO-1 as well as in permeability.

Fig. 4

Effects of SB203580 on the morphological changes induced by CVB3. a: F-actin; b: ZO-1; and c: merged image of a and b.

Fig. 5

A: Effects of SB203580 on MFI of CVB3 infected endothelial F-actin; and B: effects of SB203580 on MFI of CVB3 infected endothelial ZO-1 proteins. *P<0.05 vs normal control°P<0.05 vs CVB3 control.

We next examined the effect of p38 MAPK inhibition on endothelial permeability by the treatment of endothelial monolayer with SB203580 prior to CVB3 exposure. We found that only 25μmol/L and 50μmol/L SB203580 could reduce CVB3-induced endothelial hyperpermeability. While 0.25μmol/L and 2.5μmol/L SB203580 had no significant effect on CVB3-induced permeability (Fig. 6).

Fig. 6

Effect of SB203580 on permeability of CVB3 infected endothelial cells. *P<0.05 vs normal control.

4 Discussion

At present, it is generally accepted that, during the CVB3 invasive process, the virus must first enter into the circulation, and then leave the circulation though TJ to gain access to its final targets (Carson et al., 1999; Saijets et al., 2003). However, little attention has been given to evaluate the mechanism by which CVB3 directly crosses the TJ to reach the target organs.

Physiologically, TJ has two fundamental functions, i.e. one is a gate function, which controls the paracellular pathway; the other is a fence function, which maintains the cell polarity. Only when the TJ possesses the well-organized structure and tightly-regulated mechanism does the TJ exert its perfect functions. Actually, the functions are not only physiologically regulated but also dynamically modulated by a variety of agents as well as different infectious conditions (Nitz et al., 2003; Kiely et al., 2003). It has been reported that ZO-1, a peripheral phosphoprotein polypeptide localized exclusively to TJ, is able to organize TJ components and link them to the cytoskeleton via the interactions with transmembrane protein as well as cytoskeleton, which in turn modulate the polarity and the paracellular pathway (Fanning et al., 1998; Castillo et al., 2002). Therefore, it is conceivably expected that the structural/functional alterations in TJ complexes by attacking F-actin as well as its related molecules can contribute to infectious processes by diverse kinds of pathogens including different types of viruses.

As the primary cultures of HUVEC can express CVB receptor protein (Carson et al., 1999), which directly or indirectly associates with ZO-1 (Cohen et al., 2001), so, it is plausible to utilize HUVEC monolayers as an experimental model to explore the changes in expression and localization of ZO-1 and F-actin in response to CVB3 infection. In the present study, we clearly demonstrated that the F-actin and ZO-1 patterns displayed local discontinuities when HUVEC exposed to CVB3 for 12h, 24h, 48h compared with untreated cells. Moreover, cells showed the thickening of actin bundles, the formation of stress fibers, and numerous sawtooth-shaped structures which were often pertinent to the formation of intercellular gaps, indicating that CVB3 can directly induce reorganization of F-actin fibers and redistribution of ZO-1, which is physically connected to actin in vascular endothelia, in a time-dependent fashion.

Meanwhile, we found that the expression of F-actin, but not ZO-1, was significantly increased compared with normal cells in a time dependent manner, suggesting that CVB3 may firstly affect F-actin and, consequently, influence ZO-1 distribution via changes in F-actin. In parallel, the alterations in F-actin and ZO-1 were associated with increased albumin permeability across the monolayers. All of these data suggested that F-actin cytoskeleton rearrangement might be fundamental to the changes in cell morphology in response to CVB3, which finally resulted in the permeability alterations. This is consistent with previous studies, which showed that the increased permeability was due to the F-actin reorganization followed by the cell contraction and intercellular gap formation (Lum and Malik, 1996; van Hinsbergh, 1997; Mark et al., 2001; Kiemer et al., 2002; Morigi et al., 2006).

It has been confirmed that activation of p38 mitogen-activated protein kinase (p38 MAPK) via phosphorylation plays a key role in a number of pathophysiological processes including cytoskeletal reorganization (Obata et al., 2000). However, less information is available on whether CVB3-mediated cytoskeletal reorganization has involved p38 MAPK signaling transduction pathway. In the current study, we found that SB203580, a specific inhibitor of p38 MAPK, was able not only to reverse the CVB3-induced F-actin polymerization and ZO-1 redistribution changes, but to decrease the expression of F-actin as well. Additionally, SB203580 could reduce CVB3-induced endothelial permeability. Our data indicate that that activation of p38 MAPK participates in CVB3-induced F-actin reorganization and endothelial permeability.

In conclusion, the present study demonstrates that CVB3-induced alterations in permeability may be attributed to disruption of F-actin and ZO-1 arrangements that lead to an increase in permeability and that SB203580 can reverse these effects. The precise mechanisms underlying the CVB3-mediated effects on HUVEC need to be studied further.


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Received 31 January 2007/12 March 2007; accepted 3 April 2007


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