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Coxsackievirus B (CVB), a member of the enterovirus family, targets the polarized epithelial cells lining the intestinal tract early in infection. Although the polarized epithelium functions as a protective barrier, this barrier is likely exploited by CVB to promote viral entry and subsequent egress. Here we show that, in contrast to nonpolarized cells, CVB-infected polarized intestinal Caco-2 cells undergo nonapoptotic necrotic cell death triggered by inositol 1,4,5-trisphosphate receptor–dependent calcium release. We further show that CVB-induced cellular necrosis depends on the Ca2+-activated protease calpain-2 and that this protease is involved in CVB-induced disruption of the junctional complex and rearrangements of the actin cytoskeleton. Our study illustrates the cell signaling pathways hijacked by CVB, and perhaps other viral pathogens, to promote their replication and spread in polarized cell types.
Enteroviruses, including coxsackievirus B (CVB), are lytic viruses that destroy the host cell membrane for progeny release. Lytic viruses often develop highly efficient strategies to tightly regulate host cell death pathways to avoid killing the host cell prematurely (and terminating viral replication). Studies on the mechanism(s) by which CVB induces host cell death have identified apoptotic signaling, mediated by proapoptotic caspase-3, following CVB infection in HeLa cells (Carthy et al., 1998 , 2003 ; Yuan et al., 2003 ). CVB encounters the polarized epithelium lining the gastrointestinal tract early in infection, and viral replication in the mucosa is likely followed by epithelial cell lysis and subsequent viremia (Morens and Pallansch, 1995 ). The precise mechanism(s) by which CVB induces cell death in polarized epithelial cells remains unclear, however.
Viruses can trigger both apoptotic (programmed cell death) and nonapoptotic (necrosis, autophagy) pathways during the course of infection (Agol et al., 1998 ; Barco et al., 2000 ; Lopez-Guerrero et al., 2000 ). Apoptosis is a tightly controlled process of “cell suicide” that is associated with well-characterized morphological and biochemical changes that include fragmentation of DNA, activation of the caspase family of cysteine proteases, and the translocation of phosphatidylserine from the inner to the outer membrane leaflet. Necrosis generally lacks the changes associated with apoptosis, and is instead characterized by irreversible swelling of the cytoplasm and organelles and ultimate lysis of the plasma membrane. Necrosis results in the release of cellular material (often including degradative enzymes) from the cell into the surrounding area that can induce cellular damage to neighboring non-necrotic cells (reviewed in Festjens et al., 2006 ; Zong and Thompson, 2006 ).
Many pathogens target intracellular signaling molecules to disrupt normal cell function to facilitate many aspects of their infectious life cycles. The link between alterations in Ca2+ homeostasis and cell death pathways has been well established (Trump and Berezesky, 1996 ; Lee et al., 1999 ; Mattson, 2000 ; Sattler and Tymianski, 2000 ; Xu et al., 2001 ; Orrenius et al., 2003 ) and has been used by several viruses to promote their infections (reviewed in Zhou et al., 2009 ). Modulation of intracellular Ca2+ ([Ca2+]i) signaling has been implicated in both pro- and antiapoptotic pathways and is a key component in the activation of necrotic cell death and autophagy (Hoyer-Hansen et al., 2007 ; Hoyer-Hansen and Jaattela, 2007 ; Cardenas et al., 2010 ). Thus levels of Ca2+ are tightly regulated by the cell as a means to control both the induction and inhibition of numerous types of cell death. The release of Ca2+ by inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)-dependent mechanisms can induce apoptosis through mitochondrial transfer of Ca2+ (and the subsequent release of proapoptotic factors into the cytoplasm) or through the activation of Ca2+-sensitive proapoptotic enzymes within the cytoplasm. In some cases, the expression of IP3R is involved for the induction of apoptosis: Reducing IP3R expression in both chicken lymphoma (Sugawara et al., 1997 ) and human T-cells (Jayaraman and Marks, 1997 ) renders them resistant to apoptosis.
The link between Ca2+ release and the activation of necrotic cell death is also striking. Like apoptosis, necrotic cell death is often initiated by dramatic alterations in mitochondrial Ca2+ homeostasis resulting from endoplasmic reticulum (ER)-localized Ca2+ release and the concomitant activation of the Ca2+-activated calpain family of cysteine proteases. Members of the calpain family can be categorized into two subfamilies: μ-calpains (or calpain-1, are activated by micromolar concentrations of Ca2+) and m-calpains (or calpain-2, are activated by millimolar concentrations of Ca2+) (reviewed in Liu et al., 2004 ). After being activated by autocatalytic hydrolysis, calpains translocate from the cytosol to intracellular membranes, where they are primed to cleave a number of diverse substrates. Calpain substrates can include cytoskeletal proteins, adhesion molecules, membrane proteins, kinases, phosphatases, ion transporters, and phospholipases (Rami, 2003 ). The mechanisms by which the cell “decides” to undergo either apoptosis or necrosis may depend on the nature of the insult and/or on the signaling pathways involved in modulating the various cell death pathways.
Here we show that, in contrast to nonpolarized cells, polarized intestinal Caco-2 cells do not undergo apoptosis in response to CVB infection and instead undergo a caspase-independent form of necrotic cell death. Whereas release of progeny virus from nonpolarized cells occurred by a caspase-dependent process, CVB release from Caco-2 cells occurred in a caspase-independent manner and was instead mediated specifically through calpain-2. We further show that CVB infection of Caco-2 cells results in an early (~2 h postinfection [p.i.]) increase in [Ca2+]i that is followed by [Ca2+]i depletion occurring between 2 and 3 h p.i. CVB-induced Ca2+ release required the activity of phospholipase C (PLC) and the expression of IP3Rs, and inhibiting the activity or expression of these factors prevented CVB progeny release. Taken together, these findings indicate that the host cell factors manipulated by CVB to induce cell death and subsequent progeny release may differ among cell types and that CVB exploits PLC-dependent Ca2+ release to promote its escape from polarized intestinal cells.
Proapoptotic signals mediated by activated caspase-3 have been associated with CVB-induced cell death in nonpolarized cell types (Carthy et al., 1998 ; Yuan et al., 2003 ). Consistent with this, we found that CVB infection of HeLa cells led to the activation of caspase-3 (as determined by the appearance of cleaved caspase-3 by immunoblotting) within 3–4 h p.i. (Figure 1A). In contrast, we found no enhancement in cleaved caspase-3 in CVB-infected polarized intestinal epithelial Caco-2 cells (Figure 1A). Both CVB–infected HeLa and Caco-2 cells died within 7–10 h after infection, showing gross morphological changes and loss of membrane integrity as detected by uptake of propidium iodide (PI) (Figure 1B). Whereas CVB-infected HeLa cells displayed changes consistent with apoptosis, including externalization of phosphatidylserine (detectible by Annexin V binding), CVB-infected Caco-2 cells did not exhibit any detectable Annexin V binding (Figure 1B). This effect was not specific to Caco-2 cells as we observed the lack of Annexin V binding to other CVB-infected polarized epithelial cells (HCT-116 and HT-29; Supplemental Figure 1). Moreover, we also found that CVB-infected Caco-2 cells did not exhibit apoptosis-associated DNA fragmentation as assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, whereas infected HeLa cells did (Supplemental Figure 2A). We confirmed that Caco-2 cells were capable of undergoing apoptosis by incubating cells with the apoptosis-inducing agent staurosporine, which led to enhanced Annexin V binding (Supplemental Figure 2B).
We also found that whereas the caspase inhibitor Z-VAD-FMK prevented Annexin V binding and PI uptake in CVB-infected HeLa cells (Figure 1C), it had no effect on PI uptake in CVB-infected Caco-2 cells (Figure 1D). Furthermore, we found that whereas Z-VAD-FMK inhibited viral egress from CVB-infected HeLa cells, it had no effect on CVB release from infected Caco-2 cells (Figure 1, E and andF).F). Taken together, these data indicate that, in contrast to nonpolarized cells, the loss of membrane integrity in CVB-infected Caco-2 cells occurs via a caspase-independent mechanism.
Necrotic cell death fails to display many of the classical features (such as phosphatidylserine externalization) associated with apoptotic cell death, but is still associated with the loss of plasma membrane integrity. The activation of Ca2+-sensitive calpain proteases often contributes to the onset of necrosis (Wang, 2000 ; Liu et al., 2004 ). Because CVB appeared to kill Caco-2 cells by a nonapoptotic mechanism, we tested whether calpain-dependent necrosis might be important. We found that treatment of Caco-2 cells with the calpain inhibitor Z-Val-Phe-CHO inhibited the increase in membrane permeability associated with cell death induced by CVB infection (as assessed by PI uptake) (Figure 2A) but had no effect on CVB replication (Figure 2, A and andB).B). Furthermore, we found that Z-Val-Phe-CHO prevented the release of CVB particles from infected Caco-2 cells but had minimal effect on virus titers (Figure 2C).
To identify the time period during which calpain activity was required to induce CVB-mediated increases in membrane permeability, we performed a time-of-addition experiment in which Z-Val-Phe-CHO was added at various times p.i. We found that Z-Val-Phe-CHO exerted its most potent inhibitory effects when added to CVB-infected cultures before 2–3 h p.i. (Figure 2D). Whereas Z-Val-Phe-CHO partially inhibited CVB-induced PI uptake at 4 h p.i., it had no effect when added at >5 h p.i. (Figure 2D). Consistent with this result, we found a slight elevation in overall calpain activity in cells infected with CVB that peaked at 2–3 h p.i., but returned to control levels by 4 h p.i. (Supplemental Figure 4). Taken together, these data indicate that calpains are likely activated early in CVB infection (~2–3 h p.i.).
We found that calpains were required for alterations in membrane permeability required for CVB egress in Caco-2 cells (Figure 2, A and andC).C). Members of the calpain family can be categorized into two subfamilies: μ-calpains (or calpain-1) and m-calpains (or calpain-2) based on their sensitivity to Ca2+ (reviewed in Liu et al., 2004 ). To determine which member of the calpain family was involved in CVB-induced necrosis, we down-regulated either calpain-1 or -2 expression in Caco-2 cells by RNAi and determined the effects of this on CVB-induced membrane permeability. We found that whereas down-regulation of calpain-1 had no effect on PI uptake in CVB-infected cells, calpain-2 small interfering RNA (siRNA) significantly reduced PI uptake while having no effect on CVB replication (Figure 2E). Furthermore, we found that down-regulation of calpain-2 expression by siRNA transfection significantly inhibited CVB release from infected cells (Figure 2F). These data point to a role for calpains in CVB-mediated escape from infected Caco-2 cells.
Polarized epithelial cells are characterized by the presence of distinct apical and basolateral domains separated by junctional complexes composed in part by the apical tight junction (TJ) complex. We found that CVB infection of Caco-2 cells led to a loss of TJ integrity as assessed by decreased transepithelial resistances (TERs) within 4 h p.i. (Figure 3A) and relocalization of the TJ-associated component zonula occludens-1 (ZO-1) (Figure 3B). Activated calpains target a number of molecules associated with maintaining polarized cell architecture, such as components of apical TJ complexes and components associated with actin cytoskeletal stability (Rios-Doria et al., 2003 ; Franco et al., 2004 ; Lebart and Benyamin, 2006 ). We found that incubation of cells with Z-Val-Phe-CHO inhibited CVB-induced decreases in TER and ZO-1 relocalization (Figure 3, A and andB),B), indicating that this process requires calpain activity. We also found pronounced alterations in the actin cytoskeleton in CVB-infected cells that were characterized by loss of junction-associated actin and increased stress fiber formation (Figure 3C). CVB-induced alterations in actin cytoskeleton architecture were also inhibited by Z-Val-Phe-CHO (Figure 3C), indicating that calpains also mediate the modulation of actin cytoskeletal architecture.
Calpains regulate a variety of actin-dependent cellular processes and have been implicated in the maintenance of cell adhesion and in the control of cellular motility. The direct proteolysis of actin- and junction-associated cellular components is likely central to calpain-dependent regulation of these processes. Calpains have been shown to cleave (in vitro and/or in vivo) the cytoskeletal proteins talin, vinculin, and paxillin and the junction-associated proteins occludin, E-cadherin, and β-catenin (Rios-Doria et al., 2003 ; Franco et al., 2004 ; Lebart and Benyamin, 2006 ; Chun and Prince, 2009 ). Because we observed calpain-dependent alterations in TER and rearrangements of the actin cytoskeleton, we investigated the expression pattern of known calpain substrates in CVB-infected Caco-2 cells. We found that many calpain substrates were unaffected by CVB infection including Ezrin, paxillin, talin, vinculin, and ZO-1 (Figure 4A). We observed the appearance, however, of a distinct cleavage fragment of the TJ-associated transmembrane protein occludin in CVB-infected cultures that could be blocked by treatment of cells with Z-Val-Phe-CHO (Figure 4A). In addition, we found that CVB infection elicited significant decreases in the expression of both β-catenin and E-cadherin that were restored when cells were infected in the presence of Z-Val-Phe-CHO (Figure 4A). We also found that both occludin cleavage and E-cadherin down-regulation occurred in a time-dependent manner in CVB-infected cells and that both events were evident by 3–4 h p.i. but were most prominent at 6 h p.i. (Figure 4B). In contrast, the expression of ZO-1 did not exhibit any significant decreases, nor did we detect any cleavage fragments in response to CVB infection (Figure 4, A and andB).B). Taken together, these data indicate that CVB infection elicits the cleavage and/or down-regulation of several junction-associated membrane components and that calpains likely play a central role in these processes.
We also observed extensive rearrangements of occludin and E-cadherin (unpublished data) localization including a loss of junctional association and corresponding appearance of intracellular occludin-containing vesicles in CVB-infected cells (Figure 4C). The redistribution of occludin was inhibited in CVB-infected cells treated with Z-Val-Phe-CHO, indicating that calpains likely mediate some event in the cleavage and subsequent relocalization of occludin.
As we observed alterations in two components of the junctional complex in CVB-infected Caco-2 cells, we next examined the subcellular localization of calpains in infected cultures. Calpains localize predominantly to the cytosol but, upon exposure to Ca2+, translocate to a variety of intracellular membranes where they undergo autolysis and subsequent activation (Michetti et al., 1996 ; Glading et al., 2001 ; Leloup et al., 2010 ). Relocalization of activated calpains thus serves to facilitate substrate cleavage. We found that CVB infection of Caco-2 cells induced the cleavage and/or down-regulation of β-catenin, E-cadherin, and occludin, all components known to localize to cell–cell junctions. Interestingly, we observed the relocalization of calpain-2 to sites of cell–cell contact and to intracellular vesicles where it colocalized with occludin within 4 h following CVB infection (Figure 4D). We also found that Z-Val-Phe-CHO lost its inhibitory effect when added 2–3 h p.i. (Figure 2D). Taken together, these data indicate that calpain-2 is likely activated before to 4 h p.i. and is then translocated to cellular junctions where it cleaves several components of the junctional complex, thus disrupting junctional architecture, cell–cell contacts, and cell polarity.
Because alterations in [Ca2+]i homeostasis are essential for calpain activation, we next determined the effect of a panel of pharmacological inhibitors known to modulate intracellular calcium signaling on CVB-induced necrosis and identified a number of inhibitors that prevented cell death but had no effect on CVB infection (Figure 5A). These included Bapta-AM, a [Ca2+]i chelator; caffeine, which causes release of Ca2+ stores by activating ryanodine receptors; cyclopiazonic acid (CPA), an inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)-mediated Ca2+ uptake; and thapsigargin, a specific inhibitor of SERCAs that results in depletion of ER-derived Ca2+ stores. In contrast, cyclosporine A (CSA), an inhibitor of the mitochondrial permeability transition pore, had no effect on either necrosis or virus replication (Figure 5A).
These results point to a role for ER-derived release of Ca2+ in CVB-induced necrosis and suggest that the release of mitochondrial Ca2+ stores did not play a significant role in this process. Consistent with this result, we found that 2-APB, an inhibitor of IP3Rs, and U73122, an inhibitor of PLC, also prevented CVB-induced necrosis (Figure 5B).
Release of ER Ca2+ stores was also required for alterations in actin cytoskeletal and TJ integrity, as we found that thapsigargin inhibited CVB-induced reorganization of ZO-1 (Supplemental Figure 3) and occludin (Figure 5C). Furthermore, thapsigargin, U73122, and 2-APB all inhibited CVB release from infected Caco-2 cells (Figure 5D). These data point to a role for PLC-dependent activation and subsequent release of Ca2+ via ER-localized IP3R channels in alterations in membrane integrity required for CVB release from Caco-2 cells.
To better define the time period during which Ca2+ signaling was required to induce necrosis, we performed a time-of-addition experiment in which thapsigargin, U73122, and 2-APB were added at various times p.i. We found that all three drugs maintained their inhibitory effects when added to cells 2–3 h p.i., but lost their effectiveness when added at 4 h p.i. (Figure 5E). Interestingly, these kinetics were similar to the pattern we had observed with a calpain inhibitor (Figure 2D). Taken together, these data suggest that alterations in cellular Ca2+ signaling likely occur early in CVB infection, by 3–4 h p.i.
To more precisely measure the effect of CVB infection on ER Ca2+ stores, we monitored the level of Ca2+ release in response to the addition of thapsigargin at various times p.i. Whereas uninfected control cells exhibited pronounced release of Ca2+ stores upon exposure to thapsigargin (Figure 6, A and andB),B), cells infected with CVB for 3 h displayed a marked decrease in Ca2+ release in response to thapsigargin (Figure 6, A and andB).B). At 2 h p.i., we also observed a decrease in Ca2+ release compared with uninfected controls (Supplemental Figure 5). After 3 h p.i., we were unable to measure Ca2+ levels in CVB-infected cells because necrosis-induced increases in membrane permeability made it impossible to load cells with calcium-sensitive dye.
Next we monitored real-time Ca2+ signaling in cells infected with CVB. As we had observed a reduction in thapsigargin-sensitive Ca2+ release in CVB-infected cells at 3 h p.i., we monitored real-time changes in [Ca2+]i over the course of infection over 1 h (spanning from 2 to 3 h p.i.). We found that whereas Ca2+ levels remained stable in uninfected cells over the course of 1 h, there was a pronounced decrease in Ca2+ in CVB-infected cells between 2 and 3 h p.i. (Figure 6, C and andD).D). These data indicate that alterations in ER Ca2+ stores occur early in CVB infection (between 2 and 3 h p.i).
To determine whether CVB-induced depletion of ER Ca2+ stores involved IP3 production and/or IP3Rs, we tested the effects of pharmacological inhibitors of PLC (U73122) and IP3R function (2-APB). Cells were treated with inhibitors and infected with CVB for 3 h, then thapsigargin was added and ER-derived Ca2+ release was measured. In the absence of inhibitors, CVB-infected cells exhibited significantly smaller changes in [Ca2+]i in response to thapsigargin than did uninfected controls (Figure 7, A and andB).B). In contrast, after treatment with U73122 or 2-APB, the response to thapsigargin was the same in both CVB-infected and uninfected control cells (Figure 7, A and andBB).
To further define the role of IP3Rs in CVB-induced alterations in [Ca2+]i, we transfected cells with siRNAs targeted against IP3R-1 and -3, which are expressed in Caco-2 cells (there were no detectable levels of IP3R-2 found in these cells; unpublished data) and measured the effect of combined IP3R-1 and -3 depletion on thapsigargin-sensitive Ca2+ stores in CVB-infected cells. Similar to our findings with 2-APB, we found that transfection of cells with IP3R-1 and -3 siRNAs prevented the depletion of thapsigargin-sensitive ER Ca2+ stores in CVB-infected cells (Figure 7, C and andD).D). Moreover, we found that depletion of IP3R-1 and -3 expression by RNAi prevented the release of CVB progeny from infected Caco-2 cells but had no effect on virus titers (Figure 7, E and F).
Although pathogens likely hijack multiple host cell signaling cascades during infection, the precise cascade of signals that culminates in the release of enteroviruses from host polarized cells have remained unclear. Here we show that, in polarized intestinal epithelial cells, CVB infection leads to the release of ER-derived Ca2+ to induce a cascade of events resulting in the destruction of the host cell membrane as a means to promote progeny release. CVB specifically exploits IP3R-dependent Ca2+ release to induce nonapoptotic necrotic cell death and involves the activation of the Ca2+-activated protease calpain-2. Activated calpain-2 is involved in the cleavage and/or down-regulation of β-catenin, E-cadherin, and occludin, all components of epithelial TJs and cell–cell contacts, which likely contributes to CVB-induced loss of cell polarity and adhesion. These findings point to a novel role for [Ca2+]i signaling in the release of CVB from polarized epithelia and suggest that the signals necessary for multiple steps in the virus life cycle may differ between polarized and nonpolarized cells types.
Alterations in cellular Ca2+ homeostasis play a fundamental role in the response of many tissue types to injury or assault. We found that CVB infection induced the depletion of thapsigargin-sensitive Ca2+ stores by 3 h p.i. and that several pharmacological inhibitors targeting [Ca2+]i signaling lost their inhibitory effects between 2 and 3 h p.i. Other investigators have reported that the CVB protein 2B perturbs Ca2+ homeostasis in HeLa cells by directly modifying ER membrane permeability to promote virus release (van Kuppeveld et al., 1997 ). The effects we observe on Ca2+ homeostasis occurred by 2–3 h p.i., a time before the detection of newly synthesized viral RNA and protein (which generally occurs at ~4 h p.i. in Caco-2 cells; Figure 1A and unpublished data), which would indicate that low levels of 2B are likely present at these times. Moreover, our observations suggest that the depletion of ER Ca2+ stores induced by CVB infection of Caco-2 cells involves the initiation of PLC-dependent signaling cascades, as we observed that pharmacological inhibitors of PLC, an IP3R antagonist (2-APB), and RNAi-mediated down-regulation of IP3R-1 and -3 inhibited CVB-induced Ca2+ release. Although CVB 2B may contribute to further Ca2+ alterations induced at later stages of CVB replication, our data indicate that PLC-dependent activation of IP3Rs contributes primarily to the release of Ca2+ required to initiate necrosis and facilitate calpain activation in Caco-2 cells.
Calpains are ubiquitously expressed, Ca2+-dependent proteases that are categorized based on the Ca2+ concentration required for their activation. Our results indicate that calpain-2 plays a key role in promoting CVB-induced host cell necrosis, as inhibition of calpain activity or RNAi-mediated silencing of calpain-2 prevented CVB egress from polarized epithelial cells. Interestingly, apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii, have also been shown to use calpains to facilitate their egress from infected cells, although the mechanism by which they activate these proteases remains unclear (Chandramohanadas et al., 2009 ). Our data indicate that calpain activity is required to induce a loss of polarity and junctional integrity that accompanies CVB infection of Caco-2 cells, indicating that activated calpains target key components of epithelial architecture during the course of CVB infection. Although a recent study has implicated a role for calpains in echovirus replication (Upla et al., 2008 ), we found no evidence that calpains were specifically involved in CVB replication in Caco-2 cells as evidenced by our finding that calpain inhibitors and calpain depletion with siRNAs did not decrease titers of newly replicated virus. Although we have previously shown that calpain 2 regulates CVB trafficking in polarized endothelial cells (Bozym et al., 2010 ), we did not observe any effects of calpain inhibition on CVB entry into Caco-2 cells, suggesting that the role of calpains in facilitating CVB infection may differ between polarized cells types.
Polarized epithelial cells regulate the flow of ions and macromolecules across the epithelium by the presence of junctional complexes located at their apicolateral poles (Schneeberger and Lynch, 2004 ). We found that CVB infection induced a loss of junctonal integrity (as assessed by decrease in TER) within 4 h p.i. and that this change required calpain activity. We also found that CVB infection led to the relocalization of ZO-1, occludin, and several other junction-associated components such as β-catenin and E-cadherin (unpublished data). In addition, we found that occludin undergoes cleavage in CVB-infected cells and that both β-catenin and E-cadherin expression levels are diminished by CVB infection. These events require calpain activity as they were all blocked by a pharmacological inhibitor of calpains. Cleavage of occludin by calpains is also important in leukocyte transmigration across polarized airway epithelium (Chun and Prince, 2009 ), and calpains target both β-catenin (Benetti et al., 2005 ) and E-cadherin (Rios-Doria et al., 2003 ; Chun and Prince, 2009 ).
The relocalization of calpains from the cytoplasm to intracellular membranes has been speculated to play an important role in their activation (Leloup et al., 2010 ). Consistent with this finding, attachment of a farnesyl anchor to calpain-2 triggers the strong induction of calpain activity (Leloup et al., 2010 ). We found that calpain-2 relocalized from the cytoplasm to both the junctional complex and to internalized occludin-containing vesicles within 3 h following CVB infection. Interestingly, this time point corresponded with a significant decrease in [Ca2+]i levels in CVB-infected cells and the loss of an inhibition of necrosis by small molecule inhibitors of calpains and those that disrupt [Ca2+]i signaling. Taken together, these data suggest that calpains are activated within 2–3 h p.i. in CVB-infected cells that likely initiate a cascade of events, including cleavage of cell–cell contacts and junctional components, that ultimately leads to loss of membrane integrity and virus escape.
The mechanism of cell death we observed in several polarized intestinal epithelial cell lines differs markedly from the apoptotic cell death induced by CVB in nonpolarized cells [such as HeLa cells (Carthy et al., 1998 , 2003 ; Yuan et al., 2003 ), pancreatic β-cells (Rasilainen et al., 2004 ), and neurons (Joo et al., 2002 ; Feuer et al., 2003 )]. The reasons for the induction of divergent cell death pathways between polarized and nonpolarized cells are likely complex and may be due to inherent differences in Ca2+ regulation and/or signaling (such as in differences in the expression and cellular localization of IP3Rs; Colosetti et al., 2003 ) in the expression, localization, and/or activation of innate immune-associated components (Hershberg, 2002 ; Cario et al., 2007 ) or in the expression of signaling molecules that are associated with the regulation of cell death. Another possibility is that the composition and/or regulation of paracellular junctions of polarized epithelia play key roles in the regulation of cell death pathways in response to microbial assault. For example, as enterocytes display high rates of turnover, they have developed highly regulated means of cell death regulation, which have been linked to the integrity of their cell–cell contacts and most notably to the presence of E-cadherin. For example, loss of E-cadherin localization at epithelial junctions induces anoikis (Fouquet et al., 2004 ; Lugo-Martinez et al., 2009 ), a form of apoptosis induced by loss of cell–matrix interactions. Interestingly, the expression of a calpain-mediated cleavage fragment of E-cadherin potentiates cell death in epithelial cells (Rios-Doria and Day, 2005 ), and cleavage of the β-catenin binding domain of E-cadherin is associated with the induction of apoptosis (Vallorosi et al., 2000 ). We observed that cleavage of occludin and decreases in E-cadherin and β-catenin expression occurred in a calpain-dependent manner in CVB-infected Caco-2 cells. Although we do not know whether these events are responsible for the induction of necrosis in CVB-infected polarized epithelia or are merely nonspecific events associated with calpain activation, it is attractive to speculate that the divergent mechanism of cell death observed in polarized versus nonpolarized epithelial cells may be the result of calpain-mediated cleavage of junction-associated components that serve to induce alternative mechanisms of cell death.
The results of our study indicate that cell death in CVB-infected Caco-2 cells occurs by a caspase-independent mechanism that instead depends on the activity of PLC, subsequent release of [Ca2+]i from IP3Rs, and the activation of calpains. These findings illustrate the unique mechanisms by which enteroviruses, and perhaps other viral pathogens, coopt intracellular signaling pathways in polarized cell monolayers to promote their entry, replication, and eventual spread.
Caco-2 (ATCC) and HeLa (CCL-2) cells were cultured in modified Eagle's medium (MEM) supplemented with 10% fetal bovine serum, nonessential amino acids, sodium pyruvate, and penicillin/streptomycin. Caco-2 (BBE clone) cells were grown in DMEM-H supplemented with 10% fetal bovine serum and penicillin/streptomycin. CVB3-RD was expanded and purified as described previously (Coyne and Bergelson, 2006 ). Unless otherwise stated, all infections were performed with a multiplicity of infection (MOI) of 1–5 particle-forming units (PFU)/cell.
Mouse anti–enterovirus VP1 (Ncl-Entero) was obtained from Novocastra Laboratories (Newcastle upon Tyne, UK). Alexa Fluor–conjugated secondary antibodies, PI, phalloidin, mouse or rabbit anti–ZO-1, -occludin, or -β-catenin, and Annexin V were purchased from Invitrogen (Carlsbad, CA). Rabbit anti–caspase-3 and E-cadherin antibodies were purchased from Cell Signaling (Danvers, MA). Mouse anti-paxillin antibody was purchased from Abcam (Cambridge, MA); mouse anti-talin and mouse anti-vinculin antibodies were purchased from Sigma (St. Louis, MO); and goat anti–calpain -1 and -2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
CSA (5 μM), Z-Val-Phe-CHO (5 μM), and Z-VAD-FMK (20 μM) were purchased from Calbiochem (Gibbstown, NJ); U73122 (1 μM), Bapta-AM (10 μM), 2-APB (30 μM), caffeine (1 mM), CPA (10 μM), ruthenium red (5 μM), and thapsigargin (3 μM) were purchased from Sigma.
TER measurements were performed using an ohmmeter (EVOM; World Precision Instruments, Sarasota, FL) on cells grown on Transwell-collagen (COL) inserts for a minimum of 3 d. When cells exhibited RT values of at least 600 Ω-cm2, monolayers were infected with CVB (5–10 PFU/cell) at 37°C. At the indicated times, cells were removed and TER measured. All measurements were background corrected using a blank insert without cells.
Caco-2 monolayers grown in collagen-coated chamber slides (BD Biosciences, San Jose, CA) were exposed to CVB at the indicated MOIs for 8 h (or the indicated time) at 37°C. The cells were then washed and fixed with ice-cold methanol or paraformaldehyde and permeabilized with Triton X-100. Monolayers were incubated with primary antibody, washed, and incubated with Alexa Fluor–488– or -594–conjugated secondary antibodies, washed, and then mounted with Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI). PI and Annexin V staining was performed following the manufacturer's protocol. Briefly, cells were washed with phosphate-buffered saline (PBS), Annexin V-488 conjugate, and/or PI were added in annexin binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) for 15 min at room temperature. Cells were rinsed in annexin binding buffer and then fixed with methanol and mounted with Vectashield containing DAPI. Images were captured with an Olympus IX81 inverted microscope equipped with a motorized stage or with an Olympus (Tokyo, Japan) Fluoview 1000 laser scanning microscope. Images of infected cells (PI and Annexin V) were taken using an Olympus Pan Apo 10×/0.28 NA dry objective, whereas all other images were taken with an Olympus PlanApo 60×/1.42 NA oil objective. Quantification of percent positive cells was performed using ImageJ (National Institutes of Health) analysis, where a minimum of three fields per condition were counted (at least 600 cells total). Cells were counted via DAPI staining, and positive cells were counted in the appropriate channel for the condition.
Cells grown on collagen-coated, glass-bottom, 35-mm dishes (MatTek, Ashland, MA) were loaded with Fura-2 AM (1 μM; Invitrogen) for 30 min at 37°C. Cells were rinsed three times and bathed in a final volume of 1 ml of Ca2+- and Mg2+-free PBS. Images were captured on an Olympus IX81 motorized inverted microscope equipped with a Hamamatsu (Shizuoka, Japan) Orca-R2 CCD camera, a Sutter Lambda (Novato, CA) 10-3 High Speed filter wheel system, and an Olympus UApo/340 20× objective with an NA of 0.75 . Images were acquired using Slidebook 5.0 advanced imaging software. Selected cells were chosen [60 regions of interest (ROI)/dish], and images were captured at both excitation 340 and 380 nm every 10 s for 10 min (experiments were performed a minimum of three times). Thapsigargin was added to dishes after baseline was established (t = 50 s). Intensity ratios for selected ROIs were calculated using Slidebook 5.0, and representative traces for each experiment were plotted as a function of time (Figures 6A and 7, A and andC).C). Images were pseudocolored (using Slidebook 5.0) to better visualize [Ca2+]i mobilization with blue (low Cai2+) and red (high Cai2+). Overall changes in fluorescence intensity ratio (340/380) were calculated by subtracting the resting intensity ratio from the maximum intensity ratio achieved after thapsigargin addition.
Caco-2 cells were grown to confluence on collagen-coated, glass-bottom, 35-mm dishes as stated earlier in the text. For virus experiments, CVB was added to the dish at MOI = 5 and placed at 37°C for 1.5 h. At this time, Fura-2 AM was added to the dish, and infection was allowed to continue for 30 min at 37°C. Medium was then removed, and cells were washed three times and placed in a final volume of 1 ml of Ca2+/Mg2+-free PBS. Dishes were placed on a 37°C temperature-controlled stage insert (Bioptechs, Butler, PA) mounted over an Olympus IX81 microscope (described in detail earlier in the text). Images were captured (from ~60 ROIs per experiment) at both 340 and 380 nm every 10 min for 1 h. Intensity ratios were calculated using Slidebook 5.0, with representative traces plotted as a function of time (Figure 6C). For uninfected control experiments, cells were incubated for 1.5 h in the absence of CVB, incubated with Fura-2AM for 30 min at 37°C, rinsed, and imaged as described earlier in the text.
siRNAs against calpain-1 (5′-GGCAGCUUUCGCUUGUUCCtt-3′) and calpain-2 (5′-GGCAGCUUUCGCUUGUUCCtt-3′) were synthesized by Integrated DNA Technologies (Newark, NJ). siRNAs to IP3R-1 and -3 have been described (Bozym et al., 2010 ). Cells were transfected using HiPerfect (Qiagen, Chatsworth, CA), according to the manufacturer's protocol or were delivered by nucleofection with an Amaxa nucleofection device (solution T, program B-24).
Data are presented as mean ± SD. One-way analysis of variance and Bonferroni's correction for multiple comparisons were used to determine statistical significance (p < 0.05 or < 0.001).
We are grateful to Kevin Foskett for helpful advice and suggestions. This work was supported by funding from the National Institutes of Health (R01AI081759 to C.B.C. and R01AI52281 to J.M.B.).
This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E11-02-0094) on July 7, 2011.