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To whom correspondence should be addressed: Division of Pulmonary, Critical Care and Occupational Medicine, C-33 GH, University of Iowa Hospitals and Clinics, Iowa City, IA 52242. Tel.: 319-356-1620; Fax: 319-353-6406
* This work was supported by a Veterans Affairs Merit Review grant, National Institutes of Health Grant E509607 (to G. W. H.), Environmental Protection Agency Grant R826711 (to G. W. H.), and the Childhood Asthma Center, University of Iowa.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Both authors contributed equally to this work.
Respiratory syncytial virus (RSV) infects airway epithelial cells, resulting in cell death and severe inflammation through the induction of NF-κB activity and inflammatory cytokine synthesis. Both NF-κB activity and apoptosis regulation have been linked to phosphatidylinositol 3-kinase (PI 3-K) and its downstream effector enzymes, AKT and GSK-3. This study evaluates the role of PI 3-K and its downstream mediators in apoptosis and inflammatory gene induction during RSV infection of airway epithelial cells. Whereas RSV infection alone did not produce significant cytotoxicity until 24–48 h following infection, simultaneous RSV infection and exposure to LY294002, a blocker of PI 3-K activity, resulted in cytotoxicity within 12 h. Furthermore, we found that RSV infection during PI 3-K blockade resulted in apoptosis by examining DNA fragmentation, DNA labeling by terminal dUTP nick-end labeling assay, and poly(ADP-ribose) polymerase cleavage by Western blotting. RSV infection produced an increase in the phosphorylation state of AKT, GSK-3, and the p85 regulatory subunit of PI 3-K. The activation of PI 3-K by RSV and its inhibition by LY294002 was confirmed in direct PI 3-K activity assays. Further evidence for the central role of a pathway involving PI 3-K and AKT in preserving cell viability during RSV infection was established by the observation that constitutively active AKT transfected into A549 cells prevented the cytotoxicity and apoptosis of combined RSV and LY294002 treatment. Finally, both PI 3-K inhibition by LY294002 and AKT inhibition by transfection of a dominant negative enzyme blocked RSV-induced NF-κB transcriptional activity. These data demonstrate that anti-apoptotic signaling and NF-κB activation by RSV are mediated through activation of PI 3-K-dependent pathways. Blockade of PI 3-K activation resulted in rapid, premature apoptosis and inhibition of RSV-stimulated NF-κB-dependent gene transcription.
). The mechanisms and signaling pathways activated by RSV that result in airway epithelial cell death and inflammation are not completely understood. In this study, we investigated a novel mechanism through which RSV inhibits apoptotic cell death and regulates the activity of nuclear transcription factor NF-κB.
The earliest pathologic findings in RSV-associated bronchiolitis are airway epithelial necrosis and accumulation of inflammatory cells including neutrophils, lymphocytes, and macrophages (
) has demonstrated the direct stimulation of IL-8 synthesis and release from airway epithelial cells by RSV. RSV induces the synthesis and release of IL-8 through activation of multiple transcription factors including NF-κB (
). Therefore, the activation of NF-κB is a central determinant of the inflammatory response provoked by RSV infection.
In addition to its role in regulating cytokine synthesis, NF-κB also functions as an important mediator in apoptotic signaling pathways. Inhibition of NF-κB activation has been associated with increased apoptotic cell death initiated by a variety of stimuli including TNF, ionizing radiation, and chemotherapeutic agents (
). NF-κB appears to mediate the suppression of apoptosis through stimulus-specific induction of inhibitor proteins, including IAP (IAP1, IAP2, and XIAP), TRAF, (TRAF1 and TRAF2), Bcl-2 (A1/Bfl-1 and Bcl-xL), and A20 (
), NF-κB activity promotes apoptosis. Overall, the pro-apoptotic or anti-apoptotic effects of NF-κB appear to be determined by both the type of stimulus and the cell type. The impact of NF-κB up-regulation by RSV with respect to apoptosis has not been categorized in airway epithelial cells.
NF-κB activity is regulated at multiple levels both in the cytoplasm, where it is sequestered in an inactive state, and in the nucleus following translocation. The NF-κB superfamily of transcriptional activators form homodimers or heterodimers composed of subunits of the Rel family (p65/RelA, p50/NF-κB1, p52/NF-κB2, RelB, and c-Rel), which are sequestered in the cytoplasm by the inhibitor proteins IκBα and IκBβ. NF-κB is released and translocates to the nucleus when IκB is phosphorylated by IκB kinase and targeted for degradation (
). In addition to its direct role in apoptosis and metabolic signaling, there is evidence that PI 3-K may play a role in NF-κB regulation. PI 3-K has been shown to mediate NF-κB up-regulation during IL-1 and bradykinin stimulation of cultured epidermoid and airway epithelial cells, respectively (
). Whereas the precise mechanism of NF-κB up-regulation by PI 3-K remains to be identified, the effects may be partially mediated through AKT (protein kinase B). The enzyme activity of AKT, a serine/threonine kinase, is regulated by the phosphoinositide products of PI 3-K both directly by binding of these lipids to its pleckstrin homology domain (
). Furthermore, AKT has been shown specifically to contribute to NF-κB regulation through association with and activation of IκB kinase during TNF signaling in 293 cells and platelet-derived growth factor signaling in primary fibroblasts (
). Additionally, AKT may contribute to NF-κB regulation through p65/RelA phosphorylation as observed in HepG2 cells with IL-1 stimulation, an effect that did not appear dependent on IκB kinase activation and IκB degradation (
). Therefore, the PI 3-K/AKT pathway provides multiple potential links between survival or apoptosis signaling and NF-κB regulation.
We have undertaken these studies to define further the mechanisms of inflammatory gene induction and cell death during RSV infection. Because cultured airway epithelial cells display little or no cytotoxic effects early in the course of RSV infection, we hypothesized that RSV activates cell survival and gene transcription pathways that maintain cell viability until mature viral production has been accomplished. In A549-cultured airway epithelial cells, we have found that simultaneous RSV infection and PI 3-K blockade by chemical inhibition with LY294002 results in premature and exaggerated cell death in comparison to RSV alone. Furthermore, the death produced by concurrent RSV infection and LY294002 has features of apoptosis including characteristic DNA fragmentation and poly(ADP-ribose) polymerase (PARP) cleavage. RSV infection produces an early activation of PI 3-K as measured directly by PI 3-K activity assays and indirectly by phosphorylation of the PI 3-K p85 regulatory subunit. Additionally, the RSV-induced PI 3-K activity correlates with phosphorylation of the downstream effectors, AKT and GSK-3, at regulatory-specific residues. The phosphorylation was blocked by pretreatment with LY294002. The role of the PI 3-K/AKT pathway in preserving cellular viability during infection was further supported by the observation that constitutively active AKT attenuated the cytotoxicity and apoptotic effect of chemical PI 3-K blockade. Finally, PI 3-K inhibition by LY294002 or AKT inhibition by transfection of a dominant negative AKT blocked RSV-induced NF-κB transcriptional activity. These observations suggest that RSV activates the PI 3-K/AKT survival pathway, which promotes cellular survival and contributes to NF-κB activation.
Our observation that RSV does not cause significantly increased cell cytotoxicity until 48 h following inoculation suggested that the virus promotes cellular survival during the early course of infection. Our initial experiments showed that LY294002 pretreatment caused a significant and premature increase in the cytotoxicity associated with RSV. By DNA fragmentation analysis, TUNEL analysis, and PARP cleavage analysis, we further demonstrated apoptosis during RSV infection in the setting of LY294002 treatment. Infection of the cultured cells with an adenoviral vector containing a constitutively active AKT enzyme prevented the cytotoxicity and apoptosis observed under these conditions. By direct kinase activity assays, we have shown that RSV activates PI 3-K within 30 min of infection, an effect that parallels the changes in the phosphorylation status of the PI 3-K p85 regulatory subunits, AKT and GSK-3. From these experiments we have concluded that RSV activates both pro- and anti-apoptotic pathways in A549 cells. The anti-apoptotic effects of RSV during the first hours of infection appear to be mediated through PI 3-K or possibly the downstream mediators, AKT and GSK-3. Under our experimental conditions, the inhibition of the PI 3-K pathway resulted in unopposed pro-apoptotic effects and rapid host cell apoptosis.
Previous studies have demonstrated induction of IL-1β-converting enzyme (ICE) and the transcriptional activator, interferon regulatory factor 1 (IRF-1), within 7 h of infection by RSV in A549 cells (
), the investigators were unable to demonstrate activation of caspase-3 or apoptosis at 36 or 48 h post-infection. RSV has also been found to induce expression of other apoptosis-associated proteins including functionally active Fas (CD95) in A549 cells (
). This study, in contrast to the previous findings, provided evidence that RSV alone resulted in apoptotic cell death at 72 h following infection; this effect was further increased by cross-linking Fas with anti-Fas antibodies (
). Our data suggest that RSV infection of A549 cells produces early anti-apoptotic signaling which, when inhibited, results in RSV-driven apoptosis within 6 h. Our conclusions that RSV possesses both apoptotic and anti-apoptotic properties are consistent with prior observations that RSV induces Fas, ICE, and IRF-1 but does not necessarily result in apoptosis, even within the same cell line. In our system, it is possible that the early effect of PI 3-K activation decreases over time as viral protein synthesis consumes cellular resources and induces cellular response genes. In fact, the phosphorylation of PI 3-K, AKT, and GSK-3 in our experiments was observed to decrease by 24 h post-infection. Thus the early anti-apoptotic effect of PI 3-K activation by RSV in A549 cells may represent a transient response that may not confer apoptosis protection at late time points.
Although the existence of the PI 3-K/AKT pathway of cell survival has been carefully investigated in cytokine and TNF signaling, very little previous work has examined the effects of activation of PI 3-K during active viral infection on cell survival. Here we demonstrated increased kinase activity of PI 3-K and phosphorylation of the regulatory sites on PI 3-K, AKT, and GSK consistent with activation (PI 3-K, AKT) and inhibition (GSK-3) by RSV. Epstein-Barr virus glycoprotein gp350 has been shown to activate TNF-α gene transcription through pathways involving PKC and PI 3-K; however, the effects of this viral gene product on cell cycle and survival were not described (
). Unlike these previous observations, however, activation of PI 3-K by RSV appears to have a different and novel functional significance including the maintenance of cell survival during acute infection and the regulation of cell synthetic response through NF-κB.
LY294002 inhibits phosphatidylinositol (PI) kinases by competitively occupying the ATP-binding site and blocking kinase activity (
). In addition to blocking the p85/p110 PI 3-kinases, however, this compound and the related compound wortmannin have been shown to variably inhibit the activity of other members of the subfamily of PI kinase-related kinases including the targets of rapamycin (TORs), (
). Thus, the effects of LY294002 by itself are not sufficient to establish PI 3-K as the mediating target in human airway epithelial cells. Although the effects of LY294002 on survival could have been mediated through mammalian TOR (mTOR)-dependent pathways, this is unlikely because mTOR is both inhibited by LY294002 (
). If mTOR was the primary survival factor blocked by LY294002, then constitutively active AKT should not have maintained cellular viability following LY294002. Our observations that RSV infection leads to activation and specific regulatory phosphorylation of immunoprecipitated p85/p110 PI 3-K and also that constitutively active AKT prevented LY294002-induced apoptosis suggest that the pathway involving p85/p110 and AKT is responsible for mediating cellular survival during early RSV infection.
The mechanism of activation of NF-κB by RSV has been shown previously to involve regulation of phosphorylation or degradation of the inhibitor peptides IκBα and IκBβ. Bitko and Barik (
) have presented data which demonstrated that the early activation of NF-κB in A549 cells by RSV coincided with the phosphorylation and degradation of IκBα, while at later time points under-phosphorylated IκBβ appeared to contribute to persistent NF-κB activation. The role of IκBα in RSV-mediated NF-κB up-regulation was further established by the observation that the simultaneous transfection of an adenovirus vector containing a mutated, nondegradable form of IκBα and RSV infection blocked NF-κB activation and RANTES production in BEAS2B, normal human bronchial epithelial cells and A549 cells (
) have also presented data from A549 cells showing that IκBα is phosphorylated and degraded within 24 h of RSV infection. In these studies, however, blockade of proteolysis of IκBα was not able to reverse completely RSV-mediated NF-κB activation (
). Thus, RSV activates NF-κB through IκB phosphorylation and degradation, but additional factors may be involved in regulating and maintaining NF-κB activity.
In addition to its role in preservation of cell viability and interference with apoptosis, we have reported evidence that the PI 3-K/AKT pathway is involved in the regulation of NF-κB activity. LY294002 abolished the induction of NF-κB-driven gene transcription during RSV infection. This occurred despite the lack of effect of LY294002 on nuclear translocation and DNA binding of NF-κB as measured by electromobility shift assays. This observation is in contrast to previous studies in A549 cells, which have shown that wortmannin and LY294002 blocked NF-κB activation by bradykinin at the level of nuclear translocation (
). These different results suggest the possible existence of more than one pathway in A549 cells through which PI 3-K activity can modulate NF-κB-dependent gene transcription. In addition to sequestration in the cytoplasm, the transcriptional activity of the p65/RelA NF-κB subunit can be mediated by phosphorylation at its C-terminal transactivation domain (
). We have observed that a dominant negative AKT isoform was also able to block RSV-mediated NF-κB transcriptional activity. Therefore, we can postulate that RSV not only stimulates the nuclear translocation of NF-κB through IκB-mediated effects, which has been shown by other authors, but also that it may impose an additional level of regulation through a phosphorylation pathway involving PI 3-K and AKT.
During PI 3-K blockade, RSV infection results in the early apoptosis of A549 cells. This effect could be mediated by the loss of anti-apoptotic signaling at the level of the downstream kinases AKT or GSK-3 or, alternatively, as the result of failure of NF-κB activation. We have shown that AKT was serine-phosphorylated in a PI 3-K-dependent mechanism during RSV infection. Furthermore, constitutively active AKT prevented the cytotoxicity and apoptosis observed with PI 3-K blockade. Phosphorylation and activation of AKT have been implicated in protection of cells from apoptosis through multiple potential mechanisms, including phosphorylation of the pro-apoptotic Bcl-2 family protein Bad (
). Furthermore, AKT is involved in phosphorylation and inactivation of GSK-3. GSK-3 has been found to have apoptotic effects in staurosporine or heat shock stress of the neuroblastoma cell line SH-SY5Y (
). Finally, the anti-apoptotic effects of RSV could be mediated through NF-κB-driven gene transcription, possibly including induction of synthesis of Bcl-2 proteins. Although the precise mechanism of the anti-apoptotic effect of PI 3-K-AKT–GSK-3–NF-κB pathway in RSV infection remains to be elucidated, the demonstration of apoptosis during its inhibition establishes the functional role of this pathway.
In summary, we have shown that RSV inhibits apoptosis and activates NF-κB through a PI 3-K-dependent pathway (Fig.7). The prevention of apoptosis in airway epithelial cells may function to preserve host cell integrity until the replication phase of the virus is completed. The activation and regulation of NF-κB by RSV has been shown previously to be of critical importance in mediating the inflammatory response to infection. The precipitation of apoptosis and the inhibition of NF-κB transactivation through blockade of PI 3-K suggest that activation of the PI 3-K pathway itself is a critical determinant of the disease manifestation of RSV infection.