Abstract
Respiratory syncytial virus (RSV) infects airway epithelial cells, resulting in cell death and severe inflammation through the induction of NF-kappaB activity and inflammatory cytokine synthesis. Both NF-kappaB 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-kappaB transcriptional activity. These data demonstrate that anti-apoptotic signaling and NF-kappaB 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-kappaB-dependent gene transcription.
Highlights
From the ‡Department of Internal Medicine, University of Iowa College of Medicine and Veterans Administration Medical Center, Iowa City, Iowa 52242
In A549-cultured airway epithelial cells, we have found that simultaneous Respiratory syncytial virus (RSV) infection and phosphatidylinositol 3-kinase (PI 3-K) blockade by chemical inhibition with LY294002 results in premature and exaggerated cell death in comparison to RSV alone
This trend became statistically significant at 24 h (62.4% versus 9.5%, p Ͻ 0.005). These data suggest that chemical blockade of PI 3-K activity during RSV infection results in premature cell death
Summary
Vol 277, No 1, Issue of January 4, pp. 492–501, 2002 Printed in U.S.A. Respiratory Syncytial Virus Inhibits Apoptosis and Induces NF-B Activity through a Phosphatidylinositol 3-Kinase-dependent Pathway*. Respiratory syncytial virus (RSV) infects airway epithelial cells, resulting in cell death and severe inflammation through the induction of NF-B activity and inflammatory cytokine synthesis Both NF-B activity and apoptosis regulation have been linked to phosphatidylinositol 3-kinase (PI 3-K) and its downstream effector enzymes, AKT and GSK-3. AKT has been shown to contribute to NF-B regulation through association with and activation of IB kinase during TNF signaling in 293 cells and platelet-derived growth factor signaling in primary fibroblasts [48, 49] Together, these findings are consistent with the observation that that PI 3-K inhibition can prevent the downstream activation of both AKT and NF-B in pervanadate-stimulated T cells [50]. The PI 3-K/AKT pathway provides multiple potential links between survival or apoptosis signaling and NF-B regulation We have undertaken these studies to define further the mechanisms of inflammatory gene induction and cell death during RSV infection. These observations suggest that RSV activates the PI 3-K/AKT survival pathway, which promotes cellular survival and contributes to NF-B activation
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