Respiratory syncytial virus infection induces a reactive oxygen species-MSK1-phospho-Ser-276 RelA pathway required for cytokine expression

Abstract

Respiratory syncytial virus (RSV) is a human pathogen that induces airway inflammation, at least in part, by modulating gene expression programs in airway epithelial cells. The presence of RSV replication is detected by the intracellular retinoic acid-inducible gene I (RIG-I) RNA helicase that forms a productive signaling complex with the mitochondrion-anchored MAVS protein, resulting in nuclear translocation of the NF-kappaB transcription factor. Although nuclear translocation is a prerequisite for activation of the innate inflammatory response, recent studies show that separate pathways governing RelA activation are also required for target gene expression. In this study, we examine the mechanism of RelA phosphorylation and its requirement for RSV-induced gene expression. RSV infection produced a time-dependent RelA phosphorylation on serine (Ser) residues Ser-276 and Ser-536 in parallel with enhanced reactive oxygen species (ROS) stress. Inhibition of RSV-induced ROS inhibited formation of phospho-Ser-276 RelA without affecting phospho-Ser-536 RelA formation. RSV potently induced activation of cytoplasmic mitogen- and stress-related kinase 1 (MSK1) in an ROS-dependent manner. Inhibition of MSK1 using H89 and small interfering RNA knockdown both reduced RSV-induced phospho-Ser-276 RelA formation and expression of a subset of NF-kappaB-dependent genes. Direct examination of the role of phospho-Ser-276 in target gene expression by expression of a RelA Ser-276-to-Ala site mutation in RelA(-/-) mouse embryonic fibroblasts showed that the mutation was unable to mediate RSV-induced NF-kappaB-dependent gene expression. We conclude that RSV induces RelA activation in the innate inflammatory response via a pathway separate from that controlling RelA cytoplasmic release, mediated by ROS signaling to cytoplasmic MSK1 activation and RelA Ser-276 phosphorylation.

FIG. 1.

RSV induces RelA phosphorylation in A549 cells. (A) Human type II alveolar carcinoma A549 cells were infected with RSV (MOI, 1.0) for 0, 6, 15, and 24 h, and WCEs were prepared. Equal amounts of lysate (100 μg protein) were separated on an 8% SDS-PAGE gel, transferred to a polyvinylidene difluoride membrane, and probed with anti-phospho-Ser-276 RelA (p-RelA Ser²⁷⁶) or phospho-Ser-536 RelA (p-RelA Ser⁵³⁶) Abs. The membrane was incubated with anti-RelA Ab (Total RelA) to show equal loading. (B) RSV-infected A549 cells were fractionated into cytoplasmic (Cyt) and nuclear (Nuc) preparations for analysis by Western blotting using the indicated Abs. Each experiment was repeated twice, and data from a representative experiment were shown.

FIG. 2.

RSV induces ROS stress in a time-dependent manner. (A) RSV-induced changes in cellular ROS levels. At the times indicated, RSV-infected cells were loaded with H₂DCF-DA, and changes in DCF fluorescence were assessed by flow cytometry. A.U., arbitrary units. (B) Kinetic changes in GSH oxidation in RSV-infected cells. GSH/GSSG ratios were determined, as described in Materials and Methods. (C, D) RSV-infected cells were treated with DMSO (2%) or NAC (15 mM), and GSH/GSSG ratios were determined. All results are the means ± SEM (n = 4 to 7). *, P > 0.05; **, P = 0.01; ***, P > 0.001.

FIG. 3.

RSV-inducible phospho-Ser-276 RelA formation is antioxidant sensitive. (A) A549 cells preincubated with NAC (15 mM; top) or DMSO (2%; bottom) for 1 h were RSV infected for 15 h (MOI, 1). A Western immunoblot of RelA Ser-276 phosphorylation is shown. Total RelA was assayed as a loading control. p-RelA Ser²⁷⁶, phospho-Ser-276 RelA; p-RelA Ser⁵³⁶, phospho-Ser-536 RelA. (B) Cells treated as described in Fig. 3A were fractionated by SDS-PAGE, and formation of RelA Ser-536 phosphorylation was measured by Western immunoblotting. (C) NAC (15 mM)- or DMSO (2%, vol/vol)-pretreated A549 cells were RSV infected, and total cellular RNA was extracted. Results from the qRT-PCR assay for expression of RSV N transcripts are shown. The bars represent means ± SEM of results from triplicate samples. (D) A549 cells were treated as described in Fig. 3A, and nuclear extracts were prepared. An autoradiogram from an EMSA using the radiolabeled NF-κB duplex is shown. The identities of the NF-κB complexes are indicated (p50/p50 and p50/RelA).

FIG. 4.

RSV-induced NF-κB-mediated chemokine gene expression is antioxidant sensitive. (A) A549 cells treated as described in Fig. 3 were RSV infected at the indicated times prior to total RNA extraction. Measurement of IL-8 mRNA expression by qRT-PCR is shown. Each bar represents the mean ± SEM of results from triplicate samples from an experiment repeated twice. *, P < 0.01; **, P < 0.001. (B) Gro-β expression was measured in the same samples. (C) IκBα expression was measured in the same samples.

FIG. 5.

RSV infection induces MSK1 activity. (A) A549 cells were infected with RSV for 0, 6, 15, and 24 h. Top, 100 μg of WCE was fractionated by SDS-PAGE, and phospho-Ser-376 MSK1 (p-MSK1) formation was determined by Western immunoblotting using a phospho-specific Ab (top, left). TNF-α stimulation (20 ng/ml) was assayed at the indicated times as a positive control (top, right). NS, nonspecific band. The locations of molecular mass markers are shown on the left. Total MSK1 was measured as a loading control. Middle, WCEs from the same experiment were assayed for phospho-Ser-276 (p-RelA Ser²⁷⁶) and total RelA using cognate Abs. Bottom, the ratio of phospho-MSK was determined as a percentage of total MSK1 staining for each blot. (B) RSV-induced MSK1 kinase activity. IP-kinase activity of WCEs from control cells, RSV-infected cells, or RSV-infected cells in the presence of NAC (15 mM) or DMSO (2%). Incorporation of [γ-³²P]ATP into CREBtide is reported as a percentage of that from uninfected WCEs. Each bar is the mean ± SEM of results from three replicates. The experiment was repeated twice. **, P < 0.001. (C) MSK1 subcellular distribution. Cytoplasmic (Cyt) and sucrose cushion-purified nuclear (Nuc) extracts were prepared from RSV-infected A549 cells at the indicated times (MOI, 1). Equal cellular equivalents of protein were fractionated by SDS-PAGE. A Western immunoblot for phospho-Ser-376 MSK1 and total MSK1 abundance is shown. Bottom, the same membrane was probed with lamin B Ab as a marker for nuclear extract integrity.

FIG. 6.

Specificity of MSK1 induction by ROS. (A) A549 cells were stimulated with TNF-α (20 ng/ml) at the indicated times and loaded with H₂DCF-DA, and changes in DCF fluorescence were measured. Relative changes in DCF oxidation by flow cytometry are shown. *, P < 0.01; A.U., arbitrary units. (B) ROS induction by 100 μM H₂O₂. DCF oxidation was measured as described in panel A. (C) Induction of MSK1 by TNF-α and H₂O₂. WCEs were fractionated by SDS-PAGE. Abundance of total MSK1, phospho-Ser-376 MSK1 (p-MSK1), and phospho-Ser-276 RelA (p-RelA Ser²⁷⁶) was measured by Western blotting. n.s., nonspecific band.

FIG. 7.

MSK1 inhibitor H89 inhibits RSV-induced MSK1 phosphorylation and RelA Ser-276 phosphorylation. (A) Cells were preincubated with H89 at the indicated concentrations for 1 h and RSV infected (MOI, 1; 15 h). MSK1 phosphorylation (p-MSK1) and RelA phosphorylation (p-RelA) were determined by Western blotting with specific Abs. The same blots were probed to detect total MSK1 or RelA, respectively. (B) Effect of H89 on RSV-inducible IL-8 expression. A549 cells treated as described in Fig. 6A were extracted for total cellular RNA. The results of qRT-PCR for IL-8 mRNA are shown. *, P < 0.05; **, P < 0.01 (compared to RSV-infected cells); Con, control. (C) Effect of H89 on RSV-inducible Gro-β. The experiment was performed as described in Fig. 6B, but with qRT-PCR testing for Gro-β.

FIG. 8.

Effect of siRNA-mediated MSK1 knockdown. (A) A549 cells were transfected with 100 nM scrambled siRNA (Con siRNA) or siRNA directed against MSK1 (MSK1 siRNA) for 48 h, followed by RSV infection (MOI, 1; 15 h). Top two panels, 100 μg WCE was assayed for phospho-Ser-376 MSK1 (p-MSK1) abundance by Western blotting. A separate blot loaded with extract from the same experiment was probed with anti-phospho-Ser-276 RelA or pan-RelA (p-RelA) Ab. Bottom panel, effect of MSK1 knockdown was determined by Western blotting. Three plates of A549 cells were transfected with 100 nM scrambled or MSK1 siRNA. One hundred micrograms of WCE was assayed for total MSK1 and β-actin staining by Western blotting. (B) A549 cells treated as described in Fig. 7A were extracted for total cellular RNA. IL-8 expression was measured by RT-PCR. Each bar represents the mean ± SEM of results from triplicate samples. (C) Gro-β expression was measured in the samples, as described in Fig. 7B. Each experiment was repeated twice.

FIG. 9.

Requirement of RelA Ser-276 in RSV-inducible cytokine expression. RelA^−/− MEFs stably transfected with eukaryotic expression vectors encoding the RelA WT or RelA Ser-276-to-Ala mutation were RSV infected at the indicated times. Gro-β expression in total cellular RNA was measured by qRT-PCR. Inset, Western immunoblot of RelA WT and RelA Ser-276-to-Ala expression. −, no transfection; WT, WT RelA; 276A, the Ser-276-to-Ala RelA mutant.