Ribosomal Protein S3 Gene Silencing Protects Against Cigarette Smoke-Induced Acute Lung Injury

Abstract

Chronic obstructive pulmonary disease (COPD) is estimated to be the third leading cause of death by 2030. Transcription factor NF-κB may play a critical role in COPD pathogenesis. Ribosomal protein S3 (RPS3), a 40S ribosomal protein essential for executing protein translation, has recently been found to interact with the NF-κB p65 subunit and promote p65 DNA-binding activity. We sought to study whether RPS3 gene silencing could protect against cigarette-smoke (CS)-induced acute lung injury in a mouse model. Effects of an intratracheal RPS3 siRNA in CS-induced lung injury were determined by measuring bronchoalveolar lavage (BAL) fluid cell counts, levels of inflammatory and oxidative damage markers, and NF-κB translocation. Lung RPS3 level was found to be upregulated for the first time with CS exposure, and RPS3 siRNA blocked CS-induced neutrophil counts in BAL fluid. RPS3 siRNA suppressed CS-induced lung inflammatory mediator and oxidative damage marker levels, as well as nuclear p65 accumulation and transcriptional activation. RPS3 siRNA was able to disrupt CS extract (CSE)-induced NF-κB activation in an NF-κB reporter gene assay. We report for the first time that RPS3 gene silencing ameliorated CS-induced acute lung injury, probably via interruption of the NF-κB activity, postulating that RPS3 is a novel therapeutic target for COPD.

Keywords: NF-KB; chronic obstructive pulmonary disease; cigarette smoke; ribosomal protein S3; siRNA.

Figure 1

Gene Silencing Effects of RPS3 siRNA on RPS3 mRNA and Protein Levels in RAW 264.7 and LA-4 Cells (A) Total mRNAs were extracted 24 hr after siRNA transfection (n = 3 separate experiments). (B) Total protein lysates were prepared 24, 48, and 72 hr after siRNA transfection (n = 3 separate experiments per time point). The immunoblot band intensities were analyzed using ImageJ software and normalized to endogenous control β-actin. (C) Effects of RPS3 siRNA on cell viability was detected by MTS assay (n = 6 separate experiments). Veh, Lipofectamine 2000; Con, control siRNA. Values are expressed as means of triplicate ± SEMs. *Significant difference from control siRNA, p < 0.05.

Figure 2

Gene Silencing Effects of RPS3 siRNA on CSE-Induced RPS3 and Inflammatory Biomarker Expressions In Vitro (A) CSE significantly increased RPS3 mRNA expression in RAW 264.7 and LA-4 cell lines, and the RPS3 mRNA levels were significantly downregulated by RSP3 siRNA (n = 6 separate experiments). Gene silencing by RPS3 siRNA significantly decreased CSE-induced gene expression of inflammatory markers in RAW 264.7 cells (B) and LA-4 cells (C). In addition, RPS3 siRNA significantly suppressed CSE-induced increases in supernatant levels of cytokines in RAW 264.7 cells (B) and LA-4 cells (C) (n = 6 separate experiments). CSE upregulated RPS3 mRNA level (D) and RPS3 protein level (E) in BEAS-2B cells (n = 6 separate experiments). Values are shown as means of duplicate ± SEMs of 6 separate experiments. *Significant difference from control siRNA (Con), p < 0.05; ^#significant difference from control media group, p < 0.05.

Figure 3

Regulation of RPS3 Levels by CS and Characterization of RPS3 siRNA In Vivo (A) Representative immunoblots showing the duration of gene silencing effects of RPS3 siRNA (5 nmol) in mouse lungs for 24, 48, and 72 hr after intratracheal administration (n = 3 mice per group). Naive mice are mice without CS exposure. (B) Comparison of serum cotinine levels between sham-air-exposed (SA) and CS-exposed (CS) mice (n = 3 mice per group). (C) A 2-week CS-induced acute lung injury mouse model based on our previous report. Mice were given six consecutive daily intratracheal doses of RPS3 siRNA to silence the RPS3 gene in the lungs. (D) Gene silencing effects of RPS3 siRNA (5 nmol) in CS-exposed mouse lungs (n = 6 mice per group). Control siRNA (Con) refers to 5 nmol non-targeting siRNA. (E) Distribution of Cy5-labeled RPS3 siRNA (5 nmol, red) in CS-exposed mouse lungs 1 hr and 12 hr after intratracheal siRNA administration (n = 3 mice per group). Lung sections were prepared and probed with Alexa Fluor 488-conjugated anti-CD68 antibody and DAPI. Values are shown as means of triplicate ± SEMs. *Significant difference from control siRNA, p < 0.05; ^#significant difference from the SA group, p < 0.05.

Figure 4

Anti-inflammatory Effects of RPS3 siRNA in CS-Induced Acute Lung Injury In Vivo (A) BAL fluid total and differential cell counts from CS-exposed (CS) mice with RPS3 siRNA (1 nmol and 5 nmol) or control siRNA (5 nmol) treatment (n = 9 mice per treatment group). SA, sham air exposed; Eos, eosinophil; Mac, macrophage; Neu, neutrophil; Lym, lymphocyte. (B) Representative H&E-stained lung sections at 200× magnification. Quantitative analysis of epithelium thickness was performed blinded (n = 4 mice per treatment group). Values are shown as means ± SEMs. *Significant difference from control siRNA, p < 0.05; ^#significant difference from the SA group, p < 0.05.

Figure 5

Gene Silencing of RPS3 on BAL Fluid Cytokine and Oxidative Damage Marker Levels and Lung Tissue Inflammatory Gene Expression in CS-Induced Acute Lung Injury (A) BAL fluid cytokine levels of IL-1β, IL-6, KC, IL-12, and TNF-α. SA, sham air exposed; CS, CS exposed. (B) BAL fluid oxidative damage markers 3-NT, 8-isoprostane, and 8-OHdG. (C) Lung tissue inflammatory gene expression modulated by RPS3 siRNA at 5 nmol. The relative quantity of target gene expression was normalized to that of β-actin as an internal control. Values are expressed as means of triplicate ± SEMs of 9 mice per treatment group. *Significant difference from control siRNA, p < 0.05; ^#significant difference from the SA group, p < 0.05.

Figure 6

Effects of RPS3 Gene Silencing on NF-κB Translocation and Activity (A) Nuclear translocation of p65 induced by CSE was captured using immunofluorescence staining in RAW 264.7 cells. Percentage of cells with p65 nuclear staining was quantified (n = 4 separate experiments). (B) Immunoblot of nuclear NF-κB subunit p65 and RPS3 accumulation. Mouse lung nuclear proteins were separated by 10% SDS-PAGE, and probed with anti-p65, anti-RPS3, or anti-TATA binding protein (TBP) mAbS. TBP was used as a nuclear protein loading control (n = 9 mice per treatment group). SA, sham air exposed. CS, CS exposed. (C) Nuclear p65 DNA-binding activity was determined using a TransAM p65 transcription factor ELISA kit (n = 9 mice per treatment group). (D) NF-κB reporter gene assay in NF-κB/SEAP reporter RAW 264.7 cells pre-treated with RPS3 siRNA and then stimulated with CSE. Results are expressed as fold change relative to media control. The SEAP assay was conducted in duplicate with three independent experiments. Values are shown as means of triplicate ± SEMs. *Significant difference from control siRNA, p < 0.05; ^#significant difference from the SA or control media group, p < 0.05.