Oxidative stress induced interleukin-32 mRNA expression in human bronchial epithelial cells

Abstract

Background: Chronic obstructive pulmonary disease (COPD) is characterized by airflow obstruction and persistent inflammation in the airways and lung parenchyma. Oxidative stress contributes to the pathogenesis of COPD. Interleukin (IL)-32 expression has been reported to increase in the lung tissue of patients with COPD. Here, we show that IFNγ upregulated IL-32 expression and that oxidative stress augmented IFNγ-induced-IL-32 expression in airway epithelial cells. We further investigated transcriptional regulation responsible for IFNγ induced IL-32 expression in human airway epithelial cells.

Methods: Human bronchial epithelial (HBE) cells were stimulated with H2O2 and IFNγ, and IL-32 expression was evaluated. The cell viability was confirmed by MTT assay. The intracellular signaling pathways regulating IL-32 expression were investigated by examining the regulatory effects of MAPK inhibitors and JAK inhibitor after treatment with H2O2 and IFNγ, and by using a ChIP assay to identify transcription factors (i.e. c-Jun, CREB) binding to the IL-32 promoter. Promoter activity assays were conducted after mutations were introduced into binding sites of c-Jun and CREB in the IL-32 promoter. IL-32 expression was also examined in HBE cells in which the expression of either c-Jun or CREB was knocked out by siRNA of indicated transcription factors.

Results: There were no significant differences of cell viability among groups. After stimulation with H2O2 or IFNγ for 48 hours, IL-32 expression in HBE cells was increased by IFNγ and synergistically upregulated by the addition of H2O2. The H2O2 augmented IFNγ induced IL-32 mRNA expression was suppressed by a JNK inhibitor, but not by MEK inhibitor, p38 inhibitor, and JAK inhibitor I. Significant binding of c-Jun and CREB to the IL-32 promoter was observed in the IFNγ + H2O2 stimulated HBE cells. Introducing mutations into the c-Jun/CREB binding sites in the IL-32 promoter prominently suppressed its transcriptional activity. Further, knocking down CREB expression by siRNA resulted in significant suppression of IL-32 induction by IFNγ and H2O2 in HBE cells.

Conclusion: IL-32 expression in airway epithelium may be augmented by inflammation and oxidative stress, which may occur in COPD acute exacerbation. c-Jun and CREB are key transcriptional factors in IFNγ and H2O2 induced IL-32 expression.

Figure 1

Cell viability and IL-32 expression in IFNγ and H₂O₂ + IFNγ stimulated HBE cells. HBE cells were incubated with 10 ng/ml IFNγ or 250 μM H₂O₂ and 10 ng/ml IFNγ for 48 hours. The viability of the cells was examined using MTT assay (A). HBE cells were incubated with or without 2 hour-H₂O₂ pretreatment and then with or without IFNγ stimulation for 0, 4, 8, and 24 hours. The expression levels of IL-32 mRNA adjusted with expression levels in non-stimulated cells in each time point were determined by quantitative real-time PCR. HBE cells stimulated with H₂O₂, with IFNγ, and with IFNγ and H₂O₂, were presented in square plots, triangle plots, and inverted-triangle plots, respectively in graph (B). The bars show the means ± SE from data performed on 3 different individuals. *p < 0.01 significantly different. (C) After 48 hours of IFNγ treatment 20 μg of cell lysates were subjected to Western blotting. The 22 kDa band represents IL-32β/δ protein, and the 26 kDa band represents IL-32γ protein. The results shown are representative of 3 independent experiments.

Figure 2

Influence of MAPK inhibitors on H₂O₂ + IFNγ induced IL-32 mRNA expression in HBE cells. After treatment with the JNK inhibitor, the MEK inhibitor, the p38 inhibitor for 24 hours, IL-32 mRNA expression in H₂O₂ and IFNγ stimulated HBE cells (A) and the effect of JNK inhibitor (B) or JAK inhibitor I (C) upon IL-32 expression stimulated by IFNγ with or without H₂O₂ in HBE cells were examined by quantitative real-time PCR. All mRNA quantities were adjusted to the quantities at 0 hour control without stimulation. In graph (B) and (C), the closed bars represent the results of vehicle control and the open bars represent the results of JNK inhibitor (B) and JAK inhibitor (C). The bars show the means ± SE from 3 different individuals. *p < 0.05 significantly different.

Figure 3

Transcription factors that bind to the IL-32 gene promoter. HBE cells were stimulated with H₂O₂, IFNγ, H₂O₂ + IFNγ, or vehicle for 30 minutes. The ChIP assay was performed to identify which transcription factors bind to the IL-32 gene promoter. Binding activity was compared using quantitative real-time PCR of the IL-32 promoter in DNA from chromatin complexes immunoprecipitated by antibodies to c-Jun (A), CREB (B), and RNA polymerase II (C). The bars show the means ± SE from 3 different individuals. *p < 0.01 significantly different.

Figure 4

Transcriptional activity of promoter of IL-32 gene after IFNγ or H₂O₂ + IFNγ stimulation. HBE cells were transfected with a luciferase promoter vector without mutations (pWild-Luc), closed bars, or the same vector with mutations in the c-Jun/CREB binding site (pMutant-Luc), open bars. Cells were stimulated with H₂O₂, IFNγ, H₂O₂ + IFN, or vehicle. Six hours after indicated stimulation, the cells were lysed and luciferase activity was measured. Luciferase activity in the cells was normalized to Renilla luciferase activity. The bars represent the means ± SE from 3 different individuals. *p < 0.05 significantly different.

Figure 5

Knockdown efficiency of c-Jun and CREB mRNA and IL-32 expression induced by IFNγ and H₂O₂ in HBE cells transfected with c-Jun or CREB siRNAs. c-Jun (A) and CREB (B) mRNA expression levels examined by quantitative real time PCR in HBE cells transfected with indicated siRNAs. Expression levels of c-Jun (A) and CREB (B) by each siRNA transfection were looked by quantitative real time PCR. IL-32 expression was examined by real time PCR in HBE cells transfected with control-siRNA, closed bars, c-Jun-siRNA, open bars, or CREB-siRNA, hatched bars, respectively. Then 48 hours after transfection, cells were stimulated with H₂O₂ and/or IFNγ, followed by IL-32 quantitative real time PCR of RNA extracted 24 hours after the stimulation (C). The bars represent the means ± SE from 3 different individuals. *p < 0.05 significantly different.

Figure 6

Major transcription factor binding sites in 1500 bps upstream from transcription start site of IL-32 gene promoter. NF-kappa B binding sites at nucleotide -9 to +5 and -361 to -352, CREB binding site at nucleotide -34 to -19, c-Jun/CREB binding site is at nucleotide -30 to -23, ATF-2 binding sites at nucleotide -33 to -20 and, -769 to -759, and TATA boxes at nucleotide -1215 to -1206 and -1502 to -1497 relative to the transcription start site at +1. These binding sites were cited by using sequence searching software TFSEARCH^(TM)