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

Kudo M, Ogawa E, Kinose D, Haruna A, Takahashi T, Tanabe N, Marumo S, Hoshino Y, Hirai T, Sakai H, Muro S, Date H, Mishima M - Respir. Res. (2012)

Bottom Line: The cell viability was confirmed by MTT assay.There were no significant differences of cell viability among groups.Further, knocking down CREB expression by siRNA resulted in significant suppression of IL-32 induction by IFNγ and H2O2 in HBE cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Respiratory Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan.

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.

Show MeSH

Related in: MedlinePlus

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)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3361495&req=5

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)

Mentions: We have searched transcription factor binding sites existing on IL-32 promoter using sequence retrieval software TFSEARCH(TM) [34], and two adjacent binding sites that could be responsible for the downstream of JNK signaling pathway are those of c-Jun and CREB, which are located between nucleotides -30 to -23 and -34 to -19, respectively, as a transcription start site at +1 (Figure 6). As shown in Figure 6, there are consensus binding sites of ATF and NFκB other than c-Jun and CREB on IL-32 promoter region. The cellular signaling pathway induced by IFNγ has been investigated in bronchial epithelial cell line (BEAS-2B) [35], which revealed that the signal was dependent on IKKB1/2 but not the NFκB pathway. In addition, IL-32 expression was not suppressed by an inhibitor of p38 which is supposed to be one of the kinases upstream of NFκB. For these reasons, NFκΒ was thought to be not associated with IL-32 expression induced by IFNγ (Figure 2A).


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

Kudo M, Ogawa E, Kinose D, Haruna A, Takahashi T, Tanabe N, Marumo S, Hoshino Y, Hirai T, Sakai H, Muro S, Date H, Mishima M - Respir. Res. (2012)

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)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3361495&req=5

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)
Mentions: We have searched transcription factor binding sites existing on IL-32 promoter using sequence retrieval software TFSEARCH(TM) [34], and two adjacent binding sites that could be responsible for the downstream of JNK signaling pathway are those of c-Jun and CREB, which are located between nucleotides -30 to -23 and -34 to -19, respectively, as a transcription start site at +1 (Figure 6). As shown in Figure 6, there are consensus binding sites of ATF and NFκB other than c-Jun and CREB on IL-32 promoter region. The cellular signaling pathway induced by IFNγ has been investigated in bronchial epithelial cell line (BEAS-2B) [35], which revealed that the signal was dependent on IKKB1/2 but not the NFκB pathway. In addition, IL-32 expression was not suppressed by an inhibitor of p38 which is supposed to be one of the kinases upstream of NFκB. For these reasons, NFκΒ was thought to be not associated with IL-32 expression induced by IFNγ (Figure 2A).

Bottom Line: The cell viability was confirmed by MTT assay.There were no significant differences of cell viability among groups.Further, knocking down CREB expression by siRNA resulted in significant suppression of IL-32 induction by IFNγ and H2O2 in HBE cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Respiratory Medicine, Kyoto University Graduate School of Medicine, Kyoto, Japan.

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.

Show MeSH
Related in: MedlinePlus