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. 2013 Mar 6:2:78.
doi: 10.12688/f1000research.2-78.v1. eCollection 2013.

Anacardic acid, a histone acetyltransferase inhibitor, modulates LPS-induced IL-8 expression in a human alveolar epithelial cell line A549

Tetsuo Yasutake  1 , Hiroo Wada  1 , Manabu Higaki  1 , Masuo Nakamura  1 , Kojiro Honda  1 , Masato Watanabe  1 , Haruyuki Ishii  1 , Shigeru Kamiya  1 , Hajime Takizawa  1 , Hajime Goto  1
Affiliations

Anacardic acid, a histone acetyltransferase inhibitor, modulates LPS-induced IL-8 expression in a human alveolar epithelial cell line A549

Tetsuo Yasutake et al. F1000Res. .

Abstract

Objective and design: The histone acetylation processes, which are believed to play a critical role in the regulation of many inflammatory genes, are reversible and regulated by histone acetyltransferases (HATs), which promote acetylation, and histone deacetylases (HDACs), which promote deacetylation. We studied the effects of lipopolysaccharide (LPS) on histone acetylation and its role in the regulation of interleukin (IL)-8 expression.

Material: A human alveolar epithelial cell line A549 was used in vitro.

Methods: Histone H4 acetylation at the IL-8 promoter region was assessed by a chromatin immunoprecipitation (ChIP) assay. The expression and production of IL-8 were evaluated by quantitative polymerase chain reaction and specific immunoassay. Effects of a HDAC inhibitor, trichostatin A (TSA), and a HAT inhibitor, anacardic acid, were assessed.

Results: Escherichia coli-derived LPS showed a dose- and time-dependent stimulatory effect on IL-8 protein production and mRNA expression in A549 cells in vitro. LPS showed a significant stimulatory effect on histone H4 acetylation at the IL-8 promoter region by ChIP assay. Pretreatment with TSA showed a dose-dependent stimulatory effect on IL-8 release from A549 cells as compared to LPS alone. Conversely, pretreatment with anacardic acid inhibited IL-8 production and expression in A549 cells.

Conclusion: These data suggest that LPS-mediated proinflammatory responses in the lungs might be modulated via changing chromatin remodeling by HAT inhibition.

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Conflict of interest statement

Competing interests: No relevant competing interests were disclosed.

Figures

Figure 1.
Figure 1.. The dose- and time- dependent interleukin-8 (IL-8) release from A549 cells in vitro.
A549 cells were cultured until 80% confluence and stimulated by different concentrations of lipopolysaccharide (LPS) (10 ~ 200μg/ml). LPS showed a time- and dose-dependent stimulatory effect on IL-8 release at each concentration. Data were expressed in mean±SEM. *P<0.05, compared to LPS unstimulated cells at each concentration and time point (Mann-Whitney U test), n=4.
Figure 2.
Figure 2.. Time course analysis of lipopolysaccharide (LPS)-mediated interleukin-8 (IL-8) gene expression in A549 cells in vitro.
A549 cells were cultured until subconfluence and stimulated by LPS at 10μg/ml. After 0 ~ 8 hrs, IL-8 mRNA expression was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The IL-8 mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. Data were expressed in mean±SEM. *P<0.05: compared to LPS 0hr stimulated cells (Mann-Whitney U test), n=4.
Figure 3.
Figure 3.. Time course analysis of lipopolysaccharide (LPS)-induced histone H4 acetylation at the promoter region of the interleukin-8 (IL-8) gene in A549 cells in vitro.
A549 cells were stimulated 0 ~ 3 hr with LPS (10μg/ml). The results presented are from ChIP analyses using anti-acetyl H4 antibodies. All PCR signals from immunoprecipitated DNA were normalized to PCR signals from non-immunoprecipitated input DNA. Results are expressed as percentage of the input. Data were expressed in mean±SEM. *P<0.05: compared to LPS 0hr stimulated cells (Mann-Whitney U test), n=4.
Figure 4.
Figure 4.. Effect of trichostatin A (TSA) on lipopolysaccharide (LPS)-stimulated interleukin-8 (IL-8) release from A549 cells in vitro.
TSA at 1 ~ 1000nM treated 1 hr before LPS stimulation (10μg/ml) showed a significant stimulatory effect on LPS-induced IL-8 release. Data were expressed as mean±SEM. *P<0.05: compared to LPS-stimulated cells (Mann-Whitney U test), n=4.
Figure 5.
Figure 5.. Time course analysis of trichostatin A (TSA) on lipopolysaccharide (LPS)-stimulated interleukin-8 (IL-8) gene activation from A549 cells in vitro.
TSA at 10nM treated 1 h before LPS stimulation (10μg/ml) tended to show a stimulatory effect on LPS-induced IL-8 gene activation. After 0 ~ 8 h, the levels of IL-8 mRNA were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The IL-8 mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels, n=4.
Figure 6.
Figure 6.. Effect of anacardic acid on lipopolysaccharide (LPS)-stimulated interleukin-8 (IL-8) release from A549 cells in vitro.
Anacardic acid at 10 ~ 100μM treated 1 hr before LPS stimulation showed a significant inhibitory effect on LPS-induced IL-8 release. Data were expressed as mean±SEM. DMSO: dimethyl sulfoxide used for solvent. *P<0.05: compared to LPS (10μg/ml) stimulated cells (Mann-Whitney U test), n=4.
Figure 7.
Figure 7.. Time course analysis of anacardic acid on lipopolysaccharide (LPS)-stimulated interleukin-8 (IL-8) gene activation in A549 cells in vitro.
Anacardic acid at 100μM treated 1 hr before LPS (10μg/ml) stimulation showed a significant inhibitory effect on LPS-induced IL-8 gene activation. After 0 ~ 8hrs, the levels of IL-8 mRNA were analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The IL-8 mRNA levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. Data were expressed as mean±SEM. *P<0.05 (Mann-Whitney U test), n=4.
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