Trichostatin A, a histone deacetylase inhibitor, down-regulates interleukin-12 transcription in SV-40-transformed lung epithelial cells

Cellular Immunology 218 (2002) 26–33 www.academicpress.com

Trichostatin A, a histone deacetylase inhibitor, down-regulates interleukin-12 transcription in SV-40-transformed lung epithelial cells Kyoko Iwata, Katsuyuki Tomita,* Hiroyuki Sano, Yoshihiro Fujii, Akira Yamasaki, and Eiji Shimizu Third Department of Internal Medicine, Faculty of Medicine, Tottori University, 36-1 Nishi-machi, Yonago-shi, Tottori-ken 683-8504, Japan Received 21 March 2002; accepted 16 August 2002

Abstract Inhibition of histone deacetylation results in increased gene expression. Trichostatin (Ts)A, a specific histone deacetylase (HDAC) inhibitor, up-regulates transcription of some genes but represses expression of others. We quantified histone acetylation in SV-40-transformed lung epithelial cells using flow cytometry. Further, to evaluate the effect of TsA on transcription of genes associated with airway inflammation, we measured interleukin (IL)-8 production by enzyme-linked immunosorbent assay as well as IL-12 transcription by reverse transcription-polymerase chain reaction, in the transformed cells after stimulation with lipopolysaccharide (LPS) in the presence of TsA. Pretreatment of cells with TsA before LPS stimulation induced hyperacetylation of histones (especially in the S phase of the cell cycle), enhanced IL-8 production, and suppressed IL-12p35 and IL-12p40 mRNA accumulation. Thus we have demonstrated a useful way to detect hyperacetylation at the single-cell level, as well as the ability of an HDAC inhibitor to repress genes in epithelial cells. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Trichostatin A; HDAC inhibitor; IL-12; Histone; Acetylation; Transcription; LPS; Chromatin

1. Introduction The structure of chromatin has an important influence on the key nuclear process of transcription [1,2]. Chromatin structure and binding of proteins to DNA can be modulated by reversible acetylation of lysine residues within the N-terminal tails of core histones. In the resting cell, DNA is tightly compacted to prevent transcription factor accessibility. During activation of the cell, this compact inaccessible DNA is made available to transcription factors through histone acetylation. This chemical modification is respectively carried out and counteracted by histone acetyltransferases (HATs) and histone deacetylases (HDACs) [3–5]. Inhibitors of histone deacetylase such as trichostatin (Ts) A act to favor the acetylated state, altering chromatin structure *

Corresponding author. Fax: +81-859-34-8098. E-mail address: [email protected] (K. Tomita).

and thus up-regulating transcription of gelsolin [6], histone H1 [7], cytokeratin A [8], c-fos, c-myc [9], hsp70 [10], and HDAC mRNAs [11] as well as p21cip1 [12]. In addition, HDAC inhibition by TsA has paradoxical effect, leading causing down-regulation of transcription of several genes such as cyclin B1 and cyclin A [13], as well as interleukin (IL)-12 [14]. The conventional method for assessing the balance between HAT and HDAC involves measuring the release of [3 H]acetate from hyperacetylated histones [15]. Flow cytometry for detection of activated proteins is an important alternate approach that permits evaluation of different intracellular signaling pathways using a combination of monoclonal reagents that are specific for unmodified or activation-modified proteins [16–18]. As HAT/HDAC activity is dependent on cell-cycle phases, we have developed a flow cytometric technique to quantify acetylated lysine to detect acetylation at a single cell level, while assessing the cell cycle.

0008-8749/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 0 8 - 8 7 4 9 ( 0 2 ) 0 0 5 2 3 - 3

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IL-12 is a disulfide-linked heterodimer composed of unrelated 40-kDa (p40) and 35-kDa (p35) subunits [19]. The genes encoding the two chains of IL-12, p40, and p35 are located on different human chromosomes [20]. IL-12 is a critical link between innate and adaptive immunity and can be used as a potent vaccine adjuvant administered via the airway. The priming effect of interferon (IFN)-c that augments IL-12 production represents one mechanism by which IL-12-induced T-helper (Th) 1 responses are maintained in vivo. Impaired IL-12 production has been reported to contribute to the pathogenesis of asthma [21]. Sodium butyrate, another HDAC inhibitor, enhanced IL-10 and IL-4 secretion but reduced IL-12 production [14]. Since the functional characteristics of epithelial cells determine the nature of the developing immune response, we evaluated the effects of TsA on production of the cytokines IL-8 and IL-12 in human SV-40-transformed lung epithelial cell lines stimulated with lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-a.

temperature. Cells then were fixed in 4% paraformaldehyde (Wako Life Science, Osaka, Japan) for 10 min, and then permeabilized in ice-cold acetone-methanol (50/50, w/w; )20 °C) for 10 min. Coverslips were airdried and incubated with blocking buffer [20% normal swine serum (Dako, Kyoto, Japan) in phosphatebuffered saline (PBS), with 0.4% Triton X] for 20 min. Next, a 1-h incubation was carried out with primary antibody solution including PBS, 0.4% Triton X, and 0.1% bovine serum albumin (BSA); a rabbit antibody against acetylated lysine (New England Biolabs, Beverly, MA) were used at a 1:50 dilution. Coverslips were washed twice and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA; 1:50) for 45 min. Sections were washed twice more before counterstaining with 1 ng/ml of 40 -6-diamidino-2-phenylindole-2HCl (DAPI), and mounted on slides. Stained cells were observed by fluorescence microscopy. Normal rabbit IgG (Santa Cruz Biotechnology) was substituted for the primary antibody as a control.

2. Materials and methods

2.3. Image acquisition of acetylated lysine in histones

2.1. Cell line

Image acquisition was performed using a laser confocal scanning microscope (Leica, Heidelberg, Germany). Single optical sections were excited by the instrumentÕs argon–krypton laser at 488 nm. DAPI staining, shown in Fig. 1, was visualized with this ultraviolet (UV) laser light. Before analyzing double staining, the intensity of excitation wavelengths and the power of photodetectors were adjusted to avoid cross talk. Signals from both fluorochromes were recorded simultaneously in one scan, and saved separately in two channels to be processed independently.

BEAS-2B cells, which are lung epithelial cells transformed by the virus SV-40 [22], were obtained from the American Type Culture Collection (Manassas, VA). These cells (passages 32–70) were cultured in serum-free keratinocyte basal medium (Sigma, St. Louis, MO) supplemented with epithelial growth factor (0.2 ng/ml; Gibco BRL, Grand Island, NY) and 20 lg=ml bovine pituitary extract (Gibco BRL). The WI-26VA4 cell line, another SV-40-transformed cell originating from human lung epithelial cells, was provided by the Human Science Research Resource Bank (Osaka, Japan). These cells were cultured in DulbeccoÕs modified EagleÕs medium (DMEM; Iwaki, Tokyo, Japan) supplemented with 10% fetal calf serum (FCS; Nippon Gene, Tokyo, Japan), amino acids, vitamins, penicillin, and streptomycin. For all experiments, quiescent BEAS-2B cells or WI-26VA4 cells, respectively were deprived of growth factors or FCS for 48 h. 2.2. Immunohistochemical detection of acetylated lysine in histones BEAS-2B cells (5
106 ) were cultured on coverslip in six-well microtiter plates. After SV-40-transformed lung epithelial cells were stimulated with LPS derived from Escherichia coli 026: B6 (10 lg=ml; Sigma–Aldrich, Tokyo, Japan) in the presence or absence of 10 ng/ml TsA (Sigma–Aldrich), acetylated lysines in histone were identified by immunohistochemistry. Cells were washed with HanksÕ solution and air-dried for 30 min at room

2.4. Quantitative analysis of acetylated lysine using a fluorescence-activated cell sorter (FACS) For analyzing dose-response characteristics of TsA effect, BEAS-2B cells were treated with various doses of TsA. For analyzing the time course of the response to TNF-a, cells were stimulated with 10 ng/ml TNF-a, and treated with 10 ng/ml TsA 30 min prior to each time point. Trypsinized cells were fixed in 4% paraformaldehyde (Wako Life Science) for 10 min at room temperature. After cells were washed three times with PBS containing 0.4% Triton X and 0.1% BSA, cells were labeled with polyclonal rabbit antibody against acetylated lysine (New England Biolabs) at a dilution of 1:50 for 1 h at 4 °C. Next, cells were incubated with an FITCconjugated polyclonal anti-rabbit antibody for at least 30 min at 4 °C and then resuspended in 1 ml of PBS containing 0.5 mg of RNase A (Sigma–Aldrich) and 50 lg=ml of propidium iodide (PI; Sigma–Aldrich). Fluorescence of acetylated lysine was determined using a

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Fig. 1. Immunocytochemical detection of acetylated lysine. BEAS-2B cells were treated (C, F, and I) or untreated (B, E, and H) with trichostatin (Ts) A (10 ng/ml) for 1 h. Nuclear localization of acetylated lysine residues (B and C) was demonstrated. A rabbit polyclonal antibody against acetylated lysine was used at 1:50 dilution. Fluorescein isothiocyanate-conjugated anti-rabbit IgG (green) was the secondary antibody. Nuclear DNA was visualized by 40 -6-diamidino-2-phenylindole-2HCl (DAPI) counterstaining (blue; D–F). Two images of nuclear DNA and acetylated lysine were overlapped in each view (G–I). Nonspecific binding was determined using normal rabbit IgG as a control for the primary antibody (A). Samples were analyzed by confocal laser scanning microscopy. Results are representative of four independent experiments.

FACScan device (Becton–Dickinson, Mountain View, CA) with a laser setting of 495 nm. Mean fluorescence intensity (MFI) expressed in arbitrary units, was recorded as a ratio of the fluorescent signal. 2.5. Assay of IL-8 in supernatants BEAS-2B cells were stimulated for 24 h with 10 ng/ml TNF-a or 10 lg=ml LPS in the presence or absence of TsA at 37 °C in an atmosphere containing 5% CO2 . Culture supernatants were assayed for IL-8 using a commercial enzyme-linked immunosorbent assay (ELISA; Human IL-8 Duo Set, R&D Systems, Minneapolis, MN). ELISA was performed according to the manufactureÕs instructions. Absorbance (or optical density,

OD) was measured at 450 nm on a microtiter plate reader (Anthos reader 2001; Anthos Labtec Instrument, Salzburg, Australia). Concentrations were obtained by interpolation from standard curves using Graph Pad software. Final concentrations in each sample were calculated as the mean of the results at the proper sample dilution yielding ODs in the linear parts of the calibration curves. The limit of detection for IL-8 was 31.2 pg/ml. 2.6. RNA isolation and purification WI-26VA4 cells (5
106 ) were cultured in FCS-free medium for 72 h, pretreated in the presence or absence of 10 ng/ml TsA for 30 min, and then stimulated with

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10 lg=ml LPS for 6 h before harvesting. Total RNA was isolated using guanidinium isothiocyanate buffer as previously described. RNA was resuspended in RNasefree water, and treated with 10 U of DNase I (Promega, Madison, WI) for 1 h at 37 °C to remove contaminating genomic DNA. RNA was further purified by standard phenol–chloroform extraction and precipitated overnight in isopropanol at )20 °C. RNA pellets were resuspended in RNase-free water containing RNase inhibitor and stored at )80 °C.

IL-12p40:

IL-12p35:

GAPDH :

2.7. Transcript amplification Expression of mRNA encoding IL-12p35 and IL12p40 in SV-40-transformed epithelial cells stimulated with LPS was determined using reverse transcriptionpolymerase chain reaction (RT-PCR). RT-PCR was used to asses changes in IL-12p40 and IL-12p35 mRNA expression. RT-PCR mixtures typically contained 2.5 mM MgCl2, 0.2 mM dNTP, 2 U Taq polymerase, and 20 pM 50 - and 30 -oligonucleotide primers (Life Technologies). The cDNAs were denatured for 10 min at 95 °C and amplified for various numbers of cycles under the following conditions: 95 °C, 1 min; 61 °C, 1 min; and 72 °C, 1 min, followed by a final elongation step at 72 °C for 5 min. Reaction products stored at 4 °C until electrophoresis. Different numbers of cycles were tested to ensure linear-phase amplification of the cDNA. Based on this preliminary assessment, IL-12 and the internal standard, glyceraldehydes3-phosphate dehydrogenase (GAPDH), were amplified for 33 cycles. Reactions were conducted for a total of 33 cycles, consisting of denaturation at 95 °C for 10 min and annealing/extension at 72 °C for 1 min. Sequences of primer pairs used, based on previous descriptions, were as follows:

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forward, 50 -GAGAAATGGTGGTCCT CACCTGTG-30 ; reverse, 50 -GAGTGTAGCAGCTCCGC ACGTC-30 . forward, 50 -GTCTGCATCCAGCGGC TCGCCCTG-30 ; reverse, 50 -GGGTAGCGACAAGGAG GAGGCTCG-30 . forward, 50 -CCACCCATGGCAAATT CCATGGCA-30 ; reverse, 50 -TCTAGACGGCAGGT CAGGTCCACC-30 .

Reaction products were electrophoresed on agarose gels and visualized by ethidium bromide staining. 2.8. Data analysis Group data are expressed as the mean (SD). Differences between groups were tested using the Mann– Whitney U test.

3. Results 3.1. Hyperacetylation in BEAS-2B cells treated with TsA We initially sought to determine whether pretreatment with TsA induced hyperacetylation at lysine sites of histones in human epithelial lung cell lines. To observe the effect of TsA, we visualized acetylated lysine in BEAS-2B cells pretreated with TsA by immunofluorescence analysis. TsA pretreatment induced hyperacetylation of lysine in all nuclei of SV-40transformed lung epithelial cells (Figs. 1C and I). This represented a reversible accumulation of highly acet-

Fig. 2. Acetylation of lysine detected by fluorescent cell sorting (FACS). Representative dot plot of acetylated lysine immunostaining and fluorescent DNA staining. Cells were incubated with a fluorescein isothiocyanate-conjugated polyclonal anti-rabbit antibody and stained with propidium iodide (PI). Fluorescence data were plotted as a dot plot of DNA content versus fluorescence of acetylated lysine.

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Table 1 Acetyated lysine staining profile for each cell phase

Negative control FCS ()) FCS ()) and TsA *

G0=G1

S

G2=M

98:7  36:1 221:7  86:3 278:3  134:2

78:4  24:6 154:5  41:2 261:9  49:3

76:7  31:4 211:3  52:2 305:2  64:9

Data were indicated as mean fluorescent intensity. P < 0:05, compared to FCS ()).

ylated histones in nuclei that persisted up to 16 h after treatment. 3.2. FITC-based determination of acetylation using FACS As a new method for detecting acetylation, we used FACS analysis to quantify cell-cycle-related intensity of acetylated lysine staining using a specific antibody. The result demonstrated by dot plots and also by separate acetylated lysine staining profiles for cell cycle showed increased intensity of fluorescence, especially in the S phase, of the cell cycle compared to untreated cells (P < 0:05) (Fig. 2 and Table 1). To determine dose and time characteristics of TsA action, cells were treated with different concentrations of TsA (0.1–100 ng/ml) for 30 min. The result indicated that histone acetylation, shown as mean fluorescence intensity of acetylated lysine in cells, increased with TsA pretreatment in a dose-dependent manner (Fig. 3A). In addition, we determined the effect of TNF-a on acetylation for different times of exposure. Acetylated lysine showed an increase at 1 h after treatment with TNF-a, and this activity persisted until 12 h (Fig. 3B). Treatment with TsA 30 min prior to each time point when cells were harvested increased acetylation (Fig. 3B).

were observed in epithelial cells stimulated with LPS (Fig. 5). To determine whether IL-12p40 and IL-12p35 mRNA were inhibited in cell lines pretreated with TsA, we performed RT-PCR using IL-12p40 and IL-12p35 isoform-specific oligonucleotide primers. All expression was normalized to that of GAPDH mRNA. As shown in Fig. 5, treatment with TsA alone induced IL-12p35 mRNA but not IL-12p40 mRNA. Additionally, induc-

3.3. Enhanced IL-8 in supernatants of BEAS-2B cells treated with TsA To confirm increases in IL-8 after treatment of TsA, secretion of IL-8 was measured by ELISA in supernatant fluid from cultures. Under the experimental conditions, TNF-a and LPS-induced production of IL-8 both were enhanced by pretreatment with TsA (Fig. 4). 3.4. TsA inhibition of IL-12p40 and IL-12p35 mRNA Secretion of bioactive IL-12p70 requires production of both p40 and p35 IL-12 subunits. Genes encoding these two IL-12 chains are located on different human chromosomes. Together, p40 and p35 form biologically active IL-12. To determine whether TsA inhibited IL-12 transcription, we analyzed the effect of TsA on expression of IL-12p40 and IL-12p35 mRNA in LPSstimulated epithelial cells. Both p40 and p35 expression

Fig. 3. (A) Dose-dependent effect of trichostatin (Ts)A on hyperacetylation. BEAS-2B cells were pretreated with various doses of TsA for 30 min. (B) Time-dependent effect of tumor necrosis factor (TNF)-a on hyperacetylation. BEAS-2B cells were stimulated with 10 ng/ml TNF-a and treated with 10 ng/ml TsA prior to each time point. Cells were labeled with anti-acetylated lysine polyclonal rabbit antibody at a dilution of 1:50. Cells then, were incubated with a fluorescein isothiocyanate-conjugated polyclonal anti-rabbit antibody and stained with propidium iodide (PI). Fluorescence data were plotted as a dot plot of DNA content versus fluorescence of acetylated lysine. The results are the means of three separate experiments; bars indicate SD. *P < 0:05 vs the cells untreated with TsA. **P < 0:05 vs the cells untreated with TNF-a. MFI, mean fluorescence intensity.

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Fig. 4. Effect of trichostatin (Ts)A on interleukin (IL)-8 production in supernatants. IL-8 was detected by enzyme-linked immunosorbent assay (ELISA) in supernatants on BEAS-2B cells treated with TsA and tumor necrosis factor (TNF)-a or lipopolysaccharide (LPS). Cells were stimulated for 24 h with 10 lg=ml LPS or 10 ng/ml TNF-a in the presence or absence of TsA. After stimulation, supernatants were assayed for IL-8. *P < 0:05 vs the cells untreated with TsA.

Fig. 5. Reverse transcription-polymerase chain reaction (RT-PCR) for trichostatin (Ts) A-pretreated lipopolysaccharide (LPS)-stimulated epithelial cells. SV-40-transformed epithelial cells were pretreated with TsA for 30 min, following by incubation with LPS for 6 h. The cDNA derived from total RNA obtained from cell cultures was analyzed using RT-PCR. All expression was normalized to glyceraldehydes-3phosphate dehydrogenase (GAPDH) expression to ensure equivalence in amounts of template.

tion of mRNA encoding both IL-12p40 and IL-12p35 in response to LPS was down-regulated in cells pretreated with TsA. These results indicate that TsA inhibits LPSinduced transcription of both IL-12 chains in SV-40transformed lung epithelial cells (WI-26VA4).

4. Discussion Histone acetylation and deacetylation are linked to cell cycle progression, and have been correlated with repair and recombination events as well as gene tran-

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scription [4,23]. Highly acetylated nucleosomes are associated with transcriptionally active chromatin, while hypoacetylated histones often are found in inactive chromatin. Recent discovery of the enzymes controlling histone acetylation and deacetylation confirmed that acetylation of histones is an important step in transcription [3–5]. DNA is packaged into chromatin, a highly organized and dynamic protein–DNA complex. The fundamental subunit of chromatin, the nucleosome, is composed of an octamer of four core histones; an H3/ H4 tetramer, and two H2A/H2B dimers, surrounded by 146 bp of DNA [24,25]. The N-terminal tails of histones contain lysine residues that represent sites for posttranscriptional acetylation. This is a dynamic process that occurs on actively transcribed chromatin, both cell cycle-dependently and -independently [26]. In the present study, confocal analysis showed that TsA induced hyperacetylation of lysines in all histones in all cells. Culture of epithelial cells with TsA resulted in an increase in the amount of acetylated histone detected by immunoblotting. We used paraformaldehyde for fixation of cells because procedures involving paraformaldehyde fixation have been found to preserve the structure of acetylated chromatin organization according to indirect immunofluorescence [27]. Our report also describes development and validation of an immunofluorescence-based flow cytometric assay that detects acetylated histone in cell nuclei. Hoffmann developed a nonisotopic assay for HDAC using a fluorescent derivative of epsilon-acetyl lysine, quantitating the fluorescent substrate by high-performance liquid chromatography (HPLC) [28]. Other reports have shown that using flow cytometry for quantitative assessment of signal transduction-associated protein is both a more sensitive and a less time-consuming way to detect of phosphorylation STAT-1 [16] and STAT-4 [17]. We developed a flow cytometric procedure using an FITC-conjugated anti-rabbit antibody to detect priming antibody specific for acetylated histones. Acetylation of histones is governed by a balance of the activities of HAT and HDAC. Our results suggested that induction of acetylation by TNF-a might be attributed mainly to HAT activity, and that the effects of TsA might result from inhibition of HDAC. Our method showed a limited capacity of TsA to inhibit HDAC, since TsA alters mRNA abundance for up to 2% of the genes expressed in mammalian cells [29]. However, our method shows certain clear advantages for assessment of hyperacetylation. First, detection of acetylated histones by flow cytometry should be useful for screening agents such as HDAC inhibitors. Another benefit of using flow cytometry for detection of acetylation is that double staining using acetylated lysine antibody also PI staining could assess cell cycle-dependence of acetylation of histones. We found that TsA induced hyperacetylation particularly in the S phase (Fig. 2), which is consistent

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with some previous reports. Inhibition of histone deacetylase by TsA showed that the dynamics of acetylation of histone H4 in heterochromatin show restriction to lys 5 and 12, and to the time of DNA replication [30,31]. In G2, acetylated groups have been found to be removed by HDAC, yielding bulk chromatin that contains baseline activity of HAT [31]. Although many studies have correlated function of HDAC inhibitors with the hyperacetylation of histones, few studies have specifically addressed the issue of whether accumulation of acetylated histones, as a result of HDAC inhibition is responsible for transcription activation and repression. TsA is widely used to study the role of histone acetylation in gene expression, since genes with expression regulated by histone acetylation may be up-regulated when TsA is abundant. In our study, TsA enhanced IL-8 production in SV-40-transformed epithelial cells. TsA-induced hyperacetylation of the NF-jB-dependent receptor on the IL-8 promoter has been reported, resulting in increased IL-8 mRNA transcription [32]. Recently, sodium butyrate, another inhibitor of histone deacetylase enzymes, was found to induce histone acetylation and inhibit IL-12 production by suppression of both IL-12p40 and IL-12p35 mRNA accumulation in stimulated human monocytes [14]. TsA also has been reported to have contradictory effects on transcription of cyclin B1 and cyclin A [13], c-myc, bag-1, and LC-PTP [14]. In our results, TsA given without other additives induced transcription of IL-12p35 but not IL-12p40. As it was reported that TsA treatment did not affect the expression of house keeping gene such as GAPDH [33], we have normalized transcript levels with internal standard GAPDH. The 50 -flanking region of IL-12p35 has sequences, such as an Sp1 site, that differ from those of IL12p40. Accordingly, TsA has been suggested to affect the Sp1 site and thus induce transcription of IL-12p35, similarly to events in p21cip1 expression [12]. We also showed that TsA repressed transcription of IL-12 mRNA. Three possible explanations can be offered for repression of IL-12 mRNA in LPS-stimulated cells by TsA. One possibility is that pretreatment with TsA inhibited TNF-a-induced nuclear translocation of the proinflammatory transcription factor NF-jB by changing amounts of intracellular IjB and/or the rate of IjB-a degradation. The result would be a reduction in cellular proteasome activity [34], leading to repression of gene expression for IL-12. Another possibility is that inhibition of HDAC activity led to loss of PU.1 transcription factor, causing loss of expression of its target genes such as Toll-like receptor 4 (TLR4) [35]. As TLR4 is the receptor for LPS [36], IL-12 expression decreased might result from loss of TLR4-mediated LPS signaling. Thus, IL-12 gene expression could be regulated by PU.1 transcription factor. Last, acetylation of the GATA sequence in the IL-12 promoter, termed GA-12, which is

important in repressing IL-12 promoter activity [37]. Accordingly, GA-12 might mediate repression of IL12p40 and IL-12p35 mRNA. Epithelially derived IL-12 is involved in modifying the intensity of airway inflammation during mucosal defense reaction and disease [21]. IL-12 is the most important factor governing differentiation and magnitude of a Th1 response and is important in the defense against microbial infections. Marrow-derived antigen-presenting cells (APC) such as monocytes, macrophages, and dendritic cells (DC), are believed to be major sources of IL-12 in vivo. In addition, IL-12 expressed in human airway epithelial cells modifies the degree of airway inflammation [38]. We have analyzed IL-12 production in vitro using epithelial cells. In the present study, epithelial cells stimulated with LPS in the presence of TsA showed reduced transcription of IL-12 despite undiminished production of IL-8. As a hypothesis, a deficiency of innate immunity to endotoxin may result in dysregulated production of Th1 cytokines and polarized production of Th2 cytokines, culminating in the allergic inflammation of asthma [39]. Our present results, then, suggest that hyperacetylation of genes in asthmatic patients could result in an imbalance of Th1 and Th2 cytokines via down-regulation of IL-12 transcription. In summary, we presently demonstrate that TsA induced hyperacetylation of histone and inhibited transcription of the pro-inflammatory cytokine IL-12 in spite of increased production of IL-8 in epithelial cells stimulated with LPS. Future studies will be needed using chromatin immunoprecipitation assay to prove directly whether the treatment with TsA induces acetylated histone on IL-12 genes.

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