Neutrophil Elastase Represses IL-8/CXCL8 Synthesis in Human Airway Smooth Muscle Cells through Induction of NF-κB Repressing Factor

Shu-Chuan Ho, Kang-Yun Lee, Yao-Fei Chan, Lu-Wei Kuo, Kazuhiro Ito, Ian M. Adcock, Bing-Chang Chen, Joen-Rong Sheu, Chien-Huang Lin and Han-Pin Kuo
J Immunol July 1, 2009, 183 (1) 411-420; DOI: https://doi.org/10.4049/jimmunol.0803729

Abstract

NF-κB repressing factor (NRF), a nuclear inhibitor of NF-κB, is constitutively expressed and is implicated in the basal silencing of specific NF-κB targeting genes, including IFN-β, IL-8/CXCL8, and iNOS. Little is known about the regulation of NRF and its role in response to stimuli. Airway smooth muscle (ASM) is a rich source of inflammatory mediators that may regulate the development and progression of airway inflammation. We have previously reported that NE activates NF-κB in primary human ASM (hASM), leading to induction of TGF-β1. In this study, we describe that, instead of inducing the NF-κB response gene IL-8/CXCL8, NE suppressed IL-8/CXCL8 release and mRNA expression in hASM cells. Transcriptional blockade studies using actinomycin D revealed a similar degradation rate of IL-8/CXCL8 mRNA in the presence or absence of NE, suggesting an involvement at the transcription level. Mechanistically, the NE repressive effect was mediated by inducing NRF, as shown by RT-PCR and Western blotting, which was subsequently recruited to the native IL-8/CXCL8 promoter leading to removal of RNA polymerase II from the promoter, as demonstrated by chromatin immunoprecipitation assays. Knockdown of NRF by small interfering RNA prevented NE-induced suppression of IL-8/CXCL8 expression. In contrast, NE did not induce NRF expression in A549 and Beas-2B cells, where NE only stimulates NF-κB activation and IL-8/CXCL8 induction. Forced expression of NRF in A549 cells by an NRF expression plasmid suppressed IL-8/CXCL8 expression. Hence, we describe a novel negative regulatory mechanism of NE-induced NRF, which is restricted to hASM and mediates the suppression of IL-8/CXCL8 expression.

Airway smooth muscle (ASM)3 has long been regarded as having mainly contractile properties in response to many proinflammatory mediators and neurotransmitters. Studies now show that ASM is also a source of inflammatory mediators and cytokines through its synthetic functions (1, 2). Cytokine-stimulated ASM produces large quantities of inflammatory mediators and cytokines, such as IL-8/CXCL8 (3), growth-related oncogene-α (4), RANTES (5), and GM-CSF (6), which regulate the development and progression of airway inflammation. In the absence of cytokine stimulation, ASM is able to maintain human lung mast cell survival and induce its rapid proliferation via expression of membrane-bound stem cell factor (7). In addition to proinflammatory mediators, anti-inflammatory mediators such as prostaglandin E2 (8, 9) are also produced by ASM, suggesting a dual role in inflammation (10).

Neutrophils are recruited to the airways and play a role in the pathogenesis of chronic airway diseases, such as chronic obstructive pulmonary disease, cystic fibrosis, and more severe forms of asthma (11). Neutrophil-derived elastase (NE), one of the major proteases produced by neutrophils, is a serine protease involved in host defense against bacterial pathogens (12). In addition to its direct antibacterial effects, increasing evidence indicates that NE can provoke a variety of cellular responses in respiratory epithelial cells. By activating specific intracellular signal pathways, including NF-κB, MAPK, and protein kinase C (13, 14, 15, 16, 17), NE induces expression of a number of genes, such as IL-8/CXCL8 (13, 14, 18), cathepsin B, matrix metalloprotease-2 (15), and MUC5AC (19). NE has been shown to activate NF-κB through the TLR4/MyD88/TRAF6 pathway, leading to induction of IL-8/CXCL8 expression in bronchial and lung epithelial cells (14, 20, 21). NE-induced IL-8/CXCL8 therefore recruits additional neutrophils to the bronchial surface and supports a self-perpetuating inflammatory process (18, 22). In contrast to the comprehensive understanding of the NE-induced biological effect on respiratory epithelium, little is known about that on ASM. Previously, we have reported that NE also stimulates the TLR4/MyD88/TRAF6/NF-κB pathway in primary human ASM (hASM) cells, leading to induction of TGF-β1 transcription (17). However, despite NE activation of NF-κB in hASM cells, NE was found to suppress the basal secretion of IL-8/CXCL8 in our preliminary study.

NF-κB repressing factor (NRF) is a transcriptional silencer which is encoded by the gene located on the X chromosome and its mRNA is constitutively expressed in variable amounts in all human tissues tested (23). Using anti-sense RNA, endogenous NRF has been reported to be implicated in the basal silencing of specific NF-κB targeting genes, including IFN-β, IL-8/CXCL8, iNOS, and HIV type 1 (HIV-1) long terminal repeat (LTR) (23, 24, 25, 26). This silencing effect is mediated by NRF binding to negative regulatory elements, or NREs, in their promoters (23, 24, 25, 26). NRF binding to DNA specifically abolishes the transcriptional activity of the bordering NF-κB binding sites by a noncompeting, distance, and position-independent mechanism (23). Studies using a HIV-1 LTR reporter showed that NRF was constitutively binding to LTR and inhibiting its transcription activity. However, NRF seems to become required for transcriptional activation of the HIV-1 LTR upon stimulation with PMA and ionomycin (26), in which the level of NRF binding to the LTR is not changed. It is therefore suggested that endogenous NRF plays a dual role in gene transcription (24). It is not clear whether NRF itself is regulable and, if so, what functional roles inducible NRF plays.

In this study, we report that NE inhibits IL-8/CXCL8 gene expression and protein release specifically in hASM cells. This hASM-specific effect of NE is mediated by a novel negative regulatory mechanism through NE-induced NRF up-regulation. The differential effects of NE on hASM cells and respiratory epithelial cells are also described.

Materials and Methods

Cell culture and stimulation

Primary hASM cells were purchased from Cambrex (hBSMC) and prepared according to the manufacturer’s instruction. Cells were grown as described previously (17). Confluent cells at passages 3 through 7 were serum deprived for 24 h in serum-free medium (SFM) containing DMEM supplemented with 4 mM l-glutamine, 100 U/ml penicillin, 2.5 μg/ml amphotericin B, 1% insulin-transferrin-selenium-X, 1/100 nonessential amino acids, and 0.1% BSA. After serum deprivation, cells were incubated for various times (0–24 h) in fresh SFM containing NE (0.05–1.0 μg/ml) in the presence or absence of Elastase inhibitor II (NEI, 10 μM) or actinomycin D (5 μg/ml) as indicated in individual experiments. The MTT assay was used to measure the viability of cells.

A549 cells and human bronchial epithelial (Beas-2B) cells obtained from American Type Culture Collection were grown to 60% confluence in DMEM containing 10% FCS before incubation for 24 h in SFM. After serum deprivation, cells were incubated for various times (0–6 h) in the presence or absence of NE (0.5 μg/ml).

Reagents

Human NE and actinomycin D was purchased from Sigma-Aldrich. NEI were purchased from Calbiochem, and the recombinant human IL-8/CXCL8 was purchased from Roche Applied Science. The anti-NRF Ab was obtained from Genesis Biotech. This Ab was produced by immunizing rabbits with the synthetic peptide corresponding to aa 18–45. The Ab specific for human RNA polymerase II (RNA Pol 2) used in chromatin immunoprecipitation (ChIP) assays was from Abcam and that for p65 from Santa Cruz Biotechnology. siGENOME SMARTpools targeting human NRF (siRNA directed against NRF (siNRF)), and siCONTROL nontargeting siRNA no. 1 (siControl) were purchased from Dharmacon.

Elastase activity

Thirty microliters of cultured supernatants at variable times (0, 2, 6, 24 h) was added in triplicate to ELISA plates, followed by 240 μl of reaction buffer (0.45 M Tris-HCl and 2 M NaCl (pH 8.0)). Then, 30 μl of 0.2 mM Elastase substrate I (MeOSuc-AAPV-pNA; Calbiochem) was added in reaction buffer, and samples were incubated at 25°C. Elastase activity was measured by monitoring the change in absorbance at 410 nm. One unit of elastase activity is defined as the amount of enzyme that will hydrolyze 1.0 μmol of the substrate.

ELISA

IL-8/CXCL8 protein concentrations in the supernatants from cells treated with NE in the presence or absence of NEI (10 μM) were measured by a commercial ELISA kit (R&D Systems) according to the manufacturer’s instructions. To determine the direct effect of NE on IL-8/CXCL8, cell-free culture medium containing recombinant human IL-8/CXCL8 (750 and 250 pg/ml) in the presence or absence of NE (0.5 μg/ml) was incubated for variable times (6 and 24 h) and was determined for IL-8/CXCL8 concentrations.

RT-PCR

Total RNA was isolated from cells using TRIzol reagent (Life Technologies) according to the manufacturer’s instruction. cDNA was reverse-transcribed from isolated RNA by incubating 200 ng of DNase-treated RNA with the first-strand synthesis kit from Advanced Biotechnologies, following the manufacturer’s instructions. PCR was run-in amplification buffer containing 1.5 mM MgCl2, 10 pmol each of forward and reverse primers, 2.5 U of KlenTaq polymerase (BD Clontech), 1 mM dNTP, and 10 μl of the reverse transcriptase reaction products in a 25-μl volume. Most of he samples were denatured at 94°C for 5 min, followed by 35 cycles of annealing and extension at 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, and extra-extension at 72°C for 10 min. The melting temperature for NRF was 60°C. For GAPDH, which was used as internal control, 25 cycles were used. The PCR primers for IL-8/CXCL8 mRNA (forward, 5′-AGA TCT GAA GTG TGA TGA CTC AGG-3′, and reverse, 5′-GAA GCT TGT GTG CTC TGC TGT CTC-3′), NRF mRNA (forward, 5′-CCA AAT TCC ATG CGA GAC CTC G-3′, and reverse, 5′-TAT TTT TGG GGA TGT CGG CAG G-3′), and GAPDH mRNA (forward, 5′-CCA TGG AGA AGG CTG GGG-3′, and reverse, 5′-CCA AGT TGT CAT GGA TGA CC-3′) were synthesized by Life Technologies. The amplified products were run on a 2% agarose gel. The gel was stained with ethidium bromide and photographed.

Quantitative real-time PCR (qPCR)

qPCR was performed in a LightCycler 2.0 System (Roche Applied Science) using LightCycler DNA Master SYBR Green I (Roche Applied Science). PCR mixtures contained 0.5 μM each of forward and reverse primers. The samples were denatured at 95°C for 10 min, followed by 45 cycles of annealing and extension at 95°C for 15 s, 60°C for 5 s, and 72°C for 10 s. Melting curves were obtained at the end of amplification by cooling the samples to 65°C for 15 s, followed by further cooling to 40°C for 30 s. Data were analyzed by standard curve method of relative quantification using the LightCycler analysis software. In RT-qPCR, data were normalized to GAPDH. In ChIP assays, values were normalized to input DNA and were expressed as relative values to the control. qPCR products were further confirmed by gel electrophoresis and melting curve analysis. The primers used in RT-qPCR were as follows: IL-8/CXCL8 mRNA, forward, 5′-AGA TCT GAA GTG TGA TGA CTC AGG-3′, and reverse, 5′-GAA GCT TGT GTG CTC TGC TGT CTC-3′; GAPDH, forward, 5′-CCA TGG AGA AGG CTG GGG-3′, and reverse, 5′-CCA AGT TGT CAT GGA TGA CC-3′. Those used in ChIP assays were as follows: the IL-8/CXCL8 promoter, forward, 5′-GGG CCA TCA GTT GCA AAT C-3′, and reverse, 5′-TTC CTT CCG GTG GTT TCT TC-3′; and the IL-8/CXCL8 3′-untranslated region (3′-UTR), forward, 5′-AGG TTC AAG CAG TTT TCC-3′, and reverse, 5′-CTG TAA TCT CAG CAC TTT GG-3′.

Western blot analysis

Cytoplasmic and nuclear protein fractions were separated as described previously (27). Proteins were subjected to 10% SDS-PAGE and blotted onto nitrocellulose filters. NRF was detected with the specific anti-NRF Ab and an alkaline phosphatase-conjugated anti-rabbit secondary Ab (1/10,000 dilution; Calbiochem). Blots were incubated with ECL solution (LumiGLO; Amersham Bioscience). Images were acquired and analyzed using G:BOX (Syngene). Cell-free culture medium (100 μl) containing recombinant human IL-8/CXCL8 (1 and 0.1 μg) in the presence or absence of NE (0.5 μg/ml) was incubated for 6 h. The cultured medium was concentrated by using Microcon centrifugal filter devices (Millipore), according to the manufacturer’s protocol, and was detected for IL-8/CXCL8 with the specific human IL-8/CXCL8 Ab (Millipore) using 18% SDS-polyacrylamide gel.

NF-κB DNA binding ELISA

DNA binding activity of NF-κB p65 was measured using TransAM NF-κB p65 kits (Active Motif), according to the manufacturer’s instructions. Briefly, 20 μg of nuclear protein samples was incubated for 1 h in a 96-well plate coated with an oligonucleotide that contains a NF-κB consensus binding site (5′-GGGACTTTCC-3′), to which NF-κB contained in nuclear extracts specifically binds. After washing, an Ab specific for p65 (1/1000 dilution) was added to these wells and incubated for 1 h. After incubation for 1 h with a secondary HRP-conjugated Ab (1/1000 dilution), specific binding was detected by colorimetric estimation at 450 nm with a reference wavelength of 655 nm.

Immunostaining and confocal laser microscopic analysis

hASM cells were plated onto 24-mm-diameter round coverslips in 6-well plates. After stimulated with 0.5 μg/ml NE for1 h, cells were fixed in methanol at −20°C for 5 min. The cells were then blocked with 1% BSA/PBS at room temperature for 30 min and incubated with the rabbit anti-human p65 Ab at room temperature for 1 h. After washing, the cells were incubated with a Cy3-conjugated anti-rabbit Ab (Chemicon International) at room temperature for 1 h. After washing, the cells were incubated with Hoechst dye (Sigma-Aldrich) at room temperature for 2 min. After washing and air-drying, the cells were mounted on slides with anti-fade mounting medium (DakoCytomation). Images were acquired with a confocal laser-scanning microscope (Leica) and analyzed by Metamorph Image Analysis (Universal Imaging).

ChIP assay

ChIP assays were preformed as described previously (28). After stimulation, protein-DNA complexes were cross-linked at 37°C for 10 min by formaldehyde (1% final concentration). Cells were resuspended in 200 μl of SDS lysis buffer (50 mM Tris (pH 8.1)/1% SDS/5 mM EDTA/complete proteinase inhibitor mixture) and subjected to five cycles of sonication on ice with 10-s pulses. This sonication is optimal for shearing DNA to 150-1000 bp pieces (∼500 bp on average). The soluble chromatin solution was further diluted with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl (pH 8.1), and 167 mM NaCl) to a final volume of 2 ml. Twenty microliters (1%) of the ChIP dilution solution was kept as input control. The remained diluted solution was precleared by incubating with 80 μl of salmon sperm DNA/protein A-agarose-50% slurry for 30 min at 4°C on a rotator. After centrifuge, 900 μl of the supernatant was immunoprecipitated at 4°C overnight on a rotator by using Abs specific for NRF (5 μg), RNA Pol 2 (5 μg; Abcam), or p65 (5 μg; Santa Cruz Biotechnology), followed by 1 h of incubation for 30 min at 4°C with 60 μl of salmon sperm DNA/protein A-agarose-50% slurry. Protein-bound immunoprecipitated DNA (IP-DNA) was sequentially washed once with low-salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), and 150 mM NaCl), high-salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), and 500 mM NaCl), LiCl immune complex wash buffer (0.25 M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, and 10 mM Tris (pH 8.1), and twice with TE buffer (10 mM Tris and 1 mM EDTA (pH 8.0)). Immune complexes were eluted twice by adding 250 μl of elution buffer (1% SDS/0.1 M NaHCO3) at room temperature for 15 min. DNA-protein cross-links were reversed by incubation for 4 h at 65°C in 200 mM NaCl/1% SDS, and proteins were digested by incubation for 1 h at 45°C with 70 μg/ml proteinase K (Sigma-Aldrich). DNA was isolated with phenol/chloroform, precipitated with ethanol/0.3 M NaHCOOH/20 μg of glycogen and was resuspended in 50 μl of nuclease- free water. qPCR was performed with 7 μl of DNA sample for quantification. Preimmune rabbit IgG (IgG control; Santa Cruz Biotechnology) was used to demonstrate the specificity of the Abs. Primer pairs amplifying an unrelated site in the 3′-UTR of the IL-8/CXCL8 gene were also used to confirm the site specificity of the assay.

Plasmids construction

The full-length NRF expression vector pNRF was obtained from OriGene Technologies, which was constructed by inserting human NRF cDNA (accession no. NM_017544.1) into pCMV6-XL4 vector (pCMV).

Transfection of siRNA and plasmids

siRNA (siNRF and siCONTROL) and plasmin DNA (pCMV and pNRF) were introduced into hASM cells and A549 cells by nucleofection using hASM and A549 nucleofector kits, respectively (Amaxa), following the manufacturer’s optimized protocols. After nucleofection, cells were incubated in complete medium for 48 h (for siNRA) or 24 h (for plasmid DNA) before stimulation with NE (0.5 μg/ml) for 6 h (for siRNA).

Statistics

Data were analyzed with the GraphPad Prism 5.01 software package (GraphPad Software). Results were expressed as the mean ± SEM from at least three independent experiments. Data for hASM cells were the mean ± SEM from at least three independent experiments established from different donors. Results were analyzed by Mann-Whitney U test. In the time-course studies, one-way ANOVA with Dunnet’s multiple comparison posttest was used to validate the trend. Differences were considered significant when the p value was ≦ 0.05.

Results

Differential effects of NE on expression of IL-8/CXCL8 in hASM and respiratory epithelial cells

We have previously showed that NE is able to stimulate NF-κB activation in hASM cells, leading to an induction of TGF-β1 expression (17). In an attempt to screen other NF-κB response genes by using an array system, our preliminary data unexpectedly revealed that IL-8/CXCL8 gene expression was reduced in NE-stimulated hASM cells (data not shown). To confirm this finding and its functional effect, we set out to determine the release of IL-8/CXCL8 in the cultured supernatants with a commercialized ELISA kit. As NE stimulates IL-8/CXCL8 in respiratory epithelial cells, we also compared its effects on these two airway structure cells. As shown in Fig. 1⇓A, incubation of hASM with NE (0.5 μg/ml) for 6 h remarkably suppresses IL-8/CXCL8 release (190.6 ± 35.0 pg/ml vs 50.6 ± 5.4 pg/ml; p < 0.01). In contrast, NE stimulates its release from the alveolar epithelial A549 cells (466.7 ± 54.3 pg/ml vs 597.6 ± 33.1 pg/ml; p < 0.05) and the bronchial epithelial Beas-2B cells (450.0 ± 41.6 pg/ml vs 651.6 ± 10.1 pg/ml; p < 0.05). The differential effects of NE on IL-8/CXCL8 expression were also confirmed at the mRNA level. RT-PCR analysis revealed that levels of IL-8/CXCL8 mRNA were suppressed by NE at 2 h in hASM cells and induced in A549 and Beas-2B cells (Fig. 1⇓B).

FIGURE 1.
FIGURE 1.

Differential effects of NE on IL-8/CXCL8 expression in hASM and in respiratory epithelial cells. A, ELISA analysis of IL-8/CXCL8 release from hASM, A549, and Beas-2B cells stimulated with NE (0.5 μg/ml) or left unstimulated for 6 h. Results are expressed as mean ± SEM. *, p < 0.05; **, p < 0.01 vs the unstimulation control. B, RT-PCR analysis of IL-8/CXCL8 mRNA expression in hASM, A549, and Beas-2B cells stimulated with NE (0.5 μg/ml) or left unstimulated for 2 h. A representative picture of three independent experiments with similar results was shown.

NE-repressed IL-8/CXCL8 expression in hASM at the transcription level

We next studied the effect of NE on IL-8/CXCL8 expression in hASM cells in detail. As shown in Fig. 2⇓A, NE suppresses hASM release of IL-8/CXCL8 in a concentration-dependent manner; incubation of hASM cells with NE (0.05, 0.1, 0.5, and 1.0 μg/ml) for 6 h significantly represses IL-8/CXCL8 release at the concentrations of 0.5 and 1.0 μg/ml (85.3 ± 6.9 pg/ml and 57.2 ± 7.5 pg/ml, respectively, n = 5–6) compared with controls (198.8 ± 42.9 pg/ml, n = 5, p < 0.05). Fig. 2⇓B shows that the repressive effect of NE persists for at least 24 h. The NE activity declined with time of culture by ∼50% at 2 h; however, ∼45 and 36% of NE activity remained at 6 and 24 h of culture, respectively. Cytotoxicity, measured by MTT assay, indicated that NE had no significant effect on cell viability at concentrations up to 0.5 μg/ml (Ref. 17 and data not shown). To avoid the potential confounding influence of cytotoxicity, all subsequent experiments were performed with 0.5 μg/ml NE, being equivalent to 0.33 ± 0.02 U/ml. To see whether NE also modulates IL-8/CXCL8 production under inflammatory conditions, hASM cells were stimulated with TNF-α (1 ng/ml) for 6 h. Fig. 2⇓C shows that the robust TNF-α-stimulated IL-8/CXCL8 release is attenuated by NE (1055.0 ± 58.4 pg/ml vs 682.6 ± 16.6 pg/ml; p < 0.01).

FIGURE 2.
FIGURE 2.

NE suppresses IL-8/CXCL8 expression in hASM cells at the transcription level. A and B, Concentration response and time course of NE on IL-8/CXCL8 secretion. hASM cells were stimulated with NE (0, 0.05, 0.1, 0.5, and 1.0 μg/ml) for 6 h (A) or were stimulated with NE (0.5 μg/ml) for the times indicated (B) or left untreated. Concentration of IL-8/CXCL8 in the supernatants was determined with ELISA. C, Effect of NE on TNF-α-stimulated IL-8/CXCL8 secretion. hASM cells were stimulated with TNF-α (1 ng/ml) or left untreated for 6 h in the presence or absence of NE (0.5 μg/ml). Concentration of IL-8/CXCL8 in the supernatants was determined with ELISA. D, Effect of the neutrophil elastase inhibitor NEI on NE-induced suppression of IL-8/CXCL8 release. hASM cells were stimulated with NE (0.5 μg/ml) or left untreated for 6 h in the presence or absence of NEI (10 μΜ). Concentration of IL-8/CXCL8 in the supernatants was determined with ELISA. E, Effect of trypsin on IL-8/CXCL8 secretion. hASM cells were stimulated with trypsin (0, 0.01, 0.03, and 0.1 μg/ml) for 6 h. Concentration of IL-8/CXCL8 in the supernatants was determined with ELISA. F, Direct effect of NE on IL-8/CXCL8 protein. Cell-free medium containing recombinant human IL-8/CXCL8 (750 or 250 pg/ml, upper panel; 1 or 0.1 μg/ml, lower panel) in the presence or absence of NE (0.5 μg/ml) was incubated for 6 h. The incubated medium was determined for IL-8/CXCL8 with ELISA (upper panel) or Western blotting (lower panel). All ELISA results are expressed as mean ± SEM. *, p < 0.05; **, p < 0.01 vs basal levels (A, C, D, and E) or time control levels (B). ##, p < 0.01 vs TNF-α only (C). G, NE decreases IL-8/CXCL8 mRNA expression in hASM. hASM cells were stimulated with NE (0.5 μg/ml) or left untreated for 2 h. Total RNA was isolated, and IL-8/CXCL8 mRNA was determined by RT-qPCR. Data are normalized by GAPDH and are expressed as mean ± SEM relative to the unstimulation control. **, p < 0.01 vs the unstimulation control. H, Effect of NE on the rate of IL-8/CXCL8 mRNA degradation. Cells were pretreated with actinomycin D (5 μg/ml) for 1 h and were incubated for the times indicated in the presence or absence of NE (0.5 μg/ml). Levels of IL-8/CXCL8 mRNA were determined by RT-qPCR. Data are normalized by GAPDH and are expressed as mean ± SEM relative to time 0. Solid line (NE) and broken line (control) are the calculated rates of mRNA degradation.

To confirm that the repressive effect is NE induced, a specific elastase inhibitor elastase inhibitor II (NEI) was used. Pretreatment with NEI prevented the repressive effect of NE (0.5 μg/ml) on IL-8/CXCL8 release at 2 h (85.3 ± 6.9 pg/ml) and at 6 h (257.2 ± 7.5 pg/ml; Fig. 2⇑C) when compared with that of NE alone (22 ± 6.7 pg/ml and 102.5 ± 3.4 pg/ml, n = 5–6, respectively; p < 0.05) (Fig. 2⇑D). To exclude the repressive effect of NE not commonly shared with other serine proteases, trypsin was used to stimulate hASM cells. Trypsin at a range of concentrations does not suppress IL-8/CXCL8 release. Instead, it induces IL-8/CXCL8 secretion at 0.01 and 0.03 μg/ml (p < 0.05 and p < 0.01 vs baseline, respectively) (Fig. 2⇑E).

To exclude the possibility that the reduction in the measured levels of IL-8/CXCL8 might be just because of a direct degradation of the protein or modifications of the epitopes the assay recognized, cell-free medium with variable amount of rIL-8/CXCL8 (750 and 250 pg/ml) was incubated in the presence or absence of NE (0.5 μg/ml). As shown in Fig. 2⇑F, upper panel, the measured levels of IL-8/CXCL8 in the NE-treated medium are not different from those in the corresponding controls at 6 h (Fig. 2⇑F, upper panel) and 24 h (data not shown). The negative effect of NE on IL-8/CXCL8 protein degradation was also confirmed by SDS-PAGE with a specific Ab for IL-8/CXCL8 (Fig. 2⇑F, lower panel).

We next asked whether NE represses the IL-8/CXCL8 gene expression at the transcription level. To precisely quantify the levels, RT-qPCR was used to determine the expression of IL-8/CXCL8 mRNA at 2 h in hASM cells in the presence or absence of NE (0.5 μg/ml). As shown in Fig. 2⇑G, NE decreases the levels of IL-8/CXCL8 mRNA by ∼50% (p < 0.01).

A reduction in the level of mRNA might be through enhanced degradation instead of decreased production. To exclude this possibility, we conducted transcriptional blockade studies. The RNA Pol 2 inhibitor actinomycin D (5 μg/ml) was added to hASM cell culture for 1 h before incubation in the presence or absence of NE (0.5 μg/ml). Levels of IL-8/CXCL8 mRNA were analyzed by RT-qPCR after 0, 1, 2, and 4 h of NE treatment, and the rate of GAPDH-normalized IL-8/CXCL8 mRNA degraded per hour relative to baseline (set as 100%) was measured as described previously (29). As shown in Fig. 2⇑H, in the presence of NE, the levels of IL-8/CXCL8 mRNA were not different from those of the controls at the time points studied, with a degradation rate (26.1 ± 3.2% h−1) not different to that of the controls (24.6 ± 3.0% h−1). These data support that NE suppresses IL-8/CXCL8 transcription.

NE stimulates NF-κB p65 activation in hASM and A549 cells

NF-κB activates transcription from the IL-8/CXCL8 promoter (30, 31). NE is able to induce phosphorylation and degradation of IκB proteins and nuclear translocation of NF-κB in bronchial epithelial cells (14) and hASM (17). We next asked whether NE differentially affect the activity of NF-κB p65 in the nucleus. To examine the DNA binding activity of the nuclear p65, NF-κB TransAM was used. As shown in Fig. 3⇓A, p65 DNA binding activity is both enhanced in hASM cells by 5.5-fold (p < 0.01) and in A549 cells by 1.4-fold (p < 0.01) upon stimulation with NE (0.5 μg/ml) for 1 h. Activation of p65 in hASM cells was also confirmed by confocal microscopy with a specific Ab for p65 (Fig. 3⇓B). In the absence of stimulation, most of p65 immunoactivity was detected in the cytoplasm (Fig. 3⇓B, b and e); upon stimulation with NE for 1 h, p65 was translocated into the nuclei (Fig. 3⇓B, c and f).

FIGURE 3.
FIGURE 3.

NE stimulates NF-κB activation in hASM and in respiratory epithelial cells. A, Effect of NE on p65 NF-κB DNA binding activity. p65 TransAM kit analysis of hASM and A549 cells treated with NE (0.5 μg/ml) for 1 h or left untreated. Results are expressed as mean ± SEM. **, p < 0.01 vs the unstimulation control. B, Effect of NE on p65 nuclear translocation. Confocal images of untreated and NE-treated hASM cells for 1 h with preimmune IgG (Cy3; a) or an Ab specific for p65 (Cy3; b and c). Images from nuclear stain (Hoechst; d–f) and IgG (d) or p65 (e and f) were merged to show the subcellular localization of NRF. C, Effect of NE on the IL-8/CXCL8 promoter recruitment of p65. ChIP assays were performed on hASM and A549 cells by using a p65 Ab. Cells were treated with NE (0.5 μg/ml) for 1 h or left untreated. The IP-DNA was quantified by qPCR with primer pairs specific for the κB site at the promoter. Values are normalized by input DNA. Results are expressed as mean ± SEM fold induction vs values of untreated control. *, p < 0.05 compared with values of untreated control. IgG, preimmune IgG control.

Accessibility of a promoter determines its binding by factors. For example, promoters with regulated and late accessibility require stimulus-dependent modifications in chromatin structure to make NF-κB sites accessible (32). It is therefore possible that NE might selectively prevent the binding of p65 to the IL-8/CXCL8 promoter. To determine the recruitment of p65 to the native IL-8/CXCL8 promoter, ChIP assays with an Ab specific for p65 (28) was used. The amount of IP-DNA was determined by RT-qPCR using a primer pair amplifying a region around the κB site in the IL-8/CXCL8 promoter. To demonstrate the site specificity of the assay, a primer pair amplifying an irrelevant site around the 3′-UTR was also used. As shown in Fig. 3⇑C, NE increases recruitment of p65 to the IL-8/CXCL8 promoter in hASM cells by 2.8-fold (p < 0.05) and in A549 cells by 6.7-fold (p < 0.01). In contrast, the levels of IP-DNA around the 3′-UTR were not different between NE-treated cells and control cells; both levels were similar to those of IgG controls (data not shown). Taken together, instead of inhibiting p65 signaling, NE induces p65 nuclear translocation and binding to the native IL-8/CXCL8 promoter in hASM. It is therefore inferred that mechanisms inhibiting NF-κB transactivating activity at the IL-8/CXCL8 promoter or directly suppressing the promoter confer NE the repressive effect.

NE selectively induces NF-κB repressing factor expression in hASM

NRF represses the basal transcription of the IL-8/CXCL8 promoter (24). We therefore hypothesized that NRF is involved in the repression of IL-8/CXCL8 transcription induced by NE. To this end, we first determined the expression of NRF upon NE stimulation. RT-PCR analysis showed that NE induced rapid and prolonged up-regulation of NRF mRNA in hASM cells (Fig. 4⇓A); significant induction occurred within 30 min and persisted for at least 4 h. By contrary, levels of NRF mNRA in A549 and Beas-2B cells remained unchanged during the study time course (Fig. 4⇓A). Western blotting analysis using a specific Ab directed against aa 18–45 was also used to determine the protein levels. Becaused NRF is predominantly accumulated in the nuclei (33, 34), nuclear protein was extracted. Parallel to the kinetics of mRNA, nuclear NRF protein levels in hASM cells were increased by NE in a time-dependent manner, occurring by 1 h and persisting at 2 h (Fig. 4⇓B). Again, NE did not affect the NRF protein levels in A549 and Beas-2B cells (Fig. 4⇓B).

FIGURE 4.
FIGURE 4.

NE specifically induces NRF expression in hASM. A, RT-PCR analysis of NRF mRNA levels in hASM, A549, and Beas-2B cells treated with NE (0.5 μg/ml) for the time indicated (0–4 h). Data are representative of three independent experiments with similar results. B, Western blot analysis of NRF expression in nuclear extracts from hASM, A549, and Beas-2B cells treated with NE (0.5 μg/ml) for the time indicated (0–2 h). Data are representative of three independent experiments with similar results. C and D, Effect of variable concentrations of NE on NRF expression in hASM. Western blot analysis of NRF expression in nuclear extracts from hASM cells treated with variable concentrations of NE (0–0.5 μg/ml) for 2 h (C). Densinometry of NRF expression normalized by Lamin A/C was shown (D). *, p < 0.05 vs basal levels.

As lower concentrations of NE (<0.5 μg/ml) seemed not to suppress IL-8/CXCL8 release in hASM cells (Fig. 2⇑A), we next asked whether NRF is not stimulated at such concentrations. As shown in Fig. 4⇑, C and D, by Western blotting, although NE at the concentrations of 0.05 and 0.1 μg/ml seems to have a trend of inducing NRF expression, significant amount of NRF is only enhanced by NE at the concentration of 0.5 μg/ml.

NE induces IL-8/CXCL8 promoter recruitment of NRF followed by removal of RNA Pol 2 in hASM

By binding to the NRE in the IL-8/CXCL8 promoter, NRF suppresses its basal transcription (24). We therefore asked whether NE induces recruitment of NRF to the native IL-8/CXCL8 promoter in hASM. To this end, ChIP analysis with the Ab specific for NRF was used. The IP-DNA was amplified with the same primer pairs used in Fig. 3⇑B, which also cover the NRE in the IL-8/CXCL8 promoter (24). IgG controls were used to demonstrate the specificity of the Ab. Without NE treatment, a low but consistently detected enrichment of IP-DNA over background, the IgG control, was observed (Fig. 5⇓A). Upon treatment with NE (0.5 μg/ml) for 1 h, in which time nuclear NRF had significantly increased (Fig. 4⇑B), a robust increase in the amount of IP-DNA was observed (Fig. 5⇓A). In contrast, changes of the IP-DNA were not seen when the primers for 3′-UTR were used (data not shown). This suggests that, in hASM cells, the IL-8/CXCL8 promoter recruitment of NRF is increased by NE.

FIGURE 5.
FIGURE 5.

NE induces NRF recruitment to the IL-8/CXCL8 promoter and removal of RNA Pol 2 in hASM. ChIP assay analysis of hASM cells treated with NE (0.5 μg/ml) for 1 h (A) and the time indicated (0–4 h; B), and treated with variable concentrations of NE (0, 0.1, and 0.5 μg/ml) for 2 h (C) with Abs specific for NRF and RNA Pol 2 as indicated. qPCR was used to quantify the IP-DNA with the same primer pairs used for Fig. 3C, which also cover the NRE region. Data are normalized by input control and are expressed as mean ± SEM relative to the unstimulated control. *, p < 0.05; **, p < 0.01 vs unstimulated control. IgG, preimmune IgG control.

To demonstrate the functional effect of recruited NRF, binding of RNA Pol 2 to the native IL-8/CXCL8 promoter was determined with ChIP assays using a previously proved ChIP-grade anti-RNA Pol 2 Ab (28). Fig. 5⇑B shows that NE (0.5 μg/ml) causes a time-dependent decline in the level of RNA Pol 2 at the promoter, occurring as early as at 1 h and persisting at 4 h. This indicates that NE-induced NRF recruitment is temporarily associated removal of RNA Pol 2 from the promoter. We also studied the effects of NE at lower concentrations. Fig. 5⇑C shows that NE at the concentration <0.5 μg/ml, e.g., 0.1 μg/ml, does not seem to have any suppressive effect of RNA Pol 2 recruitment. This observation is parallel to that in IL-8/CXCL8 release (Fig. 2⇑A) and in NRF induction (Fig. 4⇑, C and D).

NRF knockdown reverses the suppression of IL-8/CXCL8 by NE in hASM

To confirm the function role of NE-induced NRF in hASM cells, knockdown of NRF by RNA interference was used. Western blotting analysis using the anti-NRF Ab revealed that transfection with an siNRF for 48 h significantly decreased the level of NRF compared with that in siCONTROL nontargeting siRNA no. 1 (scramble RNA)-transfected cells (Fig. 6⇓A). Cells were then incubated for 6 h in the presence or absence of NE, and the supernatants were collected for ELISA analysis. NE was able to repress IL-8/CXCL8 expression by ∼50% (p < 0.001) in scramble RNA-transfected cells (Fig. 6⇓B). Knockdown of NRF appeared to reverse the repressive effect of NE on IL-8/CXCL8; NE stimulated IL-8CXCL8 release in NRF knockdown cells (p < 0.01; Fig. 6⇓B). In addition, we observed higher basal levels of IL-8/CXCL8 in siNRF-transfected cells compared with that in scramble RNA-transfected cells (p < 0.01; Fig. 6⇓B). Taken together, these data indicate that NE-induced NRF is functionally implicated in the repression of IL-8/CXCL8 in hASM. Our data also confirm the constitutive repression of IL-8/CXCL8 promoter by NRF.

FIGURE 6.
FIGURE 6.

NRF knockdown in hASM reverses the suppression of IL-8/CXCL8 by NE. A, Western blot analysis of NRF expression in cellular extracts from hASM cells that were transfected with siCONTROL nontargeting siRNA (scramble) or siRNA targeting NRF (siNRF) for 48 h. Data are representative of three independent experiments with similar results. B, ELISA analysis of the supernatants from the siCONTROL- or siRNA-transfected hASM cells incubated in the presence or absence of NE (0.5 μg/ml) for 6 h. Results are expressed as mean ± SEM. ***, p < 0.001; ##, p < 0.01 vs the basal levels of siCONTROL-transfected cells.

Forced expression of NRF in A549 cells represses IL-8/CXCL8 production

To confirm the importance of inducible NRF in the differential effect of NE, we next asked whether IL-8/CXCL8 expression is suppressed by forced expression of NRF in A549 cells. To this end, we use NRF overexpression plasmid (pNRF) by inserting full-length NRF cDNA into pCMV6-XL4 vector (pCMV). Fig. 7⇓A demonstrates that the expression of NRF, determined by Western blotting, is augmented in the cells transfected with pNRF for 24 h compared with that in the empty vector pCMV-transfected cells. Fig. 7⇓B shows that overexpression of NRF causes a decline in the level of IL-8/CXCL8 in the supernatant compared with that by pCMV-transfected cells (Fig. 7⇓B, lane 1 vs 3, p < 0.001). To further confirm that the transfected NRF is functional, the transfected cells were stimulated with NE (0.5 μg/ml) for 6 h. Fig. 7⇓B shows that forced expression of NRF (pNRF) mitigates the increased release of IL-8/CXCL8 by NE seen in pCMV-transfected cells. These data support that the expression of IL-8/CXCL8 can be suppressed by inducing NRF.

FIGURE 7.
FIGURE 7.

Forced expression of NRF in A549 cells represses IL-8/CXCL8 release. A, Cells were transfected with pNRF or the empty vector pCMV for 24 h. The expression of NRF in cellular extracts was determined by Western blotting. B, ELISA analysis of the supernatants from the pCMV- or pNRF-transfected hASM cells incubated in the presence or absence of NE (0.5 μg/ml) for 6 h. Results are expressed as mean ± SEM. ***, p < 0.001; #, p < 0.05 vs the basal levels of pCMV-transfected cells.

Discussion

In this study, we demonstrate differential effects of NE on the expression of IL-8/CXCL8 in hASM and in respiratory epithelial cell lines. NE inhibits IL-8/CXCL8 synthesis in hASM cells while it stimulates its expression in A549 and Beas-2B epithelial cells. Although NF-κB is activated, NE rapidly and selectively induces NRF expression in hASM and hence suppresses the IL-8/CXCL8 promoter. In contrast, NE only stimulates NF-κB signaling without inducing NRF in A549 and Beas-2B cells. In the later cellular contexts, NE stimulates the expression of IL-8/CXCL8. Our results demonstrate a novel negative regulatory mechanism whereby inducible NRF represses IL-8/CXCL8 expression.

Previous reports have shown that NE induces IL-8/CXCL8 gene expression and protein secretion in bronchial epithelial cells (14) and lung epithelial cells (13, 20). Apart from epithelial cells, ASM is also a rich source of biologically active IL-8/CXCL8, the production of which is enhanced in response to inflammatory mediators and pathogens (3, 35). We report here that NE inhibits IL-8/CXCL8 expression in hASM cells. Upon incubation with NE, levels of IL-8/CXCL8 in the cultured supernatants declined in a concentration- and time-dependent manner. Several approaches were taken to confirm this NE-suppressive effect. First, MTT assays excluded the cytotoxicity of NE at concentrations not more than 0.5 μg/ml, a concentration used in most of the experiments. Second, IL-8/CXCL8 in cell-free culture medium was directly measured in the presence of NE, which suggests no effect of NE on the level of IL-8/CXCL8 itself determined by ELISA and SDS-PAGE. Third, the suppression of IL-8/CXCL8 secretion by NE was prevented by the specific NE inhibitor NEI. The observation that another serine proteinase, trypsin, did not suppress, but instead stimulates IL-8/CXCL8 release, excludes the possibility that the effect of NE treatment is not NE-specific but commonly shared with other serine proteases. Whether the effect of elastase treatment are attributed to degradation of junctional adhesion proteins causing cell retraction and impaired function (36, 37, 38) of hASM cells deserves further investigation.

NE appears to suppress IL-8/CXCL8 expression at the transcription level. Upon stimulation with NE, levels of IL-8/CXCL8 mRNA declined. Transcriptional blockade studies revealed that the stability of IL-8/CXCL8 mRNA was not affected by NE, suggesting that NE suppresses IL-8/CXCL8 expression at the transcription level.

In contrast to previous reports (3, 35), hASM cells used in our study actively release large quantity of IL-8/CXCL8 at basal state. The activation of NF-κB and expression of IL-8/CXCL8 mRNA indicate those hASM cells were at a state of active production of IL-8/CXCL8. In this regard, our results should be interpreted as the modulation effect of NE on active synthesis and release of IL-8/CXCL8 from hASM cells, as found in airway smooth muscle of severe asthma (39), rather than physiological naive. Importantly, NE also attenuates the secretion of IL-8/CXCL8 stimulated by TNF-α, indicating the concomitant release of NE from recruited polymorphonuclear cell (PMN) to hASM may modify the cytokine-induced IL-8/XCXL8 production under inflammatory conditions. The activity of NE (0.5 μg/ml or 0.33 U/ml) used in most of our experiment is ∼1/10 as much as in sputum from cystic fibrosis patients with clinical exacerbations (3 U/ml, airway PMN burden 1.3 × 106/ml) (40, 41) and is similar to that from stable cystic fibrosis patients (airway PMN burden 1.9 × 105/ml) (41). Thus, we found a previous undiscovered role of NE in the modulation of IL-8/CXCL8 expression specifically in hASM. Although neutrophils are recruited to the inflamed airways, neutrophil-secreted NE might induce IL-8/CXCL8 release from airway epithelium to further facilitate chemotaxis of neutrophils to the epithelial layer to combat pathogens, whereas NE might attenuate IL-8/CXCL8 release, even in response to proinflammatory mediators, to lessen PMN inflammation in the smooth muscle layer.

NRF is ubiquitously and constitutively expressed in all tested human tissues, including the lung, with variable amounts (23, 42). Constitutive NRF has been implicated in repressing basal expression of IL-8/CXCL8 but conversely in induction of IL-8/CXCL8 by IL-1β in HeLa cells (24). Whether NRF itself is regulable is not clear. We report here for the first time that NRF is up-regulated by NE in hASM. Upon simulation with NE, NRF mRNA was rapidly induced in 30 min, followed by an elevation in the levels of NRF protein. Interestingly, the NE induction of NRF appears to be tissue restricted; in airway epithelial cells such as A549 and Beas-2B, the expression of NRF is not affected by NE stimulation. It is therefore suggested that induction of NRF is sophisticatedly controlled. We currently found that NE activated distinct NF-κB subunits in hASM cells from those in A549 cells; NE specifically stimulates RelB in hASM but not in A549 cells (43). Whether distinct NF-κB subunits mediate differential effects of NE on NRF expression is currently under study.

Importantly, inducible NRF appears to confer the repressive effect of NE on IL-8/CXCL8 transcription. In hASM cells, NE-induced nuclear NRF expression accompanies its binding to the promoter. The increase in promoter recruitment of NRF was associated with loss of RNA Pol 2 from the IL-8/CXCL8 promoter, suggesting an inhibition at the transcriptional initiation. The functional role of inducible NRF is further confirmed by knockdown of NRF by RNA interference, which prevented NE-induced repression of IL-8/CXCL8. Additional evidence supporting the mediated role of NRF in NE repressive effect is that NE failed to induce NRF in A549 cells and Beas-2B cells, in which conditions NE did not suppress IL-8/CXCL8 expression. The observation that forced expression in A549 cells of NRF by pNRF inhibited IL-8/CXCL8 expression indicates that increased amount of NRF by itself is enough to suppress IL-8/CXCL8 expression. Thus, NE-induced NRF appears to behave as “immediate-early” genes (44), which is rapidly induced to play a role in regulating targeted promoters.

How NRF represses this transcriptional process is not clear. It has been suggested that the repressive effect of NRF may be through protein-protein interaction with NF-κB (23). In agreement with this, we found that p65 and NRF were both recruited to the IL-8/CXCL8 promoter in hASM cells following NE stimulation. This NRF-NF-κB protein-protein interaction has been suggested to prevent recruitment of elongation factor DRB sensitivity-inducing factor and hence to interrupt the transcriptional elongation for the HIV-1 LTR reporter (26). However, such an observation was made on a synthesized promoter, where the condition of nucleosome assembled may be different from that at the native promoters or the DNA may be naked, therefore effects via the chromatin would be ignored. We took the approach by using ChIP analysis to observe the events at the native IL-8/CXCL8 promoter. Although an effect on the elongation process could not be excluded, the observation that RNA Pol 2 was removed from the promoter following recruitment of inducible NRF suggests an inhibitory effect on the transcriptional initiation. This observation suggests an NRF repressive mechanism, which involves both p65 and NRF binding to the IL-8/CXCL8 promoter and potentially replacing coactivators with corepressor proteins or by affecting the recruitment or activity of chromatin remodeling engines. Further investigations are required to address this question.

In conclusion, we report a novel negative regulatory mechanism for NE-induced suppression of IL-8/CXCL8 synthesis through induction of NRF (Fig. 8⇓). In hASM, NE not only activates NF-κB p65 but also induces NRF expression. Both p65 and NRF is recruited to the IL-8/CXCL8 promoter, leading to removal of RNA Pol 2 from the promoter and hence interrupting transcription. NRF appears to play a central role in this transcriptional repression process. In contrast, NRF is not induced by NE in respiratory epithelial cells. In the absence of inducible NRF, NE-activated p65 results in transcription of IL-8/CXCL8 mRNA. This study not only addresses a previously unknown inhibitory role of NE but also potentially provide new insight into treatment of IL-8/CXCL8-related disease, such as chronic obstructive pulmonary disease, severe asthma, or cystic fibrosis. This might be achieved by control the amount of NRF. Further study is necessary to address the molecular mechanism whereby inducible NRF is regulated.

FIGURE 8.
FIGURE 8.

Models for the NRF-mediated repression of IL-8/CXCL8 expression by NE. In hASM cells, stimulation with NE rapidly activates p65 and induces NRF expression; both are recruited to the IL-8/CXCL8 promoter. In the presence of NRF, tranactivation activity of p65 is inhibited, culminating in removal of RNA Pol 2 (RNAP2) from the promoter and hence repressing the transcription.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by grants from the National Science Council of Taiwan (NSC 93-2314-B-182-043, NSC 94-2314-B-182-007, and NSC 95-2314-B-182-001).

  • ↵2 Address correspondence and reprint requests to Dr. Han-Pin Kuo, Department of Thoracic Medicine, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, 199 Tun-Hwa North Road, Taipei, Taiwan; E-mail address: q8828{at}ms11.hinet.net; or Dr. Chien-Huang Lin, Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei 110, Taiwan; E-mail address: chlin{at}tmu.edu.tw

  • ↵3 Abbreviations used in this paper: ASM, airway smooth muscle; ChIP, chromatin immunoprecipitation; hASM, human ASM; IP-DNA, immunoprecipitated DNA; LTR, long terminal repeat; NE, neutrophil-derived elastase; NRF, NF-κB repressing factor; PMN, polymorphonuclear cell; qPCR, quantitative real-time PCR; RNA Pol 2, RNA polymerase II; SFM, serum-free medium; si, small interfering; 3′-UTR, 3′-untranslated region.

  • Received November 7, 2008.
  • Accepted April 21, 2009.

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