Mitogen-Activated Protein Kinase Phosphatase-1 Negatively Regulates the Expression of Interleukin-6, Interleukin-8, and Cyclooxygenase-2 in A549 Human Lung Epithelial Cells
ABSTRACT
Mitogen-activated protein kinase phosphatase (MKP)-1 is a protein phosphatase that regulates the activity of p38 mitogen- activated protein (MAP) kinase and c-Jun NH2-terminal kinase (JNK) and, to lesser extent, p42/44 extracellular signal-regu- lated kinase. Studies with MKP-1(—/—) mice show that MKP-1 is a regulating factor suppressing excessive cytokine produc- tion and inflammatory response. The data on the role of MKP-1 in the regulation of inflammatory gene expression in human cells are much more limited. In the present study, we investigated the effect of MKP-1 on the expression of interleukin (IL)-6, IL-8 and cyclooxygenase (COX)-2 in response to stimulation with cytokines
(tumor necrosis factor, IL-1β, and interferon-γ; 10 ng/ml each) in A549 human lung epithelial cells. Cytokines enhanced p38 and
JNK phosphorylation and MKP-1 expression. p38 MAP kinase inhibitors 4-[4-(4-fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl] phenol (SB202190) and 1-(5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)- 3(4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl)urea (BIRB 796) in-Mitogen-activated protein (MAP) kinases include extracel- lular signal-regulated protein kinases (ERKs) and stress- activated protein kinases c-Jun NH2-terminal kinase (JNK) and p38. ERKs are activated in response to various physio- logical and pathophysiological stimuli, and they regulate cel- lular proliferation and differentiation. p38 and JNK are activated in response to a wide range of stimuli, including proinflammatory cytokines, growth hormones, G protein-cou- pled receptors, and cellular stress (Johnson and Lapadat, 2002; Rinco´n and Davis, 2009). The family of p38 MAP ki- nases consists of four isoforms, namely, p38α, p38β, p38γ, and p386. p38α and p38β are widely expressed, whereas p38γ is expressed in skeletal muscle and p386 in testes, pancreas, small intestine and in CD4+ T cells. The targets of p38 include transcription factors and a wide range of other cel- lular regulatory proteins. p38 regulates cellular growth and differentiation and, importantly, immune response (Ashwell, 2006; Rinco´n and Davis, 2009). The JNK family of MAP kinases consists of three JNK isoforms, namely, JNK1, JNK2, and JNK3. JNK1 and JNK2 are ubiquitously ex- pressed, whereas the expression of JNK3 is restricted to testes, heart, and brain. JNK is involved in the regulation of various cellular functions, such as cellular growth, differen- tiation, and apoptosis (Johnson and Lapadat, 2002; Rinco´n and Davis, 2009).
Both p38 and JNK regulate immune responses. p38 and JNK have been shown to regulate the expression of cytokines and inflammatory factors, including tumor necrosis factor (TNF); interleukin (IL)-1β, IL-2, IL-6, and IL-8 (also known as CXCL8); cyclooxygenase (COX)-2; and inducible nitric- oxide synthase (Chen et al., 2000; Clark et al., 2003; Kor- honen et al., 2007). In addition, p38 and JNK are both in- volved in the activation of T helper (Th) cells, Th1 differentiation, and the production of Th1 cytokines, such as interferon (IFN)-γ, by Th1 cells. p38 and JNK also regulate cytokine production and other inflammatory cellular re- sponses of macrophages and other cells in immune response (Ashwell, 2006; Rinco´n and Davis, 2009).
MAP kinase phosphatases (MKPs) are dual-specific phos- phatases (DUSPs) that dephosphorylate tyrosine and threo- nine residues in MAP kinases and thereby inactivate them (Liu et al., 2007; Boutros et al., 2008). In mammalian cells, 11 MKPs have been described, and they differ from each other by expressional pattern, tissue distribution, cellular location, and substrate specificity. MKP-1 (also termed DUSP1) is a nuclear phosphatase that is expressed in various cell types and tissues. MKP-1 expression is induced in response to a range of stimuli, such as cellular stress, cytokines, LPS, and glucocorticoids, in several inflammatory (such as macro- phages) and noninflammatory cells. Although originally MKP-1 was described to dephosphorylate p42/44 ERK (Sun et al., 1993), MKP-1 has been shown to have specificity to- ward p38 and JNK over p42/44 ERK (Franklin and Kraft, 1997; Liu et al., 2007; Boutros et al., 2008). Data from knock- out mice suggest that MKP-1 negatively regulates innate immune response. In macrophages, defects in MKP-1 func- tion results in increased and prolonged p38 activation (Zhao et al., 2005; Hammer et al., 2006; Salojin et al., 2006), and MKP-1 knockout mice have increased expression of TNF, IL-6, IL-10, COX-2, and macrophage inflammatory pro- tein-1α in response to in vivo LPS challenge (Salojin et al., 2006). Exposure of MKP-1(—/—) mice to Staphylococcus au- reus or Gram-positive bacterial products resulted in elevated cytokine production and inducible nitric-oxide synthase ex- pression, and these mice had increased mortality rate, in- creased neutrophil infiltration in lungs, and they suffered from more severe organ damage (Wang et al., 2007). MKP-1 is involved in the anti-inflammatory effects of glucocorti- coids. The inhibition of COX-2 and E-selectin expression by glucocorticoids has been reported to be mediated by MKP-1 (Abraham et al., 2006; Fu¨ rst et al., 2007). In addition, the inhibition of growth-related oncogene protein-α (GRO-α) ex- pression by glucocorticoids has been shown to be mediated by MKP-1 in human airway smooth muscle cells (Issa et al., 2007).
Chronic inflammatory airway diseases, such as asthma and chronic obstructive pulmonary disease, are character- ized by increased infiltration of inflammatory cells and pro- duction of proinflammatory cytokines in the lungs (Barnes, 2008). Human pulmonary epithelial cells have been reported to produce IL-6 and IL-8 in a p38-dependent mechanism in response to stimulation with IL-17 (Laan et al., 2001). Be- cause the production of cytokines and the activation of MAP kinases are interrelated, we investigated the effect of MKP-1 on the p38 and JNK activation and on the regulation of cytokine production in A549 human lung epithelial cells.
Materials and Methods
Materials. Reagents were obtained as follows. Recombinant human TNF, IFN-γ and IL-1β were purchased from R&D Systems (Minneapolis, MA). SB202190 (Tocris Biosciences, Bristol, UK), BIRB 796 (Axon MedChem, Groningen, The Netherlands), and N-(4-amino-5-cyano-6-ethoxypyridin-2-yl)-2-(2,5-dimethoxyphe- nyl)acetamide (JNK inhibitor VIII; Calbiochem, Merck Chemicals, Darmstadt, Germany) were purchased as indicated. All other reagents were from Sigma-Aldrich (St. Louis, MO) unless other- wise stated below.
Cell Culture. A549 human lung epithelial cells (American Type Culture Collection, Manassas, VA) were cultured at 37°C in 5% CO2 atmosphere in Ham’s F-12K (Kaighn’s modification) medium con- taining 5% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B (all from Invitrogen, Paisley, UK). For cytokine measurements, Western blot and quantitative RT-PCR, cells were seeded on a 24-well plate at density of 4 × 105 cells/well. Cell monolayers were grown for 48 h before the experiments were started. SB202190 or JNK inhibitor VIII at concentrations indicated were added to the cells in fresh culture medium containing 5% fetal calf serum and antibiotics 30 min before the stimulation with cytokines (TNF, IFN-γ, and IL-1β; 10 ng/ml each). Cells were further incubated for the time indicated. Preparation of Cell Lysates for Western Blot Analysis. At the indicated time points, culture medium was removed. Cells were rapidly washed with ice-cold phosphate-buffered saline and solubi- lized in ice-cold lysis buffer containing 10 mM Tris-HCl, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 20 µg/ml leupeptin, 50 µg/ml aprotinin, 5 mM sodium fluoride, 2 mM sodium pyrophosphate, and 10 µM n-octyl-β-D-glucopyranoside. After incubation for 20 min on ice, lysates were centrifuged, and supernatants were collected and mixed in a ratio of 1:4 with SDS loading buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 0.025% bromphenol blue, and 5% β-mercaptoethanol) and stored at —20°C until analyzed.
Western Blot Analysis. COX-2, actin, MKP-1, phospho-c-Jun, c-Jun, JNK, lamin A/C, polyclonal anti-rabbit, and polyclonal anti- goat antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phospho-p38, p38, phospho-JNK, phospho-MAP kinase-activated protein kinase 2 (MK2), MK2 antibodies were from Cell Signaling Technology Inc. (Danvers, MA). Poly(A)-binding pro- tein 1 antibody was from Millipore (Billerica, MA). Polyclonal anti- mouse antibody was from Pierce Chemical (Rockford, IL).
Before Western blot analysis, the samples were boiled for 10 min. Equal aliquots of protein (10 –20 µg) were loaded on a 10% SDS- polyacrylamide electrophoresis gel and separated by electrophoresis. Proteins were transferred to Hybond enhanced chemiluminescence nitrocellulose membrane (GE Healthcare, Little Chalfont, Bucking- hamshire, UK) by semidry electroblotting. After transfer, the mem- brane was blocked in 20 mM Tris base, pH 7.6, 150 mM NaCl, and 0.1% Tween 20 containing 5% nonfat milk for 1 h at room tempera- ture. For detection of phosphoproteins, membranes were blocked in 20 mM Tris base, pH 7.6, 150 mM NaCl, and 0.1% Tween 20 con- taining 5% bovine serum albumin. Membranes were incubated over- night at 4°C with primary antibody, and for 1 h with secondary antibody, and the chemiluminescent signal was detected by Flu- orChem 8800 imaging system (Alpha Innotech, San Leandro, CA) and ImageQuant LAS 4000 mini (GE Healthcare). The chemiluminescent signal was quantified with FluorChem software version 3.1 and ImageQuant TL 7.0 image analysis software.
RNA Extraction and Real-Time RT-PCR. At the indicated time points, culture medium was removed and total RNA extrac- tion was carried out with GenElute Mammalian Total RNA Mini- prep kit (Sigma-Aldrich) according to the manufacturer’s instruc- tions. Total RNA (100 ng) was reverse-transcribed to cDNA using TaqMan Reverse Transcription reagents and random hexamers (Applied Biosystems, Foster City, CA). cDNA obtained from the reverse transcription reaction was diluted 1:20 with RNase-free water and was subjected to quantitative PCR using TaqMan Uni- versal PCR Master Mix and ABI Prism 7000 sequence detection system (Applied Biosystems). The primer and probe sequences and concentrations were optimized according to manufacturer’s guidelines in TaqMan Universal PCR Master Mix Protocol part 4304449 revision C and were as follows: 5′-GCCAGGGCTGAACT- TCGAA-3′ (human COX-2 forward primer; 300 nM), 5′-CAACTC- TATATTGCTGGAACATGGA-3′ (human COX-2 reverse primer; 300 nM), 5′-TGGAAGCCTGTGATACTTTCTGTACT-3′ (human COX-2 probe; 150 nM, containing 6-FAM as 5′-reporter dye and TAMRA as 3′-quencher), 5′-TCCTACCACCAGCAACCCT- GCCA-3′ [human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward primer; 300 nM)], 5′-GCAACAATATCCACTTTACCAGAGTTAA-3′ (human GAPDH reverse primer; 300 nM), and 5′-CGCCTGGTCACCAGGGCTGC-3′ (human GAPDH probe; 150 nM, containing 6-FAM as 5′-reporter dye and TAMRA as 3′-quencher). Expression of human IL-6 and human IL-8 mRNA was measured using TaqMan gene expression assays (Applied Biosystems). PCR reaction parameters were as follows: incubation at 50°C for 2 min, incubation at 95°C for 10 min, and thereafter 40 cycles of denaturation at 95°C for 15 s and annealing and exten- sion at 60°C for 1 min. Each sample was determined in duplicate. A standard curve method was used to determine the relative mRNA levels as described in the Applied Biosystems User Bulle- tin: A standard curve for each gene was created using RNA iso- lated from A549 cells stimulated with cytokines (TNF, IL-1β, and IFN-γ; 10 ng/ml each). Isolated RNA was reverse-transcribed, and a dilution series of cDNA ranging from 1 pg to 10 ng were sub- jected to real-time PCR. The obtained threshold cycle values were plotted against the dilution factor to create a standard curve. Relative mRNA levels in test samples were then calculated from the standard curve. When calculating the results, IL-6, IL-8, and COX-2 mRNA levels were normalized against GAPDH.
Down-Regulation of MKP-1 by siRNA. MKP-1 siRNA (5′-CCAAUUGUCCCAACCAUUU-3′) and MKP-1 siRNA 1 (5′-GCAUAACUGCCUUGAUCAA-3′) were purchased from Dharmacon RNA Technologies (Lafayette, CO). Lamin A/C siRNA (5′-AACUG- GACUUCCAGAAGAACA-3′) and nontargeting control siRNA (5′- AAUUCUCCGAACGUGUCACGU-3′) were purchased from QIAGEN (Valencia, CA). A549 cells were transfected with siRNA using HiPerFect transfection reagent (QIAGEN) according to the manufacturer’s instruc- tions. In brief, cells were seeded at density of 1.25 × 105 cells/well on a 24-well plate in 500 µl of medium with 5% fetal calf serum without antibiotics followed by transfection with nontargeting control siRNA (control siRNA), lamin A/C siRNA, MKP-1 siRNA 1, or MKP-1 siRNA 2. Cells were further incubated for 48 h. Fresh culture medium was changed, and cytokines were added into the culture medium. Cells were further incubated for the time indicated, and gene expression was analyzed. All the experiments were done in triplicates. Transfection efficacy was monitored with green fluorescent siRNA oligonucleotides (siGLO green indicator; Dharmacon RNA Technologies). Approxi- mately 90% of the cells emitted green fluorescence signal when transfected with siGLO and HiPerFect. <5% of the cells emitted signal when cells were incubated siGLO oligonucleotides without transfection reagent. Enzyme-Linked Immunosorbent Assay. Culture medium samples were kept at —20°C until assayed. The concentrations of human IL-6 (PeliPair ELISA; Sanquin, Amsterdam, The Netherlands) and human IL-8 (BD OptEIA set; BD Biosciences, San Diego, CA) were determined by ELISA according to the manufacturer’s instructions. Statistics. Results are expressed as the mean ± S.E.M. When appropriate, one-way ANOVA with Dunnett’s or Bonferroni’s post- test was performed using InStat version 3.05 for Windows 95/NT (GraphPad Software, Inc., San Diego, CA). Differences were consid- ered significant at *, p < 0.05, **, p < 0.01, and ***, p < 0.001. Results p38 and JNK Were Phosphorylated in Response to Cytokines in A549 Pulmonary Epithelial Cells. A549 pulmonary epithelial cells were stimulated with a cytokine mixture (TNF, IFN-γ, and IL-1β; 10 ng/ml each), and cells were harvested for protein extraction at time points indi- cated (Fig. 1). Phosphorylation of p38 MAPK was detected in response to stimulation with a cytokine mixture at time point of 30 min, and it was reverted near the basal level at 1 h. Another submaximal increase in p38 phosphorylation was observed at 2 h after the stimulation with a cytokine mixture, which was reversed near the basal level at 3 h and remained at that level up to the 5-h follow-up (Fig. 1A). Marked phos- phorylations of JNK p54 and p46 were observed at 30 min after cytokine treatment, and it was reverted near the basal level at 1 h (Fig. 1B). Fig. 1. Phosphorylation of p38 and JNK in response to cytokine mixture in A549 cells. Cells were stimulated with cytokine mixture (CM: TNF, IFN-γ, and IL-1β; 10 ng/ml each) for the time indicated, and cells were then harvested for protein extraction. Phosphorylation of p38 (A) and JNK (B) in response to cytokines was determined by Western blot. The gel is a representative of six separate experiments with similar results. Chemiluminescent signal was quantified and phospho-p38, phospho-p46, and phospho-p54 expression was normalized against p38, p46, and p54, respectively. Phosphorylation levels are expressed in arbitrary units, unstimulated cells set as 1, and the other values related to that. Results are expressed as mean ± S.E.M.; n = 6. One-way ANOVA with Dunnett’s post-test was performed, and statistical significance was indicated with **, p < 0.01 compared with unstimulated cells. p38 Inhibitors SB202190 and BIRB 796 and JNK In- hibitor VIII Inhibited the Phosphorylation of MK2 and c-Jun, Respectively, in A549 Cells. p38 inhibitors SB202190 and BIRB 796 have been reported to effectively inhibit p38 at 1 µM and 100 nM concentrations, respectively (Bain et al., 2007). The effect of SB202190 and BIRB 796 on the phosphorylation of p38 substrate MAP kinase-activated protein kinase 2 (MK2) in A549 cells was investigated. A549 cells were preincubated with p38 MAPK inhibitors SB202190 or BIRB 796 for 30 min and stimulated then with the cyto- kine mixture (TNF, IFN-γ and IL-1β; 10 ng/ml each) for 30 min. The phosphorylation of MK2 was detected by Western blot. SB202190 and BIRB 796 inhibited the MK2 phosphor- ylation at concentrations of 1 µM and 100 nM, respectively (Fig. 2, A and B). The IC50 values of JNK inhibitor VIII for JNK1 and JNK2 in kinase assay have been reported to be 2 and 4 nM, respec- tively, and the EC50 value for c-Jun phosphorylation in HepG2 cells has been reported to be 920 nM (Szczepank- iewicz et al., 2006). The effect of JNK inhibitor VIII on the phosphorylation of JNK substrate c-Jun was tested in A549 cells. Cells were preincubated with JNK inhibitor VIII for 30 min and stimulated then with the cytokine mixture for 30 min. The phosphorylation of c-Jun was detected by Western blot. JNK inhibitor VIII inhibited c-Jun phosphorylation clearly at 1 µM drug concentration and completely at 10 µM drug concentration (Fig. 2C). p38 MAPK Inhibitors SB202190 and BIRB 796 Reg- ulated IL-6, IL-8, and COX-2 Expression in Response to Cytokines in A549 Cells. The effects of p38 MAPK inhibitors SB202190 and BIRB 796 or JNK inhibitor VIII on the expression of IL-6, IL-8 and COX-2 in A549 cells were investigated. A549 cells were preincubated with SB202190, BIRB 796 or JNK inhibitor VIII for 30 min and stimulated with a cytokine mixture (TNF, IFNγ and IL-1β, 10 ng/ml each) for 24 h. Supernatants were collected and total cellular proteins were extracted, and IL-6, IL-8, and COX-2 expression was determined. Unstimulated cells did not express measurable levels of IL-6, IL-8, or COX-2 and pretreatment with SB202190, BIRB 796, or JNK inhibitor VIII alone did not induce IL-6, IL-8, or COX-2. Cytokine mixture induced the production of IL-6 and IL-8, and their synthesis was inhibited in a dose-dependent manner by SB202190 and BIRB 796. In contrast, JNK inhibitor VIII did not inhibit the production of IL-6 or IL-8 (Fig. 3, A and B). COX-2 expression was also induced in response to cytokines. BIRB 796 dose-dependently inhibited COX-2 expression and SB202190, at 1 µM concentration, inhib- ited cytokine-induced COX-2 expression. JNK inhibitor VIII (1 µM) did not have effect on COX-2 expression (Fig. 3C). Fig. 2. Effect of SB202190 and BIRB 796 and JNK inhibitor VIII on the phosphorylation of MK2 and c-Jun in response to cytokine mixture in A549 cells. A549 cells were preincubated with SB202190 (A), BIRB 796 (B), or JNK inhibitor VIII (C) at concentrations indicated for 30 min and stimulated with cytokine mixture (CM: TNF, IFN-γ, and IL-1β; 10 ng/ml each) for 30 min, and the phosphorylation of MK2 (A and B) or c-Jun (C) was detected by Western blot. The gels are representatives of six separate experiments with similar results. The expression of IL-6, IL-8, and COX-2 mRNAs over time was investigated. A549 cells were stimulated with the cyto- kine mixture (TNF, IFN-γ, and IL-1β; 10 ng/ml each) for 0 to 4 h, and IL-6, IL-8, and COX-2 mRNA levels were measured. Cells were preincubated with SB202190 and BIRB 796 for 30 min and stimulated with cytokines. IL-6 mRNA expression was increased in response to cytokines up to 4 h, and IL-8 and COX-2 mRNA expression reach maximal levels at 3 h (Fig. 4). The effect of SB202190 and BIRB 796 on mRNA levels of IL-6, IL-8, and COX-2 were investigated at a time point of 3 h, because all IL-6, IL-8, and COX-2 expressed maximal or submaximal mRNA levels at 3 h. SB202190 and BIRB 796 markedly inhibited the expression of IL-6, IL-8, and COX-2 mRNAs (Fig. 5). MKP-1 Regulated the Phosphorylation of p38 MAPK and JNK in A549 Pulmonary Epithelial Cells. At first, we investigated the expression of MKP-1 in A549 cells. Cells were stimulated with the cytokine mixture (TNF, IFN-γ, and IL-1β; 10 ng/ml each), and cells were harvested for protein extraction at time points indicated (Fig. 6A). Unstimulated cells showed low-level basal MKP-1 protein levels, and MKP-1 protein expression was markedly increases in re- sponse to cytokines at 1 h. MKP-1 expression was returned to basal level at 2 h and remained at that level up to 5 h (Fig. 6A). To investigate whether MKP-1 regulated the phosphor- ylation of p38 or JNK in A549 cells, we used siRNA to down-regulate MKP-1 expression. In cells transfected with MKP-1-specific siRNA (MKP-1 siRNA 1 and 2), the protein levels of MKP-1 were reduced compared with the cells trans- fected with a nontargeting control siRNA (control siRNA) showing that siRNA effectively down-regulated MKP-1 in A549 cells (Fig. 6B). We proceeded to investigate the effect of the down-regulation of MKP-1 on the phosphorylation of p38 and JNK. The down-regulation of MKP-1 by siRNA resulted in an increased p38 and JNK phosphorylation in response to cytokine stimulation at 1 h (Fig. 7A). Lamin A/C-specific siRNA reduced lamin A/C protein expression (100 ± 4.8 versus 45.3 ± 4.0% in cells transfected with nontargeting control siRNA and lamin A/C-specific siRNA, respectively; n = 3) but did not affect on p38 or JNK phosphorylation in A549 cells (Fig. 7). These data show that the reduction of MKP-1 by siRNA was functional at the level of p38 phosphor- ylation and that MKP-1 regulated the phosphorylation of both p38 and JNK in A549 cells. MKP-1 Regulated the Expression of IL-6, IL-8, and COX-2 in A549 Cells. The effect of MKP-1 on the expression of IL-6, IL-8, and COX-2 in response to stimulation with cytokine mixture (TNF, IFN-γ, and IL-1β; 10 ng/ml each) in A549 cells was investigated with siRNA. Cells were trans- fected with MKP-1-specific siRNA 1 and 2 to down- regulate MKP-1. Cells were stimulated with the cytokine mixture for 3 and 24 h for mRNA and protein analyses, respectively. In cells transfected with MKP-1 siRNA 1 and 2, the expression of IL-6, IL-8, and COX-2 mRNA was increased compared with the cells transfected with either nontargeting control siRNA or lamin A/C-specific siRNA (Fig. 8). Down-regulation of MKP-1 by siRNA resulted in increased IL-6, IL-8, and COX-2 protein expression (Fig. 9). Lamin A/C-specific siRNA did not affect mRNA or protein levels of IL-6, IL-8, or COX-2, showing that the increased expression of IL-6, IL-8, and COX-2 observed with MKP-1 siRNA 1 and 2 was not due to a general activation of RNA- induced silencing complex pathway. Discussion In the present study, we investigated the effect of MKP-1 on the expression of IL-6 and IL-8, and COX-2 in response to stimulation with the combination of cytokines TNF, IL-1β, and IFN-γ in human A549 lung epithelial cells. IL-6, IL-8, and COX-2 mRNA and protein expression were inhibited by p38 inhibitors SB202190 and BIRB 796, but not with the JNK inhibitor VIII. Stimulation of A549 cells with cytokines induced also the expression of MKP-1. Down-regulation of MKP-1 by siRNA resulted in enhanced p38 and JNK phosphorylation and increased IL-6, IL-8, and COX-2 mRNA and protein expression. Two structurally distinct p38 inhibitors SB202190 and BIRB 796 inhibited IL-6, IL-8, and COX-2 expression in A549 cells. This is in line with previous reports, in which p38 inhibitors SB202190 and SB203580, when used at higher concentrations (10 –30 µM), have been shown to inhibit IL-6, IL-8, and COX-2 expression in A549 cell (Huang and Zhang, 2003; Kuwahara et al., 2006; Chen et al., 2007). The IC50 value for SB202190 in p38 inhibition has been reported to be approximately 100 nM and effective p38 inhibition takes place at 100 to 300 nM drug concentrations (Bain et al., 2007). In our study, SB202190 inhibited IL-6 and IL-8 mRNA and protein expression clearly at the 1 µM concentration, but the reduction in IL-6 and IL-8 protein expression with 100 nM SB202190 was statistically not significant. Therefore, we tested another structurally unrelated p38 inhibitor BIRB 796, which has been reported to effectively inhibit p38 at the 100 nM concentration (Bain et al., 2007). BIRB 796 inhibited IL-6 and IL-8 production in a dose-dependent manner, and the maximal inhibition of IL-6 and IL-8 mRNA and protein expression was observed at the 100 nM drug concentration. To further investigate whether the SB202190 and BIRB 796 inhibited p38, the effect of these compounds on the phosphor- ylation of a p38 substrate MK2 was investigated. SB202190 and BIRB 796 abrogated the phosphorylation of MK2 at concentrations of 1 µM and 100 nM, respectively, showing that these compounds were p38 inhibitors at the concentra- tion used. These data suggest that the reduction of IL-6 and IL-8 expression by SB202190 and BIRB 796 was due to p38 MAPK inhibition and not due to nonspecific effects of these compounds in A549 cells. In A549 cells, SB203580, a p38 inhibitor structurally re- lated to SB202190, has been reported to inhibit COX-2 ex- pression at 1 to 10 µM concentrations (Newton et al., 2000), and SB203580 has been shown to partially inhibit hsp27 phosphorylation (a p38 MAPK substrate) at the 1 µM con- centration and completely at the 10 µM concentration (King et al., 2009b). In our experiments, SB202190 inhibited COX-2 mRNA and protein expression at 1 µM concentration, which also abrogated the phosphorylation of MK2. In addition, BIRB 796 inhibited the expression of COX-2 in a dose-depen- dent manner, with maximal inhibition seen with the 100 nM drug concentration. These data suggest that, in our study, the reduction in COX-2 expression observed with SB202190 and BIRB 796 was probably due to the inhibition of p38 and that p38 regulates COX-2 expression in A549 cells. Taken together, these results suggest that p38 MAPK regulates the expression of IL-6, IL-8, and COX-2 in A549 pulmonary epi- thelial cells. We also investigated the effect of JNK pathway on the expression of IL-6, IL-8, and COX-2 in response to stimula- tion with the proinflammatory cytokines. In these experi- ments, we used a recently described aminopyridine-based compound JNK inhibitor VIII (Szczepankiewicz et al., 2006). This inhibitor has been shown to have >1000-fold selectivity for JNK over other MAP kinases. JNK inhibitor VIII inhib- ited the phosphorylation of c-Jun, a direct substrate of JNK, and the submaximal inhibition in phosphorylation was seen at the 1 µM drug concentration. With this inhibitor, we did not see a reduction in IL-6 and IL-8 production or COX-2 expression in response to the cytokines in A549 cells. Using overexpression of dominant-negative JNK and antisense method in human keratinocytes, JNK has been shown to regulate IL-6 and IL-8 production (Krause et al., 1998). Stretch-induced IL-8 mRNA expression has been reported to be inhibited with a JNK inhibitor SP600125 at a concentra- tion of 5 µM (Li et al., 2003). SP600125 has also been re- ported to inhibit COX-2 expression in response to inflamma- tory stimulation (Nieminen et al., 2006; Chen et al., 2007). The results obtained with the inhibitor SP600125 may be complicated by the fact that SP600125 has been shown to inhibit other kinases also (Bain et al., 2007). Cell type-spe- cific differences in the role of JNK on the expression of inflammatory genes seem also to exist. In vivo administra- tion of SP600125 did not inhibit IL-6 mRNA expression in mice with ozone-induced airway inflammation (Williams et al., 2007), and yet in human primary eosinophils, SP600125 inhibited IL-25-induced IL-6 and IL-8 production (Wong et al., 2005). Taken together, our results with a novel compound JNK inhibitor VIII suggest that JNK does not regulate the expression of IL-6, IL-8, and COX-2 in A549 pulmonary epi- thelial cells.
We used siRNA to investigate the effect of MKP-1 on p38 and JNK phosphorylation and expression of IL-6, IL-8, and COX-2. Down-regulation of MKP-1 with two different siRNAs reduced cytokine-induced MKP-1 expression in A549 cells. In addition,the down-regulation of MKP-1 with two different siRNAs in- creased p38 phosphorylation at 1 h after cytokine stimulation, showing that the reduction of MKP-1 expression by siRNA was functional at the level of MAP kinase activation. The substrate specificity of MKP-1 has been reported to vary between cell types. MKP-1 was found to regulate p38 in macrophages (Salojin et al., 2006), JNK in airway smooth muscle cells (Issa et al., 2007), and both p38 and JNK in endothelial cells (Zakkar et al., 2008). We found an increase in both p38 and JNK phosphor- ylation in A549 pulmonary epithelial cells with MKP-1-specific siRNA 1 and 2. This suggests that MKP-1 regulates the phos- phorylation of both p38 and JNK in A549 cells. Lamin A/C- specific siRNA was used as a positive control in siRNA experi- ments. Lamin A/C-specific siRNA down-regulated lamin A/C protein expression approximately by 50%, showing that lamin A/C siRNA was functional. Lamin A/C-specific siRNA did not have effect on p38 and JNK phosphorylation or IL-6, IL-8, or COX-2 expression, showing that the effects seen with MKP-1- specific siRNA were probably due to specific down-regulation of MKP-1 and not due to general activation of RNA-induced si- lencing complex pathway per se.
IL-8 is an important chemokine in the regulation of neu- trophil migration (Donnelly and Barnes, 2006). Prominent neutrophil infiltration, along with other inflammatory cells, characterizes both chronic obstructive pulmonary disease and severe asthma (Barnes, 2008). We found that MKP-1 negatively regulated the expression of IL-8, which is in line with the reports showing that p38 positively regulates IL-8 expression (Kuwahara et al., 2006). In a recent report using overexpression of MKP-1, MKP-1 has been shown to nega- tively regulate IL-8 mRNA expression in human BEAS-2B bronchial epithelial cells (King et al., 2009a). After intraperi- toneal administration of bacterial products from Gram-posi- tive bacteria, MKP-1(—/—) mice showed increased neutrophilic infiltration in the lung (Wang et al., 2007). IL-17 has been found to be present in eosinophils of asthmatic subjects and has been shown to induce the expression of IL-6, IL-8, and GRO-α in bronchial epithelial cells and fibroblasts (Mo- let et al., 2001). In addition to its effect on IL-8 found in the present study, MKP-1 has been reported to regulate the expression of GRO-α, another neutrophil chemokine, in hu- man pulmonary smooth muscle cells (Issa et al., 2007), suggesting that MKP-1 is an important negative factor in the regulation of inflammatory cell migration. MKP-1 is possibly an important regulatory factor in the pathogenesis of chronic obstructive pulmonary disease or neutrophilic airway inflam- mation related to severe asthma, and increased MKP-1 ex- pression may provide a rationale drug target for the treat- ment of airway inflammation.
p38 MAPK has been reported to regulate the expression of both IL-6 and COX-2 (De Cesaris et al., 1998; Dean et al.,1999). In our experiments, IL-6 production and COX-2 ex- pression were inhibited by p38 inhibitors SB202190 and BIRB 796 in A549 cells. IL-6 and COX-2 expression have also been shown to be increased in LPS-stimulated macrophages from MKP-1(—/—) mice (Abraham et al., 2006; Hammer et al., 2006; Wang et al., 2007). In our experiments, down- regulation of MKP-1 by siRNA resulted in increased expres- sion of IL-6 and COX-2 in A549 cells, which is in line with the previous data from mouse macrophages.
In conclusion, our results suggest that the inhibition of p38 regulates the expression of IL-6, IL-8, and COX-2 in response to cytokines in A549 human lung epithelial cells. MKP-1 negatively regulated the phosphorylation of p38 and JNK. Results also suggest that MKP-1 negatively regulates the expression of IL-6, IL-8, and COX-2 in response to cytokine stimulation by inhibiting the p38 MAPK phosphorylation. Our results suggest that MKP-1 is an important negative regulator of inflammatory gene expression in human lung epithelial cells, and compounds that enhance MKP-1 may have anti-inflammatory effects and control inflammatory re- sponse in the human lung.