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Proteasome Inhibitors Stimulate Interleukin-8 Expression via Ras and Apoptosis Signal-Regulating Kinase-dependent Extracellular Signal-Related Kinase and c-Jun N-Terminal Kinase Activation

Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan

Address correspondence to: Wan Wan Lin, Ph.D., Department of Pharmacology, College of Medicine, National Taiwan University, Taipei 100, Taiwan. E-mail: wwl{at}ha.mc.ntu.edu.tw

	Abstract

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In this study, we investigated the effects of proteasome inhibibors (MG132 and lactacystin) on interleukin (IL)-8 induction. In human epithelial A549 cells, MG132 and lactacystin induced IL-8 release within the range of 0.1–30 µM. The effect of MG132 resulted from IL-8 gene transcription and was blocked by PD 98059, but was unaffected by GF109203X, Ro 31–8220, or SB 203580. Mutational analysis of the 5' flanking region of the IL-8 gene revealed that activator protein (AP)-1–binding element, but not that element responsive to nuclear factor (NF)–IL-6 or NF-B, was necessary for MG132 stimulation. Consistent with this, MG132 and lactacystin increased the DNA-binding and reporter activities of AP-1, but reduced cytokine-elicited B activation. Moreover, AP-1 stimulation was associated with increased extracellular signal-related kinase (ERK), mitogen-activated protein/ERK kinase (MEK), and c-Jun N-terminal kinase (JNK) phosphorylation, whereas IL-8 activity was sensitive to the dominant-negative mutants of JNK1, JNK2, SEK, ASK, ERK2, and Ras, but not those of MEKK1, TAK, and p38 mitogen-activated protein kinase. In addition, activations of the IL-8 gene and AP-1 by MG132 and lactacystin were inhibited by GSH and NAC. Herein we present a novel action of proteasome inhibitors, possibly through ROS production, of targeting the upstream signaling molecules, ERK and JNK, which leads to AP-1 activation and IL-8 gene expression.

Abbreviations: activator protein 1, AP-1 • Dulbecco's modified Eagle's medium, DMEM • enhanced chemiluminescence, ECL • enzyme-linked immunosorbent assay, ELISA • electrophoretic mobility shift assay, EMSA • extracellular signal-related kinase, ERK • fetal bovine serum, FBS • fluorescein isothiocyanate, FITC • glutathione, GSH • interleukin, IL • c-Jun N-terminal kinase, JNK • mitogen-activated protein kinase, MAPK • mitogen-activated protein/ERK kinase, MEK • carbobenzoxy-Leu-Leu-leucinal, MG132 • N-acetylcysteine, NAC • nuclear factor, NF • reactive oxygen species, ROS • reverse transcriptase–polymerase chain reaction, RT-PCR • Tris-buffered saline containing 0.1% Tween 20, TBST • tumor necrosis factor-, TNF-

	Introduction

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Accumulating evidence has confirmed the crucial roles of ubiquitination and/or proteolytic degradation of proteins by proteasomes in regulation of many fundamental biologic functions (1). Conjugation to the short polypeptide ubiquitin has been shown to be a mandatory step in degradation of many proteins by 26S proteasome. Proteins which undergo regulation by this mechanism include cyclins, Bcl-2, p53, c-Fos, c-Jun, PPAR, IB, nuclear factor (NF)-B precursor, STAT, HIF-1, retinoid X receptors, and estrogen receptors. It is thus conceivable that a great variety of cellular regulatory mechanisms, ranging for example from the progression of the cell cycle to the pathways controlling signal transduction and metabolism, are controlled by the ubiquitin/proteasome system.

Proteasome dysfunction by proteasome inhibitors can lead to multiple changes in cell function through modulation of signaling pathways. For example, the inhibitory protein of NF-B, IBs, can be phosphorylated, ubiquitinated, and then degraded by proteasome, which results in the upregulation of NF-B–mediated gene expression of many inflammatory mediators. The inhibition of IB degradation by proteasome inhibitors has allowed a novel strategy to be set up for anti-inflammatory and anticancer drug development (2, 3). Although little information is currently available, it has been demonstrated that proteasome inhibitors can also trigger activation of signal transduction pathways, and thus are valuable new tools for cell biologists (4, 5). In this aspect, proteasome inhibitors can induce cell apoptosis either through Bcl2 phosphorylation (5) or c-Jun N-terminal kinase (JNK) activation (6). Induction of neurite outgrowth through stress-activated protein kinase activation by proteasome inhibitor has also been reported (7). This information attracted our attention and led us to study whether proteasome inhibitors can trigger other signaling transduction pathways and whether or not the action mechanism is related to protein degradation by proteasomes.

Interleukin (IL)-8 is a CXC chemokine, which stimulates chemotactic action in neutrophils, T cells, and basophils. It has been implicated in several inflammatory diseases, such as cystic fibrosis, bronchiectasis, and chronic bronchitis (8). Increased levels of IL-8 have been demonstrated by bronchial epithelial cells in patients with asthma, and it is thus implicated in inflammatory cell chemotaxis in asthma. IL-8 is secreted in a stimulus-specific manner by a wide variety of cell types and is regulated primarily at the level of gene transcription. Several studies have shown that the sequence spanning nucleotides -1 to -133 within the 5' flanking region of the IL-8 gene are essential and sufficient for transcriptional regulation of the gene. This promoter region of IL-8 contains DNA binding sites for inducible transcription factors, including NF-B, activator protein-1 (AP-1), and NF–IL-6. Despite the existence of tissue-specific differences in their dependence on IL-8 induction, all three transcription factors can act in concert to synergistically activate IL-8 promoter, especially the preferred cooperative interaction between AP-1 and NF-B (9–12). Mutation of the NF–IL-6 site had minimal effect in the presence of intact binding sites for NF-B and AP-1 (11). Despite the cooperative interaction between AP-1 and NF-B, the requirement of a single transcription factor or both of them may vary depending on the individual stimulus. For example, the induction of IL-8 gene expression by the proinflammatory cytokines IL-1ß and tumor necrosis factor- (TNF-) (13, 14) requires NF-B and AP-1. In contrast, acting as an oxidant stress–responsive target gene (15), IL-8 gene expression by oxidant stress in airway epithelial cells requires only AP-1 (14).

Although previous studies have reported that MG132 (carbobenzoxy-Leu-Leu-leucinal), identified as a peptide aldehyde inhibitor of proteasome, can increase JNK activity and upregulate AP-1–dependent gene expression, such as monocyte chemoattractant protein 1, stromelysin, and mitogen-activated protein kinase (MAPK) phosphatase 1 (16), the mechanism has still not been clearly elucidated. In this study, we investigated the effects and signaling mechanism of MG132 on IL-8 release from human pulmonary epithelial A549 cells and human embryonic kidney HEK293 cells. The former cell line has been widely used to study IL-8 release in response to different stimuli and represents a good model system to investigate pathologic changes with airway inflammation. The effects of MG132 on inflammatory cytokine IL-8 release may provide additional information for understanding its beneficial use in anti-inflammatory therapies.

	Materials and Methods

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Reagents
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were obtained from Gibco BRL (Grand Island, NY). Rabbit polyclonal Abs against active (phosphorylated) ERK1/2, mitogen-activated protein/ERK kinase (MEK), p38 MAPK, and JNK1/2 were purchased from New England Biolabs (Beverly, MA). Horseradish peroxidase–coupled anti-mouse and anti-rabbit Abs and the enhanced chemiluminescence (ECL) detection agent were purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). Rabbit polyclonal antibodies specific for ERK2, p38 MAPK, and JNK1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). MG132, lactacystin, fMLP, Ro31–8220, PD98059, SB203580, GF109203X, glutathione (GSH) and N-acetylcysteine (NAC) were purchased form Sigma (St. Louis, MO). The enzyme-linked immunosorbent assay (ELISA) kit was purchased from R&D systems (Minneapolis, MN). Fluorescein isothiocyanate (FITC)-phalloidin was purchased from Molecular Probes Inc. (Eugene, OR). All materials for SDS-PAGE were obtained form Bio-Rad Laboratories (Hercules, CA).

Plasmids
Luciferase expression vectors containing the 5'-flanking region of the IL-8 gene (-133 to -50) and specific mutants of NF-B, AP-1, and NF–IL-6 were provided by Dr. N. Mukaida (Kanazawa University, Ishikawa, Japan). AP-1–Luc construct was provided by Dr. G. Haegeman (University of Ghent-VIB, Ghent, Belgium). Negative mutants of JNK1, SEK, and MEKK were provided by Dr. T. H. Tan (Baylor College of Medicine, Houston, TX). Negative mutants of JNK2, ERK2, Ras (Asn17), p38 MAPK, and ASK were respectively provided by Dr. M. Kracht (Medical School, Hannover, Germany), Dr. D. Ann (University of Southern California, Los Angeles, CA), Dr. H. F. Yang-Yen (Academia Sinica, Taipei, Taiwan), Dr. J. Han (The Scripps Research Institute, La Jolla, CA), and Dr. S. L. Hsieh (National Yang-Ming University, Taipei, Taiwan).

Cells
Human pulmonary epithelial A549 cells, and human embryonic kidney 293 cells obtained from American Type Culture Collection (Manassas, VA) were grown at 37°C in 5% CO₂ using DMEM containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Neutrophils were isolated from citrate anticoagulated venous blood (60 ml) obtained from healthy volunteers. Blood cells were first separated from plasma by centrifugation, and leukocytes were separated from erythrocytes by differential sedimentation using 1.5% dextran. Granulocytes were then separated from monocytes and lymphocytes by centrifugation through a Ficoll-Hypaque gradient. Granulocytes were harvested from the interface of the gradient, and contaminating erythrocytes were removed by hypotonic water lysis. Neutrophil preparation contained > 95% neutrophils, of which > 99% viable as determined by Trypan blue dye exclusion. Freshly isolated neutrophils were resuspended in RPMI 1640 medium supplemented with 10% FBS and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin).

IL-8 ELISA and Chemotaxis of Neutrophils
Cells were cultured in 24-well plates. IL-8 content in conditioned medium collected from A549 and HEK293 cells treated with vehicle (DMSO for the MG132 group and H₂O for the lactacystin group), MG132, or lactacystin for 24 h was assayed using an IL-8 ELISA kit.

In some experiments, the chemotactic function of released IL-8 was assessed by neutrophil migration through a Boyden chamber. Chemoattractant fMLP or culture medium obtained from A549 cells under basal condition or stimulated by proteasome inhibitors was added to the lower chamber. One polycarbonate nucleopore membrane (13 mm diameter, 3.0 µm pore size; Neuro Probe Inc., John, MD) was placed between the lower and upper chamber. After adjusting the neutrophil density to 5x10⁶ cells/ml, 200 µl of cell suspension was added to the upper chamber, and the chamber was subsequently incubated at 37°C. Thirty minutes later, the membrane was removed, and cells which had migrated to the lower surface of the membrane were fixed and stained with FITC-phalloidin or crystal violet.

Transfection and Reporter Gene Assay
For transfection assays, 5 x 10⁵ HEK293 cells were seeded into six-well plates. Cells were transfected the following day using the calcium phosphate precipitation method. DNA was premixed with 33.4 µl 0.1x TE buffer and 12.6 µl 1 M CaCl₂ in an Eppendorf tube for each well, then this was slowly mixed with 46 µl 2x Hanks' balanced salt solution for 25 s. The mixture was incubated for 25 min at room temperature and was then added into each well. After a 24-h incubation, transfection was complete, and cells were incubated with the indicated concentrations of proteasome inhibitors. After another 24-h incubation, the media were removed, and the cells were washed once with cold phosphate-buffered saline. To prepare lysates, 100 µl of reporter lysis buffer (Promega, Madison, WI) were added to each well, and cells were scraped from dishes. The supernatant was collected after centrifugation at 13,000 rpm for 30 s. Aliquots of cell lysates (5 µl) containing equal amounts of protein (10–20 µg) were placed into the wells of an opaque, black 96-well microplate. An equal volume of luciferase substrate (Promega) was added to all samples, and the luminescence was measured in a microplate luminometer (Packard, Meriden, CT). Luciferase activity values were normalized to transfection efficiency monitored by the cotransfected ß-galactosidase expression vector (pCR3lacZ; Pharmacia, Uppsala, Sweden), and was presented as the percentage of luciferase activity measured in the presence of proteasome inhibitors, relative to the activity of control cells with no stimulation.

Preparation of Protein Lysates and Western Blot Analysis
After incubation with the proteasome inhibitor MG132 in 35-mm dishes, cells were washed twice with ice-cold phosphate-buffered saline. Then, 100 µl lysis buffer (1% Triton X-100, 125 mM NaCl, 1 mM MgCl₂, 25 mM ß-glycerophosphate, 50 mM NaF, 100 µM sodium orthovanadate, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 20 mM Tris-HCl, pH 7.5) was added to each dish; cells were scraped from the dishes, and then incubated in Eppendorf for 30 min at 4°C. This was centrifuged at 14,000 rpm and at 4°C for 30 min, and the supernatant was collected. Aliquots were either frozen at -20°C or immediately mixed with an equal volume of a protein sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 5% ß-mercaptoethanol, 0.00125% bromophenol blue, and 1% [vol/vol] glycerol) and boiled for 5 min. Protein samples ( 80 µg) were separated through SDS-10% polyacrylamide gels. After electrophoresis, proteins were transferred to NC paper using a semi-dry electrophoretic transfer. Membranes were blocked in Tris-buffered saline containing 0.1% Tween 20 (TBST), and 5% dry milk for 1 h, rinsed, and incubated with primary antibodies in TBST overnight with gentle shaking at 4°C. The primary antibody was removed, membranes were washed four times in TBST, and 0.1 µg/ml of a peroxidase-labeled secondary antibody was added for 1 h. After four washes in TBST, bands were visualized by incubation in ECL reagents following the manufacturer's instructions, and then exposed to X-ray film.

Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assays (EMSA)
In NF-B and AP-1 binding tests, binding reaction mixtures (15 µl) contained 0.25 µg of poly(dI-dC) (Amersham Pharmacia Biotech) and 20,000 dpm of ³²P-labeled DNA probe in binding buffer consisting of 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 4% Ficoll, 1 mM dithiothreitol, and 75 mM KCl. The binding reaction was started by the addition of nuclear extracts and was allowed to continue for 30 min. Samples were analyzed on native 5% polyacrylamide gels. Oligonucleotides corresponding to NF-B and AP-1 binding element were synthesized on a PS 250 CRUACHEM DNA synthesizer (Cruachem, Glasgow, UK), using the cyanoethyl phosphoroamidate method, and purified by gel filtration. Sequences of the double-stranded oligonucleotides used to detect the DNA-binding activities were 5'-GATCAGTTGAGGGGACTTTCCCAGGC-3' for NF-B and 5'-GATCCGCTTGATGACTCAGCCGGAA-3' for AP-1.

Reverse Transcription-Polymerase Chain Reaction
The expression of IL-8 mRNA was determined by reverse transcription–polymerase chain reaction (RT-PCR) analysis. After treatment with MG132 for different periods, A549 cells were homogenized with 1 ml of RNAzol B reagent (Gibco), and total RNA was extracted using the acid guanidinium thiocyanate-phenol-chloroform extraction. RT was performed using a StrataScript RT-PCR Kit, and 10 µg of total RNA was reverse-transcribed to cDNA following the manufacturer's recommended procedures. RT-generated cDNA encoding IL-8 and ß-actin genes were amplified using PCR. The oligonucleotide primers used corresponded to human IL-8 (5'-AGC TCT GTG TGA AGG TGC AGT T-3' and 5'-ACC CTC TGC ACC CAG TTT TCC T-3') and mouse ß-actin (5'-GAC TAC CTC ATG AAG ATC CT-3' and 5'-CCA CAT CTG CTG GAA GGT GG-3'). PCR was performed in a final volume of 50 µl containing Taq DNA polymerase buffer, all four dNTPs, oligonucleotide primers, Taq DNA polymerase, and RT products. After initial denaturing for 2 min at 94°C, 35 cycles of amplification (94°C for 50 s, 55°C for 50 s, and 72°C for 80 s) were performed followed by a 10-min extension at 72°C. PCR products were analyzed on 2% agarose gel. The mRNA of ß-actin served as an internal control for sample loading and mRNA integrity.

Statistical Evaluation
Values were expressed as the mean ± SEM of at least three experiments, which was performed in duplicate. ANOVA was used to assess the statistical significance of the differences, and a P value < 0.05 is considered statistically significant.

	Results

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MG132 and Lactacystin Increased IL-8 Release in A549 and HEK293 Cells
Treatment of human pulmonary A549 cells with MG132 led to a dramatic increase in IL-8 production. We noted that after 6 h of incubation with MG132 at 10 µM, the level of IL-8 release continuously rose, and reached an 3.6-fold enhancement after a 24-h incubation with 10 µM MG132 (Figure 1A) . This stimulatory effect of MG132 (0.1–30 µM) displayed a concentration dependency within the 24-h incubation (Figure 1B). These results demonstrate that MG132-induced IL-8 release in A549 cells is concentration- and time-dependent. Similar to the action of MG132, another proteasome inhibitor, lactacystin, was also found to produce a 3-fold increase in IL-8 release at 10 µM (Figure 1B). Enhanced IL-8 production with MG132 treatment was accompanied by a time-dependent increase in IL-8 mRNA within 4–14 h (Figure 1C).

View larger version (77K): [in this window] [in a new window]   Figure 1. Proteasome inhibitors increase IL-8 production in A549 cells in concentration- and time-dependent manners. Confluent A549 cells grown in 24-well plates were treated with vehicle or 10 µM MG132 for different periods (A) or with proteasome inhibitors at indicated concentrations for 24 h (B). After incubation, culture medium was collected for IL-8 assay with ELISA kits. Each data represents the mean ± SEM of at least three independent experiments performed in duplicate. In C, IL-8 mRNA level was determined by RT-PCR from cells treated with vehicle or 10 µM MG132 for 4–14 h. The ß-actin mRNA level was used as an internal control. In D and E, cell culture medium after vehicle, 10 µM MG132, or 10 µM lactacystin treatment for 24 h was collected to determine neutrophil migration. For the positive control experiment, lower chamber medium containing 1 µM fMLP was used. After a 30-min migration period, the neutrophils which had migrated to the lower surface of the membrane were counted by FITC-phalloidin (D) or crystal violet (E) staining. Data in A, B, and E represent the mean ± SEM of at least three independent experiments performed in duplicate. Data in C and D represent a typical experiment, which was repeated four times with similar results. (A) Open circles, MG132 (10 µM); closed circles, control. (B) Closed circles, MG132; open triangles, lactacystin.

In addition to exerting its stimulating effect on A549 cells, MG132 also caused a concentration-dependent increase in IL-8 production in HEK 293 cells, which was seen after incubation for 24 h at concentrations above 0.3 µM (Figure 2) . Concerning cell viability with MG132 treatment, both MTT assay and annexin V staining, which are respective indices of mitochondrial dehydrogenase activity and a hallmark of early apoptosis, were included. As a result, we detected no significant loss of mitochondrial activity or the appearance of phosphatidylserine exposure after 24 h of incubation with 30 µM MG132 in either A549 or HEK293 cell types (data not shown).

View larger version (11K): [in this window] [in a new window]   Figure 2. MG132 increases IL-8 production in HEK293 cells in a concentration-dependent manner. Confluent HEK293 cells grown in 24-well plates were treated with vehicle or MG132 at indicated concentrations for 24 h. After incubation, culture medium was collected for IL-8 assay with ELISA kits. Each data represents the mean ± SEM of at least three independent experiments performed in duplicate.

View larger version (24K): [in this window] [in a new window]   Figure 3. PD98059, but not PKC and p38 MAPK inhibitors, reduced MG132-mediated IL-8 release from A549 cells. A549 cells were pretreated with vehicle, PD98059 (50 µM), GF109203X (1 µM), Ro 31–8220 (1 µM), or SB 203580 (3 µM) for 30 min before MG132 (10 µM) treatment. After a 24-h incubation, medium was collected for IL-8 assay. Each value represents the mean ± SEM of at least three independent experiments performed in duplicate. *P < 0.05, compared with the control MG132 response without protein kinase inhibitor pretreatment. Closed bars, control; open bars, MG132 (10 µM).

View larger version (35K): [in this window] [in a new window]   Figure 4. MG132 increased IL-8 promoter activity through AP-1 in HEK 293 cells. (A) MG132 increased luciferase activity of IL-8 (-133) promoter in a concentration-dependent manner. As described in MATERIALS AND METHODS, IL-8 (-133) plasmid (0.5 µg) was cotransfected with ß-gal-lacZ (0.5 µg) in HEK 293 cells. Twenty-four hours later, the vehicle or indicated concentrations of MG132, IL-1ß, or PMA were added and incubated for another 24 h, after which cell lysates were prepared and IL-8 promoter activity was assessed by measurement of the luciferase activity. Closed bars, MG132; open bars, lactacystin. (B) IL-8 (-133) plasmid (0.5 µg) or its mutant constructs, i.e., AP-1 mutant (2 µg), NF–IL-6 mutant (1 µg), and NF-B mutant (1 µg), was cotransfected with lacZ (0.5 µg) in HEK 293 cells. After a 24-h incubation with MG132 (10 µM), cells were lysed, followed by the measurement of luciferase activity. Luciferase activity was then normalized for transfection efficiency with ß-gal, and the increased luciferase activity by MG132 on IL-8 reporter transfected cells was expressed as a percentage of the control group transfected with empty vector. Each value represents the mean ± SEM of at least three independent experiments performed in duplicate. *Statistically significant inhibition (P < 0.05) as compared with the wild-type IL-8 (-133) luciferase activity as indicated.

Proteasome Inhibitors Activated AP-1 but Inhibited NF-B
Following the finding indicating AP-1–dependent effect of proteasome inhibitors, we performed an AP-1 luciferase assay to further confirm this notion, and found that both MG132 and lactacystin increased the reporter activity of AP-1 in HEK293 cells (Figure 5A) . In terms of potency and extent, these responses of proteasome inhibitors paralleled with their stimulation of IL-8 release and gene induction. The results using EMSA to examine nuclear AP-1 binding with specific DNA element also support the stimulating effect of proteasome inhibitors on AP-1 signaling (Figure 5C).

View larger version (23K): [in this window] [in a new window]   Figure 5. Proteasome inhibitors stimulated AP-1, but reduced NF-B, DNA binding activity. (A) filled bars, MG132; open bars, lactacystin. As described in Materials and Methods, AP-1 reporter gene (0.5 µg) (A) or B reporter gene (0.5 µg) (B) was cotransfected with ß-gal-lacZ (0.5 µg) in HEK 293 cells. Twenty-four hours later, vehicle or indicated concentrations (0.01–10 µM) of proteasome inhibitor alone or in the presence of TNF- (50 ng/ml) or IL-1ß (10 ng/ml) were incubated for another 24 h, and then cell lysates were prepared for reporter activity assay. Concentrations of proteasome inhibitor: filled bars, control; thinly striped bars, 0.01 µM; dotted bars, 0.1 µM; thickly striped bars, 1 µM; open bars, 10 µM. Data represent mean ± SEM of at least three independent experiments performed in duplicate. *Statistically significant inhibition (P < 0.05) as compared with the control cytokine-mediated luciferase activity in the absence of MG132 and lactacystin. In C, nuclear extracts were prepared from cells treated with vehicle, MG132, or lactacystin for 1 or 2 h, followed by incubation with specific DNA-binding probes for AP-1 or NF-B. The data represent a typical experiment, which was repeated four times with similar results.

MG132 Stimulated ERK, p38 MAPK, and JNK
Because a number of studies have demonstrated that AP-1 activation needs MAPK activation (20, 21), we are curious about the effect of MG132 on ERKs, JNKs, and p38 MAPK activation. To elucidate the effect of MG132 on MAPK activity, phosphorylated MAPKs, which are indices of their activated state, were examined by immunoblotting with specific antibodies recognizing their phosphorylated forms. Figure 6 showed that in A549 cells, MG132 (10 µM) rapidly induced the phosphorylation of three MAPKs within 60 min of treatment. During this period, the total protein level of each MAPK did not change. Furthermore, we showed that the upstream kinase transducing ERK phosphorylation, MEK, also underwent phosphorylation (Figure 6B). The stimulatory effects of MG132 on the phosphorylation of these three MAPKs were similarly observed in HEK293 cells (data not shown).

View larger version (42K): [in this window] [in a new window]   Figure 6. MG132 activates MAPKs. A549 cells were treated with vehicle or 10 µM MG132 and cell extracts were prepared at the indicated times. Levels of phosphorylated ERK, p38 MAPK, JNK, and MEK were determined by immunoblot analysis. The amount of phosphorylated proteins was quantified after normalization of the phosphorylated kinase corresponding to total kinase at each time point, and expressed as a percentage of the control group before MG132 addition. Results in the lower panel show the mean of two or three experiments.

View larger version (29K): [in this window] [in a new window]   Figure 7. ERK, JNK, and ROS are involved in proteasome inhibitor–stimulated IL-8 reporter activity. (A) HEK293 cells were cotransfected with wild-type cDNA construct of the IL-8-promoter (0.5 µg), with ß-gal-lacZ (0.5 µg), and with empty vector or expression vectors for the dominant-negative p38 (0.5 µg), JNK (0.5 µg), ERK (0.5 µg), MEKK1 (0.5 µg), TAK (0.5 µg), SEK (0.5 µg), ASK (0.5 µg), or Ras (0.5 µg). Twenty-four hours later, vehicle (closed bars) or 10 µM MG132 (open bars) was added and incubated for another 24 h, and then cell lysates were prepared for the assessment of IL-8 reporter activity. (B) After IL-8 promoter and ß-gal-lacZ transfection, NAC (3 mM; lightly striped bars) or GSH (5 mM; darkly striped bars) was incubated together with MG132 (10 µM) or lactacystin (10 µM) for 24 h. Open bars, control. Each value represents the mean ± SEM of at least three independent experiments performed in duplicate. *Statistically significant inhibition (P < 0.05) as compared with IL-8 luciferase activity without inclusion of the negative mutant signal proteins (A) or NAC and GSH treatment (B).

Involvement of Reactive Oxygen Species in IL-8 Expression by Proteasome Inhibitors
Because reactive oxygen species (ROS) are potent secondary messengers known to transduce many signaling pathways, including MAPK signaling cascades (23–25), we used two antioxidants, GSH and NAC, to explore the role of ROS in the action of proteasome inhibitors. We found that IL-8 luciferase activities caused by MG132 and lactacystin were inhibited in the presence of 3 mM NAC or 5 mM GSH (Figure 7B), suggesting the involvement of ROS in this action.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is known that the proteasome system plays important roles in regulating expression levels of signal transduction molecules at post-transcriptional level. One of the intriguing functions following signal regulation by proteasome is the activation of NF-B (26). Because NF-B activation has been implicated in many inflammatory responses (3), the discovery of new anti-inflammatory drugs based on proteasome inhibition has begun (2). MG132 and lactacystin are reversible, potent proteasome inhibitors, and have been proved to inhibit NF-B activation and many NF-B–mediated cellular responses. In light of their potential uses in anti-inflammation, it is crucial to understand the cellular functions of proteasome inhibitors, both beneficial and deleterious, in their anti-inflammatory actions.

IL-8 induction represents an important response to many inflammatory inducers and has been implicated in a variety of inflammatory diseases. In its allergic respiratory response, IL-8 is a potent granulocyte chemoattractant. Several investigators have shown that the levels of IL-8 in bronchoalveolar lavage fluid are higher in individuals with asthma than in those without asthma. In patients with asthma, the levels of IL-8 correlated with the number of neutrophils present in lavage fluid (27). As current studies show, IL-8 gene regulation is under complex transcriptional control, particularly by the major transcriptional factor AP-1 and NF-B (9–12, 28, 29). Coordinated activation of both transcription factors produces the full activity of IL-8 transcription in different cell types (11, 12, 28). Although NF-B has been reported to be essential in IL-8 expression for many activators, NF-B–independent induction of IL-8 has recently been demonstrated (14, 29). For example, H₂O₂ induced IL-8 expression in A549 cells in the absence of increased NF-B–binding activity (14). Consistent with this finding, we herein indicate that IL-8 gene expression and IL-8 release can be induced by MG132 and lactacystin through a pathway independent of NF-B activation, but which occurs through MAPK-dependent AP-1 activation. NF-B being unnecessary for MG132 action was evidenced by similar stimulation degrees of IL-8 reporter activity in conditions when the 5'-flanking region of the IL-8 gene containing the B binding site was or was not deleted. In addition, in this study, we confirm previous data that MG132 and lactacystin are potent NF-B inhibitors, which can inhibit cytokine-induced B luciferase activity as well as basal NF-B activity.

The pivotal roles of PKC and MAPKs in AP-1 activation and IL-8 gene expression have been documented (20). However, depending on the stimuli and cell types, PKC and three MAPKs have also been reported to differentially mediate IL-8 gene expression (14, 30, 31). In this study, we show that MG132 indeed can rapidly stimulate three types of MAPK activation in A549 and HEK293 cells. Despite this, we provide evidence leading to the conclusion that MG132-activated IL-8 production depends on ERK and JNK, but not on p38 MAPK. First, MG132-induced IL-8 release was reduced by a MEK inhibitor (PD98059), but not by a p38 MAPK (SB203580) inhibitor. Second, transient transfection with dominant-negative mutants of ERK2, JNK1, or JNK2, but not of p38 MAPK, attenuated IL-8 reporter activity stimulated by MG132. Third, extending this analysis to elucidate upstream targets, we found that Ras-dependent MEK/ERK signal and ASK-dependent SEK/JNK signal together mediate MG132 action on IL-8 production.

Although previous reports have implicated MG132-induced JNK signal transduction cascade in neuronal differentiation (7), apoptosis (6), and monocyte chemoattractant protein 1 expression (16), the exact mechanism by which proteasome inhibition activates JNK remains clear. Thus, in this study, we have addressed for the first time the upstream signaling mechanism by which MG132 activates JNK. Furthermore, we also for the first time have demonstrated the involvement of Ras-dependent ERK activation, but not p38 MAPK activation, in MG132 action. The noninvolvement of p38 MAPK in MG132 action observed in the present study differs from cytokine actions. According to previous study (31) and our unpublished data on A549 cells, p38 MAPK mediates the stimulating action of IL-1ß and TNF- on IL-8 expression. Thus, we again convincingly conclude that the upstream signal transduction for IL-8 gene expression varies among different activating inducers.

In this study, we also unexpectedly found enhancement of IL-8 promoter activity, under both basal and MG132-stimulated conditions, by overexpressing cells with the dominant negative mutant of p38 MAPK (Figure 7A). Consistent with this result, although the p38 and p38ß MAPK inhibitor SB203580 did not change IL-8 release (Figure 3), it did increase IL-8 promoter activity. The mechanism by which p38 MAPK inhibition leads to enhancement of IL-8 transcription possibly involves the negative regulatory role of p38 MAPK on Ras/ERK singalling pathway, as previously demonstrated (32, 33). However, despite enhancement of IL-8 promoter activity by SB203580 and p38 (DN), MG132-mediated IL-8 release was unaffected by SB203580 (Figure 3). With respect to these differential and contradictory results in transcription and post-transcription level of IL-8 induction, although we currently have no direct evidence to explain these phenomena, several reasons possibly exist and require further investigation. First, p38 and p38ß isoforms might exert distinct action in regulating IL-8 gene expression. Second, SB203580 might have p38 MAPK-independent nonspecific action, which possibly can influence IL-8 mRNA stability or IL-8 protein translation and/or stability (34).

ROS have been implicated in mediating MAPK signal transduction by a variety of stimuli. In MAPK signaling pathway, Ras (23), ASK1-JAK/p38 MAPK (24, 35), and tyrosine phosphatase (25) are common targets for ROS and can sense the cellular redox status. To explore the role of ROS in mediating MG132 and lactacystin-induced IL-8 gene expression, we tested antioxidants GSH and NAC. As our results show, because IL-8 and AP-1 luciferase activities are elevated by proteasome inhibitors to lesser extents in the presence of antioxidants, the involvement of redox changes in MG132 and lactacystin action is suggested.

In summary, we conclude that proteasome inhibitors MG132 and lactacystin can elicit dual actions on two crucial transcription factors, namely, NF-B inhibition and AP-1 activation. For the first time, we demonstrate the upstream signaling mechanism by which MG132 activates AP-1 and upregulates IL-8 gene expression. Activation of Ras and ASK respectively accounts for ERK- and JNK-dependent AP-1 stimulation. The therapeutic benefits using proteasome inhibitors as anti-inflammatory drugs might be compromised by their stimulatory action on AP-1, and the consequent induction of some gene products deleterious to inflammatory state.

Received in original form December 4, 2001

Received in final form April 15, 2002

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