Mitogen-activated protein kinase inhibitors differently affect the growth inhibition and death of a proteasome inhibitor,MG132-treated human pulmonary fibroblast cells

Abstract

Carbobenzoxy-Leu-Leu-leucinal (MG132) as a proteasome inhibitor can induce growth inhibition and death in lung cancer or normal cells. However, little is known about relationship between proteasome inhibition and mitogen-activated protein kinase (MAPK) signaling in normal lung cells. Thus, in the present study, we investigated the effects of MAPK inhibitors on MG132-treated human pulmonary fibroblast (HPF) cells in relation to cell growth inhibition, cell death, reactive oxygen species (ROS) and glutathione (GSH). Treatment with 15 μM MG132 increased ROS levels including mitochondrial O_2•⁻ and GSH depleted cell numbers in HPF cells at 24 hours. MAP kinase or ERK kinase (MEK) inhibitor did not significantly affect cell growth inhibition, cell death, the loss of mitochondrial membrane potential (MMP; ΔΨ_m), ROS level and GSH depletion in MG132-treated HPF cells. c-Jun N-terminal kinase (JNK) inhibitor attenuated the growth inhibition and death by MG132. This inhibitor also significantly decreased O_2•⁻ level in MG132-treated HPF cells. Although p38 inhibitor slightly enhanced HPF cell growth inhibition by MG132, this inhibitor and siRNA prevented HPF cell death induced by MG132. p38 inhibitor also attenuated d O_2•⁻ level and GSH depletion. Moreover, extracellular signal regulated kinase (ERK), JNK or p38 siRNA did not strongly affect ROS levels in MG132-treated HPF cells. ERK and JNK siRNAs decreased anonymous ubiquitinated protein levels in MG132-treated HPF cells. In conclusion, MAPK inhibitors differently affected the growth inhibition and death of MG132-treated HPF cells. Especially, p38 inhibitor attenuated HPF cell death by MG132, which was in part related to changes in ROS and GSH levels.

Introduction

The main function of the ubiquitin-proteasomal system is to degrade unneeded or damaged proteins by proteolysis in eukaryotic cells.^1,2 Many proteins degraded by proteasome are implicated in crucial processes: for example, cell-cycle-regulatory proteins and apoptotic-related proteins. ³ Apoptosis in cancer cells is closely connected with the activity of ubiquitin/proteasome pathways.^4,5 Transformed cells including cancer cells accumulate more misfolded/mutated/damaged proteins due to the high replication rate of malignant cells, which are disposed of by the proteasome. ³ Accordingly, the prevention of proteasome function has emerged as a useful strategy to maneuver apoptosis in cancer cells. The peptide aldehyde carbobenzoxy-Leu-Leu-leucinal (MG132) efficiently prevents the proteolytic activity of the proteasome complex. ⁶ Proteasome inhibitors including MG132 have been shown to induce apoptotic cell death through the production of reactive oxygen species (ROS).^7,8 ROS including hydrogen peroxide (H₂O₂), superoxide anion (O_2•⁻) and hydroxyl radical (^•OH) are implicated in the regulation of many important cellular events such as differentiation, cell death and cell proliferation.^9,10 The principal metabolic pathways include superoxide dismutases (SODs), expressed as extracellular, cytoplasmic, and mitochondrial isoforms, ¹¹ which metabolize O_2•⁻ to H₂O₂. Further metabolism by peroxidases, including catalase and glutathione (GSH) peroxidase, yields O₂ and H₂O. ¹² ROS formation and GSH depletion due to proteasome inhibitors may cause mitochondrial dysfunction and subsequent cytochrome C release, which leads to cell viability loss.^13,14 A change in the redox state of the tissue and cell influences an alteration in the generation or metabolism of ROS. The mechanism underlying ROS generation after inhibition of the proteasome is still imprecise.

The mitogen-activated protein kinases (MAPKs) as a huge family of serine/threonine kinases mainly consist of three subfamilies: the extracellular signal regulated kinase (ERK1/2), the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and the p38. ¹⁵ These MAPK signaling pathways is tightly involved in cell proliferation, differentiation and cell death. ¹⁶ Each MAP kinase pathway has different upstream stimuli and specific substrates. ¹⁷ Numerous evidences reveal that JNK and p38 are strongly activated by ROS or by a mild oxidative shift of the intracellular redox state, leading to apoptosis.^18–20 ROS also can regulate the activation of the extracellular signal regulated kinase (ERK1/2)-activating kinase (MEK) and ERK.^21,22 In most instances, MEK–ERK signaling play a role in a pro-survival function rather than a pro-apoptotic effect. ²³ Since diverse ROS levels and different functions of MAPKs regulated by ROS have opposite effects even in the same type of cells, the relationship between ROS and MAPKs in view of cell survival or cell death signaling needs further clarification.

We recently demonstrated that MG132 reduced the growth of Calu-6 and A549 lung cancer cells via apoptosis and GSH depletion.^24,25 Treatment with p38 inhibitor (SB203580) is involved in the prevention of Calu-6 and A549 lung cell death by MG132.^26,27 In addition, we observed that MG132 induced the growth inhibition and death in human pulmonary fibroblast (HPF) cells via a caspase-independent manner (unpublished data). However, little is known about relationships between MG132 and MAPK inhibitors in normal lung cells in relation to ROS and GSH levels. Therefore, in the present study, we investigated the effects of MAPK inhibitors or siRNAs on cell growth, cell death, ROS and GSH levels in MG132-HPF cells.

Materials and methods

Cell culture

The HPF cells from PromoCell GmbH (Heidelberg, Germany) were maintained in humidified incubator containing 5% CO₂ at 37°C. HPF cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (GIBCO BRL, Grand Island, NY, USA). HPF cells were used between passages four and eight.

Reagents

MG132 was purchased from Calbiochem (San Diego, CA, USA) and was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich Chemical Company, St. Louis, MO, USA) solution buffer. MEK inhibitor (PD98059), JNK inhibitor (SP600125) and p38 inhibitor (SB203580) obtained from Calbiochem (San Diego, CA, USA) were dissolved in DMSO. Cells were pretreated with each MAPK inhibitor for 1 hour before MG132 treatment. Based on the previous experiment, ²⁸ 10 μM of each MAPK inhibitor was used as an optimal dose in this experiment. DMSO (0.2%) was used as a control vehicle and it did not appear to affect cell growth or death.

Cell growth assays

The cell growth inhibition effects by drugs were determined by measuring the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich Chemical Company) dye absorbance as previously described. ²⁹ In brief, cells 3 × 10⁴ cells per well were seeded in 96-well microtiter plates (Nunc, Roskilde, Denmark) for MTT assays. After exposure to 15 μM MG132 and/or a given MAPK inhibitor for 24 hours, 20 μl of MTT solution (2 mg/ml in phosphate buffered saline [PBS]) were added to each well of the 96-well plates. The plates were incubated for four additional hours at 37°C. Media in plates were withdrawn by pipetting and 200 μl of DMSO was added to each well to solubilize the formazan crystals. Optical density was measured at 570 nm using a microplate reader (Spectra MAX 340, Molecular Devices Co, Sunnyvale, CA, USA).

Annexin V/PI staining for cell death detection

Apoptosis was determined by staining cells with annexin V-fluorescein isothiocyanate (FITC, Ex/Em = 488 nm/519 nm; Invitrogen Molecular Probes, Eugene, OR, USA) and propidium iodide (PI, Ex/Em = 488 nm/617 nm; Sigma-Aldrich). Annexin V-FITC can be used to identify the externalization of phosphatidylserine during the progression of apoptosis. PI is used to differentiate dead and viable cells. In brief, 1 × 10⁶ cells in 60 mm culture dish (Nunc) were incubated with the indicated doses of MG132 with or without a given MAPK inhibitor or MAPK-related siRNA duplex for 24 hours. Cells were washed twice with cold PBS and then resuspended in 500 μl of binding buffer (10 mM HEPES/NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) at a concentration of 1 × 10⁶ cells/ml. Five microliters of annexin V-FITC and/or PI (1 μg/ml) were then added to these cells, which were analyzed with a FACStar flow cytometer (Becton Dickinson).

Measurement of mitochondrial membrane potential [MMP (ΔΨm)]

MMP (ΔΨ_m) levels were measured using a rhodamine 123 fluorescent dye (Sigma-Aldrich; Ex/Em = 485 nm/535 nm) as previously described. ³⁰ In brief, 1 × 10⁶ cells in 60 mm culture dish (Nunc) were incubated with 15 μM MG132 and/or a given MAPK inhibitor for 24 hours. Cells were washed twice with PBS and incubated with the rhodamine 123 (0.1 μg/ml) at 37°C for 30 min. Rhodamine 123 staining intensity was determined by flow cytometry (Becton Dickinson). An absence of rhodamine 123 from cells indicated the loss of MMP (ΔΨ_m) in HPF cells.

Detection of intracellular ROS and O_2•⁻ levels

Intracellular ROS were detected by means of an oxidation-sensitive fluorescent probe dye, 2′,7′-dichlorodihydrofluorescein diacetate (H₂DCFDA, Ex/Em = 495 nm/529 nm; Invitrogen Molecular Probes) as previously described. ³¹ H₂DCFDA is poorly selective for superoxide anion radical (O_2•⁻). In contrast, dihydroethidium (DHE; Ex/Em = 518 nm/605 nm; Invitrogen Molecular Probes) is a fluorogenic probe that is highly selective for O_2•⁻ among ROS as previously described. ³¹ Mitochondrial O_2•⁻ levels were detected using MitoSOX^TM Red mitochondrial O_2•⁻ indicator (Ex/Em = 510 nm/580 nm; Invitrogen Molecular Probes) as previously described. ³¹ In brief, 1 × 10⁶ cells in 60 mm culture dish (Nunc) were incubated with the indicated doses of MG132 with or without a given MAPK inhibitor or MAPK-related siRNA duplex for 24 hours. Cells were then washed in PBS and incubated with 20 µM H₂DCFDA, 20 µM DHE or 5 µM MitoSOX^TM Red at 37°C for 30 min. DCF, DHE and MitoSOX^TM Red fluorescences were detected using a FACStar flow cytometer (Becton Dickinson). ROS and O_2•⁻ levels were expressed as mean fluorescence intensity (MFI), which was calculated by CellQuest software (Becton Dickinson).

Detection of the intracellular glutathione

Cellular glutathione (GSH) levels were analyzed using a 5-chloromethylfluorescein diacetate dye (CMFDA, Ex/Em = 522 nm/595 nm; Invitrogen Molecular Probes) as previously described. ³¹ In brief, 1 × 10⁶ cells in 60 mm culture dish (Nunc) were incubated with 15 μM MG132 and/or a given MAPK inhibitor for 24 hours. Cells were then washed with PBS and incubated with 5 µM CMFDA at 37°C for 30 min. CMF fluorescence intensity was determined using a FACStar flow cytometer (Becton Dickinson). Negative CMF staining (GSH depleted) cells were expressed as the percent of (-) CMF cells.

Western blot analysis

The patterns of ubiquitinated proteins were evaluated using Western blot analysis. In brief, 1 × 10⁶ cells in 60 mm culture dish (Nunc) were incubated with 30 μM MG132 with or without MAPK-related siRNA duplex for 24 hours. The cells were then washed in PBS and suspended in five volumes of lysis buffer (20 mM HEPES. pH 7.9, 20% glycerol, 200 mM KCl, 0.5 mM EDTA, 0.5% NP40, 0.5 mM DTT, 1% protease inhibitor cocktail). Supernatant protein concentrations were determined using the Bradford method. Samples containing 40 μg total protein were resolved by 12.5% Sodium dodecyl sulfate polyacrylamide gel electropho (SDS-PAGE) gels, transferred to Immobilon-P Polyvinylidene Fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) by electroblotting and then probed with anti-ubiquitin and anti-β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies. Blots were developed using an ECL kit (Amersham, Arlington Heights, IL, USA).

Transfection of cells with MAPK-related siRNAs

Gene silencing of ERK, JNK and p38 was performed as previously described. ³² A nonspecific control siRNA duplex [5′-CCUACGCCACCAAUUUCGU(dTdT)-3′], ERK siRNA duplex [5′-CACCAUUCAAGUUCGACAU(dTdT)-3′], JNK siRNA duplex [5′-CUGGAUAUAGCUUUGAGAA(dTdT)-3′] and p38 siRNA duplex [5′-CAAAUUCUCCGAGGUCUAA (dTdT)-3′] were purchased from the Bioneer Corporation (Daejeon, South Korea). In brief, 2.5 × 10⁵ cells in six-well plates (Nunc) were incubated in RPMI-1640 supplemented with 10% FBS. The next day, cells (approximately 30%–40% confluence) in each well were transfected with the control or each MAPK siRNA (80 picomole in Opti-MEM [GIBCO BRL]) using LipofectAMINE 2000, according to the manufacturer’s instructions (Invitrogen, Brandford, CT, USA). One day later, cells were treated with or without 30 μM MG132 for additional 24 hours. The transfected cells were collected and used for annexin V-FITC/PI staining, ROS level and Western blotting.

Statistical analysis

The results represent the mean of at least three independent experiments (mean ± SD). The data were analyzed using Instat software (GraphPad Prism4, San Diego, CA, USA). The Student’s t-test or one-way analysis of variance (ANOVA) with post hoc analysis using Tukey’s multiple comparison test was used for parametric data. Statistical significance was defined as p < 0.05.

Results

Effects of MAPK inhibitors on cell growth in MG 132-treated HPF cells

We examined the effect of MAPK inhibitors on the growth of MG132-treated HPF cells. For this experiment, 15 μM MG132 as a suitable dose was used to differentiate the levels of cell growth inhibition or death in the presence or absence of a given MAPK inhibitor. Based on an MTT assay, 15 μM MG132 inhibited the growth of HPF cells about 70% at 24 hours (Figure 1). MEK inhibitor did not affect the growth inhibition by MG132 (Figure 1). While JNK inhibitor slightly prevented the growth inhibition of MG132, p38 inhibitor seemed to enhance the growth inhibition (Figure 1). p38 inhibitor alone increased the growth of HPF control cells (Figure 1).

Figure 1.

Effects of mitogen-activated protein kinase (MAPK) inhibitors on cell growth in carbobenzoxy-Leu-Leu-leucinal (MG132)-treated human pulmonary fibroblast (HPF) cells. Exponentially growing cells were treated with 15 μM MG132 for 24 hours following 1 hour pre-incubation of 10 μM MEK, JNK or p38 inhibitor. The graph shows cellular growth changes in HPF cells at 24 hours, as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. *p < 0.05 compared with the control group. ^#p < 0.05 compared with cells treated with MG132 only.

Effects of MAPK inhibitors on cell death and MMP (ΔΨm) in MG 132-treated HPF cells

MG132 significantly induced cell death in HPF cells, as evidenced by annexin V-FITC staining or PI staining cells (Figure 2A–D). None of MAPK inhibitors changed the number of annexin V staining cells in MG132-treated or -untreated HPF cells (Figure 2A and C). However, all the MAPK inhibitors seemed to decrease the PI staining cells by MG132 and treatment with JNK and p38 inhibitors showed significant effects (Figure 2B and D). None of each MAPK inhibitor alone altered PI staining cell number in HPF control cells (Figure 2B and D). Furthermore, MG132 significantly triggered the loss of MMP (ΔΨ_m) in HPF cells (Figure 2E). None of MAPK inhibitors affected the loss of MMP (ΔΨ_m) in MG132-treated or -untreated HPF cells (Figure 2E).

Figure 2.

Effects of mitogen-activated protein kinase (MAPK) inhibitors on cell death and mitochondrial membrane potential (MMP [ΔΨ_m]) in carbobenzoxy-Leu-Leu-leucinal (MG132)-treated human pulmonary fibroblast (HPF) cells. Exponentially growing cells were treated with 15 μM MG132 for 24 hours following 1 hour pre-incubation of 10 μM MEK, JNK or p38 inhibitor. Annexin V-FITC staining, PI staining and MMP (ΔΨ_m) level in HPF cells were measured with a FACStar flow cytometer. (A and B) Each histogram figure shows representatives for annexin V-FITC (A) and PI staining cells (B). M1 indicate annexin V-FITC positive cells (A) and PI positive cells (B). (C and D) Graphs show the percentages of M1 regions in A (C) and B (D). (E) Graph shows the percents percentage of rhodamine 123 negative (MMP (ΔΨ[ΔΨ_m)] loss) cells. *p < 0.05 compared with the control group. ^#p < 0.05 compared with cells treated with MG132 only.

Effects of MAPK inhibitors on ROS and GSH levels in MG 132-treated HPF cells

Next, we determined whether intracellular ROS and GSH levels in MG132-treated HPF cells were changed by each MAPK inhibitor. ROS (DCF) level such as H₂O₂ was increased in MG132-treated HPF cells (Figure 3A and D). None of MAPK inhibitors significantly changed ROS levels in these cells (Figure 3A and D). Only p38 inhibitor alone increased ROS level in HPF control cells (Figure 3A and D). Red fluorescence derived from DHE reflecting intracellular O_2•⁻ level was also increased in HPF cells (Figure 3B and E). While MEK inhibitor did not significantly change O_2•⁻ level in MG132-treated HPF cells, JNK and p38 inhibitors attenuated the O_2•⁻ level (Figure 3B and E). p38 inhibitor alone also increased O_2•⁻ level in HPF control cells (Figure 3B and E). Furthermore, MitoSOX Red fluorescence levels, which specifically indicate O_2•⁻ levels in the mitochondria, were strongly increased in MG132-treated HPF cells (Figure 3C and F). MEK inhibitor mildly increased mitochondrial O_2•⁻ level in MG132-treated HPF cells whereas JNK inhibitor decreased the O_2•⁻ level in these cells and MG132-untreated control cells (Figure 3C and F). In relation to GSH level, MG132 increased the number of GSH-depleted cells in HPF cells (Figure 4A and B). All the MAPK inhibitors seemed to decrease GSH-depleted cell number in MG132-treated HPF cells (Figure 4A and B).

Figure 3.

Effects of mitogen-activated protein kinase (MAPK) inhibitors on reactive oxygen species (ROS) levels in carbobenzoxy-Leu-Leu-leucinal (MG132)-treated HPF cells. Exponentially growing cells were treated with 15 μM MG132 for 24 hours following 1 hour pre-incubation of 10 μM MEK, JNK or p38 inhibitor. ROS levels in human pulmonary fibroblast (HPF) cells were measured using a FACStar flow cytometer. (A–C) Each histogram as a representative indicates DCF (ROS), DHE (O_2•⁻) and MitoSOX (mitochondrial O_2•⁻) levels, respectively. (D–F) Graphs indicate DCF (ROS) levels (%) from A (D), DHE (O_2•⁻) levels (%) from B (E) and MitoSOX (mitochondrial O_2•⁻) levels (%) from C (F), compared with MG132-untreated control cells. *p < 0.05 compared with the control group. ^#p < 0.05 compared with cells treated with MG132 only.

Figure 4.

Effects of mitogen-activated protein kinase (MAPK) inhibitors on glutathione (GSH) levels in carbobenzoxy-Leu-Leu-leucinal (MG132)-treated human pulmonary fibroblast (HPF) cells. Exponentially growing cells were treated with 15 μM MG132 for 24 hours following 1 hour pre-incubation of 10 μM MEK, JNK or p38 inhibitor. GSH levels in HPF cells were measured using a FACStar flow cytometer. (A) Each histogram as a representative shows CMF intensities in cells. M1 indicates (-) CMF (GSH depleted) cells. (B) Graph shows the percentage of (-) CMF (GSH depleted) cells (M1 region in A). *p < 0.05 compared with the control group.

Effects of MAPK-related siRNAs on cell death, ROS level and ubiquitinated proteins in MG 132-treated HPF cells

Furthermore, it was determined whether MAPK (ERK, JNK and p38)-related siRNAs changed cell death, ROS and ubiquitinated proteins levels in MG132-treated HPF cells. As shown in Figure 5, 30 μM MG132 increased the proportion of annexin V-stained cells about 12% compared with that in control siRNA-treated HPF cells. Treatment with 15 μM MG132 did not sufficiently induce HPF cell death in this system (data not shown). Probably, the agent of LipofectAMINE 2000 in medium was likely to attenuate the toxicological activity of MG132. All the MAPK siRNAs did not significantly alter annexin V-stained cell number in MG132-treated HPF cells but only p38 siRNA slightly decreased the number in these cells (Figure 5A). In relation to ROS level, none of the MAPK siRNAs significantly changed ROS (DCF) level in MG132-treated HPF cells (Figure 5B) and these siRNAs also did not affect O_2•⁻ level in these cells (data not shown). Moreover, MG132 relatively increased the level of anonymous ubiquitinated proteins in HPF cells compared with that of the control siRNA-treated HPF cells (Figure 5C). While ERK and JNK siRNAs attenuated the ubiquitinated protein level in MG132-treated HPF cells, p38 siRNA did not affect the level in these cells (Figure 5C).

Figure 5.

Effects of mitogen-activated protein kinase (MAPK) small interfering RNAs (siRNAs) on cell death, reactive oxygen species (ROS) level and ubiquitinated proteins in carbobenzoxy-Leu-Leu-leucinal (MG132)-treated human pulmonary fibroblast (HPF) cells. HPF cells (approximately 30%–40% confluence) were transfected with either nontarget control siRNA or each MAPK siRNA. One day later, cells were treated with 30 μM MG132 for additional 24 hours. (A) Annexin V-FITC and PI positive cells were measured with a FACStar flow cytometer. The number (%) in each figure indicates Annexin V-FITC-positive cells regardless of PI negative and positive cells. (B) Each histogram figure shows DCF (ROS) intensities in cells. DCF (ROS) levels were expressed as mean fluorescence intensity (MFI). (C) 40 μg samples of protein extracts were resolved by SDS-PAGE gel, transferred onto PVDF membranes and immunoblotted with the indicated antibodies against ubiquitin and β-actin.

Discussion

In the present study, we demonstrated the effects of MAPK inhibitors or siRNAs on cell growth, cell death, ROS and GSH levels in MG132-treated HPF cells. Various proteasome inhibitors including MG132 can trigger apoptotic cell death through the induction of ROS.^7,8 Likewise, MG132 increased ROS levels including O_2•⁻ and induced growth inhibition and death in HPF cells. Furthermore, the loss of MMP (ΔΨ_m) and mitochondrial O_2•⁻ level was also strongly increased in MG132-treated HPF cells, implying the possibility that the production of O_2•⁻ in MG132-treated HPF cells primarily originates from the mitochondria.

ERK activation has a pro-survival function rather than pro-apoptotic effects. ²³ According to our result, MEK inhibitor, which presumably decreased ERK activity, did not significantly affect HPF cell growth inhibition and death by MG132. MEK inhibitor alone did not change HPF growth and death. Our recent reports also demonstrated that MEK inhibitor does not affect the growth and death in MG132-treated or -untreated Calu-6 and A549 lung cancer cells.^26,27 Furthermore, ERK siRNA did not change HPF cell death by MG132. These results suggested that the inhibition of ERK signaling is not tightly related to cell growth inhibition and death in MG132-treated or -untreated lung cells. In addition, the inhibition of ERK signaling did not seem to strongly affect redox state in HPF cells because MEK inhibitor or ERK siRNA did not alter ROS levels in MG132-treated or -untreated lung cells.

Numerous evidences demonstrate that JNK or p38 signaling is related to cell death.^18,19 Our recent data also demonstrated that p38 inhibitor significantly prevented MG132-induced Calu-6 and A549 cell death.^26,27 Likewise, p38 or JNK inhibitor significantly decreased HPF cell death by MG132 in view of PI staining cells. In addition, p38 siRNA slightly decreased annexin V-stained cell number in MG132-treated HPF cells. These data imply that JNK or p38 signaling in MG132-treated HPF cells is involved in the function of cell death. In addition, JNK inhibitor slightly prevented the growth inhibition of MG132. Interestingly, p38 inhibitor seemed to enhance the growth inhibition of MG132-treated HPF cells, but this inhibitor alone increased HPF control cell growth. These results suggested that the inhibition of p38 signaling differently affects the pathways of HPF cell growth inhibition and death. In addition, JNK or p38 inhibitors did not affect the loss of MMP (ΔΨ_m) in MG132-treated or -untreated HPF cells, implying that these inhibitors were not involved in MMP (ΔΨ_m) loss in MG132-treated HPF cells.

In relation to ROS levels, JNK or p38 inhibitor did not affect ROS (DCF) level but decreased O_2•⁻ level in MG132-treated HPF cells. These results suggest the possibility that the inhibition of JNK and p38 signaling attenuates HPF cell death by MG132 via decreasing the O_2•⁻ level. On the other hand, JNK or p38 siRNA did not affect ROS levels including O_2•⁻ in MG132-treated HPF cells. p38 inhibitor alone significantly increased ROS levels in HPF control cells without the induction of cell death. In addition, this inhibitor did not significantly alter mitochondrial O_2•⁻ level in MG132-treated HPF cells. Moreover, we observed that MG132 inhibited the activity of proteasome in HPF cells. While ERK and JNK siRNAs attenuated the proteasome inhibition in MG132-treated HPF cells, p38 siRNA did not affect the inhibition. Therefore, the relationship among proteasome inhibition, MAPK signaling and ROS level changes in HPF cells needs to be further studied in relation to cell death. GSH depletion due to proteasome inhibitors can lead to cell death.^13,14 As expected, MG132 increased GSH-depleted cell number in the HPF cells. In addition, JNK or p38 inhibitor slightly decrease GSH-depleted cell number in these cells. MEK inhibitor showing a decrease in PI staining cells by MG132 mildly attenuated GSH depletion. Therefore, the intracellular GSH content seems to be a decisive role on HPF cell death.

In conclusion, MAPK inhibitors differently affected the growth inhibition and death of MG132-treated HPF cells. Especially, p38 inhibitor to some extent prevented HPF cell death by MG132, which was partially related to changes in ROS and GSH levels.