Mouse auditory HEI-OC1 cells were purchased from the House Ear Institute (Los Angeles, CA, USA) and cultured in sodium pyruvate-free Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (all obtained from Welgene, Inc., Gyeongsan, Korea) at 37°C in a humidified 5% CO₂ atmosphere. Recombinant interleukin (IL)-1β was purchased from R&D Systems, Inc. Minneapolis, MN, USA). Phorbol-12-myristate-13-acetate (PMA) was obtained from EMD Millipore (Billerica, MA, USA). Rottlerin, PD98059, SB203580 and SP600125 were purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Glutathione (GSH), N-acetylcysteine (NAC) and trolox were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyclonal mouse anti-COX-2 antibody (1:1,000, cat. no. 160106) was obtained from the Cayman Chemical Company (Ann Arbor, MI, USA). Polyclonal rabbit anti-p46/54 c-Jun N-terminal kinase (JNK, 1:700, cat. no. 9252), polyclonal rabbit anti-phospho (p)-p46/54 JNK (1:700, cat. no. 9251), polyclonal rabbit anti-p38MAPK (1:700, cat. no. 9211), polyclonal rabbit anti-p-p38MAPK (1:700, cat. no. 9212), polyclonal rabbit anti-extracellular signal-regulated kinase (ERK1/2, 1:700, cat. no. 9102) and polyclonal rabbit anti-p-ERK1/2 (1:700, cat. no. 9101) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Polyclonal rabbit anti-ATF4 (1:1,000, cat. no. sc-200) and polyclonal rabbit anti-ATF3 (1:700, cat. no. sc-188) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Polyclonal rabbit anti-regulated in development and DNA damage responses 1 (REDD1) anti body (1:1,000, cat. no. 10638-1-AP) was obtained from ProteinTech Group, Inc. (Chicago, IL, USA). Monoclonal mouse anti-actin antibody (1:15,000, cat. no. A5441) was purchased from Sigma-Aldrich.

For the western blotting, cells were washed with cold phosphate-buffered saline (PBS) and lysed on ice in modified radioimmunoprecipitation assay buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM Na₃VO₄ and 1 mM NaF) containing protease inhibitors (100 µM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml pepstatin, and 2 mM EDTA). The lysates were centrifuged at 10,000 × g for 10 min at 4°C and the supernatant fractions were collected. The protein concentration was determined using Micro BCA protein assay kit was from Pierce Chemical (Rockford, IL, USA), according to the manufacturer's protocol. An equal amount of cell lysate (60 µg) was dissolved in sample buffer, and samples were boiled for 5 min. The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes (Amersham, Uppsala, Sweden) and were subsequently blocked for 1 h at room temperature in 5% (w/v) non-fat dried milk, and then were incubated for overnight at room temperature with the aforementioned primary antibodies, followed by an incubation with biotinylated secondary antibodies. The specific proteins were detected using an enhanced chemiluminescence western blotting kit (Merck Millipore, Darmstadt, Germany) according to the manufacturer's instructions. The band intensity of COX-2 protein was measured using Image J (National Institutes of Health, Bethesda, MA, USA), and expressed as a ratio to actin band intensity.

Total RNA was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and 1 µg of total RNA was used to synthesize cDNA using M-MLV RT (Gibco; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Then, 2 µl of cDNA was amplified with Taq (Solgent, GeNetBio, South Korea) kits in a total volume of 25 µl according to the manufacturer's protocol. The PCR primers were purchased from Macrogen (Seoul, South Korea). The following primers were used for amplification: Sense 5′-ACACACTCTATCACTGGCACC-3′ and antisense 5′-TTCAGGGAGAAGCGTTTGC-3′ for COX-2; and sense 5′-ACGACATGGAGAAGATCTGG-3′ and antisense 5′-TCAGGCAGCTCATAGCTCTT-3′ for actin. The PCR amplification was performed using the following cycling conditions: 94°C for 3 min; 17 (actin) or 25 cycles (COX-2) of 94°C for 45 sec, 58°C for 45 sec, 72°C for 1 min; and a final extension step at 72°C for 10 min. The amplified products were separated by electrophoresis on a 1.5% agarose gel (Invitrogen; Thermo Fisher Scientific, Inc.) and detected under UV light.

MTT assay was used to determine the cell viability using WelCount Cell Viability Assay kit (WelGENE, South Korea). Briefly, 24 h after drug treatment the reagent was added to each well and then measured with multi-well plate reader (at 450/690 nm).

HEI-OC1 cells were seeded at a density of 1×10⁵ cells/well in 6-well culture plates one day prior to transfection to achieve 50–60% confluency. The ATF4 and green fluorescent protein (GFP) control siRNA duplexes used in the present study were purchased from Santa Cruz Biotechnology, Inc. and Invitrogen; Thermo Fisher Scientific, Inc., respectively. Cells were transfected with siRNA oligonucleotides using Oligofectamine reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Intracellular accumulation of ROS was determined using the fluorescent probe 2′, 7′-dichlorodihydrofluorescein diacetate (H2DCFDA). H₂DCFDA is commonly used to measure ROS generation (13). Cells were seeded in a 6-well plate for 24 h prior to treatment with 5 µM rottlerin, and treated with 5 µM H2DCFDA for 30 min. Cells were then trypsinized (Welgene, Inc., Gyeongsan, South Korea) and resuspended in PBS. Fluorescence was measured at the desired time intervals using a flow cytometer (BD FACSCanto II; BD Biosciences, Franklin Lakes, NJ, USA) or detected using a fluorescence microscope (Axiovert200; Zeiss GmbH, Jena, Germany).

Values are expressed as the mean ± standard deviation of at least three independent experiments. Data were analyzed with one-way analysis of variance and Student-Newman-Keuls post hoc comparisons using the Statistical Package for Social Sciences software version 22.0 (IBM SPSS, Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

To determine whether rottlerin affects the mRNA and protein expression levels of COX-2, HEI-OC1 cells were treated with various rottlerin concentrations (0.5–5.0 µM) for 12 h. As demonstrated in Fig. 1A, COX-2 protein levels were increased by rottlerin in a dose-dependent manner. To elucidate the correlation between COX-2 protein and COX-2 mRNA in HEI-OC1 cells, RT-PCR was performed. The levels of COX-2 mRNA were also increased by rottlerin in a dose-dependent manner. Additionally, the cytotoxic effects of rottlerin were evaluated by MTT assay. Cell viability following rottlerin treatment was determined to be >90% using the MTT assay (Fig. 1C).

Several previous studies have suggested that ROS are associated with signaling mechanisms that induce COX-2 expression (14,15). Therefore, the present study examined whether rottlerin induces ROS production using H2DCFDA-derived fluorescence and fluorescence-activated cells sorting detection in HEI-OC1 cells. As presented in Fig. 2A and B, compared with untreated cells, rottlerin significantly increased the intracellular ROS levels within 10 min of treatment (P<0.0001). Additionally, the present study investigated whether blockade of rottlerin-induced ROS generation affects COX-2 expression levels in HEI-OC1 cells. Compared with rottlerin treatment, pretreatment with the antioxidants NAC, GSH and trolox did not inhibit rottlerin-induced COX-2 protein expression (Fig. 2C). These results suggest that ROS are not involved in rottlerin-induced COX-2 expression.

The current study investigated the effect of rottlerin on MAPK activity in order to determine whether this signaling pathway is involved in rottlerin-induced COX-2 expression. As shown in Fig. 3A, rottlerin markedly increased the phosphorylation of p38MAPK, but not of ERK and JNK. p38MAPK activation was detected at 3 h and subsequently increased in a time-dependent manner (Fig. 3A). To determine the importance of individual MAPK pathways in rottlerin-induced COX-2 expression, the present study examined the effects of PD98059 (a potent ERK inhibitor), SB203580 (a specific p38MAPK inhibitor) and SP600125 (a potent JNK inhibitor) on rottlerin-induced COX-2 expression. As presented in Fig. 3B, pretreatment with SB203580 in the presence of rottlerin resulted in a 0.57-fold decrease in COX-2 protein expression levels compared with rottlerin treatment, but SP600125 and PD98059 did not affect COX-2 protein expression. To further investigate the correlation between the p38MAPK pathway and rottlerin-induced COX-2 expression, SB203580 dose-response experiments were conducted in rottlerin-treated HEI-OC1 cells. As demonstrated in Fig. 3C, SB203580 markedly reduced COX-2 protein expression levels in a dose-dependent manner. Together these results indicate that the activation of p38MAPK pathway is important for the regulation of rottlerin-induced COX-2 expression in HEI-OC1 cells.

A previous study demonstrated that ER stress-inducing agents can induce COX-2 expression (16). Therefore, kinetic studies were performed on rottlerin-treated HEI-OC1 cells to determine the effects of ER stress-associated transcription factors (REDD, ATF 3 and 4) on rottlerin-induced COX-2 expression. Incubation with rottlerin caused a time-dependent increase in COX-2 protein expression (Fig. 4). Notably, ATF4 protein expression levels were markedly increased by treatment with rottlerin compared with untreated cells. However, ATF3 and REDD1 levels were not identified to be increased in response to rottlerin (Fig. 4A). To examine whether rottlerin-induced COX-2 expression is dependent on ATF4 activity, an siRNA duplex targeting AFT4 mRNA was used. HEI-OC1 cells transfected with the control (GFP) or ATF4 siRNA were treated with rottlerin for 12 h. Suppression of ATF4 expression by transfection with siRNA inhibited rottlerin-induced upregulation of COX-2 protein levels compared with control siRNA (Fig. 4B). These results suggest that the expression of ATF4 is critical for rottlerin-induced COX-2 expression.

PMA and IL-1β have previously been demonstrated to induce COX-2 expression (17-23). Western blotting and RT-PCR were performed to determine the effect of rottlerin on PMA- and IL-1β-induced COX-2 expression in HEI-OC1 cells. As demonstrated in Fig. 5, treatment with rottlerin enhances PMA and IL-1β-induced COX-2 protein and mRNA expression compared with treatment with PMA/IL-1β alone.