Phorbol ester (PMA) and ionomycin (ION) were purchased from Sigma-Aldrich, Merck Millipore (Darmstadt, Germany). Murine IL-4 and IL-6 ELISA kits were purchased from eBioscience, Inc. (San Diego, CA, USA). TRIzol reagent was purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Antibodies against ERK2/1 (MK1; cat. no. sc-135900; 1:400), AKT (C-20; cat. no. sc-1618; 1:200), phosphorylated (p)-ERK (E-4) (cat. no. sc-7383; 1:400), p-AKT-Thr308 (cat. no. sc-16646; 1:200) and MMP9 (cat. no. sc-6841; 1:400), the ERK/MAPK inhibitor U0126, the AKT inhibitor MK2206 and the MMP9 inhibitor CTK8G1150, and MMP9 small interfering RNA (siRNA) (cat. no. sc-29401; 1:400) were all purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). For transient silencing of MMP9, 21-nt sequences of siRNA duplexes were synthesized (GenePharma, Shanghai, China): sense 5′-CAUUCAGGGAGACGCCCAUUUTT-3′ and antisense 5′-AAAUGGGCGUCUCCCUGAAUGTT-3′; scramble control siRNA sense 5′-UUCUCCGAACGUGUCACGUTT-3′ and antisense 5′-ACGUGACACGUUCGGAGAATT-3′. A total of 40 nM siRNA duplexes was transfected reagent on a 24-well plate. The efficiency of MMP9 transient silencing was confirmed by western blotting. Recombinant murine IL-4 (cat. no. 404-ML; 1:200) and murine IL-4 antibodies (cat. no. MAB404; 1:200); were purchased from R&D Systems, Inc. (Minneapolis, MN, USA).

The murine mast cell P815 cell line and human mast cell HMC-1 cell line were obtained from the American Type Culture Collection (Manassas, VA, USA). RPMI-1640 supplemented with L-glutamine, sodium pyruvate, non-essential amino acids, a 2-fold vitamin solution, and penicillin-streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.). Fetal bovine serum (FBS; HyClone; GE Healthcare Life Sciences, Logan, UT, USA) or horse serum (Invitrogen; Thermo Fisher Scientific, Inc.) was added to the media. All cultures were performed at 37°C in a 5% CO₂ atmosphere.

A total of 2×10⁵ P815 or HMC-1 cells were stimulated with 50 nM PMA and 500 nM ION for 24 h, and were subsequently centrifuged at 400 × g for 5 min at 25°C. Cell supernatant was collected and IL-4 and IL-6 levels were measured by ELISA, according to the manufacturer's protocol. To detect degranulation, 50 µl supernatant was removed for β-hexosaminidase measurement, and deionized water was added to the remaining cell pellets. The samples were frozen, thawed, and a second 50 µl was removed to determine the total β-hexosaminidase content. β-hexosaminidase samples (50 µl) were incubated in 0.04 M citric acid with 0.02 M Na₂HPO₄ containing 10 mM p-nitrophenyl N-acetyl-α-D-glucosaminide for 90 min at 37°C. The reaction was developed using 0.4 M glycine and the absorbance was determined at 405 nm. The release percentage was calculated as follows: [β-hexosaminidase in supernatant/(β-hexosaminidase in supernatant + total β-hexosaminidase in pellet)] ×100.

The total RNA was extracted using TRIzol reagent. and cDNA was synthesized using a PrimeScript™ RT reagent kit (Takara Bio, Inc., Otsu, Japan), according to the manufacturer's protocol. The following primers were used: β-actin, forward: 5′-CGTTGACATCCGTAAAGACC-3′ and reverse: 5′-AACAGTCCGCCTAGAAGCAC-3′; MMP9, forward: 5′-CGGCACGCCTTGGTGTAGCA-3′ and reverse: 5′-GGCGCACCAGCGGTAACCAT-3′. The following PCR conditions were used: 1 cycle of 95°C for 30 sec followed by 40 cycles of 95°C for 5 sec and 60°C for 34 sec. RT-qPCR and the 2^−∆∆Cq method was performed on an Applied Biosystems 7500 real time PCR system (Thermo Fisher Scientific, Inc.) with version 2.0.6 software (20).

To inhibit the ERK and AKT signaling pathways, the 2×10⁵ P815 cells were pre-treated for 2 h with either 100 nM U0126 or 10 nM MK2206, or both, and were then activated using 50 nM PMA and 500 nM ION for 24 h. To inhibit MMP9, the 2×10⁵ P815 cells were pre-treated with 10 nM CTK8G1150 for 2 h and subsequently activated by 50 nM PMA and 500 nM ION for 24 h. The control group was treated with DMSO.

The total proteins were extracted from cells (HMC-1 and P815) using protein extracting buffer [20 mmol/l Tris-Cl buffer, pH 7.5, containing 1 mmol/l ethylenediamine tetraacetic acid, a protease inhibitor cocktail (complete, Mini, ethylenediamine tetraacetic acid-free, 1 tablet of 10-ml buffer; Sigma-Aldrich, Merck Millipore), 1% sodium dodecyl sulfate, 10% Triton X-100, and 2 mol/l dithiothreitol]. After 30 min on ice, the samples were centrifuged at 17,600 × g for 10 min at 4°C). A total of 20 µg crude proteins extracted from cell lysates from HMC-1 and P815 cells were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were subsequently transferred onto polyvinylidene difluoride membranes (Merck Millipore). The membranes were blocked with 5% FBS in Tris-buffered saline, pH 8.0, plus 0.05% Tween-20, for 1 h at room temperature, and were then incubated with the corresponding primary antibodies at 4°C overnight. Following washing with Tris-buffered saline plus 0.05% Tween-20, the membranes were incubated with secondary antibody conjugated to horseradish peroxidase (cat. nos. P-0217, P-0161, P-0160; 1:1,000; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) was incubated for 1 h at room temperature. Proteins were visualized using SuperSignal West Femto Maximum Sensitivity Chemiluminescence substrate (Thermo Fisher Scientific, Inc.).

A gelatin zymography assay was performed to investigate the secretion of active MMP9. A total of 4×10⁴ of HMC-1 and P815 cells were grown to 70% confluence, washed twice with PBS and incubated in serum-free medium. Conditioned medium was collected 24 h following this and was concentrated with a centrifugal filter (Merck Millipore) at 6,000 × g for 15 min at 4°C Concentrated samples were prepared in non-reducing sample buffer (Invitrogen; Thermo Fisher Scientific, Inc.). Proteins (20 µl/lane) were separated by SDS-PAGE on gels containing 1 mg/ml gelatin (Novex 10% gelatin gel; Invitrogen; Thermo Fisher Scientific, Inc.). The gels were renatured for 1 h at room temperature in 1X renaturing buffer (Invitrogen; Thermo Fisher Scientific, Inc.). Following this, gels were incubated overnight at 37°C in 1X developing buffer (Invitrogen; Thermo Fisher Scientific; Inc.). Gels were stained with Coomassie blue for 2 h at room temperature. The brightness of the clear bands, where MMP9 was located and the gelatin was degraded, was analyzed according the optical density using Bio-Rad Image Lab version 4.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

MMP9 and negative control siRNA duplexes (40 nM) were transfected into cells of HMC-1 and P815 (5×10⁵/well) using 3 µl INTERFER in siRNA transfection reagent (Santa Cruz Biotechnology, Inc.) in a 24-well plate. The efficiency of MMP9 silencing was confirmed by western blot analysis.

Data are presented as the mean ± standard error. The results were compared using one-way analysis of variance in SPSS version 16 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Following stimulation with 50 nM PMA and 500 nM ION, P815 cells released significantly increased levels of IL-4, IL-6 and β-hexosaminidase compared with control cells or DMSO-treated cells (P=0.0018, P=0.0014 and P=0.0002, respectively; Fig. 1A). HMC-1 cells also had higher levels of IL-4, IL-6 and β-hexosaminidase (data not shown). Following activation, increased mRNA (Fig. 1B) and protein (Fig. 1C) expression levels of MMP9 were detected in both P815 and HMC-1 cells. To confirm an increase of MMP9 release, MMP9 activity was detected by gelatin zymography. Activated P815 and HMC-1 cells degraded visibly more gelatin compared with the control or DMSO-treated cells (Fig. 1D). These results indicated that MMP9 expression is upregulated in activated P815 and HMC-1 mast cells.

The ERK and AKT signaling pathways are involved in the activation of mast cells (21) and have also been reported to induce the expression of MMP9 (22). The present study aimed to establish whether the ERK and AKT signaling pathways are involved in MMP9 upregulation in activated mast cells. Phosphorylated ERK and AKT expression levels were visibly increased in activated P815 cells (Fig. 2A). Following treatment with the ERK-specific inhibitor U0126 or the AKT-specific inhibitor MK2206, PMA+ION treatment-induced increases in MMP9 protein levels were partially inhibited in activated P815 cells compared with DMSO-treated activated P815 cells (Fig. 2B). Combined treatment with both U0126 and MK2206 appeared to completely abolish increased MMP9 protein levels in activated P815 cells compared with DMSO-treated activated P815 cells (Fig. 2B). These results suggested that the increased MMP9 levels observed in activated mast cells are dependent on the ERK and AKT signaling pathways.

To establish the effect of MMP9 on mast cell activation, the MMP9 inhibitor CTK8G1150 was used. PMA and ION treatment-induced IL-4, IL-6 and β-hexosaminidase release significantly decreased in CTK8G1150-treated P815 cells compared with the untreated P815 cells (P=0.0495, P=0.0128, P=0.0067, respectively; Fig. 3A). Furthermore, similar results were demonstrated in activated P815 cells transfected with siRNA, with IL-4, IL-6 and β-hexosaminidase release significantly decreased in MMP9 siRNA-transfected P815 cells compared with control siRNA-transfected P815 cells (P=0.0490, P=0.0099 and P=0.0073, respectively; Fig. 3B). These results suggested that MMP9 is involved in the activation of mast cells.

IL-4 has been previously demonstrated to inhibit MMP9 expression in rat synovial membranes incubated in vitro (23) and activated mast cells produce high levels of IL-4 (24). Therefore, it was hypothesized that IL-4 was involved in the regulation of MMP9 expression in activated mast cells. The protein expression of MMP9 was visibly inhibited in 10 ng/ml IL-4-treated activated P815 cells compared with untreated activated P815 cells (Fig. 4A). Furthermore, inhibiting IL-4 signaling using 10 µg/ml IL-4 neutralizing antibody resulted in visibly increased MMP9 protein levels in activated P815 cells compared with untreated activated P815 cells (Fig. 4B). These results indicated that IL-4 negatively regulates MMP9 expression in activated mast cells.