Time-dependent activation of PLD by Der f 2 and changes in PLD expression level after treatment of BEAS-2B cells with Der f 2. A, BEAS-2B cells were labeled with 2 μCi/ml [³H]palmitic acid and then stimulated with Der f 2 (10 μg/ml) for the indicated times. PLD activities were then determined by estimating the amount of [³H]PBt in the presence of 1-butanol. Results are means ± S.D. from three independent experiments. B, cells treated with Der f 2 (10 μg/ml) for the indicated times were lysed. Each fraction, containing 30 μg of protein, was analyzed by 10% SDS-PAGE and subsequently by Western blotting using anti-PLD polyclonal antibody. Bands were shown by ECL. The intensity of the bands was quantified using Quantity One software (Bio-Rad). All determinations were carried out on three different cell lysates.

Effect of the PLCγ inhibitors U73122 and PAO on Der f 2- induced PLD activation and IL-13 production in BEAS-2B cells. A, BEAS-2B cells were treated with Der f 2 (10 μg/ml) for 1 min. Cells were harvested, and cell extracts were subjected to immunoblot analysis for active and total PLCγ1 (upper panel). Serum-starved cells were stimulated with Der f 2 (10 μg/ml) for 1, 2, or 3 min. The cells were then lysed and subjected to subcellular fractionation into cytosolic and membrane fractions by centrifugation at 100,000 × g, and each fraction containing 30 μg of protein was analyzed by 10% SDS-PAGE and subsequently by Western blotting using anti-PLC γ1 polyclonal antibody (lower panel). B, cells were labeled with 2 μCi/ml [³H]palmitic acid and then pretreated with 10 μm PLCγ inhibitor U73122 or PAO for 30 min prior to Der f 2 (10 μg/ml) stimulation. PLD activities were then determined by estimating the amount of [³H]PBt in the presence of 1-butanol. Results are means ± S.D. from three independent experiments. C, cells in 96-well culture plates were pretreated with U73122 or PAO (10 μm) for 30 min and then stimulated with Der f 2 (10 μg/ml) for 2 h. The results shown are mean values ± S.E. of the amount of IL-13 measured by ELISA for each group of samples (n = 8).

Effect of p38 MAPK inhibition on Der f 2-induced PLD activation in BEAS-2B cells. A, BEAS-2B cells treated with Der f 2 (10 μg/ml) at the indicated times were harvested, and cell extracts were subjected to immunoblot analysis for active and total MAPKs. B, cells were pretreated with SB203580 or SB202190 (50 μm) for 30 min and then stimulated with Der f 2 (10 μg/ml) for 5 min. Phosphorylated p38 MAPK was determined by Western blot analysis. The cells were labeled with 2 μCi/ml [³H]palmitic acid and then pretreated with SB203580 or SB202190 (50 μm) for 30 min before stimulation with Der f 2 (10 μg/ml) for 30 min. PLD activities were determined by estimating the formation of [³H]PBt in the presence of 1-butanol. Results are means ± S.D. from three independent experiments. C, cells in 96-well culture plates were pretreated with SB203580 or SB202190 (50 μm) for 30 min and then stimulated with Der f 2 (10 μg/ml) for 2 h. The results shown are mean values ± S.E. of the amount of IL-13 measured by ELISA for each group of samples (n = 8). D, cells were pretreated with U73122(10 μm), PAO (10 μm), RO320432 (50 μm), GO6976 (50 μm), SB203580 (50 μm), or SB202190 (50 μm) for 30 min and then stimulated with Der f 2 (10 μg/ml) for 5 min. Phosphorylated p38 MAPK was determined by Western blot analysis. The intensity of bands was quantified using Quantity One software (Bio-Rad). Data represent the results of three separate experiments. E, cells were pretreated with U73122(10 μm), PAO (10 μm), RO320432 (50 μm), GO6976 (50 μm), SB203580 (50 μm), or SB202190 (50 μm) for 30 min and then stimulated with Der f 2 (10 μg/ml) for 15 min. Total RNA was isolated using TRIzol reagent. RT-PCR analyses were performed using IL-13 and β-actin primers and real-time RT-PCR with primers for QIL-13 or GAPDH. Data are means ± S.D. of three values.

Effect of ATF-2 phosphorylation on IL-13 expression and production induced by Der f 2 in BEAS-2B cells. A, BEAS-2B cells were transiently transfected with 100 nm ATF-2 siRNAs or scrambled siRNA for 72 h and then stimulated with Der f 2 (10 μg/ml) for 5 min (for p-ATF-2) and 15 min (for IL-13 mRNA levels). Expression levels of ATF-2 and p-ATF-2 were determined by Western blot analysis (upper panel). The cells were harvested and total RNA was isolated using TRIzol reagent. RT-PCR analyses were performed using IL-13 and β-actin primers (lower panel) and real-time PCR with primers for QIL-13 or GAPDH. Data are means ± S.D. of three values. B, BEAS-2B cells on 96-well culture plates were transfected with 100 nm ATF-2 siRNAs or scrambled siRNA for 72 h and then stimulated with Der f 2 (10 μg/ml) for 2 h. The result shown are mean values ± S.E. of the amount of IL-13 measured by ELISA for each group of samples (n = 8).

Effect of PLD knockdown on Der f 2- induced IL-13 production and expression in BEAS-2B cells. A, BEAS-2B cells in 96-well culture plates were treated with Der f 2 (10 μg/ml) for the indicated times. The results shown are mean values ± S.E. of the amount of IL-13 measured by ELISA for each group of samples (n = 8). B, cells were treated with Der f 2 (10 μg/ml) for the indicated times. Total RNA was isolated using TRIzol reagent, and mRNA levels were determined by RT-PCR with primers for IL-13 or β-actin and real-time PCR with primers for QIL-13 or GAPDH. Data are means ± S.D. of three values. C, cells were transiently transfected with EGFP, DN-PLD1, PLD1, DN-PLD2, or PLD2 plasmid DNA. After transfection for 24 h, they were treated with Der f 2 (10 μg/ml) for 15 min. Total RNA was isolated using TRIzol reagent, and mRNA levels were determined by RT-PCR with primers for IL-13 or β-actin and real-time PCR with primers for QIL-13 or GAPDH. *, p < 0.05 ANOVA versus values for Der f 2-treated control. Data are means ± S.D. of three values. D, cells were transiently transfected with EGFP, DN-PLD1, or PLD1 plasmid DNA. After transfection for 24 h, they were treated with Der f 2 for 15 min. Transfected cells are defined by their green or red color under the fluorescence microscope after immunostaining using antibodies against EGFP and IL-13 (original magnification ×200). E, BEAS-2B cells on 96-well culture plates were transfected with 100 nm PLD1 siRNAs or scrambled siRNA for 72 h and then stimulated with Der f 2 (10 μg/ml) for 2 h. The results shown are the mean values ± S.E. of the amount of IL-13 measured by ELISA for each group of samples (n = 8). F, cells were transiently transfected with 100 nm PLD1 siRNAs or scrambled siRNA for 48 h and then stimulated with Der f 2 (10 μg/ml) for 15 min. The cells were harvested, and total RNA was isolated using TRIzol reagent. RT-PCR analyses were performed using IL-13 and β-actin primers and real-time PCR with primers for QIL-13 or GAPDH. Data are means ± S.D. of three values.

Effect of Der f 2 on PKCα translocation and effects of PKC down-regulation on Der f 2-induced PLD activation and IL-13 production in BEAS-2B cells. A, serum-starved cells were stimulated with Der f 2 (10 μg/ml) for 1 or 3 min. As a positive control for PKC translocation to the plasma membrane, cells were treated with 200 nm phorbol myristate acetate (PMA) for 30 min. The cells were then lysed and subjected to subcellular fractionation into cytosolic and membrane fractions by centrifugation at 100,000 × g. Each fraction containing 30 μg of protein was analyzed by 10% SDS-PAGE and subsequently to Western blotting using anti-PKCα, PKCβ1, PKCλ, and PKCγ polyclonal antibody. B, serum-starved cells were pretreated with U73122 or PAO (10 μm) for 30 min (upper panel) and RO320432 or GO6976 (50 μm) for 30 min (lower panel) before stimulation with Der f 2 (10 μg/ml) for 1 min. The cells were then lysed and subjected to subcellular fractionation into cytosolic and membrane fractions by centrifugation at 100,000 × g. Each fraction containing 30 μg of protein was analyzed by 10% SDS-PAGE and subsequently by Western blotting using anti-PKCα polyclonal antibody. The data are expressed as a fold of basal value in membrane fraction and are means ± S.D. of three experiments. C, BEAS-2B cells were labeled with 2 μCi/ml [³H]palmitic acid and then pretreated with RO320432 or GO6976 (50 μm) for 30 min before stimulation with Der f 2 (10 μg/ml) for 30 min. PLD activities were then determined by estimating the amount of [³H]PBt in the presence of 1-butanol. Results are means ± S.D. from three independent experiments. D, cells in 96-well culture plates were pretreated with RO320432 or GO6976 (50 μm) for 30 min and then stimulated with Der f 2 (10 μg/ml) for 2 h. The results shown are mean values ± S.E. of the amount of IL-13 measured by ELISA for each group of samples (n = 8).

Effect of PLD1 knockdown and exogenous PA treatment on Der f 2-induced ATF-2 phosphorylation in BEAS-2B cells. A, BEAS-2B cells were stimulated with Der f 2 (10 μg/ml) for the indicated time periods, and the amounts of total ATF-2 and phosphorylated ATF-2 (p-ATF-2) were determined by Western blot analysis (upper panel). The cells were pretreated with U73122 (10 μm), PAO (10 μm), RO320432 (50 μm), GO6976 (50 μm), SB203580 (50 μm), or SB202190 (50 μm) for 30 min and then stimulated with Der f 2 (10 μg/ml) for 5 min. Expression levels of ATF-2 and p-ATF-2 were determined by Western blot analysis (lower panel). B and C, cells were transiently transfected with 100 nm PLD1 siRNAs or scrambled siRNA for 72 h and then stimulated with Der f 2 (10 μg/ml) for 5 min. B, expression levels of ATF-2 and p-ATF-2 were determined by Western blot analysis. The intensity of bands was quantified using Quantity One software (Bio-Rad). *, p < 0.05 ANOVA versus values for Der f 2-treated control. Data represent the results of three separate experiments, C, p-ATF-2-immunostained cells were observed under a fluorescence microscope and photographed with magnification ×100. D, cells were transiently transfected with EGFP, DN-PLD1, or PLD1 plasmid DNA. After transfection for 24 h, the cells were treated with Der f 2 for 5 min. Expression levels of ATF-2 and p-ATF-2 were determined by Western blot analysis. E, cells were stimulated with indicated concentrations of PA for 5 min, and the amounts of total ATF-2 and p-ATF-2 were determined by Western blot analysis (upper panel). The cells were pretreated with SB203580 (50 μm) or SB202190 (50 μm) for 30 min and then stimulated with PA (10 μm) for 5 min (for p-ATF-2) and 15 min (for IL-13 mRNA levels). Expression levels of ATF-2 and p-ATF-2 were determined by Western blot analysis. Total RNA was isolated using TRIzol reagent. RT-PCR analyses were performed using IL-13 and β-actin primers (lower panel) and real-time RT-PCR with primers for QIL-13 or GAPDH. Data are means ± S.D. of three values. F, cells were pretreated with mepacrine (10 μm) or propranolol (50 μm) for 1 h and then stimulated with PA (10 μm) for 15 min. Total RNA was isolated using TRIzol reagent. RT-PCR analyses were performed using IL-13 and β-actin primers and real-time RT-PCR with primers for QIL-13 or GAPDH. Data are means ± S.D. of three values. G, cells were stimulated with LPA (10 μm) for the indicated time periods. Total RNA was isolated using TRIzol reagent. RT-PCR analyses were performed using IL-13 and β-actin primers and real-time RT-PCR with primers for QIL-13 or GAPDH. Data are means ± S.D. of three values.

Proposed model for the signaling pathway of Der f 2-induced IL-13 expression and production in BEAS-2B cells. Our model suggests that Der f 2 induces expression and production of IL-13 via activation of PLD1 through PLCγ/PKCα/p38 MAPK in BEAS-2B cells.