House Dust Mite Allergen Der f 2-induced Phospholipase D1 Activation Is Critical for the Production of Interleukin-13 through Activating Transcription Factor-2 Activation in Human Bronchial Epithelial Cells*

The purpose of this study was to identify the role of phospholipase D1 (PLD1) in Der f 2-induced interleukin (IL)-13 production. The major house dust mite allergen, Der f 2, increased PLD activity in human bronchial epithelial cells (BEAS-2B), and dominant negative PLD1 or PLD1 siRNA decreased Der f 2-induced IL-13 expression and production. Treatment of Der f 2 activated the phospholipase Cγ (PLCγ)/protein kinase Cα (PKCα)/p38 MAPK pathway. Der f 2-induced PLD activation was attenuated by PLCγ inhibitors (U73122 and PAO), PKCα inhibitors (RO320432 and GO6976), and p38 MAPK inhibitors (SB203580 and SB202190). These results indicate that PLCγ, PKCα, and p38 MAPK act as upstream activators of PLD in Der f 2-treated BEAS-2B cells. Furthermore, expression and production of IL-13 increased by Der f 2 were also blocked by inhibition of PLCγ, PKCα, or p38 MAPK, indicating that IL-13 expression and production are related to a PLCγ/PKCα/p38 MAPK pathway. We found that activating transcription factor-2 (ATF-2) was activated by Der f 2 in BEAS-2B cells and activation of ATF-2 was controlled by PLD1. When ATF-2 activity was blocked with ATF-2 siRNA, Der f 2-induced IL-13 expression and production were decreased. Thus, ATF-2 might be one of the transcriptional factors for the expression of IL-13 in Der f 2-treated BEAS-2B cells. Taken together, PLD1 acts as an important regulator in Der f 2-induced expression and production of IL-13 through activation of ATF-2 in BEAS-2B cells.

House dust mites (HDMs) 3 are the most important source of indoor aeroallergens that contribute to the rising incidence of allergic diseases such as allergic asthma (1,2). Allergens from two common house dust mite species are Dermatophagoides farinae (Der f) and Dermatophagoides pteronyssinus (Der p), which share a similar amino acid sequence (3,4). Recent data provide evidence that the allergens amplify allergen-induced cytokine expression (5). Indeed, several studies demonstrated that Der p 1 stimulates release and expression of cytokines in human airway epithelial cells (6), including IL-13 in cord blood mononuclear cells (7) and IL-2 and IL-4 in a cord blood T-cell phenotype with atopic dermatitis (8). Although the release of cytokines by allergen stimulation has been studied, details of the intracellular signaling mechanism, including the secretion of cytokines, remain unclear.
Phospholipase D (PLD) catalyzes the hydrolysis of phospholipids at the terminal phosphodiester bond, thereby producing phosphatidic acid (PA) and releasing the free polar head group. PA can be metabolically converted to diacylglycerol by phosphatidic acid phosphatases type 2A (PPAP2A) or to lysophosphatidic acid (LPA) and arachidonic acid by phospholipase A2 (PLA2G1B) (9). Both of these factors serve as second messengers that contribute to the effects of PLD. Recently, two PLD isozymes (PLD1 and PLD2) have been identified from mammalian cells (10,11). PLD1 is present throughout the cell, particularly in perinuclear, Golgi, and heavy membrane fractions, whereas PLD2 is located almost exclusively in the plasma membrane of light membrane "lipid raft" fractions (12). The differences in the localization of these PLDs can provide important clues, suggesting their specific roles in various situations and cell types (13). PLD and phospholipase C (PLC) ␥ play an important regulatory role in cell immune response (14,15). The activation of PLC␥1 results in the hydrolysis of phosphatidylinositol 4,5-bisphosphates (PIP 2 ) to generate inositol 1,4,5-trisphosphates (IP 3 ) and diacylglycerol (16,17). These second messengers are responsible for the activation of protein kinase C (PKC) (18). Activation and phosphorylation of PLD1 are regulated by PKC␣ in phorbol myristate acetate-treated COS-7 cells, also demonstrating the interrelationships between PLD and PKC isoforms in a variety of cell types (19). Previously, we reported that Der p 1 activates PLD in human peripheral blood mononuclear cells from allergic patients (13) and that Der f 2 activates PLD in human T lymphocytes from Der f 2-specific allergic individuals (1). However, the molecular mechanisms of PLD for the expression of cytokine in the immune response induced by Der f 2 have not been fully understood.
Recent reports show that mitogen-activated protein kinase (MAPK) activation is involved in the process of allergen-induced cytokine expression (20,21). There are three distinct MAPK signaling pathways in mammalians: extracellular signalregulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 MAPK (21,22). Among the MAPK subtypes, activation of p38 MAPK is critical for the conversion of external stimuli to proinflammatory gene expression (20,23), and it has also been implicated in the regulation of respiratory burst activity, priming, deregulation, and cytokine production (24,25). In particular, the p38 MAPK-specific inhibitor pyridinyl imidazole compound SB203580 has facilitated extensive investigation of the role of the p38 MAPK pathway in inflammatory responses (26). Commonly, p38 MAPK phosphorylates activating transcription factor 2 (ATF-2) to augment gene transcription (27). ATF-2 activity is regulated by phosphorylation of Thr-69 and Thr-71 residues in its N-terminal region (28), and the signaling to activate ATF-2 affects the selection of target genes by ATF-2. Therefore, ATF-2 may play a role in the regulation of cytokine gene expression.
IL-13 is produced when cells are exposed to a variety of environmental stimuli such as allergens, pharmacological agents, and infectious stimuli. IL-13 production is highly regulated by a variety of mediators and cytokines, thereby leading to a positive feedback loop which sustains immune responses (29). The importance of IL-13 in allergic disorders has been indicated by consistent associations between IL-13 levels of tissue and genetic variants in the IL-13 gene with asthma and related traits (30). IL-13 production has also been shown to mediate allergic responses through signaling pathways such as PKA, PKC, p38MAPK, ERK1/2, and JAK/STAT in B cells, monocytes/ macrophages, eosinophils, basophils, and airway epithelial cells (30 -34). However, the mechanisms of Der f 2-induced signal transduction and the role of PLD in mediating IL-13 production in human bronchial epithelial cells are not well defined.
In the present study, we investigated Der f 2-induced activation of PLD and its regulation by the PLC␥/PKC␣/p38 MAPK pathway. This novel mechanism regulated ATF-2-mediated expression and production of IL-13. To the best of our knowledge, this is the first report to show that PLD1 is an important regulator in the production of IL-13 through a Der f 2-stimulated PLC␥/PKC␣/p38 MAPK pathway in BEAS-2B cells.

EXPERIMENTAL PROCEDURES
Cell Culture-BEAS-2B, a human airway epithelial cell line transformed with adenovirus 12-SV40 virus hybrid, was pur-chased from the American Type Culture Collection. BEAS-2B cells were cultured in Dulbecco's modified Eagle's medium/F12 with 10% fetal bovine serum, 100 units/ml penicillin, and 100 ng/ml streptomycin (Invitrogen) at 37°C with 5% CO 2 in humidified air.
Preparation of Cytosolic and Membrane Fraction-Serumstarved cells were incubated with Der f 2 (10 g/ml) for 1 or 3 min, scraped in ice-cold phosphate-buffered saline, and harvested by microcentrifugation. The cells were then resuspended in a buffer solution (50 mM NaCl, 1 mM MgCl 2 , 2 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, 50 mM HEPES, pH 7.5), disrupted by sonication, and centrifuged at 100,000 ϫ g for 1 h. The supernatant was used as the cytosolic fraction, and the pellet (membrane fraction) was resuspended in lysis buffer containing 1% (v/v) Triton X-100.
Determination of PLD Activity-PLD activity was determined by the formation of PBt, the product of PLD-mediated transphosphatidylation, in the presence of butanol as described previously with a slight modification (35). The cells, which had been incubated for the indicated times in 6-well plates, were radioactively labeled with 2 Ci/ml of [ 3 H]palmitic acid in serum-free medium for 20 h. The cells were then pretreated with 0.3% (v/v) 1-butanol for 15 min before stimulation with Der f 2. Also, the cells were preincubated with U73122, PAO, RO320432, GO6976, SB203580, or SB202190 for 30 min after being labeled with [ 3 H]palmitic acid and serum-starved for 20 h. After treatment with Der f 2 for 30 min, the cells were quickly washed with ice-cold phosphate-buffered saline and suspended in ice-cold methanol. Lipids were extracted according to the method of Bligh and Dyer (36), and PBt was separated by TLC using a acetate/isooctane/acetic acid/water (110:50:20: 100,v/v) solvent system. The regions corresponding to the authentic PBt bands were identified with 0.002% (w/v) primulin in 80% (v/v) acetone, scraped, and counted using a liquid scintillation counter.
ELISA-The cell supernants were collected after Der f 2 treatment for the indicated times, and the levels of IL-13 were quantified by ELISA. The detection limit was 30 pg/ml. Plates were read on a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA) and analyzed with SOFTmax analysis software (Molecular Devices). The means of triplicate ELISA values for each of the dose relationships among PLD1, ATF-2 knockdown, and IL-13 protein expression were calculated by linear regression.
Small Interfering RNAs-The single siRNA for PLD1 was purchased from Dharmacon Research Inc. (Lafayette, IN), and the secondary siRNA oligonucleotide, PLD1 (5Ј-AAGGUGG-GACGACAAUGAGCA-3Ј), was synthesized by Ambion (Austin, TX). Two kinds of ATF-2 oligonucleotide siRNAs were purchased from Qiagen (Cambridge, MA) and Santa Cruz Biotechnology. Oligonucleotide siRNAs were transfected using the siPORT TM NeoFX (Ambion) reagent according to the manufacturer's instructions. At the indicated intervals following transfection, cell lysates were assayed for gene silencing effects by RT-PCR or Western blotting.
Immunocytochemistry-For immunocytochemical tests, BEAS-2B cells were plated on a glass coverslip. Cells were fixed with 4% (w/v) paraformaldehyde, 0.15% (w/v) picric acid in phosphate-buffered saline and then incubated overnight with primary antibody at 4°C. The antigen fluorescent protein (GFP) monoclonal antibody was used at 1:400 (Roche Molecular Biochemicals). For detection of the primary antibody, fluo-

FIGURE 1. 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 Ci/ml [ 3 H]palmitic acid and then stimulated with Der f 2 (10 g/ml) for the indicated times. PLD activities were then determined by estimating the amount of [ 3 H]PBt in the presence of 1-butanol. Results are means Ϯ S.D. from three independent experiments. B, cells treated with Der f 2 (10 g/ml) for the indicated times were lysed. Each fraction, containing 30 g 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. JULY 24, 2009 • VOLUME 284 • NUMBER 30

JOURNAL OF BIOLOGICAL CHEMISTRY 20101
rescently labeled (Cy3) secondary antibodies (Jackson Immu-noResearch Laboratories, West Grove, PA) were used according to the specifications of the manufacturer. Cells were mounted in Vectashield with 4,6-diamidino-2-phenylindole (mounting medium for fluorescence analysis; Vector Laboratories, Burlingame, CA) and photographed using a fluorescent microscope (Nikon).
Statistical Analysis-All experiments were performed at least three to five times, and data analyzed using one-way ANOVA were considered to be significantly different at p Ͻ 0.05.

RESULTS
Effect of Der f 2 on PLD Activity in Human Bronchial Epithelial Cells-To examine the role of PLD in human bronchial epithelial cells (BEAS-2B), we first measured PLD activity after treating the cells with Der f 2. As shown in Fig. 1A, when the cells were treated with Der f 2, PLD activity was increased by nearly 1.9-fold in 30 min. Subsequently, to examine whether the increase of PLD activity was due to increased de novo PLD synthesis, we quantitated the PLD expression by Western blot analysis after treatment of BEAS-2B cells with Der f 2. As shown in Fig. 1B, PLD1 and PLD2 protein expression levels did not change within 1 h, indicating that up-regulation of PLD activity within 1 h was not due to an increased PLD protein level but rather most likely to the presence of a PLD activation pathway.
PLD1 Regulates Der f 2-induced IL-13 Expression and Production-Many studies have revealed that allergens induce various types of cytokine expression in many mammalian cell lines (37,38). To determine whether Der f 2 could enhance IL-13 levels, BEAS-2B cells were cultured with Der f 2 for the indicated times, and the IL-13 production and expression were then examined by ELISA, RT-PCR, and real-time PCR. Der f 2induced IL-13 production and its expression reached a maximum within 30 min of the treatment (Fig. 2, A and B).
As PLD stimulates cytokine expression in mammalian cells (39,40), we investigated which isozymes of PLD in BEAS-2B cells are involved in IL-13 expression when induced by Der f 2. Thus, the cells were transfected with wild types and catalytically inactive mutants of PLD1 or PLD2 plasmid constructs. Two days after transfection, cells were treated with Der f 2 for 15 min. As shown in Fig. 2, C and D, PLD1 overexpression potentiated IL-13 expression increased by Der f 2, whereas knockdown of PLD1 using DN-PLD1 transfection completely abolished IL-13 expression induced by Der f 2 but not PLD2, indicating that PLD1 is involved in Der f 2-induced IL-13 expression. To confirm the role of PLD1 in Der f 2-induced production and expression of IL-13, we examined the effects of PLD1 siRNA transfection in BEAS-2B cells. Scrambled siRNA was transfected as a control. Two day after transfection of the cells with PLD1 siRNA or scrambled siRNA, cells were treated with Der f 2 for 15 min, and the IL-13 and PLD1 expressions were then examined by RT-PCR and real time PCR. As shown in Fig. 2, E and F, PLD1 siRNA completely abolished both Der f 2-induced IL-13 and PLD1 expression and IL-13 production. These data suggest that PLD1 activation is important for Der f 2-induced expression and production of IL-13 in BEAS-2B cells.
Der f 2-induced IL-13 Production and Expression Are Regulated by the PLC␥/PKC␣/p38 MAPK Pathway in BEAS-2B Cells-The activation of PLC is involved in allergic airway-related signal transduction (41). To investigate how Der f 2 activates PLD1, we examined PLC␥ signaling because there are studies suggesting that PLC␥ activation results in increased PLD activity (42). The mammalian PLC␥ family has two closely related members, PLC␥1 and PLC␥2. PLC␥1 is ubiquitously expressed, whereas PLC␥2 expression is limited to certain immune cell types (43). PLC␥1 phosphorylation leading to its activation is a key step in intracellular distal signal transduction. Fig. 3A shows that PLC␥1 phosphorylation induced by Der f 2 resulted in translocation of PLC␥1 from cytosol to membrane. To determine whether PLC␥1 affects Der f 2-induced PLD activation, cells were pretreated with a specific PLC␥ inhibitor, U73122 or PAO, and the effects on Der f 2-induced PLD activation and IL-13 production were examined. As shown in Fig.  3B, pretreatment with U73122 or PAO for 30 min completely inhibited Der f 2-induced PLD activation. Furthermore, when the activity of PLC␥ was blocked, a remarkable inhibition of IL-13 production was observed (Fig. 3C), indicating that PLC␥1 activation is located upstream of PLD1 and is important for Der f 2-induced IL-13 production via PLD activation in BEAS-2B cells.
We had reported earlier that Der p1 and Der f 2 can stimulate PLD activity and that PKC activation is involved in this stimulation (1,13). To assess which PKC isozymes are involved in Der f 2-induced IL-13 production, we examined the translocation of PKC isozymes from cytosol to membrane. As shown in Fig. 4A, translocation of only PKC␣ from cytosol to membrane was found when BEAS-2B cells were treated with Der f 2 for 1 min. Other PKC isozymes such as PKC␤, PKC␥, and PKC were not responsive to Der f 2 in BEAS-2B cells. To determine

Phospholipase D1 Controls Der f 2-induced IL-13 Production
whether PKC␣ translocation is related to the PLC␥ translocation, we inhibited both PKC␣ and PLC␥ with their specific inhibitors U73122/PAO and RO320432/GO6976, respectively. As shown in Fig. 4B, specific PLC inhibitors U73122 and PAO inhibited PKC␣ translocation stimulated by Der f 2 (upper panel), whereas PKC inhibitors RO320432 and GO6976 could not inhibit PLC␥ translocation (lower panel). This result showed that PLC␥ is located upstream of PKC␣ in Der f 2-stimulated BEAS-2B cells. To further elucidate the role of PKC␣ in Der f 2 induced IL-13 production via PLD1 activation, cells were pretreated with RO324032 or GO6976, and the effects of PKC inhibition on Der f 2-induced PLD activation and IL-13 production were examined. The pretreatment of the cells with RO320432 or GO6976 for 30 min inhibited Der f 2-induced PLD activation (Fig. 4C). Furthermore, when the activity of PKC␣ was blocked, a remarkable inhibition of IL-13 production was observed (Fig. 4D). Taken together, these results suggest that PKC␣ is involved in Der f 2-induced PLD activation and is important for Der f 2-induced IL-13 production via PLD1 activation in BEAS-2B cells.
One of the targets of Der f 2 is MAPK (44,45). We also confirmed that Der f 2 stimulates the phosphorylation of MAPKs. A time course analysis showed that Der f 2 induced rapid phosphorylation of p38 MAPK, reaching a maximum in 5 min, whereas the phosphorylation of SAPK/JNK and ERK did not change within 60 min (Fig. 5A). To determine whether p38 MAPK phosphorylation has any influence on Der f 2-induced PLD activation, p38 MAPK inhibitors SB203580 and SB202190 were used to block the activation of p38 MAPK. When the cells were pretreated with SB203580 or SB202190 for 30 min, Der f 2-induced PLD1 activation was abolished (Fig. 5B). Furthermore, Der f-2-induced IL-13 production was decreased by SB203580 and SB202190 (Fig.  5C). These results indicate that p38 MAPK participates in Der f 2-stimulated PLD activation and IL-13 production in BEAS-2B cells. Next, we studied whether the PLC␥/PKC␣ pathway is related to Der f-2-stimulated phosphorylation of p38 MAPK. As shown in Fig. 5D, phosphorylation of p38 MAPK induced by Der f 2 treatment was inhibited by U73122, PAO, RO320432, and GO6976, respectively. Furthermore, when treated with U73122, PAO, RO320432, GO6976, SB203580, and SB202190, respectively, IL-13 expression in the cells was significantly reduced compared with the Der f 2-treated control (Fig. 5E). These results indicate that the PLC␥/PKC␣/p38MAPK/PLD1 pathway is critical for Der f 2-induced IL-13 production and expression.

Activation of ATF-2 Is Induced by PLD1 Activation via PLC␥/ PKC␣/p38 MAPK Pathway in Der f 2-treated BEAS-2B Cells-
Activation of ATF-2 is known to regulate the production of cytokines in mammalian cell lines as a transcription factor (46,47). A time course analysis showed that Der f 2-induced phosphorylation of ATF-2, reaching a maximum in 5 min (Fig. 6A,  upper panel). When the cells were pretreated with U73122, PAO, RO320432, GO6976, SB203580, or SB202190 for 30 min, Der f 2-induced ATF-2 activation was blocked (Fig. 6A, lower  panel), indicating that the PLC␥/PKC␣/p38 MAPK pathway is involved in Der f 2-induced ATF-2 activation in BEAS-2B cells. Also, we studied the effects of PLD1 on ATF-2 phosphorylation using PLD1 siRNA transfection in BEAS-2B cells. As shown in Fig. 6, B and C, PLD1 siRNA markedly decreased Der f 2-induced ATF-2 activation. To confirm the role of PLD1 in Der f 2induced ATF-2 phosphorylation, the cells were transfected with PLD1 and DN-PLD1, respectively. As shown in Fig. 6D, overexpression of PLD1 increased the phosphorylation of

. Effect of the PLC␥ inhibitors U73122 and PAO on Der f 2induced PLD activation and IL-13 production in BEAS-2B cells.
A, BEAS-2B cells were treated with Der f 2 (10 g/ml) for 1 min. Cells were harvested, and cell extracts were subjected to immunoblot analysis for active and total PLC␥1 (upper panel). Serum-starved cells were stimulated with Der f 2 (10 g/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 ϫ g, and each fraction containing 30 g of protein was analyzed by 10% SDS-PAGE and subsequently by Western blotting using anti-PLC ␥1 polyclonal antibody (lower panel). B, cells were labeled with 2 Ci/ml ATF-2 with no treatment of Der f 2, whereas DN-PLD1 transfection completely blocked Der f 2-induced ATF-2 phosphorylation. These results not only suggest that ATF-2 activation is associated with PLD1 activation through the PLC␥/PKC␣/p38 MAPK pathway triggered by Der f2 but also show the possibility that ATF-2 activation is able to regulate Der f 2-induced IL-13 expression and production in BEAS-2B cells. Furthermore, to confirm the functional role of PLD1 activation, we treated the cells with PA, which is one of the enzymatic products of PLD. When treated with the indicated concentrations of PA for 5 min, ATF-2 activation was increased (Fig. 6E, upper panel). This result shows the possibility that PA can increase IL-13 expression via ATF-2 activation. However, there are some stud-ies to indicate that ATF-2 activation is controlled directly by p38 MAP kinase activation (27,48). Therefore, we examined whether p38 MAP kinase is located downstream of PLD1 or not. As shown in Fig. 6E (lower panel), p38 inhibitors SB203580 and SB202190 could not block ATF-2 activation and IL-13 expression, respectively, induced by exogenously added PA. These results led us to suggest that p38 MAP kinase acts upstream of PLD activation and that PLD is located upstream of ATF-2 activation. PA, an end-product of PLD, is usually degraded to diacylglycerol by PPAP2A or to LPA/arachidonic acid by PLA2G1B. To determine whether PA itself regulates IL-13 expression via ATF-2 phosphorylation, we pretreated the cells with propranolol (a PPAP2A inhibitor) or mepacrine (a

Phospholipase D1 Controls Der f 2-induced IL-13 Production
PLA2G1B inhibitor) for 1 h prior to treatment with PA for 15 min. As shown in Fig. 6F, pretreatment with propranolol or mepacrine for 1 h had no effect on IL-13 expression. Moreover, when the cells were treated with LPA for the indicated time (Fig. 6G), IL-13 expression was not increased by LPA. These results suggest that PA induces IL-13 expression directly through ATF-2 phosphorylation.
Knockdown of ATF-2 Inhibits Der f 2-induced IL-13 Gene Expression and Production-Finally, we investigated the effect of ATF-2 activation on IL-13 expression and production in Der f 2-treated BEAS-2B cells. As shown in Fig. 7, knockdown of ATF-2 using ATF-2 siRNAs transfection decreased Der f 2induced IL-13 expression and production. This result indicates that gene expression and production of IL-13 are regulated by ATF-2 transcriptional activity in BEAS-2B cells.

DISCUSSION
HDMs Der f and Der p induce expression and production of inflammatory cytokines and also participate in various signaling pathway. The regulation of HDM-induced cytokine production is a key step in immunity-related diseases. Recently, immunotherapy using mite allergen has been tried for the treatment of atopic dermatitis (49). HDMs induce the production of IL-4 and IL-13 in mite-sensitive asthmatic basophils and stimulate IL-8 release in epithelial cells (45,50,51). However, Der f 2-induced intracellular signaling pathways leading to cytokine expression are still far from clearly understood. In this study, we have provided new insight into the PLD1-regulated signaling pathway for Der f 2-induced IL-13 expression and production. In the present study, Der f 2 was found to increase PLD activity maximally at 30 min, whereas expressions of PLD1 and -2 were not changed within the indicated time (Fig. 1), indicating that activation of PLD is caused by Der f 2-regulated signaling molecules. We also found that Der f 2 increased both expression and production of IL-13. The production of IL-13 can be triggered by antigen in sensitized hosts as well as by other cytokines under certain circumstances (29). In human bronchial epithelial cells, overexpression of PLD1 or PLD2 potentiates S1P-induced cytokine production, and blockades of PLD activity inhibit cytokine production (40). These studies suggest a possible functional role for PLD in cytokine production and expression. Therefore, to investigate the role of PLD isoforms in Der f 2-induced expression and production of IL-13, we transiently overexpressed PLD1, DN-PLD1, PLD2, and DN-PLD2 in BEAS-2B cells with EGFP plasmid vector prior to Der f 2 stimulation (Fig. 2) and found that PLD1 overexpression increased expression of IL-13 induced by Der f 2. On the other hand, overexpression of PLD2 rather down-regulated IL-13 expression induced by Der f 2, and overexpression of DN-PLD2 also decreased IL-13 expression induced by Der f 2. In the present study, therefore, we have focused on the role of PLD1 in IL-13 expression induced by Der f 2. Moreover, we found that DN-PLD1 inhibited the expression of IL-13 induced by Der f 2, and PLD1 blockage by PLD1 siRNA also decreased production and expression of IL-13. These results strongly indicate that PLD1 plays an important role in Der f 2-induced expression and production of IL-13 in BEAS-2B cells.
Proinflammatory cytokine secretion and inflammatory mediator-induced cytokine expression are dependent on PLC in human epithelial and synovial fibroblasts cell, suggesting that activation of PLC is required in signal transduction events, leading to inflammatory response (15,52). Several studies suggest that PLC␥1 regulates PLD activity in intracellular signal transduction (42,53,54). Thus, we tried to determine whether PLC␥ signaling pathway is involved in Der f 2-induced PLD activation and found that pretreatment with U73122 or PAO, specific PLC␥ inhibitors, completely blocked Der f 2-induced PLD1 activation and IL-13 production in BEAS-2B cells, suggesting that PLC␥ activation is related to PLD activation and IL-13 production.
There are many evidences to indicate that PKC is a major mediator in activating PLD in response to various agonists (19). Moreover, many studies have reported the relationship between PLD and PKC isoforms in a variety of cell types (55)(56)(57)(58)(59), suggesting that small molecular weight G proteins such as ARF (ADP-ribosylation factor) and RhoA are involved in agonist-induced PLD activation in several cell types (36); it is not yet clear whether these small molecular weight G proteins may be the regulator of Der f 2-induced PLD activation. As we demonstrated earlier that Der f 2 activates PLD through PKC activation (1,13), in this study we focused on the PKC-dependent pathway related to Der f 2-induced PLD activation. Among PKC isoforms, translocation of only PKC␣ occurred from cytosol to membrane by treatment with Der f 2, and inhibition of PKC␣ with RO320432 and GO6976 blocked Der f 2 -induced PLD activation and IL-13 production. Therefore, we suggest that Der f 2-induced PLD activation is mediated by PKC␣ in BEAS-2B cells. Also, we have shown that Der f 2-induced PKC␣ translocation in BEAS-2B cells is dependent on PLC ␥1. Although translocation of PLC␥1 to the membrane was not inhibited by PKC inhibitors such as RO320432 and GO6976, PKC␣ translocation was significantly inhibited by PLC␥ inhibitors U73122 and PAO. These results demonstrate that PLC␥ acts as an upstream component of Der f 2-induced PKC␣ activation in BEAS-2B cells.
Cross-talk between PKC and MAPKs by agonists has recently been reported in various cell systems (60 -62). Specifically, many groups of researchers have demonstrated that the p38 signaling pathway possibly controls inflammatory responses. p38 MAPK plays a role in transducing the mitogenic signal in T-cells in response to IL-2 and IL-7 (63). P38 MAPK also mediates the production of IL-10 and IL-1␤ in lipopolysaccharide-stimulated monocytes, and it regulates IL-5 synthesis and tumor necrosis factor-␣ mRNA stability in human T-cells (61). However, these authors did not examine the relationship between p38 MAPK and the PLC␥/PKC␣ pathway, which regulates PLD activation in immune response. In the present study, we found that Der f 2 stimulated the activation of p38 MAPK but not SAPK/JNK or ERK1/2. The failure of Der f 2 to activate ERK1/2 and SAPK/JNK indicates that Der f 2 specifically activates p38 MAPK. Our observation led us to assume that Der f 2-induced p38 MAPK activation is also involved in PLD activation. As expected, the p38 MAPK inhibitors SB203580 and SB202190 attenuated not only Der f 2-induced PLD activation but also Der f 2-induced IL-13 production, thus raising the possibility that p38 MAPK regulates PLD activation and IL-13 production induced by Der f 2 in BEAS-2B cells. Furthermore, we have shown that inhibition of PLC␥1 with U73122 and PAO, or of PKC␣ with RO320432 and GO6976, blocked Der f 2-induced p38 MAPK activation. Therefore, it is highly likely that Der f 2-induced p38 MAPK activation is dependent on the PLC␥/PKC␣ pathway in BEAS-2B cells. Next, we tested whether the PLC␥/PKC␣/p38 MAPK pathway was involved in Der f 2-induced IL-13 expression and found that U73122, PAO, RO320432, GO6976, SB203580, and SB202190 blocked the expression of IL-13, which was elicited by Der f 2, suggesting that the PLC␥/PKC␣/p38 MAPK pathway is critical for Der f 2-induced IL-13 expression. Such an observation appears to be similar to the PLC-dependent pathway in rLZ-induced cytokines in human T-cells (64).
What kind of transcription factor is involved in the expression of IL-13 via PLC␥, PKC␣, p38 MAPK, and PLD activation in BEAS-2B cells? Previous studies have indicated that PKC leads to ATF-2 activation and that PKC inhibitors completely block ATF-2 phosphorylation (47). Furthermore, activation of transcription factor ATF-2 in the p38 MAPK pathway for cytokine production has also been reported previously (20,46). The possibility that PLD might be related to ATF-2 phosphorylation has never been considered before. However, based on the present results, it does not seem to be far-fetched to suggest the possibility of the involvement of PLD in ATF-2 activation, which increases IL-13 expression.
In this study, we have proved that the phosphorylation of ATF-2 is required for Der f 2-induced expression and production of IL-13 through PKC␣, p38 MAPK, and PLD activation. First, pretreatment with U73122, PAO, RO320432, GO6976, SB203580, or SB202190 decreased the phosphorylation of ATF-2 induced by Der f 2. Second, PLD1 siRNAs and DN-PLD1 inhibited Der f 2-induced phosphorylation of ATF-2. Third, PA (the functional and enzymatic product of PLD) directly increased the phosphorylation of ATF-2 and IL-13 expression. Fourth, ATF-2 siRNAs reduced the Der f 2triggered elevation of expression and production of IL-13. However, Der f 2-induced IL-13 production was not completely reduced by ATF-2 siRNA, whereas ATF-2 siRNA treatment completely blocked IL-13 expression as well as the ATF-2 phosphorylation induced by Der f 2. This could be explained by the fact that ATF-2 is not the only transcription factor for IL-13 production in Der f 2-treated BEAS-2B cells, and it is quite possible that there are other hitherto unknown transcription factors.
In conclusion, the present study provides evidence that Der f 2-induced PLD activation is mediated by the PLC␥/ PKC␣/p38 MAPK pathway and that activation of PLD1 is critical for the expression and production of IL-13 through activation of ATF-2 by providing PA in BEAS-2B cells (Fig. 8). Further studies, such as the elucidation of molecular mechanisms involved between PLD1 and transcription factors in BEAS-2B cells, are expected to provide deeper insight into the understanding of Der f 2-induced PLD activation and cytokine production.