Abstract
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 Cgamma (PLCgamma)/protein kinase Calpha (PKCalpha)/p38 MAPK pathway. Der f 2-induced PLD activation was attenuated by PLCgamma inhibitors (U73122 and PAO), PKCalpha inhibitors (RO320432 and GO6976), and p38 MAPK inhibitors (SB203580 and SB202190). These results indicate that PLCgamma, PKCalpha, 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 PLCgamma, PKCalpha, or p38 MAPK, indicating that IL-13 expression and production are related to a PLCgamma/PKCalpha/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.
Highlights
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
To determine whether Dermatophagoides farinae (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 examined by ELISA, RT-PCR, and real-time PCR
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 phospholipase C (PLC)␥/protein kinase C (PKC)␣/p38MAPK/phospholipase D1 (PLD1) pathway is critical for Der f 2- induced IL-13 production and expression
Summary
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. Mitogen-activated protein kinase; ERK, extracellular signal-regulated kinases; JNK, c-Jun N-terminal kinase; ATF-2, activating transcription factor-2; p-ATF-2, phosphorylated ATF-2; siRNA, small interfering RNA; EGFP, enhanced green fluorescent protein; PBt, phosphatidylbutanol; PA, phosphatidic acid; LPA, lysophosphatidic acid; PPAP2A, phosphatidic acid phosphatases type 2A; PLA2G1B, phospholipase A2, group 1B; RT, reverse transcription; ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; glyceraldehyde-3-phosphate dehydrogenase; DN, double negative; SAPK, stress-activated protein kinase; QIL, quantitative interleukin. Phospholipase D1 Controls Der f 2-induced IL-13 Production trisphosphates (IP3) and diacylglycerol [16, 17] These second messengers are responsible for the activation of protein kinase C (PKC) [18]. 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
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