Expression of IL-8 and IL-13 in Der f 2 activates phospholipase D in human bronchial epithelial cells with involvement of protein kinase C-α

초록

Phospholipase D (PLD) catalyses the hydrolysis of phosphatidylcholine (PC) and other membrane phospholipids to phosphatidic acid (PA) and choline. Two isoforms of PLD, PLD1 and PLD2, have been cloned and are being characterized for regulation and cellular function. We recently demonstrated that Der f 2 induces sensitively activation of PLD with involvement of protein kinase C-α in T cells from Der f specific atopic individuals. In airway/alveolar epithelial derived cell lines, PLD activation and IL-8 secretion enhancement are observed. However, the mechanism of the role of PLD in mediating IL-8 secretion in airway epithelial cells are not defined. The purpose of this study was to identify the role of PLD in Der f 2 induced IL-8 and IL-13 expression. The major house dust mite allergen, Der f 2, stimulates the PLD in human bronchial epithelial cell line (BEAS-2B). PLD activity was increased within 5 min after exposure of Der f 2. The well-known PLD activator PKC-α was found to be translocated to membrane from cytosol in Der f 2 treated BEAS-2B cells. To determine whether the effects of Der f 2 on PLD occurred as a consequence of PKC activation, BEAS-2B cells were pretreated for 30 min with PKC inhibitor (RO320432). RO320432 reduced the effects of Der f 2 induced PLD activation suggesting that PKC-α acts as upstream activator of PLD in Der f 2 treated BEAS-2B cells. Also, the tyrosine kinase inhibitor (Genistein) prevented PLD activation. Der f 2 enhanced IL-8 and IL-13 expressions in BEAS-2B cells. We found that the expressions of IL-8 and IL-13 were increased when PLDs were activated with Der f 2 in BEAS-2B cells. By blocking of PLD activity using 1-butanol, expressions of IL-8 and IL-13 were decreased. To confirm the role of PLD in IL-8 and IL-13 expression in BEAS-2B cells, we transfected the PLD1 and PLD2, and their dominant negative forms. Interestingly, we found that only PLD1, not PLD2, overexpressed IL-8 and IL-13. These results indicate that Der f 2 might activate PLD through an upstream tyrosine phosphorylation to elicit PKC-α activation which induces IL-8 and IL-13 expression in human bronchial epithelial cells.