Abstract
Purpose::
We previously showed that flagellin via Toll-like receptor-5 induces activation of an array of signaling pathways including NF-ΚB and PI3K in human corneal epithelial cells (HCECs). The aim of this study was to investigate the role of phosphatidylinositol 3'-kinase (PI3K) activation during flagellin-induced inflammatory response in HCECs.
Methods::
HUCL cells,a telomerase-immortalized HCEC line, were challenged with flagellin (100ng/ml) purified from Pseudomonas (P.) aeruginosa (PAO1 strain) for different time points in the presence or absence of the inhibitors of PI3K, AKT, MAPKs (ERK1/2, p38 and JNK), as well as EGFR neutralizing antibody. IΚB-α phosphorylation and degradation, p38, JNK and ERK phosphorylation in HUCL cells were detected by Western blotting. Secretion of IL-6 and IL-8 were assessed by ELISA.
Results::
Flagellin elicited 2 phases of PI3K activation in HCECs: the first within 1 h post-stimulation (p.s.) and the second at 8 h p.s. and thereafter; whereas only single peak parallel to the first phase of PI3K activation was observed for ERK1/2, p38 and JNK. Inhibition of EGFR with neutralizing antibody blocked the first phase of PI3K activation, as well as ERK1/2 activation but exhibited no effect on NF-ΚB, p38 and JNK activation. Flagellin induced activation of porinflammatory signal pathways, NF-ΚB, p38 and JNK, but not ERK1/2, was greatly elevated by PI3K inhibitor wortmannin. Flagellin-induced IL-6 and IL-8 production was further elevated by PI3K or AKT inhibitors and suppressed by MAPKs (ERK1/2, p38 and JNK) inhibitors or EGFR neutralizing antibody.
Conclusions::
PI3K is activated in response to flagellin in a TLR5- and EGFR-dependent manner and modulates the inflammatory response of HCECs by negatively regulating NF-ΚB, JNK and p38 activation.
Keywords: inflammation • signal transduction • cornea: epithelium