Abstract
Purpose::
To determine in human corneal epithelial cells the role of asialoGM1 interaction with toll like receptor 5(TLR5) in mediating mitogen- activated protein kinases (MAPKs) superfamily and Nuclear Factor kappa B (NF-ΚB) activation.
Methods::
SV40 human corneal epithelial cells (HCEC) were exposed to the asialoGM1 agonist,anti-asialoGM1antibody, at different concentrations and times with or without inhibitors (Erk1/2 inhibitor U0126 or anti-TLR5 blocking antibody). Western blot analysis was applied to evaluate the expression levels of phospho-Erk1/2, phospho-p38, phospho -JNK and phospho-IΚB.
Results::
Anti-asialoGM1 antibody increased NF-ΚB activity in a time-dependent manner. This increase occurred within 5min of stimulation with anti-asialoGM1 (F=15.99, P< 0.05; Fisher LSD P< 0.05). The magnitude of increase ranged from 25% after 5 min of stimulation to 50% after 30 min of stimulation with the asialoGM1 antibody.Neither phosphorylated-p38 nor -JNK appeared to be significantly altered in response to stimulation with the anti-asialoGM1 antibody. On the other hand, asialoGM1 antibody caused a strong activation of Erk1/2 phosphorylation in a dose dependent manner at and above dilutions of 1:640 . When compared with the untreated control, anti-asialoGM1 antibody increased pErk1/2 maximally at a dilution of 1:80 (F=43.79, P< 0.05; Fisher LSD P< 0.05 for 1:320, 1:160 and 1:80 AGM1).Up-regulation of phospho-IΚB was completely inhibited by U0126. Similarly, up-regulation of phospho-Erk1/2 and phospho-IΚB was completely inhibited by anti-TLR5 antibody (F=46.70 P< 0.05; Fisher LSD, P< 0.05). Up-regulation of phospho-Erk1/2 mediated by asialoGM1 was not inhibited by blocking TLR2 activation with the anti-TLR2 antibody (F=135.8, P< 0.05; Fisher LSD, P=0.65).
Conclusions::
In HCEC, asialoGM1 is a coreceptor, which selectively interacts with TLR5. This interaction induces innate immunity through stimulation of a signaling pathway that involves the ERK MAPK branch and NF-ΚB.
Keywords: cornea: basic science • cornea: epithelium • signal transduction