Calcium Signaling Induced by AsialoGM1 –Toll–Like Receptor 5 Interaction in Human Corneal Epithelial Cells

Abstract

Purpose: : Asialoganglioside gangliotetraosylceramide (asialoGM1) acts as coreceptors in association with different toll–like receptor subtypes (TLRs) for many pathogens, and together they have a vital role in inducing innate immunity. We determined whether calcium transients are a component of signaling pathways elicited by asialoGM1 ligation.

Methods: : The presence of asialoGM1 and TLR5 was evaluated in SV40–immortalized human corneal epithelial cells (HCECs) by Western blot and flow cytometry analyses. AsialoGM1–TLR5 interaction was assessed by coimmnoprecipitation. Calcium transients were measured with single cell fluorescence imaging of fura2–AM loaded serum–starved HCECs on the stage of an inverted microscope.

Results: : Based on coimmunoprecipitation data, asialo GM1 interacts with TLR5. Such an association promotes extracellular ATP release and increases extracellular Ca²⁺ influx through L–type voltage dependent channels. The dependence of asialoGM1 antibody induced increases in [Ca²⁺]_i on ATP release is evident since preincubation with either 10 U/mL apyrase, an ATP dephosphatase, or 100 µM ARL67156, an ectonucleotidase inhibitor,suppressed and potentiated these transients respectively. Such rises in Ca²⁺ influx occur through L–type Ca²⁺ channels since 1 µM nifedipine reduced them. Other evidence for L–type Ca²⁺ channel involvement is that membrane voltage hyperpolarization resulting from exposure to Cl^––free medium suppressed ATP induced Ca²⁺ transients. Such rises are in part dependent on stimulation of P2Y receptors since preincubation with either 100 µM suramin or 100 µM PPADS blocked these changes.

Conclusions: : AsialoGM1 is a co–receptor whose interaction with TLR5 appears to induce calcium signaling. This event is dependent on extracellular ATP release, which in turn induces calcium influx through the sequential activation of P2Y receptors and L–type Ca ²⁺channels. These events may be essential for inducing cellular responses required for the development of innate immunity.

Keywords: cornea: epithelium • signal transduction • calcium