Abstract
Purpose: :
Corneal epithelial injury induces the release of nucleotides leading to purinergic receptor activation and subsequent phosphorylation of specific sites on the EGFR. Src has been shown to play a role in purinergic signaling and the activation of EGFR. We hypothesized that Src was a necessary signaling molecule in nucleotide-mediated cell migration, and that the recruitment of downstream adaptor proteins to the EGFR after stimulation by injury and nucleotides differed from stimulation by EGF.
Methods: :
Assays were performed on human corneal limbal epithelial cells (HCLE) and rabbit primary corneal epithelial cells. Transwell and scratch wound migration assays were performed with and without the Src kinase inhibitor, SU6656. Cells treated with ATP or UTP were resuspended in media with or without SU6656 and allowed to migrate for 16 hours at 37ºC, and the number of migrated cells was determined. Immunoprecipitation and Western blot analysis were performed to determine the recruitment of adaptor molecules to EGFR in response to wounding and nucleotide treatment.
Results: :
When cells were incubated in the presence of a Src kinase inhibitor and stimulated with nucleotides, chemotactic migration was diminished by more than 50%. Immunoprecipitation of the EGFR demonstrated that PLCγ and Src are constitutively bound. Five minute stimulation with 5nM EGF, a concentration used to promote wound healing in vivo, recruited Shc and Grb2 to EGFR. In contrast, neither injury nor UTP elicited Shc and Grb2 binding. As 5nM is above physiological levels, the physiological concentration of 0.5nM EGF was tested, but did not cause the recruitment of Grb2. A one hour time point was examined to determine if recruitment of Grb2 would occur at a lower concentration. Again only 5nM EGF recruited Grb2 binding and it was greater than the five minute time point.
Conclusions: :
Src is necessary for nucleotide-induced cell migration as determined by the inhibition of chemotactic cell migration in the presence of Src kinase inhibitor. In human corneal limbal epithelial cells, Grb2 recruitment to EGFR is not elicited by wounding, nucleotides, or 0.5nM EGF. Injury and nucleotides induce purinergic receptor transactivation of the EGFR but this activation differs from that induced by the clinically used concentration of 5nM EGF.
Keywords: cornea: epithelium • wound healing • signal transduction