Abstract
Purpose :
Histatin (Hst) peptides are a family of multi-functional endogenous proteins that have been shown to have broad anti-microbial activity, immune modulation activity, and the ability to promote wound healing. Studies in other model systems have demonstrated that the wound healing function of Hst1 is dependent upon ERK activation. Moreover, corneal epithelial wound healing appears to depend, at least in part on ERK activation. In the past, it had been thought that Hst5 was a primarily anti-fungal member of this family, but recent reports suggest that Hst5 can promote wound healing. This encouraged us to determine whether Hst5 induced wound healing was dependent upon ERK activation in corneal epithelia.
Methods :
Human corneal epithelial cells (HCE) were cultured in a 24 well plate. A standard scratch assay was performed. The wounded epithelial sheets were then treated with Hst5, PD98059, an inhibitor of ERK1/2, in MEM media with reduced serum conditions (0.5% FBS). Scratches were photographed microscopically at 4x magnification every hour. The wound areas were measured using ImageJ. Relative wound closure was calculated by dividing the closure of the treated wound by that of the untreated control wound. Western blotting (WB) was performed following standard methods using primary antibody against phospho (p)ERK1/2 or total (t)Erk1/2and secondary antibody. Immunofluorescence imaging was performed in 8-well chamber slides. After formation of a confluent monolayer a standard scratch was performed. Wounded areas were then treated with Hst5 or control. Cells were subsequently incubated with primary antibodies against p-ERK1/2 and then appropriate secondary antibody.
Results :
We found that wound healing promotion by Hst5 was sensitive to ERK inhibition in standard scratch assays and that level of activation of ERK increased with Hst5 compared with control as seen in WB and immunolocalization.
Conclusions :
The results of this experiment suggest that Hst5 induced wound healing in corneal epithelia is associated with increases in cellular ERK activation, as determined by WB and immunolocalization. Moreover, these results indicate that ERK inhibition could abrogate Hst5 mediated effects in corneal epithelia. Future studies to determine the cellular receptors and other important pathways could help to explain the interesting and translationally applicable effects of histatin peptides in corneal epithelia and other tissues
This is a 2020 ARVO Annual Meeting abstract.