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Vivian Lee, Christine Marshall, Xiaoping Yang, Michael D Gober, Tzvete Dentchev, John T Seykora; Role of Src-activating and signaling molecule (Srcasm) and Src-family tyrosine kinases (SFK) in Corneal Epithelial Wound Healing. Invest. Ophthalmol. Vis. Sci. 2014;55(13):490.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies suggest activation of epithelial growth factor receptor (EGFR) and SFK may play a role in corneal epithelial wound healing. In skin keratinocytes, Srcasm has been shown to regulate EGFR and SFK signaling. Srcasm is a SFK substrate downstream of EGFR that can preferentially activate Fyn and Src. To further understand the role of Srcasm in the EGFR/SFK pathway in corneal epithelial cells, wound healing migration assays and Western blot analysis were performed using mouse corneal epithelial cultures (MCEC) derived from Srcasm and Fyn null mice.
Monolayers from primary MCEC were established and subsequently wounded from: 1) Srcasm wildtype (WT); 2) Srcasm heterozygote (Srcasm +/-); 3) Srcasm knockout (Srcasm -/-); and 4) Fyn knockout (Fyn -/-) mice. Wound migration studies were performed and analyzed using a live cell imaging system and Slidebook software. The above MCECs were also wounded for Western blot analysis to evaluate levels of activated EGFR and SFKs and phosphorylated Srcasm.
Wound migration studies showed increased migration of corneal epithelial cells established from Srcasm -/- mice and decreased migration of cells from Fyn -/- mice compared to WT. The results from the wound migration studies correlated with increased levels of activated EGFR and SFKs in Srcasm -/- MCEC compared to WT MCEC, and decreased levels of activated EGFR and SFKs in Fyn -/- MCEC.
Wounded corneal epithelial cells demonstrate enhanced migration activity with loss of Srcasm, paralleling the increased activation of EGFR and SFK seen on Western blot analysis. Attenuated migration activity, on the other hand, is observed with knockout of Fyn, correlating with decreased EGFR and SFK activation. Results from this study suggest a critical role for EGFR and SFK activation in corneal epithelial wound healing with Srcasm potentiating EGF-dependent signaling through activation of SFK. Studies to determine how Srcasm regulates activation of EGFR and SFK in corneal epithelial cells should be conducted.
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