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Adna Halilovic, Daohong Lin, Gregory Joseph, Brian Shkolnik, Michal Schwartzman; HO-2 knockdown delays wound healing in Human Corneal Epithelial (HCE) cells by altering the signaling of EGFR and FAK mediated pathway. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2572.
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Heme oxygenase (HO) represents an intrinsic cytoprotective and anti-inflammatory system. Inhibition of HO activity significantly impairs wound healing in human corneal epithelial (HCE) cells. We have shown that HO-2 knockdown in vitro attenuates corneal wound healing and that HO-2 null mice display an aberrant corneal wound healing following injury. In this study, we investigated the mechanisms that may contribute to HO-2 cytoprotective role in the corneal epithelium.
HCE cells were stably transfected with lentivirus HO-2 shRNA or non-target shRNA plasmids. Modified Boyden Chamber assay was used to study the migration of HO-2 knockdown cells. Flow cytometry cell cycle analysis was done using propidium iodide staining. Western blot was performed to evaluate the expression of proteins involved in migration and proliferation in HO-2 shRNA and non-target shRNA cells.
HO-2 knockdown, using virus-mediated shRNA, impaired HCE wound healing by 35% and 25% at 24h and 48h after injury, respectively. Furthermore, HO-2 knockdown inhibited basal and EGF(10ng/mL)-stimulated HCE migration by 50% and 40%, respectively and was associated with a 35% decrease in p-Akt and 45% decrease in p-EGFR levels. Flow cytometric analysis showed that EGF-stimulated cell cycle progression was attenuated in HO-2 knockdown cells. Immunofluorescence of HO-2shRNA and control transfected cells revealed a cytoplasmic distribution of p-FAK. Upon injury, control cells displayed typical p-FAK as focal adhesion points in the membrane, whereas in HO-2 knockdown cells p-FAK had no focal distribution.
Knockdown of HO-2 results in attenuated corneal epithelial wound healing. Deficiency of HO-2 impairs wound healing by a mechanism that involves alterations in ERK, Akt and FAK signaling pathways, all of which have been shown to play an important role in growth factor-mediated cell migration and proliferation following injury.
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