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Association of Protein Tyrosine Phosphatases (PTPs)-1B with c-Met Receptor and Modulation of Corneal Epithelial Wound Healing

From the Department of Ophthalmology and Neuroscience Center of Excellence, Louisiana State University Health Sciences Center, New Orleans, Louisiana.

PURPOSE. The purpose of this study was to investigate the expression and activity of protein tyrosine phosphatases (PTPs) in epithelium during corneal wound healing and to investigate how PTPs regulate activation of the c-Met receptor and the receptor’s proximal signaling.

METHODS. Rabbit corneas were injured by gentle scraping of the surface, leaving the limbal epithelium intact, and epithelium was collected at 1, 2, 3, and 7 days after injury. In organ culture models, epithelium was removed and corneas were incubated with hepatocyte growth factor (HGF), with or without the PTP inhibitor bpV(phen), and the PI-3K inhibitors wortmannin and LY294002. Human corneal epithelial (HCE) cells were stimulated with HGF with or without bpV(phen). Total cell lysates and cytosolic and membrane fractions were analyzed by Western blot. PTP activities were measured with specific substrates. PTP1B and SHP-2 genes were knocked down by interference RNA (siRNA).

RESULTS. PTP activity and expression increased during wound healing. The most abundant were SHP-2, PTP1B, and PTEN. HGF activated the c-Met receptor in HCE cells up to 30 minutes and was downregulated by 2 hours. Inhibition of PTPs increased HGF-promoted wound healing, HGF-activated phosphorylation of c-Met, and its downstream signal PI-3K/Akt, but not ERK1/2 or p70S6K. PTP1B and SHP-2 were bound to the c-Met. Part of the c-Met was colocalized in the endoplasmic reticulum with PTP1B. PTP1B phosphorylation increased when the c-Met receptor was deactivated, and gene knockdown of PTP1B increased c-Met activation. SHP-2 phosphorylation and binding to c-Met was higher during receptor activation, and SHP-2 gene silencing decreased receptor phosphorylation.

CONCLUSIONS. Inhibition of PTP activity mimics the effect of HGF by activating the PI-3K/Akt signal involved in wound healing. PTP1B and SHP-2 are bound to the c-Met receptor to control its activity. Although the binding of PTP1B increases when there is a decrease in c-Met activation and acts as a negative regulator of the receptor, the increased binding and phosphorylation of SHP-2 coincide with maximal stimulation of c-Met, acting as a positive regulator.