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A.H. Kakazu, G.D. Sharma, H.E. P. Bazan; Protein Tyrosine Phosphatase 1B (PTP1B) Is Activated During Corneal Epithelial Wound Healing and Modulates HGF Signaling . Invest. Ophthalmol. Vis. Sci. 2005;46(13):2140.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: During corneal epithelial wound healing, activation of the PI–3Kinase signaling by HGF is maintained for some time and then switched off, probably to avoid overactivation (Curr Eye Res, 18:168,1999) Protein tyrosine phosphatases (PTPs) are a family of enzymes that catalyze the de–phosphorylation of tyrosyl phosphorylated proteins. The purpose of this study was to investigate whether there are changes in the expression or activity of three non–transmembrane PTPs, PTP1B, 1C, and 1D, during corneal wound healing, and how they affect signal–transduction pathways stimulated by HGF. Methods: Total de–epithelialization was performed in New Zealand rabbits and epithelium was collected at 1, 2, 3, and 7 days after injury. In corneal organ–culture experiments, the epithelium was removed from a 7–mm–diameter central area and corneas were incubated with HGF for 1, 2, or 3 days. In additional experiments PTP inhibitors were added and the wound was monitored. Corneal epithelial cells in culture (CEC) were stimulated with HGF with or without the PTP inhibitor bpV(phen) (10 µM). Total cell lysate and cytosolic and membrane fractions were collected and proteins were analyzed by Western blot using specific antibodies. PTP 1C and 1D activity was measured in immunoprecipitates with p–NPP as substrate. For PTP1B a specific peptide substrate was used. Results: Expression of PTP1C and 1D was found mainly in the cytosolic fraction. PTP1B was mostly expressed in the membrane fraction. In vivo, 48 and 72 hours after injury there was increased PTP1B expression that returned to control levels at 72 hours. In organ culture, HGF stimulation of the injured corneas induced the expression of PTP1B in the membrane fraction of the epithelial cells at 24 and 48 hours after injury. No changes were noted with PTP1D or 1C.Stimulation of CEC with HGF for 5 min increased the activity of PTP1B by 25 %. There was no change in PTP1C or 1D activity under the same conditions. Blocking the activity of PTP1B with bpV(phen) increased the phosphorylation of the HGF receptor c–Met, the p85 subunit of PI3–K, and the downstream kinases Akt and p70S6K. The PTP inhibitor significantly increased the rate of epithelial wound healing in corneas in organ culture. Conclusions: PTP1B is the main PTP in corneal epithelium. Our results suggest that during HGF stimulation, the phosphatase could be complexed to the HGF receptor to control its activity. The PTP inhibitor bpV(phen) mimics the effect of HGF by activating the PI–3K signal involved in wound healing.
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