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A. H. Kakazu, H. E. P. Bazan; Protein Tyrosine Phosphatase 1B (PTP1B) Modulates Cell Proliferation, Migration, and Survival Through c-Met in Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2951.
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© ARVO (1962-2015); The Authors (2016-present)
Hepatocyte growth factor (HGF) is an important paracrine growth factor released after injury that activates the c-Met receptor and promotes wound healing and cell survival in corneal epithelial cells (JBC 278:21989,2003; IOVS 45:3492,2004). We had previously reported that PTP1B binds to c-Met and inhibition of its activity affect downstream signaling pathways stimulated by HGF (ARVO2006-2007). Here, by specificly knocking down the PTP1B gene, we investigate how PTP1B regulates the phosphorylation of c-Met and affects cells proliferation, migration, and survival of human corneal epithelial cells.
Immortalized human corneal epithelial cells (HCEC) transfected with PTP1B siRNA were used in the experiments. Cells were stimulated with 20-40 ng/ml HGF without or with the PTPs inhibitor bpV(phen) (4 or 6 uM) and analyzed by western blot for PTP1B and phospho-c-Met (p-c-Met). Cell proliferation was evaluated by counting the cells after incubations for 48 h with HGF. For cell migration a linear scraping was made to the center of confluent cultures with a sterile pipet tip and migration was assayed after 14 h incubation in presence or not of 20 ng/ml HGF and 5 mM hydroxyurea. Apoptosis was induced by staurosporine (100-500 ng/ml) in the presence of HGF. Apoptotic cells were detected by Hoechst staining and DNA laddering after agarose gel electrophoresis.
HCEC transfected with PTP1B siRNA showed a 70 % decreased in PTP1B expression. Gene knock down significantly increased the phosphorylation of c-Met at Tyr 1234 and Tyr 1235 in cells stimulated with HGF and bpV(phen). Cell proliferation and cell migration also increased in cells depleted of PTP1B and stimulated with HGF. HCEC apoptosis induced by staurosporine was protected by HGF (18%), and this protection increased to 29 % when the gene was knocked down.
PTP1B decreases the phosphorylation of tyrosine residues of activated c-Met and as a consequence modulates the action of HGF on corneal epithelial cell proliferation, migration, and survival, demonstrating a key role of PTP1B in HGF-stimulated epithelial wound healing.
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