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DL Harris, NC Joyce; Phosphotyrosine Phosphatases (PTPs) and Contact-dependent Inhibition of Proliferation in Rat Corneal Endothelium . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3180.
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Purpose: To investigate the role of PTPs in maintaining cell-cell contacts and preventing receptor tyrosine kinase (RTK)-induced proliferation in cultured rat corneal endothelium. Methods: Cells were grown to confluence and then incubated under one of the following conditions: 1) + 10% FBS; 2) - FBS; 3) 50 uM sodium orthovanadate (SOV, a general PTP inhibitor) alone; and 4) pre-incubated for 30 min in 5 mM EGTA (a calcium chelator) and then incubated - FBS. Cultures were fixed, prepared for immunolocalization (ICC) of ZO-1 or ß-catenin at 24 hrs to compare junctional integrity or Ki67 at 48 hrs to compare induction of cell cycle entry, and then mounted in medium containing propidium iodide to visualize nuclei. To determine the effect of the RTK, epidermal growth factor (EGF), and SOV on net tyrosine phosphorylation (Tyr-PO4), confluent cultures were treated for 24 hrs with 10 ng/ml EGF alone, 50 uM SOV alone, or EGF + SOV. Total protein was extracted, electrophoresed, and Western blots were prepared using anti-phosphotyrosine as the probe. Results: Monolayer integrity was maintained in the presence and absence of FBS, indicated by intense staining for ZO-1 at cell borders and by membrane-associated localization of ß-catenin. SOV and EGTA caused visible breaks in the endothelial monolayer and loss of membrane-associated ZO-1 and ß-catenin staining. Cultures maintained in the absence of serum or treated with EGTA alone showed no positive Ki67 staining. Cells incubated in SOV alone showed positive Ki67 staining. In Western blots, EGF and SOV alone showed several positive bands, indicating moderate levels of net Tyr-PO4. In contrast, treatment of confluent cells with EGF + SOV yielded several intense bands, including EGF receptor, indicating high levels of Tyr-PO4. Conclusion: Calcium chelation or SOV-induced inhibition of PTP activity can dissociate junctional proteins from the plasma membrane and disrupt the confluent monolayer. SOV-induced inhibition of PTP activity can stimulate cell cycle entry without the addition of exogenous mitogen. Results using SOV alone suggest that there is a constitutive PTP activity that must prevent Tyr-PO4-induced loss of junctional integrity and promotion of cell cycle entry. The high levels of Tyr-PO4 when SOV and EGF were added together to confluent cells suggests that inhibiting PTP activity might enhance EGF-stimulation of cell cycle entry in confluent corneal endothelium.
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