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W. Chen, D.L. Harris, N.C. Joyce; Identification of Phosphotyrosine Phosphatase (PTP) Subtypes in Rat Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4333.
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Purpose: PTPs are a diverse group of enzymes that negatively regulate tyrosine phosphorylation of a variety of signaling molecules. Preliminary studies indicate that treatment of confluent cultures of rat corneal endothelial cells with sodium orthovanadate (SOV), a general PTP inhibitor, induces release of cell-cell contacts and cell cycle entry in the absence of serum, suggesting a role for PTPs in contact-dependent inhibition of proliferation. The current studies were conducted to identify specific PTPs expressed in rat corneal endothelium. Methods: Immunocytochemistry (ICC) and Western blots were tested for expression of SH-PTP1, SH-PTP2, PTP-1B, PTEN, and PTP-mu, in the endothelium of rat cornea and in cultured rat corneal endothelial cells. Established protocols were used for tissue procurement, culture, ICC, and Western blotting. Antibodies did NOT cross-react with other PTP subtypes. A431 cells acted as positive controls. For ICC, negative controls included primary antibody pre-incubated with specific competitive peptides (when available) or incubation with secondary antibody alone. Western blots verified protein expression using extracts of cultured endothelial cells. Results: SH-PTP1 and SH-PTP2 are non-transmembrane PTPs that help mediate the interaction of receptor tyrosine kinase (RTK) receptors. Endothelial cells in corneal sections and in culture stained positively for both proteins. In cultured cells and in A431 controls, SH-PTP1 and -2 stained more intensely in the nucleus than in the cytoplasm. PTP-1B negatively regulates RTK receptor signaling and also inhibits tyrosine phosphorylation of ß-catenin. Confluent endothelial cultures showed positive staining in the cytoplasm beneath the plasma membrane. Nuclei of endothelial cells stained positively for PTEN, a PTP that negatively regulates cell cycle progression induced via the PI3K/Akt pathway. No staining was observed in any negative controls. In some cell types, PTPmu interacts with the cytoplasmic domain of cadherin. No staining for PTPmu was observed in either frozen sections or cultured cells, although positive staining for this PTP was observed in A431 controls. Western blots confirmed the expression of SH-PTP1, SH-PTP2, PTP-1B, and PTEN in cultured cells. Conclusion: SH-PTP1, SH-PTP2, PTP-1B, and PTEN are among the PTPs expressed in rat corneal endothelial cells. PTPmu does not appear to be expressed. Studies are ongoing to identify specific binding proteins associated with each PTP and to determine whether these PTPs play a role in regulation of contact-dependent inhibition of proliferation in corneal endothelium.
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