The corneal endothelium is responsible for maintaining corneal clarity, but these cells do not divide throughout life. Our long-term goal is to develop methods to stimulate proliferation and increase cell density in individuals at risk for vision loss due to low endothelial cell counts. Human corneal endothelial cells CAN divide in organ culture, suggesting that multiple factors together exert a strong anti-proliferative pressure in vivo. Contact-dependent inhibition of proliferation appears to be an important anti-proliferative factor, which is regulated, in part, by phosphotyrosine phosphatases (PTPs). Activation of receptor tyrosine kinases, such as the EGF receptor, leads to tyrosine phosphorylation (Tyr-PO4)-induced cell cycle entry. Cell-cell contacts can be disrupted by Tyr-PO4 of cadherin-associated proteins. PTPs inhibit both Tyr-PO4 events, suggesting that PTP activity helps maintain contact-dependent inhibition of proliferation. Release of cell-cell contacts in confluent endothelium does NOT stimulate cell cycle entry in the absence of mitogens. Sodium orthovanadate (SOV), a PTP inhibitor, releases cell contacts and promotes cell cycle entry without mitogens, suggesting that inhibition of PTP activity might augment the proliferative effect of mitogens, such as EGF, in confluent endothelium. We hypothesize that, by down-regulating the protein expression or inhibiting the activity of specific PTPs in the endothelium, we will be able to make the cells more responsive to mitogenic stimulation, thus providing a method to increase cell density in individuals at risk for vision loss due to low endothelial cell counts.
Specific Aims proposed to test this hypothesis include: I. Identify specific PTPs expressed in corneal endothelial cells; II. Determine the importance of each PTP in regulating EGF-driven mitogenic signaling; III. Determine whether the cadherin-catenin complex is a target for PTP regulation in corneal endothelium; and IV. Test if down-regulating the expression or inhibiting the activity of specific PTPs will promote proliferation and increase cell density in human corneal endothelial cells in situ. Immunoprecipitation, Western blotting, RT-PCR, immunocytochemistry, flow cytometry, antisense methods, and expression of dominant-negative mutants will be used to accomplish the objectives of this proposal.
Showing the most recent 10 out of 20 publications
