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DA Blake, TS Coston; Activated Integrins on Early Passage Human Corneal Endothelial Cells and a Virally Transduced Cell Line . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3187.
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Purpose: Integrins are plasma membrane heterodimeric receptors critical in mediating the processes of cell adhesion, migration, and proliferation. In many cell types integrin function is constitutively repressed and receptor activation requires cell stimulation. In this study, integrin expression and activation was studied in early passage human corneal endothelial cells and in a virally transduced cell line. Methods: Early passage cultures of human corneal endothelial (HCE) cells and the virally transduced cell line (E6/E7) were established as previously described (Blake et al (1997) IOVS 38:1119 and Wilson et al (1995) IOVS 35:32). The cell surface expression of individual integrin subunits was assessed using specific antibodies and flow cytometry. Integrin activation was determined by studying the cell adhesion to increasing concentrations of immobilized, purified extracellular matrix (ECM) components. Cell adhesion data was fit to the Hill equation. Results: The α2, α3, α5, and ß1 integrin subunits were expressed on early passage HCE cells; expression of α4, αv, and ß3 subunits was below the limit of detection. E6/E7 cells showed a similar patterns of integrin expression except for α4, which was detectable at low levels on E6/E7 cells. Early passage HCE cells showed a strong apparent positive cooperativity in binding to immobilized fibronectin (FN) and a recombinant fragment of FN that contains the RDG domain. E6/E7 cells showed this apparent positive cooperativity in their interactions with FN, the FN fragment, and type I collagen. Conclusion: The highly cooperative binding to some, but not other immobilized ECM components suggests that binding of a few activated cell surface receptors to the immobilized substratum increases the chances that additional cell surface receptors will be engaged. Analysis of the nature of these binding interactions can be used to determine the extent of integrin activation on cultured cells.
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