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D.A. Blake, H. Yu, T.S. Coston; Adhesion to Fibronectin Changes Phosphorylation of Signal Transduction Proteins in Human Corneal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4735.
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Purpose: Previous studies from our laboratory (Blake et al. (1997) IOVS 38:1119) have shown that both extracellular matrix (ECM) and growth factors are required to stimulate the proliferation of human corneal endothelial (HCE) cells. This study was initiated to determine if adhesion to fibronectin (FN) alters the patterns of protein phosphorylation in early passage cultures. Methods: Preliminary experiments were performed to determine which ECM components were most efficient in supporting HCE cell proliferation in vitro. Second passage HCE cells (3 y/o donor) were removed from the culture dish with trypsin. The cells were washed in complete culture medium and half of the cells were subsequently maintained in suspension while the others were allowed to adhere to immobilized fibronectin. After 20 min, cellular proteins were harvested and phosphorylation patterns were compared using Kinexus technology. Results: Purified ECM proteins stimulated proliferation with the following efficacy: FN = type I collagen > type IV collagen > laminin. Adhesion to FN was most efficient in stimulating phosphorylation of ERK1(T202/Y204), ERK2(T185/Y187), SRC48 (Y529), and adducin α (S724); negative changes in phosphorylation were most evident in SRC48(Y418), RAF1(S259), and STAT1(S701). Changes in phosphorylation patterns were also observed in a number of as yet unidentified cross-reactive proteins. Conclusions: Previous reports have demonstrated that adhesion to FN stimulates MAP kinases and SRC-related oncogenes in a wide variety of cell types, and this study confirms that HCE cells share at least a portion of these signal transduction pathways. A novel finding in the present study is the large increase in adducin α phosphorylation upon adhesion to FN, and studies are in progress to understand the role of adducin α in the adhesion/proliferation of HCE cells.
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