GROWTH-FACTORS AND CORNEAL ENDOTHELIAL-CELLS .2. CHARACTERIZATION OF EPIDERMAL GROWTH-FACTOR RECEPTOR FROM BOVINE CORNEAL ENDOTHELIAL-CELLS

Epidermal growth factor (EGF) is a potent mitogen for corneal endothelial cells and may play a role in endothelial wound healing. To further characterize the interaction of EGF with endothelial cells, we measured biochemical parameters of I-125-EGF binding to cultured bovine corneal endothelial cells (BCEC), determined the pattern of EGF-induced protein phosphorylation, and investigated the influence of retinoic acid (RA) and transforming growth factor-beta (TGF-beta) on EGF-induced DNA synthesis and receptor levels. Binding of I-125-EGF to BCEC was dependent on time, reaching a plateau after approximately 2 h at 37-degrees-C, was specific for EGF, and had high affinity (K(d) = 100 pM) with approximately 21,000 receptors per cell. Cellular substrates for the EGF receptor kinase, which may function as initial second messengers for EGF, were detected by autoradiography of sodium dodecyl sulfate polyacrylamide gels of P-32-labeled BCEC proteins. EGF stimulated phosphorylation of 170, 37, 21 and 20-kDa proteins. Addition of 1 nM, 100 nM, and 10-mu-M RA to BCEC cultured in serum-free medium for 24 h progressively inhibited DNA synthesis by up to 80% compared with control cultures. However, when added in combination with 5 nM EGF, 1 nM and 100 nM RA synergistically stimulated DNA synthesis by up to 80% above the level of EGF stimulation without altering EGF receptor levels or binding affinity. Thus, short-term exposure of BCEC to RA potentiated EGF-stimulated DNA synthesis, most likely by acting at a postreceptor step. Addition of 10 nM TGF-beta to BCEC cultured in serum-free medium for 24 h stimulated low levels of DNA synthesis and did not significantly alter EGF-induced DNA synthesis nor alter EGF-induced protein phosphorylation patterns. These results demonstrate that EGF receptor in BCEC has biochemical characteristics that are similar to EGF receptors in other mammalian cells including stimulation of phosphorylation of several cellular proteins that may mediate the biological action of EGF in BCEC, and that EGF may interact synergistically with other agents that influence corneal endothelial cell mitosis.