Growth factors and corneal endothelial cells: II. 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 125I-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 125I-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 (Kd = 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 32P-labeled BCEC proteins. EGF stimulated phosphorylation of 170, 37, 21 and 20-kDa proteins. Addition of 1 nM, 100 nM, and 10 microM 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Woost
P G
PG
Department of Biochemistry, University of Louisville, Kentucky.
Jumblatt
M M
MM
Eiferman
R A
RA
Schultz
G S
GS
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004247:DNA / Q000096:biosynthesis; D004591:Electrophoresis, Polyacrylamide Gel; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism*; D007700:Kinetics; D010766:Phosphorylation; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014212:Tretinoin / Q000494:pharmacology
D016895:Culture Media, Serum-Free; D011994:Recombinant Proteins; D016212:Transforming Growth Factor beta; D014212:Tretinoin; D004815:Epidermal Growth Factor; D004247:DNA; D066246:ErbB Receptors