Modulation of the Epidermal Growth Factor Receptor of Human Keratinocytes by Calcium Ion

Human keratinocytes grown in medium containing reduced calcium concentrations (0.07 mM) have been found to show altered morphology and decreased differentiation in comparison with cells grown in medium with physiologic calcium concentrations (1–2 mM). Since such alterations could be mediated by growth factors, we measured binding of [125I]epidermal growth factor (EGF). Neonatal keratinocytes were subcultured without feeder layers and grown to confluence in 1.1 mM calcium. Medium was changed and cells incubated for various periods in reduced calcium prior to binding assays. Scatchard plots of binding data showed a 5-fold increase in receptor number with no change in affinity (3 × 10-9 M) after 4–24 h at 37°C. Maximal binding occurred at 0.02–0.04 mM calcium and decreased sharply with increasing calcium concentrations. The increase could be prevented by calcium added soon after binding began to increase but was altered less after substantial elevation had occurred, although morphologic changes at reduced calcium concentrations were reversed within several hours. Substantial increases in binding of [125I]somatomedin C and [125I]concanavalin A were detected, but binding of [125I]pindalol, a beta-receptor ligand, was changed little. Keratinocytes at reduced calcium concentrations responded to added EGF by decreasing surface EGF receptors briskly in a time- and temperature-dependent fashion. The data suggest that keratinocytes show enhanced binding of EGF and some other cell surface ligands under conditions in which differentiation is retarded