Cell density-dependent mitogenic effect and -independent cellular handling of epidermal growth factor in primary cultured rat hepatocytes

Aims: Mitogenic effect and cellular handling of epidermal growth factor (EGF) were analyzed in primary cultured rat hepatocytes at several cell densities. Methods/Results: DNA synthesis, assessed by the incorporation of ¹²⁵I-deoxyuridine, was accelerated by EGF at a low cell density while that stimulated by EGF was relatively low at the highest cell density, suggesting a cell density-dependent regulation of mitogenic response to EGF. An equilibrium binding study of ¹²⁵I-EGF in the presence of various concentrations of unlabeled EGF at 0°C revealed that the dissociation constant (K(d)) was 0.47-0.88 nM while the specific binding capacity (n) was 86-96 fmol/mg protein at each cell density. No significant difference was observed in the time profiles of the surface-bound, internalized, and degradation products of ¹²⁵I-EGF, assessed per mg protein, between different cell densities. Based on a kinetic analysis of the time-profiles, the internalization rate constant and the degradation rate constant were found to be independent of cell density. Conclusions: These results indicate that the cellular binding and disposition of EGF are not regulated by cell density, and that the cell density-dependence of the mitogenic effect cannot be attributed to differences in the affinity or capacity of the EGF receptor, internalization, or degradation of EGF. We speculate that the cell density-dependent mitogenic response may be accounted for by the difference in other factors such as the signal transduction processes induced by the receptor binding of EGF, or the translocation of a small fraction of the total EGF to hepatocyte nuclei.