AUTONOMOUS GROWTH OF HUMAN KERATINOCYTES REQUIRES EPIDERMAL GROWTH-FACTOR RECEPTOR OCCUPANCY

In the studies reported here, we demonstrate that transforming growth factor alpha (TGF-alpha) or epidermal growth factor (EGF) is required for the establishment of small colonies of human keratinocytes at clon al densities, but once small (10-15 cells) colonies have formed, the c ontinued growth of these colonies can proceed in the absence of exogen ous TGF-alpha or EGF. Equivalent receptor-binding concentrations of TG F-alpha and EGF were equipotent in stimulating colony formation. We al so demonstrate that the growth of keratinocytes at high densities proc eeds in the absence of exogenous peptide growth factors or hormones. T he expression of TGF-alpha mRNA and protein is regulated by both cell density and the presence of exogenous growth factors. The addition of an antibody which blocks the mitogenic effect of mature TGF-alpha had no effect on the autocrine/paracrine growth of these cells at either d ensity. However, monoclonal antibodies which antagonize ligand activat ion of the EGF receptor inhibit the autonomous proliferation of kerati nocytes at high density and abrogate the exogenous TGF-alpha/EGF-indep endent expansion of colonies at clonal density. The results of these e xperiments are among the first evidence to demonstrate that normal hum an epithelial cells in culture exhibit autocrine/paracrine-mediated pr oliferation. Exogenous growth factors initiate colonies of human kerat inocytes that become self-perpetuating in culture. Keratinocytes regul ate production of the mitogenic ligand, TGF-alpha, through a density-d ependent mechanism, and cell density stringently controls proliferatio n.