Role of the degradation process in t growth factor (mitogen degradation/leupeptin/granulosa cells/cell proliferatior

The protease inhibitor leupeptin inhibits the degradation process of l25I-labeled epidermal growth factor (12 I-EGF) by cultured bovine granulosa cells. At 80 /g/ml, leupeptin inhibited the appearance of degradation products of i25I-EGF in the medium by 95% during 1 hr of incubation and by 90% during 24 hr of incubation when the cells were exposed to 5 ng of 125I-EGF per ml. In contrast, cultures exposed to either saturating (10 ng/ml) or nonsaturating (0.1 ng/ml) concentra- tions of EGF in the presence of leupeptin (80 Ag/ml) exhibited an increase in DNA synthesis that was 70-80% that of cultures exposed to EGF alone. Cultures responded to either EGF or fi- broblast growth factor with a logarithmic increase in cell number and, over a period of 8 days, the number of cells in- creased 10- to 18-fold. Addition of leupeptin did not diminish the growth rate of the cultures. In the presence of leupeptin, 1251-EGF accumulated within the granulosa cells and was in a form that was precipitable with antiserum against EGF and that comigrated on isoelectric focusing with native 125I-EGF. That a full mitogenic response can be obtained despite a 90-95% inhibition of EGF degradation at either saturating or nonsatu- rating concentrations of the mitogen suggests that a proteolytic degradation of a given mitogen may not be involved in the in- duction of a proliferative response. Cultured bovine granulosa cells derived from medium-sized ovarian follicles are highly responsive to both epidermal (EGF) and fibroblast (FGF) growth factors (1). Carpenter and Cohen (2) have demonstrated in cultured fibroblasts that, after binding of human EGF to specific cell surface receptor sites, the re- ceptor-EGF complexes are internalized and degraded by ly- sosomal proteases. Similar results have been obtained by