Evidence that a variety of cultured cells secrete protease nexin and produce a distinct cytoplasmic serine protease‐binding factor

Abstract

Four criteria were used to examine serum‐free conditioned cell culture medium for protease nexin (PN):(1) formation of SDS‐stable ∼77 K Da complexes between a medium component and [¹²⁵l]thrombin; (2) acceleration by heparin of the rate of formation of these complexes; (3) cellular binding of these complexes; and (4) inhibition by heparin of the cellular binding of complexes. Listed in order of decreasing PN production, PN was detected in media conditioned by the following cell types: human foreskin fibroblasts (0.18 μg/10⁶ cells), rat embryo heart muscle cells (0.13 μg/10⁶ cells), mouse myotubes (0.1 μg/10⁶ cells), monkey kidney epithelial cells, human fibrosarcoma cells, human lung fibroblasts, simian virus 40 (SV‐40)‐transformed human fibroblasts, human epidermoid carcinoma cells, bovine aortic endothelial cells (only after phorbol ester treatment), and mouse myoblasts. No PN was found in medium conditioned by mouse 3T3 cells, SV40 virus‐transformed 3T3 cells, human lymphoblasts, or mouse leukemia cells. Eleven of the cell types examined for secretion of PN were also examined for the presence of cytoplasmic thrombin‐binding factors. Lysates from all of these cell types contained a factor that formed ∼60–65 K Da sodium dodecyl sulfate (SDS)‐stable complexes with [¹²⁵l] thrombin. This MW is significantly lower than that of [¹²⁵l] thrombin‐PN complexes, indicating that the factor is distinct from PN. Nevertheless, PN and the cytoplasmic factor share similarities. Production of both PN (by HF cells and WI‐26 cells) and the cytoplasmic factor (by HF cells and 3T3 cells) are stimulated by epidermal growth factor and phorbol myristate acetate. Also, both PN and the cytoplasmic factor complex trypsin, plasmin, urokinase, and thrombin, but not pancreatic elastase. Because a number of the cells that produce PN or the cytoplasmic serine protease‐binding factor are known to produce plasminogen activators, both PN and the cytoplasmic factor could regulate plasminogen activator activity.