Tetradecanoyl phorbol acetate stimulates latent collagenase production by cultured human endothelial cells

Angiogenesis is associated with the fragmentation of blood vessel basement membranes. Since collagen is a major constituent of basement membranes, cultured human endothelial cells derived from umbilical cord veins were assayed for their ability to produce collagenase. Unstimulated cultured human endothelial cells did not secrete detectable levels of active collagenase into the culture medium. However, if the post-culture medium was treated with trypsin or plasmin, low levels of collagenolytic activity were detected, indicating that endothelial cells secrete small amounts of latent collagenase. Addition of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) to the culture medium stimulated the secretion of collagenase by endothelial cells 5–30 fold. More than 90% of the collagenase was secreted in the latent form. Stimulation of collagenase production was detected at 10^?9 M TPA and was maximal at 10^?8 M TPA. An increase in the rate of collagenase production could be detected within 3 hr after the addition of TPA, and full induction occurred by 12 hr. Cycloheximide (3 μg/ml) or actinomycin D (0.1 μg/ml) inhibited both basal levels of collagenase production and the stimulation of collagenase production by TPA. Phorbol-12,13-didecanoate (PDD), a tumor-promoting analog of TPA, also stimulated collagenase production when administered at the same concentrations that were effective for TPA. However, 4-O-methyl TPA and 4-αPDD, two analogs of TPA which are not tumor promoters, did not stimulate collagenase production at concentrations up to 10^?7 M. The collagenase produced by endothelial cells was a typical vertebrate collagenase as judged by the following criteria: it cleaved collagen into only two fragments which were three quarters and one quarter of the length of the intact molecule; it was inhibited by EDTA and human serum; it was not inhibited by inhibitors of serine, thiol or aspartate proteases. Thus TPA causes an increase in the production of latent collagenase by cultured human endothelial cells.