Kinetics of fibronectin release from fibroblasts in response to 12-O-tetradecanoylphorbol-13-acetate and retinoic acid.

We have studied the effects of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and its in vivo and in vitro antagonist retinoic acid (RA) on the synthesis and release of a major extracellular glycoprotein, fibronectin (FN), in human lung fibroblasts (HLF). The studies reported here investigate the question of whether the increased amounts of FN released by TPA treatment are cell-surface derived or require de novo synthesis of FN. Untransformed HLF continuously released FN into the medium. Addition of TPA rapidly enhanced this release of FN into the culture medium, as shown with enzyme-linked immunosorbent assay. RA, given simultaneously with TPA, prevented the increased release and resulted in a normal FN accumulation in the medium. RA alone did not influence FN release. To study the effect of TPA or RA on synthesis rates of FN, HLF were labeled metabolically: FN synthesis rates with or without TPA, RA, or with TPA and RA were similar, as judged by assaying cell-layer-associated and medium [3H]FN. Pre-existing (unlabeled) FN accumulated in the medium as a result of TPA treatment at a time when newly synthesized (labeled) FN was still intracellular. Cycloheximide, in concentrations which inhibited protein synthesis by 95%, did not prevent but only reduced the normal FN release, nor did it prevent the effect of TPA at this reduced level. We conclude that phorbol ester action and RA counteraction on the release of FN takes place on a cell-surface target; that FN which is released into the medium by TPA is derived from pre-existing FN; that RA specifically antagonizes TPA action. No protein synthesis is required to release FN, to mediate enhanced FN release by TPA, or to counteract the enhanced release by RA.