Surface-bound plasmin induces selective proteolysis of insulin-like-growth-factor (IGF)-binding protein-4 (IGFBP-4) and promotes autocrine IGF-II bio-availability in human colon-carcinoma cells.

Limited proteolysis of insulin-like-growth-factor (IGF)-binding proteins (IGFBPs) represents a key process to modulate IGF bio-availability at the cellular level. In human colon carcinomas, urokinase-type plasminogen activator (u-PA) produced by stroma cells can bind to cancer-cell-associated u-PA receptor (u-PAR), and then catalyze the conversion of plasminogen (Pg) into plasmin (Pm). We therefore investigated the interplay between the IGF and Pm systems in the HT29-D4 human colon-carcinoma-cell model. HT29-D4 cells secreted IGF-II totally complexed to IGFBP-2, IGFBP-4 and IGFBP-6. Approximately 15% of IGFBP-4 was associated with the extracellular matrix. HT29-D4 cells produced neither u-PA- nor IGFBP-specific proteases. However, activation of Pm at the HT29-D4 cell surface obtained by the sequential addition of exogenous u-PA and Pg to mimic the stromal complementation induced selective proteolysis targeted to IGFBP-4 only (>95%). IGFBP-2 and IGFBP-6, though sensitive to proteolysis by soluble Pm, were not altered by cell-bound Pm. IGFBP-4 proteolysis yielded 18- and 14-kDa immunoreactive fragments which were not detectable by Western ligand blotting, indicating that they bound IGF-II with poor affinity. Release of IGF-II from IGF-II-IGFBP complexes after IGFBP-4 proteolysis by cell-bound Pm was indicated by the observation that approximately 20% of the 125I-IGF-II initially associated with endogenous IGFBP in reconstituted complexes was transferred to HT29-D4 cell-surface IGF-I receptors. These results suggest that IGFBP-4 proteolysis by cell-bound Pm can promote autocrine/paracrine IGF-II bio-availability in colon-cancer cells. This may have important consequences on the behavior of cancer cells at the interface between stroma and malignant cells in carcinomas of the colon in vivo.