Insulin-like growth factor binding protein (IGFBP)-3 levels in conditioned media of Hs578T human breast cancer cells are post-transcriptionally regulated

Hs578T human breast cancer cells secrete insulin-like growth factor binding protein (IGFBP)-3 (41-kDa and 39-kDa) and IGFBP-4 (24-kDa) as major BP species. In addition, cell surface-associated IGFBP-3 is demonstrable by use of cell monolayer affinity cross-linking or by employing immunoperoxidase staining of the cell surface with specific polyclonal anti-human IGFBP-3 antibodies (αIGFBP-3gl and αIGFBP-3ngl). In this study, we have demonstrated that regulation of Hs578T IGFBP-3 by IGF peptides is specific, non-receptor mediated, and post-translational by showing: 1) dose-dependent increase of IGFBP-3 in conditioned media (CM) following addition of IGF-I and IGF-II (maximum 8-13-fold increase at 100 ng/ml concentration), but not by insulin up to 1 μg/ml; 2) confirmation of IGF-induced increases in CM concentrations of IGFBP-3 by means of Western ligan blot, affinity cross-linking, and IGFBP-3-specific radioimmunoassay; 3) increase of IGFBP-3 in CM in addition of IGF analogs which retain full affinity for IGFBPs ([Leu²⁷]IGF-II and [Tyr⁵⁵, Gln⁵⁶]IGF-I], but not by IGF analogs which have significantly decreased affinity for IGFBPs ([Gln³, Ala⁴, Tyr¹⁵ Leu¹⁶]IGF-I, [Gln⁶, Ala⁷, Tyr¹⁸, Leu¹⁹, Leu²⁷]IGF-II and IGF-I/insulin hybrid); 4) no change in IGFBP-3 mRNA following addition of IGFs; 5) existence of metal ion-dependent IGFBP-3 specific protease in CM and protection of IGFBP-3 from protease by formation of [IGF:IGFBP-3] complexes; and 6) release of cell surface-associated IGFBP-3 into CM by addition of IGF peptides. These studies demonstrate that IGF peptides regulate CM concentrations of IGFBP-3 through non-receptor mediated events, including dissociation of cell surface-associated IGFBP-3 and protection of IGFBP-3 from protease activity.