INSULIN-LIKE GROWTH-FACTOR-I (IGF-I) REGULATES IGFBP-3 AND IGFBP-4 BYMULTIPLE MECHANISMS IN A549 HUMAN ADENOCARCINOMA CELLS

The insulin-like growth factors (IGF-I and IGF-II) participate in the control of cell proliferation in normal and neoplastic lung cells. To examine the role of IGF binding proteins (IGFBPs) in modulating IGF ac tions in lung, we examined the production and regulation of IGFBPs fro m A549 cells, a human adenocarcinoma-derived lung cell line. Ligand bl ot and immunoblot analysis of conditioned media (CM) from A549 cells d emonstrated IGFBP bands of relative molecular mass (M(r)) similar to 3 9-43,000 (IGFBP-3), 34,000 (IGFBP-2), 30,000 (IGFBP-1), and 24,000 (IG FBP-4), IGFBP-3 abundance in A549 cell CM increased following exposure to IGF-I and IGF-II (3.0- and 1.8-fold, respectively) without a chang e in IGFBP-3 transcript abundance, suggesting IGFBP-3 is post-transcri ptionally regulated. Cycloheximide almost completely abrogated the IGF -I-stimulated increase in CM IGFBP-3, suggesting that ongoing protein synthesis is necessary for the IGF-I-stimulated increase in IGFBP-3 ab undance, Increases in IGFBP-3 occurred by at least two mechanisms, thr ough activation of the type 1 IGF receptor and by a type 1 IGF recepto r independent mechanism. The increase in IGFBP-3 was due, in part, to activation of the type 1 IGF receptor because blocking type 1 IGF rece ptor activation with an antibody (alpha IR3) diminished the IGF-I-indu ced increase in IGFBP-3 and insulin, at doses that stimulate the type 1 IGF receptor, increased IGFBP-3 abundance. The increase in IGFBP-3 w as partially independent of type 1 IGF receptor activation because [QA YL]-IGF-I, an analog of IGF-I that binds the type 1 IGF receptor but n ot IGFBP-3, was less potent than IGF-I in stimulating IGFBP-3 abundanc e, and IGF-II, which binds IGFBP-3 normally, but binds the type 1 IGF receptor with lower affinity than IGF-I, was nearly equipotent to IGF- I in its stimulation of IGFBP-3 accumulation at low concentrations. Th ese results suggest that ligand binding decreases IGFBP-3 clearance or increases IGFBP-3 accumulation in CM, IGF-I decreased IGFBP-4 abundan ce in A549 cell CM without decreasing IGFBP-4 mRNA transcripts and wit hout increasing the amount of cell-associated IGFBP-4, To determine wh ether the decrease in IGFBP-4 was due to increased degradation, cell-f ree CM was incubated with and without IGF-I, and IGFBP-4 abundance mea sured by ligand and immunoblot analyses. The 24,000 M, IGFBP-4 band de creased following the addition of IGF-I or IGF-II but not insulin or [ QAYL]-IGF-I to cell-free A549 CM samples, suggesting that binding of l igand to IGFBP-4 accelerates its clearance. Likewise, incubation of ce ll-free A549 CM containing purified IGFBP-1 with IGF-I resulted in a d ecrease in immunoreactive IGFBP-4. The disappearance of IGFBP-4 could be inhibited by lowering the temperature of incubation to 4 degrees C, by including the protease inhibitor benzamidine (25 mM), or by includ ing the divalent cation chelators EDTA (2 mM) or 1, 10-phenanthroline (5 mM), suggesting that the IGFs may activate an IGFBP-4 protease pres ent in A549 cell CM. Taken together, these findings demonstrate that I GF-I and -II regulate the abundance of IGFBPs in A549 cells through mu ltiple mechanisms. These changes in IGFBP abundance may help modulate the actions of the IGFs in these cells.