Induction of Apoptosis in Human Prostate Cancer Cells by Insulin-Like Growth Factor Binding Protein-3 Does Not Require Binding to Retinoid X Receptor-α

IGF binding protein (IGFBP)-3 can induce apoptosis in human prostate cancer cells directly without sequestering IGF-I and -II. The molecular mechanisms responsible for the IGF-independent actions of IGFBP-3 remain unclear. IGFBP-3, a secreted protein, can be internalized and translocate to the nucleus. It binds to the nuclear retinoid X receptor (RXR)-α. Binding to RXR-α has been proposed to be required for IGFBP-3 to induce apoptosis. The present study tests this hypothesis in the PC-3 human prostate cancer cell line. PC-3 cells express RXR-α, and apoptosis is induced by incubation with RXR-specific ligand. A COOH-terminal region in IGFBP-3 (residues 215–232) contains a nuclear localization signal, and binding domains for RXR-α and heparin (HBD). Different combinations of the 11 amino acids in this region that differ from IGFBP-1, a related IGFBP, which does not localize to the nucleus or bind RXR-α, were mutated to the IGFBP-1 sequence. By confocal imaging, mutation of residues 228-KGRKR-232 in nonsecreted IGFBP-3 diminished its nuclear localization. IGFBP-3 binding to glutathione S-transferase-RXR-α only was lost when all 11 sites were mutated (HBD-11m-IGFBP-3). Expressed nuclear RXR-α did not transport cytoplasmic IGFBP-3 nuclear localization signal mutants that can bind RXR-α to the nucleus even after treatment with RXR ligand. Expressed HBD-11m-IGFBP-3 still induced apoptosis in PC-3 cells in an IGF-independent manner as determined by flow cytometric analysis of Annexin V staining. We conclude that in PC-3 cells, RXR-α is not required for the nuclear translocation of IGFBP-3 and that IGFBP-3 can induce apoptosis in human prostate cancer cells without binding RXR-α.