IGFBP-3 Induced by Ribotoxic Stress Traffics From the Endoplasmic Reticulum to the Nucleus in Mammary Epithelial Cells

: IGF-binding protein (IGFBP)-3 is a multifunctional protein that can exert IGF-independent effects on apoptosis. Anisomycin (ANS) is a potent inducer of IGFBP-3 production in bovine mammary epithelial cells (MECs), and knockdown of IGFBP-3 attenuates ANS-induced apoptosis. IGFBP-3 is present in the nucleus and the conditioned media in response to ANS. The goal of this study was to determine whether ribotoxic stress induced by ANS or a second ribotoxin, deoxynivalenol (DON), specifically regulates transport of IGFBP-3 to the nucleus and to determine the pathway by which it traffics. In ribotoxin-treated cells, both endogenous IGFBP-3 and transfected IGFBP-3 translocated to the nucleus. Inhibition of the nuclear transport protein importin-β with importazole reduced ribotoxin-induced nuclear IGFBP-3. Immunoprecipitation studies showed that ANS induced the association of IGFBP-3 and importin-β, indicating that ribotoxins specifically induce nuclear translocation via an importin-β‒dependent mechanism. To determine whether secretion of IGFBP-3 is required for nuclear localization, cells were treated with Pitstop 2 or brefeldin A to inhibit clathrin-mediated endocytosis or overall protein secretion, respectively. Neither inhibitor affected nuclear localization of IGFBP-3. Although the IGFBP-3 present in both the nucleus and conditioned media was glycosylated, secreted IGFBP-3 exhibited a higher molecular weight. Deglycosylation experiments with endoglycosidase Hf and PNGase indicated that secreted IGFBP-3 completed transit through the Golgi apparatus, whereas intracellular IGFBP-3 exited from the endoplasmic reticulum before transit through the Golgi. In summary, ANS and DON specifically induced nuclear localization of nonsecreted IGFBP-3 via an importin-β‒mediated event, which may play a role in their ability to induce apoptosis in MECs.