DescriptionThe overall goal of this work was to investigate a role for endogenous insulin-like growth factor binding protein-3 (IGFBP-3) in intrinsic apoptosis in non-transformed bovine mammary epithelial cells (MEC). IGFBP-3 was produced and secreted in response to anisomycin (ANS), an activator of the intrinsic apoptotic pathway. Knock-down of IGFBP-3 with small interfering (si)RNA attenuated ANS-induced apoptosis, establishing a role for IGFBP-3 in MEC apoptosis. A nuclear function for IGFBP-3 was suggested by findings from cell fractionation experiments showing that ANS induced nuclear accumulation of IGFBP-3. In MECs, knock-down of IGFBP-3 attenuated ANS-induced phosphorylation and nuclear export of the orphan nuclear receptor Nur77. Co-immunoprecipitation experiments revealed an association between IGFBP-3 and Nur77 in ANS-treated cells, but not in untreated controls, suggesting that IGFBP-3 exerts its nuclear effects through physical association with Nur77. A second goal of the thesis was to determine the mechanism by which IGFBP-3 localizes to the nucleus in bovine MEC. An inhibitor of endocytosis had no effect on nuclear localization of IGFBP-3 while an inhibitor of secretion enhanced nuclear IGFBP-3. Together these data indicate that nuclear IGFBP-3 does not arise from secreted IGFBP-3 that is re-internalized. Since the molecular weight of glycosylated IGFBP-3 is near the cut-off for passive diffusion through nuclear pores, IGFBP-3 was tagged with GFP in order to determine if transport was a regulated process. ANS treatment of cells transfected with IGFBP-3-GFP increased the protein in the nucleus, indicating that nuclear import of IGFBP-3 is a regulated event. An antibody specific to bovine IGFBP-3 was generated to enable co-immunoprecipitation experiments. An association between IGFBP-3 and nuclear transport protein importin-β was found only in ANS-treated cells. Inhibition of importin-β attenuated nuclear import of IGFBP-3-GFP, establishing a role for importin-β in nuclear transport of IGFBP-3. In summary, these data indicate that nuclear localization of IGFBP-3 plays a role in intrinsic apoptosis in MEC and are the first to establish a mechanism for nuclear transport of endogenous IGFBP-3.