Abstract
Insulin-like growth factor binding protein-3 (IGFBP-3) is well established as a growth-inhibitory, apoptosis-inducing secreted molecule that acts via insulin-like growth factor (IGF)-independent as well as IGF-dependent pathways. Nuclear localization of IGFBP-3 has been observed and nuclear binding partners for IGFBP-3 demonstrated. However, little is known about the mechanism of IGFBP-3 internalization. We hypothesized that IGFBP-3 is first secreted then taken up again into cells and that its internalization could occur via binding to transferrin or caveolin. Incubation of cells with an IGFBP-3-neutralizing antibody demonstrated that nuclear translocation of endogenous IGFBP-3 requires IGFBP-3 secretion and re-uptake. Nuclear localization of exogenously added IGFBP-3 was rapid, occurring within 15 min, inhibited by co-incubation and extracellular sequestration with IGF-I, and dependent on the transferrin-binding C-terminal peptide region of IGFBP-3. Co-immunoprecipitation assays confirmed that IGFBP-3 binds transferrin but not directly to the transferrin receptor (TfR1); however, transferrin binds TfR1 and a ternary complex is formed. Specific binding to caveolin scaffolding docking sequence was confirmed utilizing radiolabeled IGFBP-3. Blocking TfR1-mediated endocytosis prevents both endogenous and exogenous IGFBP-3 re-uptake and inhibitors of caveolae formation also retard IGFBP-3 nuclear entry. Co-treatment with anti-transferrin receptor antibody and cholesterol depletion agents completely abolished endogenous and exogenous IGFBP-3 uptake. Suppression of IGFBP-3 internalization by TfR1 blockade inhibited IGFBP-3-induced apoptosis. Together, these data indicate that the actions of IGFBP-3 are mediated by internalization via distinct endocytic pathways.
Highlights
Insulin-like growth factor binding protein-3 (IGFBP-3) is well established as a growth-inhibitory, apoptosis-inducing secreted molecule that acts via insulin-like growth factor (IGF)-independent as well as IGF-dependent pathways
To resolve some of the issues related to the process of insulin-like growth factor binding proteins (IGFBPs)-3 internalization, we examined the effect of membrane cholesterol depletion and blockade of the transferrin receptor (to ardomain; Hsp60, heat shock protein 60; IB, immunoblot; IGF, insulinlike growth factor; IP, immunoprecipitation; MCD, methyl--cyclodextrin; NLS, nuclear localization sequence; PARP, poly(ADP-ribose) polymerase; PBS, phosphate-buffered saline; TGF-, transforming growth factor-; Transferrin Receptor-1 (TfR1), transferrin receptor; TNF-␣, tumor necrosis factor-␣; IGFBP-3, insulin-like growth factor binding protein-3; TBS, Tris-buffered saline; rh, recombinant human; DAPI, 4,6-diamidino-2-phenylindole; PVDF, polyvinylidene difluoride; Ab, antibody
Consistent with our previously published findings of a 10-fold increase in the presence of IGFBP-3 mRNA and protein in conditioned media of TGF--treated cells, treatment of PC-3 cells with 1 ng/ml TGF-1 for 48 h resulted in a 10-fold increase in IGFBP-3 secreted into conditioned media, as well as of IGFBP-3 found in the nuclear fractions and a 5-fold increase of cytoplasmic IGFBP-3 as compared with control serum-free conditions (Fig. 2A)
Summary
Insulin-like growth factor binding protein-3 (IGFBP-3) is well established as a growth-inhibitory, apoptosis-inducing secreted molecule that acts via insulin-like growth factor (IGF)-independent as well as IGF-dependent pathways. Nuclear localization of exogenously added IGFBP-3 was rapid, occurring within 15 min, inhibited by co-incubation and extracellular sequestration with IGF-I, and dependent on the transferrin-binding C-terminal peptide region of IGFBP-3.
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