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

Abstract

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.

Keywords: IGFBP-3; anisomycin; deoxynivalenol; glycosylation; nuclear localization.

Figure 1.

Bovine IGFBP-3 polyclonal antibody specifically detects IGFBP-3 in whole cell lysates (WCLs) and CM of MAC-T cells. MAC-T cells were transfected with 50 nM of IGFBP-3 or Scr siRNA for 48 h, serum starved overnight, and treated with ± 0.1 μM of ANS for 6 h (WCLs) or 18 h (CM). (a) WCLs (40 µg) and (b) CM (100 µL) were immunoblotted for IGFBP-3 [27]. HSP60 served as a loading control for WCLs. Scr, scramble.

Figure 2.

Induction of IGFBP-3 expression was required for ribotoxins to induce apoptosis. (a‒c) Confluent MAC-T cells were serum starved overnight and treated with 0.1 μM ANS, 1.0 μg/mL DON, or SF media as indicated. (a) Caspase-3/7 activation was measured with the SensoLyte Caspase-3/7 Assay (AnaSpec) after 6 hours of treatment; (b) total RNA was collected and analyzed for IGFBP-3 mRNA by Reverse transcription-quantitative polymerase chain reaction with data corrected for cyclophilin levels after 4 h of treatment. (a and b) Bars represent mean ± SEM of four individual experiments, with treatment measured in triplicate within each experiment. Data were analyzed using one-way ANOVA with the Tukey multiple comparisons post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001 compared with SF control. (c) Whole-cell lysates were collected after 6 h of treatment, and 40 μg was separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted for IGFBP-3. HSP60 was used as a loading control. Data are representative of four independent experiments. (d) MAC-T cells were transfected with 50 nM of IGFBP-3 or Scr siRNA for 48 h, serum starved overnight, and treated with 0.1 μM ANS, 1.0 µg/mL DON, or SF media for 6 h. Whole-cell lysates (40 μg) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted for IGFBP-3 and cleaved PARP, caspase-3, and caspase-7. HSP60 was used as a loading control. (e) Western blots from three experiments were quantitated by densitometry. Bars represent mean ± SEM. Data were analyzed by two-way ANOVA with the Sidak multiple comparisons post hoc test. *P < 0.05; **P < 0.01. AU, arbitrary units; Scr, scramble.

Figure 3.

ANS and DON induced nuclear localization of IGFBP-3. (a) MAC-T cells were treated for 6 h ± 0.1 µM ANS or 1 µg/mL DON, then fractionated and immunoblotted for IGFBP-3. Lamin A/C and HSP60 served as controls for cytoplasmic (cyto) and nuclear (nuc) loading, respectively. Results are representative of three independent experiments. (b) MAC-T cells transfected with IGFBP-3-GFP were treated for 2 h ± 0.1 µM ANS or 1 µg/mL DON. Cells were fixed in formalin, and then nuclei were stained with Hoechst. Images were acquired with an Olympus FSX100 microscope at 40× magnification. Images are representative of two independent experiments. (c) For the two experiments, approximately 200 transfected cells were counted to determine the percentage that contained nuclear IGFBP3-GFP. Bars represent mean ± SD. Data were analyzed using one-way ANOVA with the Tukey multiple comparisons post hoc test. *P < 0.05 compared with SF control. (d) As a control, cells were transfected with EGFP-plasmid alone and nuclei were stained with Hoechst. EGFP, pEGFP-N1 plasmid.

Figure 4.

Importazole reduced nuclear import of IGFBP-3-GFP. Cells transfected with IGFBP-3-GFP were treated for 4 h ± (a) 0.1 µM ANS or (b) 1 µg/mL DON ± 40 µM importazole. Cells were fixed in formalin, and then nuclei were stained with Hoechst. Images were acquired with an Olympus FSX100 microscope at (a) 60× and (b) 40× magnifications. Images are representative of two independent experiments. (c) For the two experiments, approximately 200 transfected cells were counted to determine the percentage that contained nuclear IGFBP3-GFP. Bars represent mean ± SD. Data were analyzed using one-way ANOVA with the Tukey multiple comparisons post hoc test. **P < 0.01; #P < 0.0001 compared with SF control. A+I, ANS + IMP; D+I, DON + IMP; DMSO, dimethyl sulfoxide; IMP, importazole.

Figure 5.

IGFBP-3 was associated with importin-β in response to ANS treatment. MAC-T cells were treated for 8 h ± 0.1 µM ANS. WCLs were collected, and IGFBP-3 and importin-β were immunoprecipitated (IP) with (a) anti‒IGFBP-3 antibody or (b) importin-β antibody, respectively. (c) MAC-T cells transfected with IGFBP-3-His were treated for 1 h ± 0.1 µM ANS. IGFBP-3 was IP with an anti‒His-tag antibody. (a) Rabbit nonimmune serum (CTL) and (b, c) mouse IgG served as controls. Results represent three independent experiments.

Figure 6.

Secreted IGFBP-3 was not reinternalized. (a) To confirm that Pitstop 2 blocks clathrin-mediated endocytosis, cells were treated for 45 min + transferrin-FITC (25 μg/mL) ± Pitstop 2. Cells were fixed in formalin, and then nuclei were stained with Hoechst. Images were acquired with an Olympus FSX100 microscope at 40× magnification. (b) Cells treated with ANS ± Pitstop 2 were fractionated and western immunoblotted for IGFBP-3 or lamin A/C. CM were collected and immunoblotted for IGFBP-3 to confirm that synthesis of IGFBP-3 was not affected. Results are representative of (a) two or (b) three experiments. DMSO, dimethyl sulfoxide.

Figure 7.

IGFBP-3 was not secreted before nuclear localization. MAC-T cells were treated with either (a) 0.1 µM ANS or (b) 1 µg/mL DON ± 10 µg/mL BFA. Cells were treated for 6 h with ANS or DON. BFA was added for the last 4 h of toxin treatment. Conditioned media were collected, and cells were fractionated. Samples were immunoblotted for IGFBP-3. PARP and HSP60 served as cytoplasmic (cyto) and nuclear (nuc) loading controls, respectively. Results are representative of three independent experiments. MAC-T cells transfected with IGFBP-3-GFP were treated with either (c) 0.1 µM ANS ± 10 µg/mL BFA for 1.5 h or (d) 1 µg/mL DON ± 10 µg/mL BFA for 2 h. Cells were fixed in formalin, and then nuclei were stained with Hoechst. Images were acquired with an Olympus FSX100 microscope at 40× magnification. Images represent two independent experiments. BFA, brefeldin A.

Figure 8.

IGFBP-3 was differentially glycosylated depending on cellular localization. (a) Cells transfected with IGFBP-3-His were treated for 6 h with ANS to induce nuclear localization of IGFBP-3 and then fractionated. Lysates were treated ± Endo H to deglycosylate proteins, separated by SDS-PAGE, and immunoblotted for IGFBP-3. PARP and HSP60 served as controls for nuclear and cytoplasmic loading, respectively. Results are representative of two independent experiments. (b) Cells were treated with 0.1 µM ANS or 1 µg/mL DON. After 6 h, cells were fractionated or lysed to collect WCLs. After 18 h, CM were collected. Membranes were immunoblotted for IGFBP-3. (c) Cells were treated for 18 h + 0.1 µM ANS or 1 µg/mL DON and CM were collected, or (d) cells were treated for 6 h + 0.1 µM ANS or 1 µg/mL DON and WCLs were collected. Samples were treated ± Endo H or PNGase F to deglycosylate proteins, separated by SDS-PAGE, and immunoblotted for IGFBP-3. cyto, cytoplasmic; nuc, nuclear.