Heat shock protein 90-mediated inactivation of nuclear factor-κB switches autophagy to apoptosis through becn1 transcriptional inhibition in selenite-induced NB4 cells

Abstract

Autophagy can protect cells while also contributing to cell damage, but the precise interplay between apoptosis and autophagy and the contribution of autophagy to cell death are still not clear. Previous studies have shown that supranutritional doses of sodium selenite promote apoptosis in human leukemia NB4 cells. Here, we report that selenite treatment triggers opposite patterns of autophagy in the NB4, HL60, and Jurkat leukemia cell lines during apoptosis and provide evidence that the suppressive effect of selenite on autophagy in NB4 cells is due to the decreased expression of the chaperone protein Hsp90 (heat shock protein 90), suggesting a novel regulatory function of Hsp90 in apoptosis and autophagy. Excessive or insufficient expression indicates that Hsp90 protects NB4 cells from selenite-induced apoptosis, and selenite-induced decreases in the expression of Hsp90, especially in NB4 cells, inhibit the activities of the IκB kinase/nuclear factor-κB (IKK/NF-κB) signaling pathway, leading to less nuclear translocation and inactivation of NF-κB and the subsequent weak binding of the becn1 promoter, which facilitates the transition from autophagy to apoptosis. Taken together, our observations provide novel insights into the mechanisms underlying the balance between apoptosis and autophagy, and we also identified Hsp90-NF-κB-Beclin1 as a potential biological pathway for signaling the switch from autophagy to apoptosis in selenite-treated NB4 cells.

FIGURE 1:

Sodium selenite specifically suppressed autophagy during apoptosis in NB4 cells. (A–C) The effect of sodium selenite on apoptosis- and autophagy-related genes in human leukemia cells. NB4, HL60, and Jurkat cells were treated with sodium selenite (20 μmol/L) for different times as indicated. Then, cleaved-PARP, cleaved-caspase3, p62, Beclin1, and LC3 were detected by Western blot. The top panels show representative Western blots, and the bottom panels show the quantification of protein levels normalized to those of the β-actin control. (D) Immunofluorescence staining of LC3 in human leukemia cells. Cells were treated with selenite (20 μmol/l) or 3-MA (1 mmol/l) for 24 h. After fixation, the cells were immunostained with anti-LC3 antibody (green). The nuclei were stained by DAPI (blue). The scale bar represents 20 μm. (E) MDC staining for visualization of autophagic vacuoles in human leukemia cells. NB4, HL60, and Jurkat cells were stained with MDC as described in Material and Methods, and staining was detected by fluorescence microscopy. The scale bar represents 100 μm. (F) The percentage of cells with punctate MDC fluorescence was calculated relative to a minimum of 100 cells per sample. Data are presented as the mean ± SD (n = 3). *p < 0.05 compared with control group.

FIGURE 2:

Autophagy antagonized selenite-induced NB4 apoptosis, and inhibition of autophagy sensitized cells to apoptosis. (A and C) An autophagy inhibitor, either 3-MA (1 mmol/l) or bafilomycin A1 (10 nmol/l) (A), or an autophagy activator, either Rapamycin (250 nmol/l) or GF109203X (1 μmol/l) (C), was added into NB4 cells for 1.5 h before selenite treatment. Then, cleaved-PARP, cleaved-caspase3, p62, Beclin1, and LC3 were detected by Western blot. The left panels show representative Western blots, and the right panels show the quantification of protein levels normalized to those of the β-actin control. (B and D) The effect of autophagy inhibitors (B) or activators (D) on selenite-induced NB4 apoptosis was analyzed on a flow cytometer using Annexin V/PI staining methods. Data are presented as the mean ± SD (n = 3). *p < 0.01 compared with control group. **p < 0.05 compared with selenite treatment group.

FIGURE 3:

Analysis of an RT-PCR autophagy array and detection of Hsp90 expression in different human leukemia cells. (A) The fold change of relative gene expression in sodium selenite–induced NB4 cell apoptotic process at 6, 12, and 24 h from the RT-PCR autophagy array, which included the autophagy-related genes ATG1, ATG3, ATG5, ATG6/BECN1, ATG7, ATG9A, ATG9B, ATG12, ATG16L, and ATG16L2 and the apoptosis-related genes P53, BAX, BAD, BCL2, and BCLXL. (B) Fold change of the relative gene expression of the chaperone molecules HSP70 and HSP90 in selenite-induced NB4 cell apoptosis. (C) Validation of the obtained microarray results by Western blot and standard PCR confirmed Hsp90 down-regulation during selenite treatment in NB4 cells. The left panel shows representative Western blots and PCR results. The middle and right panels show the quantification of normalized Hsp90 levels relative to that of the control. (D) Confirmation of Hsp90 expression by Western blot during selenite treatment in HL60 and Jurkat cells. The left panel shows representative Western blots, and the right panel shows the quantification of normalized Hsp90 levels relative to that of the control. The data are representative of at least three separate experiments.

FIGURE 4:

Hsp90 is required for selenite-induced apoptosis. (A, C, and E) NB4 cells were transfected with pCMV-Hsp90 and pCMV Blank (negative control) through electroporation (A), transfected with siRNA targeting Hsp90 and nonsilencing scrambled siRNA (C), and incubated with 0.2 or 1 μM 17-AAG for 1.5 h (E). Afterward cells were treated with 20 μM selenite or nothing for 24 h. Cell lysates were analyzed for the levels of Hsp90, cleaved-PARP, cleaved-caspase3, p62, Beclin1, and LC3 via Western blot. The left panels show the representative Western blots, and the right panels show the quantification of normalized protein levels relative to those of the β-actin control. (B, D, and F) The effect of Hsp90 on selenite-induced apoptosis was analyzed by the Annexin-V assay. Following overexpression, insufficient expression, or functional inhibition of Hsp90, NB4 cells were incubated with or without sodium selenite (20 μM) for 24 h and then measured and quantified by flow cytometry as described in Materials and Methods. Data are presented as the mean ± SD (n = 3). *p < 0.01 compared with control group. **p < 0.05 compared with selenite treatment group. The data are representative of at least three separate experiments.

FIGURE 5:

Selenite inactivated the IKK/NF-κB signaling pathway in NB4 cells. (A–C) The effect of selenite on the IKK/NF-κB signaling pathway in different cell lines. NB4, HL60, and Jurkat cells were treated with selenite (20 μM) for the indicated times. Then, IKKα, IKKβ, IκB, phospho-NF-κB, and NF-κB were detected by Western blot. The top panels show representative Western blots, and the bottom panels show the quantification of normalized protein levels relative to those of the β-actin control. (D and E) The nucleocytoplasmic translocation of NF-κB induced by selenite in different cell lines. NB4, HL60, and Jurkat cells were exposed to selenite for the indicated times, and the cytoplasmic and nuclear fractions were extracted. NF-κB was detected by Western blot. The purity of cytoplasmic and nuclear proteins was confirmed by β-actin and B23, respectively. The left panels show representative Western blots, and the right panels show the quantification of normalized protein levels relative to those of the β-actin and B23 controls. (F) Immunofluorescence staining results of NF-κB intracellular localization in NB4, HL60, and Jurkat cells treated with or without selenite. The red and blue fluorescence signals represent NF-κB and the nucleus, respectively. The scale bar represents 100 μm. The data are representative of at least three separate experiments.

FIGURE 6:

Hsp90 interacts with IKK in all three cell lines. (A) The interaction of Hsp90 with IKKα/IKKβ in NB4, HL60, and Jurkat cells as observed by coimmunoprecipitation. Protein extracts prepared from cells were immunoprecipitated with anti-Hsp90 antibodies. The immune complexes and the input (10% of the cell extracts used in the immunoprecipitation step) were analyzed by Western blot with antibodies specific to IKKα or IKKβ. The same membrane was stripped and reprobed to detect Hsp90. The left panel shows representative Western blots, and the right panel shows quantification of normalized protein levels relative to those of the input controls. (B) Indirect immunofluorescence colocalization of Hsp90 and IKKα. NB4 cells were treated with 20 μM selenite for 24 h and analyzed by immunofluorescence microscopy with antibodies to Hsp90 (green) and IKKα (red). The scale bar represents 100 μm. (C and D) The effect of Hsp90 on the activity and cellular localization of NF-κB. NB4 cells were transfected with pCMV-Hsp90 or Hsp90 siRNA by electroporation, as described previously. Then phospho-NF-κB expression from whole cell lysates (C) and NF-κB expression from the cytoplasmic and nuclear fraction (D) were detected by Western blot. The left panels show representative Western blots, and the right panels show the quantification of normalized protein levels relative to those of the β-actin and B23 controls. The data are representative of at least three separate experiments.

FIGURE 7:

NF-κB is responsible for the transcription of becn1. (A) The effect of the NF-κB inhibitor CAPE on Beclin1 expression. NB4 cells were incubated with CAPE (1 μM) combined with or without selenite (20 μM) for 24 h. Then, Beclin1, cleaved-PARP, cleaved-caspase3, p62, and LC3 were detected by Western blot. The top panel shows representative Western blots, and the bottom panel shows the quantification of protein levels normalized to those of the β-actin control. (B) NB4 cells treated with 1 μM of the NF-κB inhibitor CAPE and/or selenite were analyzed with an Annexin-V assay. Untreated NB4 cells (Con), selenite-subjected NB4 cells (Se), and inhibitor-treated NB4 cells (CAPE) served as controls. Data are presented as the mean ± SD (n = 3). *p < 0.01 compared with control group. **p < 0.05 compared with selenite treatment group. (C) The location of primer pairs used for PCR analysis following ChIP. (D) NF-κB ChIP analysis revealed the enrichment of putative NF-κB transcription factor binding sites in becn1 (κB site) in NB4 cells. The ChIP assay performed with an anti-p-NF-κB antibody was compared with normal rabbit IgG as a negative control. An equal amount (input) of DNA-protein complex was applied (left panel). Real-time PCR quantification of becn1 promoter sequences in anti-NF-κB ChIP in NB4 cells. Data are expressed as the percentage of input DNA and represent the mean ± SD of triplicate (right panel). (E and F) The effect of selenite or CAPE on the expression of components of the autophagy core complex in NB4 cells. Cells were treated with sodium selenite (20 μM) for different times as indicated (E) and treated with CAPE (1 μM) combined with or without selenite (20 μM) for 24 h (F). Then, PI3KC3, Ambra-1, and UVRAG were detected by Western blot. The top panels show representative Western blots, and the bottom panels show the quantification of normalized protein levels relative to those of the β-actin control. Data are representative of at least three independent experiments.

FIGURE 8:

The schematic diagram delineating the Hsp90–NF-κB–Beclin1 pathway involved in the selenite-induced apoptosis and autophagy of NB4 cells. Sodium selenite decreased Hsp90 expression, which occurred through some unknown transcription factors and attenuated the interaction of Hsp90 with IKK, which suppressed the activation and nuclear translocation of NF-κB. The inactivated NF-κB then suppressed becn1 transcription and anti-apoptotic genes, leading to a cell signaling switch from autophagy to apoptosis and, finally, irreversible cell death. This mechanism probably mediates the selenite-induced regulatory balance of apoptosis and autophagy in NB4 cells.