Induction of apoptosis in human prostate cancer cells by insulin-like growth factor binding protein-3 does not require binding to retinoid X receptor-alpha

Abstract

IGF binding protein (IGFBP)-3 can induce apoptosis in human prostate cancer cells directly without sequestering IGF-I and -II. The molecular mechanisms responsible for the IGF-independent actions of IGFBP-3 remain unclear. IGFBP-3, a secreted protein, can be internalized and translocate to the nucleus. It binds to the nuclear retinoid X receptor (RXR)-alpha. Binding to RXR-alpha has been proposed to be required for IGFBP-3 to induce apoptosis. The present study tests this hypothesis in the PC-3 human prostate cancer cell line. PC-3 cells express RXR-alpha, and apoptosis is induced by incubation with RXR-specific ligand. A COOH-terminal region in IGFBP-3 (residues 215-232) contains a nuclear localization signal, and binding domains for RXR-alpha and heparin (HBD). Different combinations of the 11 amino acids in this region that differ from IGFBP-1, a related IGFBP, which does not localize to the nucleus or bind RXR-alpha, were mutated to the IGFBP-1 sequence. By confocal imaging, mutation of residues 228-KGRKR-232 in nonsecreted IGFBP-3 diminished its nuclear localization. IGFBP-3 binding to glutathione S-transferase-RXR-alpha only was lost when all 11 sites were mutated (HBD-11m-IGFBP-3). Expressed nuclear RXR-alpha did not transport cytoplasmic IGFBP-3 nuclear localization signal mutants that can bind RXR-alpha to the nucleus even after treatment with RXR ligand. Expressed HBD-11m-IGFBP-3 still induced apoptosis in PC-3 cells in an IGF-independent manner as determined by flow cytometric analysis of Annexin V staining. We conclude that in PC-3 cells, RXR-alpha is not required for the nuclear translocation of IGFBP-3 and that IGFBP-3 can induce apoptosis in human prostate cancer cells without binding RXR-alpha.

Figure 1

A, Schematic diagram showing the IGFBP-3 mutants used. A COOH-terminal basic region of IGFBP-3 (residues 215–232) contains the NLS [residues 228-KGRKR-232 (25)] and confers the ability to bind RXR-α (26). Of the 18 amino acids in the region, 11 differ from the corresponding sequence of IGFBP-1. They are indicated in the HBD-11m-IGFBP-3 mutant; identical amino acids are indicated by a dash. We also examined mutants with partial substitutions of these residues (MDGEA-IGFBP-3, HBD-9m-IGFBP-3, and HBD-6m-IGFBP-3). In some experiments the NH₂-terminal IGF-binding site (black portion of the bar) also was mutated in HBD-11m-IGFBP-3 by substituting alanine for six critical amino acids [⁵⁶I, ⁵⁷Y, ⁷⁵R, ⁷⁷L, ⁸⁰L, and ⁸¹L (5)]. B, Confocal imaging of PC-3 cells transfected with WT and mutant YFP-IGFBP-3 constructs. PC-3 cells were transfected with YFP empty vector (YFP) or the indicated YFP-IGFBP-3 constructs: YFP-WT IGFBP-3 (YFP-IGFBP-3), YFP-MDGEA-IGFBP-3, YFP-HBD-11m-IGFBP-3, YFP-HBD-9m-IGFBP-3, and YFP-HBD-6m-IGFBP-3. The cells were fixed, incubated with the nuclear stain DAPI, and examined by confocal imaging. Images from representative fields show green fluorescence (YFP) (left column), blue fluorescence (DAPI) (center column), and overlay of DAPI and YFP fluorescence (right column).

Figure 2

Binding of YFP-IGFBP-3 mutant proteins to RXR-α. A, In vitro binding of YFP-IGFBP-3 mutant proteins to GST-RXR-α fusion proteins. GST-RXR-α (expressed in E. coli) or GST control was coupled to Glutathione-Sepharose-4B beads. PC-3 cells were transfected with YFP empty vector (lane 1), YFP-WT-IGFBP-3 (lane 2), YFP-MDGEA-IGFBP-3 (lane 3), YFP-HBD-6m-IGFBP-3 (lane 4), YFP-HBD-9m-IGFBP-3 (lane 5), and YFP-HBD-11m-IGFBP-3 (lane 6). After 24 h, total cell lysates were prepared and incubated overnight at 4 C with beads that had been coupled with GST-RXR or GST. Bound proteins were eluted and examined by Western blotting using antibodies to GFP, which recognize YFP (upper-left panel). Similar results were obtained in two other experiments. Lower-left panel, Lysates used for the GST pull down were blotted with anti-GFP. Anti-GFP binds to approximately 27-kDa YFP (open arrowhead, lane 1) and to the YFP moiety of the approximately 67-kDa YFP-IGFBP-3 fusion proteins (solid arrowheads, upper and lower-left panels). For clarity, lanes separating the individual lanes shown have been deleted. Lanes 1–3 and lanes 4–6 were taken from different gels. The greater intensity of the YFP-HBD-9m-IGFBP-3 lysate signal relative to the YFP-HBD-11m-IGFBP-3 lysate in this immunoblot is not consistently observed, and does not account for the inability to detect YFP-HBD-11m-IGFBP-3 by GST-RXR pull down. As an additional control, the GST-RXR and GST pull-down blots were stripped and immunoblotted with anti-RAR (upper-right panel). Endogenous RAR, a known partner of RXR, binds specifically to GST-RXR in all lysates, excluding the possibility that the inability of YFP-HBD-11m-IGFBP-3 to bind to GST-RXR was due to nonspecific inhibitors or competitors of binding to RXR-α in the cell lysates. It has been reported that IGFBP-3 can dissociate preformed RXR:RAR heterodimers (by binding to either RXR or RAR) (29). Decreased binding of RAR to GST-RXR, however, would not be expected in our experiment because of the large excess of GST-RXR. B, Coimmunoprecipitation of YFP-IGFBP-3 mutants and RXR-α from PC-3 cell lysates. PC-3 cells were transfected with pCMX-RXR-α expression vector and either YFP-HBD-11m-IGFBP-3 (lanes 1 and 2) or YFP-HBD-6m-IGFBP-3 (lanes 3 and 4). After 24 h, cell lysates were prepared, precleared, and equal aliquots were incubated with rabbit IgG control (−, lanes 1 and 3) or with antibody to RXR-α (+, lanes 2 and 4). After 16 h, Protein A/G Plus Agarose beads were added to all the samples. After high-stringency washes, the immunoprecipitated proteins were eluted and analyzed by Western blotting using antibody to GFP to identify the YFP-IGFBP-3 mutant proteins. The position of immunoreactive YFP-IGFBP-3 is indicated by a solid arrowhead. The lanes shown are from the same gel. Lanes separating lanes 1 and 2 from lanes 3 and 4 have been removed for clarity of presentation, and the images spliced to put them in closer proximity.

Figure 3

The RXR ligand LG268 activates endogenous RXR-α, and induces apoptosis in PC-3 and LNCaP human prostate cancer cell lines. A, Immunoblot of PC-3 cell lysates with anti-RXR-α antibody. Cells in the left lane (+) were transfected with pCMX-RXR-α expression vector. Cells in the right lane (−) were not transfected. The arrow on the left shows the migration of recombinant human RXR-α standard. B, RXRE luciferase assay. PC-3 cells (left) or LNCaP cells (right) were transfected with pCMX or pCMX-RXR-α as indicated, and an RXRE-luciferase reporter plasmid (TK-CRBPII-Luc) for 3 h. After incubation in medium containing 10% charcoal-treated FBS for 24 h, the cells were washed and then incubated in serum-free medium for an additional 24 h with LG268 (0.1 μm) or DMSO vehicle. Luciferase activity is expressed relative to control cells incubated with DMSO. The mean ± se of three experiments is plotted. In these experiments, 0.1 μm LG268 stimulated luciferase activity 21-fold in LNCaP cells and 5-fold in PC-3 cells that had been transfected with pCMX-RXR-α. In other experiments 1 μm LG268 stimulated luciferase activity in PC-3 cells to the same extent as 0.1 μm LG268-treated LNCaP cells (results not shown). C, LG268 induces apoptosis in untransfected PC-3 and LNCaP cells. PC-3 cells (two left lanes) and LNCaP cells (two right lanes) were incubated for 48 h in serum-free medium with (+, black bars) or without (−, gray bars) 0.1 μm LG268. Cell death was determined by Annexin V staining and flow cytometry, and is expressed relative to control cells treated with vehicle for each cell line. The mean ± se for three experiments is plotted. P = 0.05 for PC-3 cells and P = 0.06 for LNCaP cells using the Student’s t test. The ability of LG268 to induce apoptosis but not RXRE-dependent promoter activity (B) in nontransfected PC-3 and LNCaP cells may reflect differences in the experimental conditions, or may indicate that RXR can induce apoptosis by mechanisms that do not involve transcription (63).

Figure 4

Treatment of PC-3 cells with RXR ligand does not change the subcellular localization of expressed IGFBP-3 constructs and RXR-α. PC-3 cells were cotransfected for 3 h with pCMX-RXR-α and either YFP empty vector (YFP), YFP-WT IGFBP-3 (YFP-IGFBP-3), or the indicated YFP-IGFBP-3 mutants (YFP-MDGEA, YFP-HBD-11m, YFP-HBD-9m, YFP-HBD-6m). After 24 h incubation in medium containing 10% dialyzed FBS, the cells were washed and incubated in serum-free medium without (“−”) or with (“+”) 1 μm of the RXR-specific ligand, LG268, for 24 h. The cells were then stained with DAPI, fixed, incubated with mouse anti-RXR monoclonal antibody/Alexa Fluor 594 goat antimouse IgG, and examined by confocal microscopy. From left to right, the columns show blue fluorescence (DAPI), green fluorescence (YFP), red fluorescence (RXR-α), and an overlay.

Figure 5

YFP-HBD-11m-IGFBP-3 induces apoptosis in PC-3 and LNCaP human prostate cancer cells. A, PC-3 cells were transfected with YFP empty vector (lane 1), YFP-WT-IGFBP-3 (lanes 2 and 3), YFP-MDGEA-IGFBP-3 (lanes 4 and 5), YFP-HBD-11m-IGFBP-3 (lanes 6 and 7), and YFP-6m/HBD-11m-IGFBP-3 (lanes 8 and 9) for 3 h, then incubated in serum-containing medium in the presence (+) or absence (−) of the general caspase inhibitor, Z-VAD-fmk (20 μm). Cell death was determined by Annexin V staining, which was quantified using flow cytometry, and is expressed relative to YFP-empty vector in the same experiment (dashed line). The mean ± se of three experiments is plotted. B, YFP-HBD-11m-IGFBP-3 induces cell death in LNCaP cells. LNCaP cells were transfected with YFP, YFP-WT-IGFBP-3, or YFP-HBD-11m-IGFBP-3 for 3 h, and allowed to recover by incubation in DMEM:F12K medium containing 5% FBS for 24 h. After incubation for another 48 h in serum-free medium, cell death was determined by Annexin V staining and flow cytometry as described previously. The mean ± se is plotted for two experiments.