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. 2007 May 1;109(9):3849-55.
doi: 10.1182/blood-2006-11-056879. Epub 2007 Jan 16.

Nur77 converts phenotype of Bcl-B, an antiapoptotic protein expressed in plasma cells and myeloma

Frederic Luciano  1 , Maryla Krajewska, Paulina Ortiz-Rubio, Stan Krajewski, Dayong Zhai, Benjamin Faustin, Jean-Marie Bruey, Beatrice Bailly-Maitre, Alan Lichtenstein, Siva Kumar Kolluri, Arnold C Satterthwait, Xiao-Kun Zhang, John C Reed
Affiliations

Nur77 converts phenotype of Bcl-B, an antiapoptotic protein expressed in plasma cells and myeloma

Frederic Luciano et al. Blood. .

Erratum in

  • Blood. 2009 Feb 12;113(7):1613

Abstract

Defects in apoptosis mechanisms play important roles in malignancy and autoimmunity. Orphan nuclear receptor Nur77/TR3 has been demonstrated to bind antiapoptotic protein Bcl-2 and convert it from a cytoprotective to a cytodestructive protein, representing a phenotypic conversion mechanism. Of the 6 antiapoptotic human Bcl-2 family members, we found that Nur77/TR3 binds strongest to Bcl-B, showing selective reactivity with Bcl-B, Bcl-2, and Bfl-1 but not Bcl-X(L), Mcl-1, or Bcl-W. Nur77 converts the phenotype of Bcl-B from antiapoptotic to proapoptotic. Bcl-B is prominently expressed in plasma cells and multiple myeloma. Endogenous Bcl-B associates with endogenous Nur77 in RPMI 8226 myeloma cells, where RNA interference experiments demonstrated dependence on Bcl-B for Nur77-induced apoptosis. Furthermore, a Nur77-mimicking peptide killed RPMI 8226 myeloma cells through a Bcl-B-dependent mechanism. Because Bcl-B is abundantly expressed in plasma cells and some myelomas, these findings raise the possibility of exploiting the Nur77/Bcl-B mechanism for apoptosis for eradication of autoimmune plasma cells or myeloma.

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Figures

Figure 1
Figure 1
Nur77 binds selectively to Bcl-2, Bfl-1, and Bcl-B. (A) GST fusion proteins representing Bcl-2 members (2 μg) were incubated overnight at 4°C with lysates from HEK 293T cells transfected with plasmid encoding GFP-Nur77. GST proteins were recovered on glutathione-Sepharose, and associated proteins were analyzed by SDS-PAGE immunoblot using rabbit anti-GFP (top blot) and anti-GST (bottom blot) antibodies. (B) 293T cells were cotransfected with plasmids encoding either GFP or GFP-Nur77ΔDBD (a mutant of Nur77 lacking the DNA binding domain that allows Nur77 to enter the cytoplasm without requiring additional stimulation) in combination with plasmids encoding Myc-tagged versions of various Bcl-2 family members. After 24 hours, immunoprecipitation (IP) was performed using anti-Myc antibody, and immune-complexes were analyzed by immunobloting using anti-GFP (top blot) or anti-Myc (middle blot) antibodies. Cell lysates (50 μg) were analyzed directly (bottom blot). (C) For Bcl-W, we performed immunoprecipitation using anti–Bcl-W antibody, and immune-complexes were analyzed by immunobloting using anti-GFP (top blot) and anti–Bcl-W (middle blot) antibodies. BimEL serves as a positive control. Lysates (50 μg) were also analyzed directly in gels to confirm protein production (“total lysate”) (bottom). Asterisks indicate nonspecific bands.
Figure 2
Figure 2
Nur77 collaborates with Bcl-2, Bfl-1, and Bcl-B to induce apoptosis. HeLa cells in 12-well plates were cotransfected with 0.3 μg plasmids encoding either GFP or GFP-Nur77ΔDBD in combination with 1 μg plasmids encoding the 6 antiapoptotic Bcl-2 family members. After 1 day, cells were washed with PBS, fixed with 3.7% formaldehyde, and stained with DAPI to visualize nuclei by UV microscopy. The percentages of apoptotic cells were determined by counting 200 GFP-positive cells, scoring cells having nuclear fragmentation and/or chromatin condensation. Data are reported as mean ± SE (n = 3). For assessing protein expression (3 lower panels), lysates were prepared from transfected cells, normalized for total protein content, and analyzed by SDS-PAGE/immunoblotting using Bcl-2, Myc, and GFP antibodies.
Figure 3
Figure 3
Membrane targeting is required for Bcl-B–induced apoptosis with Nur77. (A) HEK293T cells were transfected with 2 μg of either pEGFP-C1 or pEGFP-Nur77ΔDBD together with 0.5 to 1 μg plasmid DNA encoding a tagged Bcl-B or Bcl-BΔTM. After 24 hours, cells were lysed and caspase activity was assessed using DEVD-AFC substrate (mean ± SD; n = 3). (B) HEK293T cells were transfected as described for panel A. After 48 hours, fixed cells were stained with DAPI to visualize nuclei by UV microscopy. The percentages of apoptotic cells were determined by counting 200 GFP-positive cells (mean ± SE; n = 3). (C) HEK293T cells were transfected with pEGFP-C1 or pEGFP-Nur77ΔDBD in combination with Myc–Bcl-B or Myc–Bcl-BΔTM in the presence of 50 μM z-VAD-fmk. Cell lysates were prepared, and immunoprecipitation was performed with monoclonal anti-Myc monoclonal antibody. The immunoprecipitates or the lysates were immunoblotted with anti-GFP or anti-Myc antibodies.
Figure 4
Figure 4
Endogenous Nur77 binds endogenous Bcl-B in cells of plasma cell lineage. (A) HEK293T cells were transfected with 1 μg plasmids encoding Myc-tagged Bcl-B, Bcl-2, Bcl-XL, Bfl-1, Mcl-1, and Bcl-W proteins. After 24 hours, cells were lysed and analyzed by SDS-PAGE/immunoblotting using anti-Myc (top blot) and anti–Bcl-B (bottom blot) antibodies. Molecular weight markers are shown in kilodaltons (kDa). (B) Immunohistochemical detection of Bcl-B in normal human bone marrow. Serial sections of bone marrow biopsy were stained with (i) preimmune serum, (ii) anti–Bcl-B antiserum, (iii) anti–Bcl-B antiserum preadsorbed with GST–Bcl-B, and (iv) anti–Bcl-B serum preadsorbed with Bcl-W peptide. Specimens were counterstained with nuclear red dye. Original magnifications are × 1000. (C) Lysates from various myeloma and nonmyeloma cell lines were analyzed by SDS-PAGE/immunoblotting using Bcl-B, Bcl-2, and tubulin antibodies. (D) RPMI 8226 cells were stimulated for 1, 3, or 5 hours with TPA (100 ng/mL) and ionomycin (1 μM). Lysates were prepared, and immunoprecipitation was performed with monoclonal anti-Nur77 monoclonal antibody or mouse IgG. Immunoprecipitates and lysates were analyzed by SDS-PAGE/immunobloting using anti-Nur77 (top blot) or anti–Bcl-B (bottom blot) antibodies. In panels C and D, “293/Bcl-B” represents lysate (20 μg) from HEK 293T cells transfected with plasmid encoding Bcl-B protein, used as a positive control. Asterisk indicates a nonspecific band. Phospho form of Nur77 is indicated by the circled P.
Figure 5
Figure 5
Endogenous Bcl-B mediates Nur77-induced apoptosis in myeloma cells. (A) RPMI 8226 myeloma cells were electroporated with either control or Bcl-B siRNA. After 72 hours, cells were harvested and lysates (100 μg) were analyzed by SDS-PAGE/immunoblotting using Bcl-B (top blot) and actin (bottom blot) antibodies. (B) RPMI 8226 cells were electroporated with either control, Bcl-B, or Bcl-2 siRNA. Then, 72 hours later, cells were transfected with plasmids (0.1 to 1 μg) encoding GFP or GFP-Nur77ΔDBD proteins. After 18 hours, fixed cells were stained with DAPI to visualize nuclei by UV microscopy and determine percentages of apoptotic cells by counting 200 GFP-positive cells (mean ± SE; n = 3). (C) RPMI 8226 cells were electroporated with either control or Bcl-B siRNA. Cells were treated 72 hours later with staurosporine (0.01 to 0.3 μM) for 18 hours. Apoptosis was determined as in panel B.
Figure 6
Figure 6
Nur77-mimicking peptide induces Bcl-B–dependent apoptosis of myeloma cells. (A) Schematic representation of Nur77 protein and the corresponding Nur77 9′mer peptide. “GX” corresponds to the linker, and “r8” corresponds to 8 arginines that endow the peptide with cell penetration activity. (B) A serial concentration of GST or GST–Bcl-2, GST–Bcl-XL, or GST–Bcl-B (left panel) or GST–Bcl-W, GST–Bfl-1, (right panel) or GST–Mcl-1 was incubated with 20 nM FITC–Nur77 peptides in PBS buffer. Fluorescence polarization was measured after 10 minutes. (C) RPMI 8226 cells were electroporated with either control or Bcl-B siRNA. After 72 hours, cells were treated for 18 hours with either Nur77 peptide or corresponding inactive mutant (3 to 12 μM). Then, cells were washed with PBS, fixed with 3.7% formaldehyde, and stained with DAPI to visualize nuclei by UV microscopy. The percentages of apoptotic cells were determined by counting 200 GFP-positive cells (mean ± SE; n = 3).
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