Characterization of MUDENG, a novel anti-apoptotic protein

Abstract

MUDENG (Mu-2-related death-inducing gene, MuD) is revealed to be involved in cell death signaling. Astrocytes, the major glial cell type in the central nervous system, are a source of brain tumors. In this study, we examined MuD expression and function in human astroglioma cells. Stimulation of U251-MG cells with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resulted in a 40% decrease in cell viability and a 33% decrease in MuD protein levels, although not in MuD mRNA levels. To study the functional relevance of MuD expression, stable transfectants expressing high levels of MuD were generated. Stimulation of these transfectants with TRAIL resulted in enhanced cell survival (77% for stable and 46% for control transfectants). Depletion of MuD led to a marked reduction upon TRAIL stimulation in cell viability (22% in MuD-depleted cells and 54% in control cells). In addition, we observed that MuD depletion increased the susceptibility of the cells to TRAIL by enhancing the cleavage of caspase-3/-9 and BH3-interacting domain death agonist (Bid). A unique 25-kDa fragment of B-cell lymphoma 2 (Bcl-2) lacking BH4 was observed 60-180 min post TRAIL treatment in MuD-depleted cells, suggesting that Bcl-2 is converted from its anti-apoptotic form to the truncated pro-apoptotic form. Importantly, the TRAIL-mediated decrease in cell viability in MuD-depleted cells was abrogated upon Bid depletion, indicating that the role of MuD in apoptotic signaling takes place at the Bid and Bcl-2 junction. MuD localizes predominantly in the endoplasmic reticulum and partly in the mitochondria and its amounts are reduced 6 h post TRAIL stimulation, presumably via caspase-3-mediated MuD cleavage. Collectively, these results suggest that MuD, a novel signaling protein, not only possesses an anti-apoptotic function but may also constitute an important target for the design of ideal candidates for combinatorial treatment strategies for glioma cells.

Figure 1

MuD domain analysis and generation of a MuD monoclonal antibody (MAb)-recognizing domain 1 of MuD. (a) The hydrophilicity score was calculated based on the methods of Goldman, Engelman and Steitz (GES) and Kyte–Doolittle (KD) using the CLC Genomics Workbench program (http://www.clcbio.com/products/clc-genomics-workbench/), which was also used for calculating the isoelectric point (pI). The hydrophilicity of the MuD amino acid sequence (NCBI Reference Sequence: NM_018229.3) was evaluated using the GES (solid black line) and KD (dotted line) methods;^, two hydrophilic regions (domain 1, pI=6.82; domain 3, pI=8.34) surround a non-hydrophilic region (domain 2, pI=4.32). (b) Domain 1 of MuD was expressed in Escherichia coli BL21 (DE3) using pET23dw-His-MuD and then purified using His-bind resin (Novagen, San Diego, CA, USA). The purified MuD protein was used to generate MAb; immunization, cell fusion and selection of hybridoma clones, and production and purification of the MAb were performed according to the standard procedures. A mouse MuD MAb (C22B3) produced from one of the hybridomas was used for the experiments. Green fluorescent protein (GFP)-tagged MuD deletion mutants (pEGFPC1-MuD AA 1-490; AA 41−490; AA 81−490; AA 121−490) were generated by PCR. All cDNA constructs were verified by DNA sequencing and expressed in HCT-116 cells obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) by transient transfection using Lipofectamine2000 (Invitrogen, Gaithersburg, MD, USA). Anti-GFP was purchased from Santa Cruz (Dallas, TX, USA) and horseradish peroxidase-conjugated goat anti-mouse IgGs were obtained from Jackson ImmunoResearch Laboratories (West Groove, PA, USA).

Figure 2

Viability and the expression level of the MuD protein, but not MuD mRNA, decreased following TRAIL treatment. U251-MG, U373-MG and U87-MG cells were obtained from Dr Benveniste EN (University of Alabama at Birmingham, Birmingham, AL, USA). A172 and T98G cells were obtained from Dr Lee JH (Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul, Korea). (a) Cell lysates (30 μg) obtained from each cells were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) then transferred onto a polyvinylidene fluoride (PVDF) membrane. The pattern of MuD protein expression was analyzed by western blot using C22B3 monoclonal antibody (MAb); anti-β-actin (Santa Cruz) was used to analyze β-actin as a loading control. (b) Cells (2 × 10⁴ cells/well) grown in 96-well plates were treated for the indicated time (0–12 h) with 200 ng/ml recombinant TRAIL (Peprotech, Rocky Hill, NJ, USA). Cell viability was measured using a colorimetric assay for 96-well plates with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) reagent (Roche Applied Science, Mannheim, Germany). Absorbance was determined at 450 nm using a microplate reader (Bio-Tek, Winooski, VT, USA). Absorbance was directly proportional to the number of viable cells, because the tetrazolium salts in the WST-1 are cleaved to formazan by mitochondrial dehydrogenases in the cells. The level of significance for comparisons between samples was determined using Student's t-test distribution. The results presented are the mean±s.d. of three experiments (significant versus control, *P<0.05). (c) Cells (3 × 10⁵ cells/well) grown in 6-well plates were treated with TRAIL (200 ng/ml) for the indicated times (0–12 h). Cell lysates were separated by 10% SDS-PAGE then transferred onto a PVDF membrane. The pattern of MuD protein expression was analyzed using C22B3 MAb; β-actin served as a loading control. (d) For RT–PCR analysis, total RNA was extracted from prepared U251-MG cells using TRIzol reagent (Ambion, Carlsbad, CA, USA). First-strand DNA synthesis was conducted with oligo (dT) and AccuPower CycleScript RT PreMix kit (BIONEER, Daejeon, Korea) followed by PCR. The amplification program was as follows: 1 cycle of 96 °C/5 min; 35 cycles of 96°C/30 s, 58 °C/30 s, 72 °C/30 s and 1 cycle of 72 °C/7 min. PCR products were detected on a 1% agarose gel with ethidium bromide and analyzed by ChemiImager 5500 (Alpha Innotech, San Leandro, CA, USA). The following primers were used: MuD sense: 5′-GGGATACAGGATTTTCTT-3′ MuD anti-sense: 5′-GAGACTCAAGCTGATGGT-3′ hGAPDH sense: 5′-GTCTTCACCCCATGGAGAAGG-3′ hGAPDH anti-sense: 5′-CGTTCAGCTCTGGGATGACCTTG-3′. NS, non-specific.

Figure 3

MuD exerts an inhibitory effect on TRAIL-induced sensitivity to death stimuli. (a) U251-MG cells stably expressing GFP alone (pEGFPC1) and GFP-MuD (pEGFPC1-MuD) were generated by transfection using Lipofectamine2000 and selected on G418 sulfate (200 μg/ml; Invitrogen). Amount of MuD was analyzed by western blot using C22B3 MAb and anti-GFP Ab (Santa Cruz). The blot was re-probed with anti-β-actin. (b) pEGFPC1 (white) and pEGFPC1-MuD (black) stable transfectants were stimulated by TRAIL for the indicated time periods (0–12 h). Cell viability was measured using WST-1. Data presented are the mean±s.d. of three experiments (significant versus control, *P<0.05). (c) pEGFPC1 and pEGFPC1-MuD transfectants were seeded at a density of 3 × 10⁶ cells in 100-mm culture dishes and incubated overnight. Cells were then treated with TRAIL (200 ng/ml) for the indicated times (0–180 min). Cell lysates were separated on 10% SDS-PAGE and analyzed by western blot with the following antibodies; C22B3 MAb, anti-Bid, anti-caspase-3 (Cell Signaling Technology, Danvers, MA, USA), anti-caspase-9, anti-Bcl-2 (Santa Cruz) and β-actin as a loading control. (d, f) MuD siRNA (sense strand: 5′-GAGCAAGUUAUGUGCCUGUdTdT-3′ anti-sense strand: 5′-ACAGGCACAUAACUUGCUC-3′), a target-specific 19-nt siRNA designed to silence the gene expression and control siRNA (sense strand: 5′-CCUACGCCACCAAUUUCGU-3′ anti-sense strand: 5′-ACGAAAUUG GUGGCGUAGG-3′) were purchased from BIONEER. U251-MG (d) and T98G cells (f) were seeded at a density of 3 × 10⁵ cells in 6-well plates, respectively. Twelve hours after seeding, cells were transfected with control and MuD siRNA duplexes (100 nM) using Lipofectamine2000. Following 36 h incubation, 2 × 10⁴ cells were then re-seeded into 96-wells, incubated for 12 h, and then treated with TRAIL (200 ng/ml) for the indicated time periods (0–12 h). Cell viability was measured using WST-1. Data presented are the mean±s.d. of three experiments (significant versus control, *P<0.05). (e, g) Each cell from the experiment presented in d and f were lysed, subjected to 10% SDS-PAGE, and amount of MuD was analyzed by western blot using C22B3 MAb. The blot was re-probed with anti-β-actin. (h) U251-MG cells were seeded at a density of 3 × 10⁶ cells in 100-mm cell culture dishes. Following overnight incubation, the cells were transfected with control and MuD siRNA duplexes (100 nM) using Lipofectamine2000, respectively. Subsequent to 36 h incubation, the cells were re-seeded into 6 wells (3 × 10⁵ cells/well), incubated for an additional 24 h, and then stimulated with TRAIL for the indicated times (0–180 min). The expression patterns of MuD and the caspases were detected by western blot analysis with the following antibodies; C22B3 MAb, anti-Bid, anti-caspase-9, anti-caspase-3, anti-Bcl-2 and β-actin as a loading control. FL, full length.

Figure 4

Bid neutralizes MuD function in TRAIL-mediated apoptotic signaling. (a, b) Bid siRNA1 (#681, sense strand: 5′-GGCAGAUUCUGAAAGUCAATT-3′ anti-sense strand: 5′-UUGACUUUCAGAAUCUGCCTT-3′) and Bid siRNA2 (#1085, sense strand: 5′-CGAUGUGGUCACAGCUGUATT-3′ anti-sense strand: 5′-UACAGCUGUGACCACAUCGTT-3′) target-specific 21-nt siRNAs designed to silence gene expression were obtained from BIONEER. Control and MuD siRNA were used as described in Figures 3d and f. pEGFPC1-MuD transfectants and T98G cells were seeded at a density of 3 × 10⁵ cells in 6-well plates, respectively, and transfected with control, MuD, and Bid siRNA duplexes and MuD plus Bid siRNA using Lipofectamine2000. Following 36 h incubation, 2 × 10⁴ cells were re-seeded into 96-wells, incubated for 12 h, and then treated with TRAIL (200 ng/ml) for an additional 12 h. Cell viability was measured using WST-1. Data presented are the mean±s.d. of three experiments (significant versus control, *P<0.05, **P<0.01). The rest of each cell were lysed, subjected to 12% SDS-PAGE, and the patterns of MuD expression were analyzed by western blot using C22B3 MAb (top panel) and anti-Bid Ab (middle panel). The blot was re-probed with anti-β-actin (bottom panel). (c) U251-MG cells were seeded at a density of 3 × 10⁵ cells in 6-well plates. The cells were transfected with control and Bid siRNA duplexes (#681), respectively, using Lipofectamine2000. Following 36 h incubation, the cells were lysed, subjected to 12% SDS-PAGE, and amount of MuD and Bid was analyzed by western blot using C22B3 MAb (top panel) and anti-Bid Ab (middle panel). The blot was re-probed with anti-β-actin (bottom panel). (d) SV-40-transformed (control), Bid^+/+ and Bid^−/− mouse embryonic fibroblast (MEF) lysates were subjected to 12% SDS-PAGE and analyzed by western blot using C22B3 MAb (top panel) and anti-Bid Ab (middle panel). The blot was re-probed with anti-β-actin (bottom panel). (e) pEGFPC1-MuD transfectants were seeded at a density of 3 × 10⁵ cells in 6-well plates and transfected with control and MuD siRNA duplexes using Lipofectamine2000. Subsequent to 36 h incubation, cells were lysed and subjected to 12% SDS-PAGE. Amount of MuD and Bid was analyzed by western blot. (f) U251-MG cells were grown to 80% confluence in 100-mm culture dishes and then stimulated with TRAIL (200 ng/ml) for the indicated times (0–12 h). The cytoplasmic, mitochondrial and nuclear fractions were isolated from U251-MG cells using a cell fractionation kit (ab109719, Abcam, Cambridge, MA, USA) according to the manufacturer's instructions. Fractions were subjected to 12% SDS-PAGE, and analyzed by western blot using C22B3 MAb to determine the subcellular localization of MuD. The blots were re-probed with anti-Cox IV, anti-tubulin (Abcam) and anti-lamin B (GeneTex, Irvine, CA, USA), respectively. (g) The microsomal fraction was isolated from the cultured cells after TRAIL treatment (200 ng/ml) for 0–12 h using an ER isolation kit according to the manufacturer's instructions (ER0100, Sigma, Saint Louis, MO, USA). Sample lysates were analyzed by 12% SDS-PAGE followed by western blot using C22B3 MAb for MuD detection. The blots were re-probed with anti-calnexin (Abcam), anti-Cox IV and anti-lamin B, respectively. (h) Proposed mechanism underlying the role of MuD in TRAIL-mediated death signaling. MuD resides at the ER and mitochondria regulates apoptotic signaling induced by the formation of TRAIL/TRAIL-R complex. Bid may be essential for the regulation of MuD expression and function. In addition, the formation of 25-kDa Bcl-2 fragments by TRAIL stimulation which converts Bcl-2 from its anti-apoptotic form to the truncated pro-apoptotic form is inhibited by MuD, affecting regulation of caspase activation which subsequently leads to apoptosis.