Assessing Bad Sub-cellular Localization Under Conditions Associated with Prevention or Promotion of Mitochondrial Permeability Transition-Dependent Toxicity

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 648)

Abstract

Cells belonging to the monocyte/macrophage lineage are in general highly resistant to peroxynitrite, a reactive nitrogen species extensively produced by these and other cell types under inflammatory conditions. Resistance is not dependent on the scavenging of peroxynitrite but is rather associated with the prompt activation of a survival signaling in response to various molecules largely available at the inflammatory sites, as arachidonic acid and products of the 5-lipoxygenase or cyclooxygenase pathways. We detected significant levels of Bad in the mitochondria of monocytes/macrophages and found that these signaling pathways converge in Bad phosphorylation, and thus in its cytosolic accumulation. Phosphorylation inhibits binding of Bad to Bcl-2, or BclXL, and promotes its translocation to the cytosol, thereby enabling Bcl-2 and BclXL to exert effects leading to prevention of mitochondrial permeability­ transition (MPT). Upstream inhibition of the survival signaling indeed promotes the mitochondrial accumulation of Bad and the rapid onset of MPT-dependent toxicity. The above results contribute to the definition of the mechanism(s) whereby monocytes/macrophages survive to peroxynitrite in inflamed tissues.

Key words

Bad Peroxynitrite Mitochondria Mitochondrial permeability transition Inflammation Sub-cellular localization Western blot analysis 

1 Introduction

The Bcl-2 family of proteins, a critical death regulator that resides immediately upstream of mitochondria, comprises anti-apoptotic/pro-survival members (Bcl-2, BclXL, Bcl-w, Mcl-1 and A1) and two groups of pro-apoptotic/pro-death members, the “multidomain” (Bax, Bak and Bok) and “BH3-domain only” members (Bim, Bad, Bid, Bik, Bmf, Puma, Noxa and Hrk). Pro- and anti-apoptotic family members may heterodimerize, thereby affecting their respective functions, and their activity is regulated either at the transcriptional or posttranslational levels.

Bad (Bcl-2 antagonist of cell death) is a positive regulator of cell death and, like other BH3-only proteins, selectively displaces Bax to heterodimerize with Bcl-2 or BclXL. Thus, Bad may activate Bax simply by freeing it from the above pro-survival proteins. The pro-death effect of Bad is dependent on its ability to dimerise with pro-survival members of the Bcl-2 family (1, 2).

In the absence of survival stimuli, endogenous Bad is dephosphorylated and localized in the outer mitochondrial membrane. In the presence of survival factors, Bad is promptly phosphorylated on serine 136 by phosphatidylinositol 3-kinase/Akt (3, 4) or on serine 112 by either protein kinase A (PKA) or protein kinase C (PKC)/p90RSK (5, 6). Phosphorylation at serine 112, or 136, allows Bad sequestration in the cytoplasm, bound to 14–3–3 scaffold proteins. PKA-dependent phosphorylation at serine 155 has also been reported and shown to reduce the affinity of Bad for pro-survival Bcl-2 family members (7).

Our previous work has shown that pro-monocytic cell lines, and human monocytes/macrophages, survive to peroxynitrite because of their ability to respond to molecules largely available in the inflammatory milieu (e.g. arachidonic acid) with the triggering of events converging in Bad phosphorylation (8, 9). In particular, we showed that 5-hydroxyeicosatetraenoic acid (5-HETE), a product of 5-lipoxygenase (5-LO), promotes the mitochondrial translocation of PKCα and the ensuing phosphorylation of Bad (10). Inhibition of the survival signaling at the level of cytosolic phospholipase A2, 5-LO or PKCα rapidly leads to the mitochondrial accumulation of Bad, associated with the rapid onset of mitochondrial permeability transition (MPT)-dependent toxicity. Cells, however, can nevertheless be rescued by supplementation of products downstream to the inhibited step or, in alternative, via EP2 receptor-mediated, PKA-dependent Bad phosphorylation elicited by exogenous PGE2 (11). A third Bad kinase, Akt, is severely inhibited by peroxynitrite in U937 cells.

In short, the survival strategy adopted by monocytes/macrophages to cope with peroxynitrite is simple, yet very effective: while committed to MPT-dependent toxicity, these cells nevertheless survive by promoting Bad phosphorylation via different pathways. It is interesting to note that these pathways are triggered by molecules extensively produced under inflammatory conditions (as peroxynitrite) by monocytes/macrophages themselves, as well as by other cell types.

2 Materials

2.1 Cell Culture and Treatments

  1. 1.

    RPMI 1640 Medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT), penicillin (100 U/ml), and streptomycin (100 μg/ml) (HyClone).

     
  2. 2.

    T-75 tissue culture flasks (Sarstedt, Nümbrecht, Germany).

     
  3. 3.

    Peroxynitrite was synthesized by the reaction of nitrite with acidified H2O2, as described previously (12), with minor modifications (13) (see Note 1).

     
  4. 4.

    Saline A: 8.182 g/l NaCl, 0.372 g/l KCl, 0.336 g/l NaHCO3, and 0.9 g/l glucose, pH 7.4 at 37°C.

     

2.2 Cell Fractionation

  1. 1.

    Extraction buffer: 20 mM HEPES-KCl (pH 7.4), 10 mM KCl, 250 mM sucrose, 1.5 mM MgCl2, 1 mM sodium EDTA, 1 mM sodium EGTA. Immediately before use add 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 10 μg/ml aprotinin.

     
  2. 2.

    Lysis buffer: 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% NP40. Immediately before use add 1 mM DTT, 1 mM PMSF, 10 mM sodium orthovanadate (Na3VO4), 10 mM sodium fluoride (NaF), 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 10 μg/ml aprotinin.

     

2.3 SDS-Polyacrylamide Gel Electrophoresis

  1. 1.

    Bio-Rad Dye Binding protein assay (Bio-Rad Laboratories, Hercules, CA).

     
  2. 2.

    Mini gel electrophoresis apparatus (e.g., BioRad Mini-PROTEAN 3 electrophoresis system).

     
  3. 3.

    Loading buffer: 125 mM Tris–HCl (see Note 2), 2% (w/v) sodium dodecyl sulphate (SDS), 10% glycerol, 0.002% bromophenol blue, pH 6.9. Prepare a 4× solution and store in aliquots at −20°C. Before use add 100 mM DTT.

     
  4. 4.

    Upper Tris (4×): 0.5 M Tris–HCl, pH 6.8. Store at 4°C (see Note 2).

     
  5. 5.

    Lower Tris (4×): 1.5 M Tris–HCl, pH 8.8. Store at 4°C (see Note 2).

     
  6. 6.

    Forty percent acrylamide/bisacrylamide solution: 39:1 w/w; this is a neurotoxin when unpolymerized and so care should be taken not to receive exposure (see Note 3).

     
  7. 7.

    Ammonium persulfate (APS): prepare a 10% solution in water (see Note 4).

     
  8. 8.

    Stacking gel solution (4%): prepare 100 ml of solution by mixing 64 ml of water, 10 ml of 40% acrylamide, 25 ml of Upper Tris, 1 ml of 10% SDS. Store at 4°C. Add 4 μl of N,N,N,N′-Tetramethyl-ethylendiamide (TEMED, Sigma-Aldrich) (see Note 5) and 40 μl of APS to 4 ml of the stacking gel solution.

     
  9. 9.

    Running gel solution (12.5%): prepare 10 ml of solution by mixing 4.3 ml of water, 3.1 ml of 40% acrylamide, 2.5 ml of Lower Tris, 100 μl of 10% SDS, 14 μl of TEMED and 60 μl of APS.

     
  10. 10.

    Pre-stained molecular weight markers: Color Burst (Sigma-Aldrich).

     

2.4 Western Blotting for Mitochondrial and Cytosolic Fractions

  1. 1.

    Blotting apparatus (e.g., Bio-Rad Mini-PROTEAN 3 full immersion electroblotting system).

     
  2. 2.

    Electrophoresis Buffer (10×): 250 mM Tris (pH 8.3), 1.92 M glycine, 0.1% SDS. Store at room temperature.

     
  3. 3.

    Transfer Buffer (10×): 250 mM Tris (pH 8.3), 1.92 M glycine. Store at room temperature, dilute 1:10 in water and add 20% (v/v) methanol prior to use (see Note 6).

     
  4. 4.

    Polyvinyldiene (PVDF) membrane (GE Healthcare).

     
  5. 5.

    Tris-buffered saline with Tween (TBS-T): prepare a 10× solution with 1.40 M NaCl, 500 mM Tris–HCl, pH 7.2. Dilute 100 ml with 900 ml water for use and complete with 0.1% Tween-20 (see Note 7).

     
  6. 6.

    Blocking buffer: 5% (w/v) nonfat dry milk in TBS-T.

     
  7. 7.

    Primary antibody dilution buffer: dilute Bad primary antibody (BD Transduction Laboratories, Lexington, KY) 1:700 in 4 ml of blocking buffer (see Note 8). Dilute anti-actin and anti-HSP-60 antibodies (Santa Cruz, Santa Cruz, CA) 1:1,000 in 4 ml of blocking buffer.

     
  8. 8.

    Secondary antibody: dilute antimouse IgG conjugate to horse radish peroxidase (Santa Cruz) 1:2,000 in 4 ml of blocking buffer.

     
  9. 9.

    Enhanced chemiluminescent (ECL) reagent (Sigma-Aldrich): mix 10 ml of solution A (1 M Tris–HCl, pH 8.6, 1.25 mM luminol) with 3.2 μl of 30% H2O2 and 50 μl of solution B (6.7 mM p-Coumaric acid in DMSO).

     
  10. 10.

    X-ray film and exposure cassette.

     

3 Methods

A significant amount of Bad is normally detected in the mitochondria of untreated U937 cells. Exposure of these cells to a non-toxic, but nevertheless MPT-committing concentration of peroxynitrite, promotes the mitochondria to cytosol translocation of Bad, an event critical for survival, as MPT-dependent toxicity is readily observed under conditions in which upstream inhibition of the survival signaling enforces the mitochondrial accumulation of Bad. In these experiments, sub-cellular localization of Bad is conveniently measured with an anti-Bad antibody. As indicated in Fig. 1, peroxynitrite promotes the cytosolic accumulation of Bad via a mechanism sensitive to either AA861 (a 5-LO inhibitor) or Gö6850 (a PKC inhibitor). Fig. 1 also shows that loss of mitochondrial Bad, associated with the cytosolic accumulation of the protein, is promptly re-established upon addition of 5-HETE (a 5-LO product) to 5-LO inhibited cells. When this signaling is intercepted downstream to 5-LO, with a PKC inhibitor, accumulation of Bad can also be promoted via a PKA-dependent signaling triggered by PGE2.
Fig. 1.

Peroxynitrite promotes the mitochondrial loss of Bad and its cytosolic accumulation, via a 5-LO/PKC-dependent mechanism mimicked by exogenous PGE2. U937 cells were treated for 3 min with 100 μM peroxynitrite and subsequently exposed for a further 7 min to 1 μM AA861 (5-LO inhibitor), 0.3 μM 5-HETE, 3 μM Gö6850 (PKC inhibitor) or 0.3 μM PGE2, as detailed in the figure. After treatments, mitochondrial and cytosolic fractions were isolated and processed for Western blot analysis using an antibody against Bad. Blots were subsequently re-probed for actin and HSP-60 to assess the purity of mitochondrial fraction and the equal loading of the lanes. Blots are representative of three separate experiments.

3.1 Preparation of Samples for Detection of Bad by Western Blotting

  1. 1.

    U937 human myeloid leukaemia cells were cultured in suspension in RPMI 1640 Medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37° in T-75 tissue culture flasks gassed with an atmosphere of 95% air-5% CO2. Isolation of cytosolic and mitochondria-enriched fractions requires at least 5 ×106 U937 cells.

     
  2. 2.

    All the materials required for treatments and cell fractionation are made ready: inhibitors of 5-LO and PKC, as well as 5-HETE and PGE2, at appropriate stock concentrations for 1:1,000 dilution into the cultures; a centrifuge tube for each sample; a vacuum aspirator; cooled centrifuges, and cold extraction and lysis buffer.

     
  3. 3.

    Treatments were performed in pre-warmed saline A containing 2.5 ×105cells/ml. The cell suspension (20 ml) was inoculated into 50 ml plastic tubes before addition of peroxynitrite. Peroxynitrite was added on the wall of these tubes and immediately mixed to equilibrate its concentration on the cell suspension (see Note 9).

     
  4. 4.

    After treatments (10 min), collect the cells by centrifugation (3 min, 4°C) at 1,300×g.

     
  5. 5.

    Add cold extraction buffer to the cell pellet (35 μl extraction buffer/1 ×106cells) and incubate for 10 min. Homogenize the cells by 40 passages through a 26-gauge needle (see Note 10). Centrifuge the cell lysate (5 min) at 1,000 ×g to remove nuclei, unbroken cells, and cell debris. The resulting supernatant should then be centrifuged (30 min) at 12,000×g to obtain the mitochondrial fraction (pellet). The cytosolic fraction is obtained from the supernatant upon further purification with an additional centrifugation (1 h) at 10,000×g. The cytosolic fraction is maintained at 4°C prior to electrophoresis. The mitochondrial fraction is suspended in 55 μl of cold lysis buffer, incubated for 20 min on ice and finally centrifuged (5 min) at 15,000×g to obtain the solubilised enriched mitochondrial fraction.

     
  6. 6.

    Protein concentration is determined with the Bio-Rad Dye Binding protein assay (see Note 11).

     
  7. 7.

    Equal amounts (20 μg, see Note 12) of the mitochondrial and cytosolic fractions are diluted in loading buffer and incubated for 5–10 min at 95°C.

     

3.2 SDS-PAGE

  1. 1.

    Carefully clean and extensively rinse the glass plates of the gel cassette.

     
  2. 2.

    Prepare a 1.0 mm thick 12.5% gel by mixing 4.3 ml of water, 3.1 ml of 40% acrylamide, 2.5 ml of Lower Tris, 100 μl of 10% SDS, and 14 μl of TEMED. 500 μl of this solution should be mixed with an excess APS (50–60 µl, promoting a rapid polymerization) to prepare a 2–3 mm thick border on the bottom of the gel cassette. Complete the running gel with the above gel solution (approximately 9.5 ml) and APS (60 µl) and immediately pour into the gel cassette (6 cm from the bottom). Overlay with isobutanol (about 700–800 µl) and allow polymerization for about 30 min.

     
  3. 3.

    Pour off the isobutanol layer, rinse twice the top of the gel with water and then carefully dry with a thin blotting-paper.

     
  4. 4.

    Add to the stacking gel solution (4 ml), prepared as detailed above (Subheading 2.3, point 8), 4 μl of TEMED and 40 μl of APS. Immediately pour this solution on top of the running gel. Insert the comb and allow polymerization for about 30 min.

     
  5. 5.

    Prepare the electrophoresis buffer by diluting 100 ml of the 10× electrophoresis buffer (Subheading 2.4, point 2) with 900 ml of water.

     
  6. 6.

    Electrophoresis was performed with a Bio-Rad Mini-PROTEAN 3 electrophoresis gel system. Place the gel cassette sandwich into the electrode assembly and then in the inner chamber of the electrophoresis module. The inner chamber is finally lowered into the mini tank.

     
  7. 7.

    Fill the inner chamber with the electrophoresis buffer and subsequently add the same solution in the mini tank (3 cm from the bottom). Carefully remove the comb and use a 50 μl syringe, fitted with a 22-gauge needle (see Note 13), to rinse the wells with the electrophoresis buffer.

     
  8. 8.

    Load each sample in a well with the same 50 µl syringe. Carefully rinse the syringe with electophoresis buffer when loading the different samples. Load one well with pre-stained molecular weight markers. The first and the last wells should be loaded with the loading buffer alone.

     
  9. 9.

    Complete the assembly of the gel unit and connect to a power supply. The gel is generally run at 55 V through the stacking gel and at 110–120 V through the running gel. In order to prevent loss of low molecular weight proteins, electrophoresis should be stopped immediately prior to or soon after the dye front runs off the gel. The dye front is blue because of the presence of bromophenol blue.

     

3.3 Western Blotting for Detection of Mitochondrial and Cytosolic Bad

  1. 1.

    After SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE), proteins are transferred to PVDF membranes using a Bio-Rad Mini-PROTEAN 3 full immersion electroblotting system.

     
  2. 2.

    Place the transfer cassette in a tray and add enough transfer buffer (Subheading 2.4, point 3) to submerge the lower side of the cassette, in which a piece of sponge sheet and three sheets of blotting-paper are sequentially layered.

     
  3. 3.

    The gel unit is disconnected from the power supply and disassembled. Remove the stacking gel and soak (30 s) the remaining portion of the gel in transfer buffer. Place the gel on top of the blotting-paper and make sure that it remains submerged in the transfer buffer.

     
  4. 4.

    Soak a PVDF membrane (approximately 6×9 cm, same size as the running gel) in 100% methanol (1 min). The membrane should then be rinsed with the transfer buffer (1–2 min) and equilibrated in the same buffer for at least 30 min. Finally, place the membrane on top of the gel in the transfer cassette.

     
  5. 5.

    Carefully lay three sheets of blotting-paper and a sponge (pre-wetted with the transfer buffer) on top of the PVDF membrane, ensuring that no bubbles are trapped in the resulting sandwich. Close the transfer cassette.

     
  6. 6.

    Place the cassette into the transfer tank. In order to prevent loss of the proteins into the transfer buffer, the PVDF membrane should be between the gel and the anode.

     
  7. 7.

    Insert the Bio-Ice cooling unit in the transfer tank. This is necessary to optimize protein transfer and to prevent damage to the electroblotting system due to heat generated during transfer.

     
  8. 8.

    Fill the transfer tank with the transfer buffer.

     
  9. 9.

    Complete the assembly of the electroblotting system and connect to a power supply (36 V for 75 min).

     
  10. 10.

    After protein transfer, the transfer cassette should be removed from the tank and immediately disassembled. Remove the upper sponge/blotting-paper sheets and lay the PVDF membrane on clean paper. The gel and blotting-paper can be discarded. Mark positions of molecular weight markers with a pen (see Note 14).

     
  11. 11.

    The membrane is then incubated for 1 h (at room temperature) in 20 ml of blocking buffer (Subheading 2.4, point 6) on a rocking platform to prevent nonspecific protein binding.

     
  12. 12.

    Discard the blocking buffer and quickly rinse the membrane prior to addition of the anti-Bad antibody (diluted as detailed in Subheading 2.4, point 7). The membrane is then incubated overnight (4°C) on a roller mixer.

     
  13. 13.

    After overnight incubation, wash the membrane with 20 ml TBS-T at room temperature (1×quick rinse, 1×15 min and 2×10 min).

     
  14. 14.

    The secondary antibody should be freshly prepared (Subheading 2.4, point 8) in each experiment. Add the antibody-containing solution to the membrane and allow incubation for 90 min (at room temperature) on a rocking platform.

     
  15. 15.

    Discard the secondary antibody and wash the membrane three times (10 min each) with 20 ml of TBS-T.

     
  16. 16.

    Soak the membrane in ECL reagent under safe light conditions. Rotate the membrane by hand for 1 min (to ensure even coverage), remove the excess ECL reagent with blotting paper and immediately place the membrane between the leaves of an acetate sheet protector (cut to the size of an X-ray film cassette) (see Note 15).

     
  17. 17.

    The final step is performed in the dark room. Place the membrane-containing acetate sheet in an X-ray film cassette. Place the film on top of the membrane for a suitable exposure time, typically 10 min. This time can be decreased or increased, depending on the results obtained.

     

3.4 Re-probing Blots for Actin and HSP-60

  1. 1.

    After film exposure, strip the membrane of the Bad signal and re-probe with anti-actin or anti-HSP-60 antibodies to assess equal loading of the lanes and the purity of the mitochondrial fractions.

     
  2. 2.

    Since the above proteins display remarkably different molecular weights, the stripping procedure is simplified as detailed below.

     
  3. 3.

    Wash the membrane with TBS-T, for 1 h, on a rocking platform with occasional change of buffer. The membrane is now ready to be re-probed with anti-actin or anti-HSP-60 (diluted as detailed in Subheading 2.4, point 7) antibodies, using the same procedure described for the anti-Bad antibody.

     

4 Notes

  1. 1.

    Solutions should be prepared in distilled or deionized water.

     
  2. 2.

    Prepare the Tris buffer by dissolving the Tris base in water and adjusting the pH with HCl. Adjust to pH 6.8 for Upper Tris and to pH 8.8 for Lower Tris. Tris base cannot be replaced by Tris–Cl, or Trizma, as the salt concentration would be too high and, under these conditions, polypeptides migrate anomalously through the gel, yielding extremely diffuse bands.

     
  3. 3.

    In solution, acrylamide and bisacrylamide are slowly converted during storage to acrylic acid and bisacrylic acid. These reactions are catalyzed by light or alkali. Check that the pH of the solution is 7.0 or less. Store the solution in dark bottles. Fresh solutions should be prepared every few months.

     
  4. 4.

    APS provides the free radicals that drive polymerization of acrylamide and bisacrylamide. A small amount of a 10% (w/v) stock solution should be prepared in water and stored at 4°C. APS decomposes slowly. Fresh solutions should be prepared weekly.

     
  5. 5.

    TEMED may decline in quality after opening (gels will take longer to polymerize).

     
  6. 6.

    Transfer buffer can be used for two transfers within 1 week.

     
  7. 7.

    Tween-20 is a viscous solution and causes problems during pipetting, partially avoided by cutting the pipette tip.

     
  8. 8.

    Once diluted, the primary antibody can be stored at −20°C for up to 3 weeks. In addition, the primary antibody can be used in three different blots with the only adjustment required for increasing length of exposure to film at the ECL step.

     
  9. 9.

    To avoid changes in pH due to the high alkalinity of the peroxynitrite stock solution, an appropriate amount of 1.5 N HCl, approximately the same amount of the peroxynitrite stock solution, normally made in 1.5 N NaOH, was also added to the wall of the tubes prior to peroxynitrite. Note that this procedure has to be fast since peroxynitrite rapidly decomposes at neutral pH.

     
  10. 10.

    Note that the numbers of passages necessary to lyse the cells may differ significantly among different cell types. The status of the cells can be conveniently monitored after addition of trypan blue under a microscope.

     
  11. 11.

    The fractionation procedure of U937 cells normally yields about 1.7 mg/ml proteins for the cytosolic fraction and 1.2 mg/ml proteins for the mitochondrial fraction.

     
  12. 12.

    Apply 5–20 μg proteins to each well of a 0.75–1.0 mm thick gel. For thicker gels (i.e. 1.5 mm), apply up to 25–40 μg in each well.

     
  13. 13.

    A syringe is normally used for 0.75–1.0 mm thick gels. Use a micropipette for 1.5 mm thick gels.

     
  14. 14.

    Interrupt the experiments if colored molecular weight markers are not clearly visible on the membrane. It indicates that something did not work during transfer.

     
  15. 15.

    Apply a luminescent sticker on the edge of the acetate sheet to provide an alignment mark for the film and the membrane.

     

Notes

Acknowledgments

This work was supported by grant from AIRC and PRIN (OC).

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Institute of Pharmacology and PharmacognosyUniversity of Urbino “Carlo Bo”UrbinoItaly

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