Transition from TNF-Induced Inflammation to Death Signaling

Abstract

Tumor necrosis factor (TNF) plays a key role in inflammatory responses and in various cellular events such as apoptosis and necroptosis. The interaction of TNF with its receptor, TNFR1, drives the initiation of complex molecular pathways leading to inflammation and cell death. RARγ is released from the nucleus to orchestrate the formation of the cytosolic death complexes, and it is cytosolic RARγ that plays a pivotal role in switching TNF-induced inflammatory responses to RIPK1-initiated cell death. Thus, RARγ provides a checkpoint for the transition from inflammatory signaling to death machinery of RIPK1-initiated cell death in response to TNF. Here, we use techniques to identify RARγ as a downstream mediator of TNFR1 signaling complex. We use confocal imaging to show the localization of RARγ upon activation of cell death. Immunoprecipitation of RARγ identified the interacting proteins.

1. Introduction

Tumor necrosis factor (TNF) plays a critical role in diverse cellular events, including inflammation, apoptosis, and necroptosis through different signaling complexes [1, 2]. The molecular mechanism involving the interaction of TNF with TNR receptor 1 (TNFR1) and recruitment of several effector proteins has been intensely investigated [1, 2]. However, the transition from inflammatory signaling to death pathways is not well understood. TNF engagement triggers the formation of a TNFR1 signaling complex (complex I) by recruiting effector molecules such as TRADD (TNFR1-associated death domain protein), RIPK1 (receptor interacting protein kinase 1), and TRAF2 (TNFR-associated factor 2) leading to inflammatory responses [3]. However, to trigger cell death pathways, this TNFR1 signaling complex (complex I) dissociates from the receptor and recruits other proteins to form different secondary complexes for apoptosis and necroptosis [4]. The TNFR1 signaling complex dissociates from the receptor and RIPK1 recruits FADD (Fas-associated death domain protein) and Caspase-8 are recruited to form complex IIa and trigger apoptosis and RIPK3 (receptor interacting protein 3) and MLKL (mixed lineage kinase-domain like) are recruited to form the necrosome to carry out necroptosis [4]. When RIPK1 is recruited to the death receptor signaling complex through its death domain interactions, it is key for mediating inflammatory responses such as NF-kB and MAP kinase activation however, for TNF-induced cell death, RIPK1, is the initiator of the death signaling process.

Our recent study demonstrated that cytoplasmic Retinoic acid receptor γ (RARγ), not the nuclear RARγ, is a key regulator of RIPK1-initiated cell death [5]. RARγ is critical for converting the inflammatory response to death signaling by mediating the formation of cytosolic death complexes. TNF-induced cell death (apoptosis and necroptosis) could be initiated by TRADD when de novo protein synthesis is blocked or by RIPK1 when IAP E3 ligases are inhibited. Previous studies suggest that blocking RIPK1 ubiquitination by cIAP1/2 leads to RIPK1 recruitment of FADD and RIPK3, which engages apoptosis and necroptosis respectively [6, 7]. However, our data found that RARγ is essential for the release of RIPK1 from TNFR1 as the nonubiquitinylated RIPK1 was found in the TNFR1 complex and no complex IIa or necrosome was formed in the absence of RARγ. In contrast, the release of TRADD from TNFR1 does not require RARγ. Therefore, RARγ is required for RIPK1-initiated, but not TRADD-initiated, cell death. The functions of RARs have been extensively studied as nuclear transcription factors and the RARs are predominantly nuclear even in the absence of their ligands [8]. To rule out the contribution of RARγ transcriptional activity in cell death, we show that the deletion of the transcription activation domain of RARγ, the DNA-binding domain, and the NLS site interact with the C-terminal of RIP1 and is sufficient to restore the sensitivity of RARγ knockdown cells to TSZ-induced necroptosis. Additionally, it is RARγ and not other RARs such as RARα, that is specifically released from the nucleus. Smac mimetic alone was found to be sufficient to induce the release of RARγ from the nucleus and suggests that cIAPs may play a role in the regulation of RARγ localization. Our in vivo studies showed that RARγ1-knockout mice are resistant to TZ, but not TG treatment, while TRADD knockout mice are resistant to TG, but not TZ treatment [5, 9]. These findings further supported our conclusion that RARγ is required for RIPK1-initiated cell death. RIPK1-initiated cell death is a vital cellular response triggered by death factors and the engagement of this pathway is finely regulated by RARγ [10].

In this chapter, we describe how to analyze the TNFR1 signaling regulated by RARγ using regular Western blot analysis. The actual procedure of immunoblotting will not be described here; however, we provide all the necessary details on how to treat the cells, how to prepare the samples for Western blot, and what antibodies to use. We focus on two methods: Confocal microscopy for detecting nuclear versus cytoplasmic RARγ localization and the immunoprecipitation (IP) of endogenous TNFR1 and specifically serial IPs to determine the composition of the death complex. By utilizing the technique of IP we can analyze the proteins that are recruited to the receptor-signaling complex.

2. Materials

2.1. Common Materials

1. PBS: 137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 2 mM KH₂PO₄.

2. Complete growth medium for cell lines: DMEM, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10% fetal bovine serum.

3. TNF-α (R&D Systems): Reconstitute and store according to the manufacturer’s instructions.

4. z-VAD-fmk (R&D Systems): Reconstitute and store according to the manufacturer’s instructions.

5. Cycloheximide (CHX) (Sigma).

6. Smac mimetic (S. Wang, University of Michigan, Ann Arbor, Michigan, USA).

7. Rotator.

8. 10-cm and 15-cm dishes.

9. 50-mL tubes.

10. 1.5-mL microcentrifuge.

2.2. Immunoprecipitation (IP) and Immunoblotting

1. Antibodies: All antibodies are at concentration of 1 μg/mL and use at 1:1000 dilution unless otherwise stated. Anti-RARγ (C-15, Santa Cruz) for human, anti-RARα (C-20, Santa Cruz), anticaspase-8 (C-20, Santa Cruz), anti-cIAP2 (H-85, Santa Cruz) and anti-Fas (C-20, Santa Cruz), anti-RIP1 (38/RIP, BD Biosciences) and anti-FADD (1/FADD, BD Biosciences), anti-RARγ1 (Abcam) for mouse, anti-RIPK3 (Abcam), anti-RIPK3 (ProSci) for mouse, anti-TRADD (Upstate), and anti-TNFR1 (R&D Systems), anti-Actin (clone AC-40, Sigma) (dilution 1:10,000), anti-FLAG (clone M2, Sigma) (dilution 1:5000) and anti-GFP (clone GFP-20, Sigma) (dilution 1:5000), anti-V5 (Invitrogen) (dilution 1:5000), anticleaved caspase-8 (clone 18C8, Cell Signaling Technology), anti-RIPK1 (Cell Signaling Technology) and anti-p-RIPK1(Cell Signaling Technology), anti-DsRed (Clontech) (dilution 1:5000).

2. Lysis buffer: M2 buffer (20 mM Tris–HCl, pH 7.5, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA) supplemented with 2 mM DTT, 0.5 mM PMSF, 20 mM β-glycerol phosphate, 1 mM sodium vanadate, 1 μg/mL leupeptin (see Note 1).

3. Protein G-agarose beads.

4. Criterion^™ TGX^™ (Tris-Glycine extended, Bio-Rad) precast gels for poly acrylimide gel electrophoresis (PAGE).

5. SDS loading buffer: 5× solution of Tris base, 10% SDS, bromophenol blue, and glycerol (Quality Biological).

6. Tris-Glycine-SDS running buffer: Purchased from Bio-Rad as a 10× premixed electrophoresis buffer, containing 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3 following dilution to 1× with water.

7. Criterion^™ wet transfer system.

8. Tri-Glycine transfer buffer: Purchased from Bio-Rad as a 10× premixed electrophoresis buffer contains 25 mM Tris, 192 mM glycine, pH 8.3, following dilution to 1× with water.

2.3. Confocal Microscopy

1. Plasmid: pRARγ-GFP.

2. HeLa cells: Medium for the culturing: DMEM, 2 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 10% fetal bovine serum.

3. 3-cm Ibidi plates (Ibidi).

4. Dimethyl sulfoxide (DMSO).

5. 3% paraformaldehyde.

6. 4′,6-diamidino-2-phenylindole (DAPI).

7. Antibodies: anti-F-actin (SIGMA) and anti-RARγ (C-15, Santa Cruz) antibodies.

8. Transfection reagent: Lipofectamine-Plus reagent (Invitrogen). Use as per manufacturer’s recommendation.

9. A confocal microscope (Carl Zeiss LSM780 confocal microscope) equipped with a Plan-Apochromat 63 Å ~ numerical aperture 1.40 DIC oil objective.

3. Methods

3.1. Treatment of Cells with Cell Death Conditions

1. Day 1: Plate MEFs at 3–5 × 10⁶ in a 10 cm dish in complete growth medium overnight at 37 °C.

2. Day 2: Treat the cells with either necroptosis or apoptosis conditions.

3. To induce necroptosis use TSZ treatment [(TNF-α (30 ng/mL), Smac mimetic (10 nM), z-VAD-fmk (20 μM)] or TCZ [(TNF-α (30 ng/mL), CHX (cycloheximide, 10 μg/mL), z-VAD-fmk (20 μM)].

4. To induce Apoptosis, treat the cells with TNF-α (30 ng /mL) and Smac mimetic (10 nM) or TNF-α (30 ng/mL) and CHX (10 μg/mL).

5. At different time points, collect the cells and wash once with PBS and use them for Western blot analysis.

3.2. Preparation of Samples for Western Blot

1. Collect cells after cell death treatment (see Note 2), at different time points depending on your experiment.

2. Prepare 1.5-mL microcentrifuge tubes on ice.

3. Collect the cells from each dish or well to a separate 1.5-mL microcentrifuge tube.

4. Centrifuge tubes for 5 min at 300 × g at 4 °C.

5. Aspirate the supernatant, add 1 mL of ice-cold PBS, and centrifuge for 5 min at 300 × g at 4 °C.

6. Aspirate supernatant and resuspend the pellet in 30 μL of M2 lysis buffer (see Note 3).

7. Rotate tubes in a rotator for 30 min at 4 °C.

8. Centrifuge tubes for 10 min at 15,000–20,000 × g at 4 °C.

9. Transfer the supernatant to a fresh 1.5-mL microcentrifuge tube without disturbing the pellet. The supernatant can be frozen at −70 °C.

10. Measure the concentration of proteins in the sample.

3.3. Immunoprecipitation (IP)

3.3.1. Preparation of the Cell Lysate

1. Plate 5–6 × 10⁶ cells in a 15-cm dish and treat with cell death conditions. (see Note 4).

2. Prepare 50-mL tube with 20 mL of ice-cold PBS.

3. After treatment, quickly transfer the cells from the plate to the tube with PBS. Collect the residual cells with an additional 10 mL of ice-cold PBS.

4. Centrifuge tubes for 5 min at 300 × g at 4 °C.

5. Aspirate supernatant and resuspend the pellet in 1 mL of M2 lysis buffer that has no DTT. This resuspended pellet can be frozen at −70 °C for future use.

6. Transfer the resuspended pellet to a 1.5-mL microcentrifuge tube.

7. Rotate samples on a rotator for 30 min at 4 °C.

8. Centrifuge tubes for 10 min at 15,000–20,000 × g at 4 °C.

9. Transfer the supernatant to a fresh 1.5-mL microcentrifuge tube without disturbing the pellet. This supernatant can be frozen at −70 °C.

10. Measure the concentration of proteins in the sample. Take 1 mg for IP and 1–3% (w/w) for the input sample (loading control).

3.3.2. Washing the Beads and Preparation of 50% Bead Slurry

1. Take enough quantity of protein G-agarose beads (see Note 5).

2. Centrifuge for 1 min at 500 × g at 4 °C.

3. Resuspend the pellet in the lysis buffer equal to the volume of beads taken. This is now 50% bead slurry.

3.3.3. Preclearance of the Lysate

1. Bring the volume of the protein lysate taken for IP to 1 mL with the lysis buffer and add 40 μL of 50% bead slurry.

2. Rotate on a rotator for 1 h at 4 °C.

3. Centrifuge for 2 min at 500 × g at 4 °C.

4. Transfer the supernatant to a fresh 1.5-mL microcentrifuge tube. Be careful not to transfer beads as the bead pellet is very loose (see Note 6).

3.3.4. Immunoprecipitation

1. Add 1 μg of the antibody (anti-TNFR1, anti- RARγ, or anticaspase 8) of the protein to be immunoprecipitated and 30 μL of 50% protein G-agarose bead slurry to 1 mL of cell lysate.

2. Rotate tubes on a rotator overnight at 4 °C.

3. Centrifuge tubes for 2 min at 500 × g at 4 °C.

4. Very carefully aspirate the supernatant leaving some liquid above the pellet and add 1 mL of M2 lysis buffer (see Note 6).

5. Rotate tubes on a rotator for 10 min at 4 °C.

6. Centrifuge tubes for 2 min at 500 × g at 4 °C.

7. Wash beads four more times (repeat steps 4–6).

8. After the final wash, aspirate as much supernatant as possible, and resuspend the beads in 40 μL of SDS loading buffer.

9. Denature the immunoprecipitated samples and input samples by heating at 100 °C for 3 min.

10. Resolve the samples in 4–20% SDS-polyacrylamide gels for western blot analysis. Use the following antibodies to detect proteins in the signaling complex: anti-TNFR1, anti-TRADD, anti-RARγ, anti-RIPK3, anti-RIPK1, and anticaspase 8 antibodies (see Note 7). Visualize the proteins by enhanced chemiluminescence.

3.4. Confocal Microscopy

1. Plasmid Expression in HeLa Cells.

   1. Culture 1 × 10⁵ HeLa cells in 3-cm Ibidi plates for 24 h.
   2. Transfect the HeLa cells with 1 μg of pRARγ-GFP by Lipofectamine-Plus reagent as per manufacturer’s recommendation (see Note 8).

2. For overexpression proteins, 24 h post transfection, treat the cells with DMSO (1%), TNF-α (30 ng/mL) or TS [(TNF-α (30 ng/mL), Smac mimetic (10 nM)] or TSZ [(TNF-α (30 ng/mL), Smac mimetic (10 nM), z-VAD-fmk (20 μM)] for 0–2 h.

3. Immunofluorescent Staining.

   1. Assess the localization of endogenous RARγ by immunofluorescent staining. Fix the treated HeLa cells with 3% paraformaldehyde by intermittent shaking for 20 min at room temperature.
   2. Stain the cells with anti-F-actin and anti-RARγ antibodies, and nucleus with DAPI and directly visualize by confocal microscopy (see Note 9).
   3. For overexpression proteins, stain the treated HeLa cells with nuclear DAPI and directly visualize by confocal microscopy.

4. Perform confocal imaging and analysis. We visualized confocal images using a Carl Zeiss LSM780 confocal microscope equipped with a Plan-Apochromat 63 Å ~ numerical aperture 1.40 DIC oil objective. Analyze acquired images by using Carl Zeiss ZEN software.

4. Notes

1. Basic M2 buffer can be prepared in a large amount and stored at 4 °C. If protease inhibitors are added, lysis buffer should be stored in aliquots at −20 °C. After thawing, discard unused buffer.

2. For early TNFR1 complexes, an early time point of 30 min should be used. For later complexes, a time of 2 h should be used.

3. Signaling complexes are formed within 5 min of treatment. It is important to keep everything on ice and lyse the cells quickly after treatment.

4. Cells should be plated the night before. The number of cells will vary based on the type of cell line. Plate them so that they will be at 60–80% confluency.

5. For each sample, you will need 40 μL of beads. Since the beads will be used twice (to preclear the lysate and for the actual IP), the total amount should be doubled.

6. The bead pellet is very loose. Be very careful. We usually use protein gel-loading tips or 30 G needles to suck the supernatant off.

7. When probing with RIPK1 antibodies in addition to the actual RIP1 band, you should be able to see high-molecular-weight smear, which corresponds to ubiquitination of RIPK1.

8. Human RARγ transcript variant 1 (NM_000966) was cloned into the mammalian expression vector pEGFP-C1 to generate pRARγ-GFP.

9. DAPI is used to visualize the nucleus and is added a few minutes before imaging if you wait too long the staining may be too strong.