Characterization of DISC formation and TNFR1 translocation to mitochondria in TNF-α-treated hepatocytes

Abstract

Tumor necrosis factor receptor 1 (TNFR1) activation in hepatocytes can trigger apoptotic or inflammatory signaling. The factors that determine which signaling pathway dominates are not clear and are thought to relate to the efficiency of death-inducing signaling complex (DISC) formation. However, the steps involved in DISC formation in hepatocytes are poorly understood. In characterizing DISC formation within cultured hepatocytes, we demonstrated that TNF-α exposure leads to the rapid formation of a DISC involving TNF-α, the TNFR-associated death domain adaptor molecule (TRADD), the Fas-associated death domain adaptor molecule (FADD), caspase-8, TNFR-associated factor 2 (TRAF2), and receptor-interacting protein (RIP). The inclusion of the sensitizing agent actinomycin D both accelerated and amplified the appearance of the DISC. Notably, TNFR1 along with some DISC components also appeared within mitochondria within 30 minutes. Whereas TNFR1 consistently co-localized with the TRADD, FADD, the caspase-8, and TRAF2 in the cytosolic fraction, TNFR1 in the mitochondria was associated only with caspase-8 after TNF-α exposure. Similar observations were made in vivo using TNF-α with D-galactosamine. Actinomycin D alone also enhanced the appearance of DISC components in both cytosol and the mitochondria. Thus the DISC that includes TNFR1 forms in the cytosol of hepatocytes under both survival and pro-apoptotic conditions. The observations also suggest that TNF-α-mediated signaling includes the translocation of TNFR1 to mitochondria.

Figure 1

Effects of TNF-α, TNF-α/Act D, and Act D on hepatocyte cell death. Primary rat hepatocytes were treated with TNF-α (2000 units/mL) ± Act D (200 ng/mL) for 3-, 6-, 9-, and 18-hour treatment(s). *P < 0.01, media group; ^†P < 0.01, group treated with TNF-α alone.

Figure 2

TNFR1 accumulates in the mitochondria following TNF-α, TNF-α/Act D, or Act D treatment in vitro and in vivo. Primary hepatocytes were treated with TNF-α (2000 units/mL) ± Act D (200 ng/mL). A: Fraction purity was assessed using antibodies to β-actin, COX IV, calreticulin, TGN-38, and lamin B. B: The level of TNFR1 protein in cytosolic or mitochondrial was determined by immunoblot (N = 3). Livers were harvested and separated into mitochondrial and cytosolic fractions from rats injected with sterile TNF-α (10 μg/kg) ± D-gal (750 mg/mL). C: Contamination assays using equal amount of proteins measured β-actin, COX IV, calreticulin, TGN-38, and lamin B by immunoblot analysis. D: TNFR1 protein in cytosolic or mitochondrial fractions were determined by immunoblot analysis.

Figure 3

Immunofluorescent confocal microscopy and immunoelectron microscopy confirmed localization of TNFR1 in TNF-α–stimulated primary hepatocytes and isolated mitochondria. Primary rat hepatocytes were treated with TNF-α (2000 units/mL) ± Act D (200 ng/mL) for 1 hour. A: Fluorescent labeling: TNFR1, red; ATP synthase, green; nucleus, gray. B: Sections were labeled with rabbit anti-TNFR1 and then labeled with 10 nmol/L gold-conjugated second antibodies. Note the labeling of TNFR1 in basal level (media) of TNFR1 in mitochondria and increased TNFR1 when treated with TNF-α, TNF-α/Act D, or Act D. Arrows point to TNFR1 positive 10-nm gold beads.

Figure 4

TNFR1 complexes isolated from hepatocytes treated with TNF-α ± sensitizing agents do not contain TRADD in the mitochondria. A: Rat primary hepatocytes were collected and subcellular populations were isolated following treatment with TNF-α (2000 units/mL) ± Act D (200 ng/mL) for the indicated times. Each fraction was analyzed by WB for TRADD. TNFR1 complex was isolated by immunoprecipitation with TNFR1 antibody and then analyzed for the presence of TRADD interaction with TNFR1 by WB. B: TNF-α (10 μg/kg) or D-gal (750 mg/mL) was injected into rats, and the livers were harvested 1 hour later. Cytosolic and mitochondrial fractions were isolated from these livers, followed by WB for TRADD. TNFR1 complex was isolated by immunoprecipitation with TNFR1 antibody and then analyzed for the presence of TRADD interaction with TNFR1 by WB. IP, immunoprecipitated protein.

Figure 5

TNFR1 complexes isolated from hepatocytes treated with TNF-α ± Act D do not contain FADD in the mitochondria. Rat primary hepatocytes were treated with TNF-α (2000 units/mL) for the indicated times ± Act D (200 ng/mL). A: Hepatocytes were then fractionated into cytosolic and mitochondrial lysates and analyzed by WB for FADD. TNFR1 complex was isolated by immunoprecipitation with TNFR1 antibody and then analyzed for the interaction with TRADD with TNFR1 by WB. B: Livers were harvested 1 hour after injections with TNF-α (10 μg/kg) or D-gal (750 mg/mL); mitochondrial and cytosolic fractions were obtained and analyzed by WB for FADD. TNFR1 complex was isolated by immunoprecipitation with TNFR1 antibody and then analyzed for presence of FADD interaction with TNFR1 by WB. IP, immunoprecipitate.

Figure 6

TNFR1 complexes isolated from hepatocytes treated with TNF-α ± D-gal or Act D contain caspase-8 in the mitochondria. Rat primary hepatocytes were collected following treatment with TNF-α (2000 units/mL) for the indicated times ± Act D (200 ng/mL). A: Cytosolic and mitochondrial hepatocyte fractions were analyzed by WB for caspase-8. TNFR1 complex was isolated by immunoprecipitation with TNFR1 antibody and then analyzed by immunoblot for presence of caspase-8 interaction with TNFR1. Livers from rats injected with TNF-α (10 μg/kg) or D-gal (750 mg/mL) were harvested after 1 hour and fractionated into cytosol and mitochondria. B: Each fraction was analyzed by WB for caspase-8. TNFR1 complex was isolated by immunoprecipitation with TNFR1 antibody and then analyzed for presence of caspase-8 interaction with TNFR1 by WB. IP, immunoprecipitated protein.

Figure 7

TNFR1 complexes isolated from TNF-α with or without hepatocytes treated with a sensitizing agent do not contain TRAF2 in the mitochondria. Rat primary hepatocytes were harvested following treatment with TNF-α (2000 units/mL) for the indicated times ± Act D (200 ng/mL). A: Cytosolic and mitochondrial fractions were analyzed by immunoblot for TRAF2. TNFR1 complex was isolated by immunoprecipitation with TNFR1 antibody and then analyzed for presence of TRAF2 interaction with TNFR1 by WB. B: Livers harvested from rats 1 hour after injection with sterile TNF-α (10 μg/kg) or D-gal (750 mg/mL) were separated into cytosolic and mitochondrial fractions and assayed by immunoblot for TRAF2. TNFR1 complex was isolated by immunoprecipitation with TNFR1 antibody and then analyzed for the presence of TRAF2 interaction with TNFR1 by WB. IP, immunoprecipitated protein.

Figure 8

TNFR1 complexes isolated from hepatocytes treated with TNF-α ± a sensitizing agent contain RIP in the mitochondria. A: Rat primary hepatocytes treated with TNF-α (2000 units/mL) for the indicated times ± Act D (200 ng/mL) were fractionated into cytosolic and mitochondrial fractions to be analyzed for RIP content by WB. TNFR1 complex was isolated by immunoprecipitation with TNFR1 antibody and then inmmunoblotted to measure TRAF2 interaction with TNFR1. Livers were harvested from rats 1 hour after injection with sterile TNF-α (10 μg/kg) or D-gal (750 mg/mL). B: Cytosolic and mitochondrial fractions were analyzed by WB for RIP. TNFR1 complex was isolated by immunoprecipitation with TNFR1 antibody and then analyzed for presence of RIP interaction with TNFR1 by WB. IP, immunoprecipitated protein.