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-␣ exposure leads to the rapid formation of a DISC involving TNF-␣, 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-␣ exposure. Similar observations were made in vivo using TNF-␣ 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-␣-mediated signaling includes the translocation of TNFR1 to mitochondria.

Novel Piperazine-based Compounds Inhibit Microtubule Dynamics and Sensitize Colon Cancer Cells to Tumor Necrosis Factor-induced Apoptosis

“…We previously showed that mitotically arrested cells have increased cell surface expression of TNFR1 (18). Increased TNF-TNFR1 interactions at the cell surface (or following TNF internalization) may increase the formation of death-inducing signaling complex and caspase-8 activation (51,52), which has been observed in arrested cells. Interestingly, AK301 was the most potent TNF-sensitizing agent tested in these studies, relative to other well studied microtubule inhibitors (colchicine, nocodazole, and vincristine).…”

A Role for cGMP in Inducible Nitric-oxide Synthase (iNOS)-induced Tumor Necrosis Factor (TNF) α-converting Enzyme (TACE/ADAM17) Activation, Translocation, and TNF Receptor 1 (TNFR1) Shedding in Hepatocytes

“…Immunofluorescence Staining and Confocal Microscopy on Liver Sections-Livers were fixed in 2% paraformaldehyde, and the previously described standardized protocol for cryopreservation was performed (7). Livers were sectioned in a cryostat and stained as follows.…”

TNFR1/Phox Interaction and TNFR1 Mitochondrial Translocation Thwart Silica-Induced Pulmonary Fibrosis

“…Ledgerwood et al (29) used immunoelectron microscopy and Western blotting techniques to demonstrate that TNF is delivered to mitochondria where it interacts with a 60-kDa protein in the inner mitochondrial membrane. Recently, Eum et al (22) reported that TNF treatment of hepatocytes induces the appearance of TNFR1 and caspase 8, but not TRADD, in mitochondria. Consistent with these observations, the current work found the expression of TNFR1, caspase 8, and BID in the mitochondria of RAW 264.7, but not IC21, macrophages (Fig.…”

“…TNF induces mtROS, and TNF is delivered to mitochondria where binding proteins have been documented (22,29). Mitochondria from RAW 264.7, but not IC21, macrophages express TNFR1 (detected by a TNFR1 25-kDa C terminus peptide Ab) (Fig.…”

“…In HeLa cells, TNFR1 activation of NADPH involves the recruitment of riboflavin kinase (RFK) to the death domain of the receptor, where it is coupled to the p22 phox subunit of the oxidase (21). The mechanisms by which TNF induces mtROS production are poorly understood, but TNFR1 translocation to mitochondria could play a role, and cells with mutations in TNFR1 exhibit increased mtROS (22,23). TNF-induced mtROS has been linked to cell death by promoting mitochondrial permeability transition pore formation, cardiolipin (CL) migration to the outer mitochondrial membrane, and cytochrome c mobilization into the cytosol (20,24).…”