Characterization of DISC Formation and TNFR1 Trans location to Mitochondria in TNF-α-Treated Hepatocytes

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 a re poorly understood. In characterizing DISC formation within cultured hepatocytes, we demonstrated that TNF-alpha exposure leads to the rapid formation of a DISC involving TNF-alpha, 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-alpha exposure. Similar observations were made in vivo using TNF-alpha 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-alpha-mediated signaling includes the translocation of TNFR1 to mitochondria. (Am J Pathol 2011, 179:1221-1229; DOI: 10.1016/j.ajpath.2011.05.046)