Tumor necrosis factor receptor 1 and its signaling intermediates are recruited to lipid rafts in the traumatized brain

Abstract

The tumor necrosis factor (TNF) ligand-receptor system plays an essential role in apoptosis that contributes to secondary damage after traumatic brain injury (TBI). TNF also stimulates inflammation by activation of gene transcription through the IkappaB kinase (IKK)/NF-kappaB and JNK (c-Jun N-terminal protein kinase)/AP-1 signaling cascades. The mechanism by which TNF signals between cell death and survival and the role of receptor localization in the activation of downstream signaling events are not fully understood. Here, TNF receptor 1 (TNFR1) signaling complexes in lipid rafts were investigated in the cerebral cortex of adult male Sprague Dawley rats subjected to moderate (1.8-2.2 atmospheres) fluid-percussion TBI and naive controls. In the normal rat cortex, a portion of TNFR1 was present in lipid raft microdomains, where it associated with the adaptor proteins TRADD (TNF receptor-associated death domain), TNF receptor-associated factor-2 (TRAF-2), the Ser/Thr kinase RIP (receptor-interacting protein), TRAF1, and cIAP-1 (cellular inhibitor of apoptosis protein-1), forming a survival signaling complex. Moderate TBI resulted in rapid recruitment of TNFR1, but not TNFR2 or Fas, to lipid rafts and induced alterations in the composition of signaling intermediates. TNFR1 and TRAF1 were polyubiquitinated in lipid rafts after TBI. Subsequently, the signaling complex contained activated caspase-8, thus initiating apoptosis. In addition, TBI caused a transient activation of NF-kappaB, but receptor signaling interacting proteins IKKalpha and IKKbeta were not detected in raft-containing fractions. Thus, redistribution of TNFR1 in lipid rafts and nonraft regions of the plasma membrane may regulate the diversity of signaling responses initiated by these receptors in the normal brain and after TBI.

Figure 1.

TBI induces TNFR1 signaling intermediates in the injured cortex. Immunoblot analysis of TNFR1, TNFR2, TRADD, TRAF1, TRAF2, RIP, FADD, cIAP-1, cIAP-2, caspase-8, IKKα, and IKKβ proteins after TBI. Rats were subjected to moderate TBI, and cerebral cortices were harvested at 5, 15, 30, and 60 min and 4 hr after injury. Levels of TNFR1, TRADD, TRAF1, TRAF2, cIAP-2, IKKα, IKKβ, and caspase-8 are increased on the side of the injury early after trauma. Asterisk denotes the first time point at which significant increases in protein levels were measured. ^*p < 0.05 versus contralateral; error bars represent ±SDs. I, Ipsilateral (injury) cortex; C, contralateral cortex.

Figure 2.

Partitioning of TNFR1 in lipid rafts. A, Cortical samples from naive and traumatized brains at 15 min after injury were lysed in 1% Triton X-100 and subjected to sucrose density centrifugation to isolate lipid rafts. Proteins of equal volume of representative collected fractions were separated by SDS-PAGE and analyzed by immunoblotting using specific antibodies against TNFR1, TNFR2, Fas, flotillin-1, and caveolin-1. B, Quantification of the total TNFR1 protein in naive and TBI groups. R, Raft; S, soluble fractions; P, pellet.

Figure 3.

TBI induces recruitment of signaling molecules into lipid rafts. Rats were subjected to moderate TBI, and cortices were harvested at 5, 15, 30, and 60 min and 4 hr after injury. Triton X-100 soluble (S) and insoluble lipid raft (R) fractions were isolated. Equal aliquots of the fractions were subjected to SDS-PAGE, and the protein distribution was assessed by immunoblotting using specific antibodies against TNFR1, TRADD, TRAF1, TRAF2, RIP, FADD, cIAP-1, cIAP-2, caspase-8, IKKα, IKKβ, flotillin-1, and caveolin-1.

Figure 4.

Association of TNFR1 with signaling intermediates in lipid rafts after TBI. Coimmunoprecipitation with TNFR1 of raft fractions of naive lysates and lysates obtained at 5, 15, 30, and 60 min and 4 hr after TBI. TNFR1 immunoprecipitates were blotted for TNFR1, TRADD, TRAF2, RIP, FADD, TRAF1, cIAP-1, cIAP-2, and caspase-8. Raft-associated protein flotillin-1 served as control. N, Naive control groups.

Figure 5.

TBI-induced TNFR1 and TRAF1 ubiquitination in lipid rafts. Lipid rafts and soluble fractions were isolated from injured cortices and immunoprecipitated with TNFR1 (A) and ubiquitin (Ubq) (B). Ubiquitinated proteins were analyzed by immunoblotting using antibodies against Ubq (A) and TRAF1 (B). Polyubiquitinated proteins are denoted as Ubq(n). N, Naive control groups.

Figure 6.

TBI induces NF-κB activation early after injury. Naive (N) and injured cortical lysates were subjected to immunoblotting, and NF-κB activation was monitored by the phosphorylation of IκB-α (P-IκB-α). The same blot was reprobed with an antibody to total IκB-α as internal control for protein loading.