Sergei A. Nedospasov

We have explored the cis-acting elements necessary for the LPS-mediated activation of the mouse TNF-alpha promoter by transfecting a set of 5' deletion mutants linked to the CAT reporter gene into primary bone marrow-derived macrophages. A major drop in inducibility by LPS was seen upon deletion of a region mapping between nt -655 and nt -451. Gel retardation assays revealed that LPS induced the appearance in this region of several specific DNA-protein complexes mapping to sequence motifs with strong homology to the kappa B enhancer. Constructs containing two or more copies of one of the kappa B enhancer motifs linked to a heterologous promoter were inducible by LPS. Additional deletion of a region between nt -301 and nt -241, which contains a MHC class II-like "Y box" and formed a Y box-specific complex with a protein whose concentration was increased by LPS, caused a nearly complete loss of inducibility by LPS. We speculate that NF-kappa B and/or related proteins are involved in the LPS-induced transcriptional activation of the TNF-alpha gene, and that factors interacting with the Y box can additionally modulate the activity of the gene in macrophages.

The mechanism of tumor necrosis factor (TNF)-induced nonapoptotic cell death is largely unknown, although the mechanism of TNF-induced apoptosis has been studied extensively. In wild-type mouse embryonic fibroblast cells under a caspase-inhibited condition, TNF effectively induced cell death that morphologically resembled necrosis. In this study, we utilized gene knockout mouse embryonic fibroblasts cells and found that tumor necrosis factor receptor (TNFR) I mediates TNF-induced necrotic cell death, and that RIP, FADD, and TRAF2 are critical components of the signaling cascade of this TNF-induced necrotic cell death. Inhibitors of NF-kappaB facilitated TNF-induced necrotic cell death, suggesting that NF-kappaB suppresses the necrotic cell death pathway. JNK, p38, and ERK activation seem not to be required for this type of cell death because mitogen-activated protein kinase inhibitors did not significantly affect TNF-induced necrotic cell death. In agreement with the previous reports that the reactive oxygen species (ROS) may play an important role in this type of cell death, the ROS scavenger butylated hydroxyanisole efficiently blocked TNF-induced necrotic cell death. Interestingly, during TNF-induced necrotic cell death, the cellular ROS level was significantly elevated in wild type, but not in RIP(-/-), TRAF2(-/-), and FADD(-/-) cells. These results suggest that RIP, TRAF2, and FADD are crucial in mediating ROS accumulation in TNF-induced necrotic cell death.