Lipopolysaccharide Signals an Endothelial Apoptosis Pathway Through TNF Receptor-Associated Factor 6-Mediated Activation of c-Jun NH2-Terminal Kinase

Abstract

Inflammatory mediators such as TNF and bacterial LPS do not cause significant apoptosis of endothelial cells unless the expression of cytoprotective genes is blocked. In the case of TNF, the transcription factor NF-κB conveys an important survival signal. In contrast, even though LPS can also activate NF-κB, this signal is dispensable for LPS-inducible cytoprotective activity. LPS intracellular signals are transmitted through a member of the Toll-like receptor family, TLR4. This family of receptors transduces signals through a downstream molecule, TNFR-associated factor 6 (TRAF6). In this study, we demonstrate that the C-terminal fragment of TRAF6 (TRAF6-C) inhibits LPS-induced NF-κB nuclear translocation and c-Jun NH2-terminal kinase (JNK) activation in endothelial cells. In contrast, LPS activation of p38 kinase is not inhibited by TRAF6-C. TRAF6-C also inhibits LPS-initiated endothelial apoptosis, but potentiates TNF-induced apoptosis. LPS-induced loss of mitochondrial transmembrane potential, cytochrome c release, and caspase activation are all blocked by TRAF6-C. We demonstrate that TRAF6 signals apoptosis via JNK activation, since inhibition of JNK activation using a dominant-negative mutant also inhibits apoptosis. JNK inhibition blocks caspase activation, but the reverse is not true. Hence, JNK activation lies upstream of caspase activation in response to LPS stimulation.

Bordetella pertussis Adenylate Cyclase Toxin Modulates Innate and Adaptive Immune Responses: Distinct Roles for Acylation and Enzymatic Activity in Immunomodulation and Cell Death

“…We observed cell death in macrophages incubated with A-CyaA or iAC-CyaA. In contrast, cell lysis was only observed with the highest dose of NA-CyaA examined and this effect was abolished, as was a proportion of the lytic activity of A-CyaA, by coincubation with polymyxin B. LPS and LPS-induced TNF-␣ have been associated with apoptosis (28) and, therefore, may have contributed to the cell death observed with NA-CyaA and CyaA. This may explain the higher levels of cell lysis reported in previous studies, where steps were not taken to remove LPS during the purification of CyaA.…”

Lipopolysaccharide signaling in endothelial cells

“…95 However, a dominant negative mutant of TRAF6 does not inhibit LPS activation of p38 in endothelial cells, suggesting that p38 does not lie downstream of TRAF6. 96 Similarly, TRAF6 is not required for LPS-induced activation of ERK in endothelial cells. 90 Nonetheless, in immune cells, the activation of p38 involves TRAF6 and apoptosis signal-regulating kinase 1 (ASK1).…”

“…100 In endothelial cells, JNK activation has been shown to require TRAF6 activation. 96 In macrophages, however, activation of JNK is dependent on interaction of the scaffolding protein JNK-interacting protein 3 (JIP3) with the cytoplasmic domain of TLR4. 101 Inhibition of JIP3 function, either by expression of a dominant negative form of the protein or using RNA interference, inhibits JNK activation in macrophages.…”

The Zinc Finger Mutation C417R of I-κB Kinase γ Impairs Lipopolysaccharide- and TNF-Mediated NF-κB Activation through Inhibiting Phosphorylation of the I-κB Kinase β Activation Loop

“…5A, there were significantly more cell deaths in THP/IKK␥C417R cells than in THP/V cells following TNF stimulation. Several studies reported that LPS also induced monocyte apoptosis through the Fas-associated death domain-containing protein-dependent mechanism (27)(28)(29). Because the percentage of cell death induced by LPS in human THP.1 monocytes was relatively low, the cell death ELISA, which accurately measured DNA fragmentation and histone release from apoptotic cells, was used.…”