Laminar flow inhibits TNF-induced ASK1 activation by preventing dissociation of ASK1 from its inhibitor 14-3-3
Yingmei Liu, … , Bradford C. Berk, Wang Min
Yingmei Liu, … , Bradford C. Berk, Wang Min
Published April 1, 2001
Citation Information: J Clin Invest. 2001;107(7):917-923. https://doi.org/10.1172/JCI11947.
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Article

Laminar flow inhibits TNF-induced ASK1 activation by preventing dissociation of ASK1 from its inhibitor 14-3-3

Abstract

The inflammatory cytokine TNF-α stimulates several presumed pro-atherogenic signaling events in endothelial cells (ECs), including activation of c-Jun NH2-terminal kinase (JNK) and induction of E-selectin. Here, we show that apoptosis signal-regulating kinase 1 (ASK1), a MAP kinase kinase kinase, is required for TNF-mediated JNK activation. TNF activates ASK1 in part by dissociating ASK1 from its inhibitor 14-3-3. Because the risk of atherosclerosis is decreased in regions of steady laminar flow, we hypothesized that laminar flow inhibits proinflammatory cytokine-mediated activation of JNK. Steady laminar flow inhibited both TNF activation of ASK1 and JNK. Inhibition of ASK1 by flow correlated with increased association of ASK1 with 14-3-3. A constitutively active form of ASK1 lacking the 14-3-3-binding site (ASK1-ΔNS967A) was not inhibited by flow. These data establish ASK1 as a target for flow-mediated inhibition of cytokine signaling and indicate a novel role for 14-3-3 as an anti-inflammatory mediator in ECs.

Authors

Yingmei Liu, Guoyong Yin, James Surapisitchat, Bradford C. Berk, Wang Min

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Figure 1

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Flow pre-exposure inhibits TNF-mediated JNK and ASK1, but not NF-κB, act...
Flow pre-exposure inhibits TNF-mediated JNK and ASK1, but not NF-κB, activation in ECs. ECs were subjected to the following “preconditioning” protocol: they were maintained in static conditions for 25 minutes (Ctrl), exposed to flow for 10 minutes and then held static for 15 minutes (Flow), maintained in static conditions for 10 minutes, followed by TNF stimulation for 15 minutes or subjected to flow for 10 minutes followed by TNF-α (100 U/ml) stimulation for 15 minutes (flow + TNF). (a) Flow inhibits JNK activation by TNF. Cell lysates were prepared and analyzed for JNK activity by an in vitro kinase assay using GST–c-Jun as a substrate. (b) Flow has no inhibitory effect on NF-κB. Cells were treated as in a. Nuclear extracts from these cells were used for EMSA with a κB probe. Specificity of NF-κB complex was verified by 50-fold molar excess of the unlabeled κB oligonucleotide in the TNF-treated sample (TNF + competitor). (c) Flow inhibits ASK1 activation by TNF. Cell lysates in a were prepared and analyzed for ASK1 activity by an in vitro kinase assay using GST-MKK4 as a substrate. Autoradiograms shown in a–c are representative of three experiments in HUVECs.