Iron-mediated H2O2 Production as a Mechanism for Cell Type-specific Inhibition of Tumor Necrosis Factor α-Induced but Not Interleukin-1β-induced IκB Kinase Complex/Nuclear Factor-κB Activation

Andreas Panopoulos,Maged Harraz,John F Engelhardt,Ebrahim Zandi

doi:10.1074/jbc.m409524200

Andreas Panopoulos, Maged Harraz + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m409524200

Copy DOI

Abstract

Coordinated and specific regulation of tumor necrosis factor (TNF) and interleukin (IL)-1 signaling pathways and how and whether they are modified by different agents are key events for proper immune responses. The IkappaB kinase complex (IKK)/NF-kappaB and JNK/AP-1 pathways are central mediators of TNF and IL-1 during inflammatory responses. Here we show that l-mimosine, a toxic non-protein amino acid that has been shown to reduce serum TNFalpha levels and affect inflammatory responses, specifically inhibits TNF-induced IKK but not JNK in a cell type-specific manner. l-Mimosine did not affect IKK and NF-kappaB activation by IL-1beta. l-Mimosine caused cell cycle arrest at G(1)-S phase, but inhibition of IKK was found to be independent of cell cycle arrest. Treatment of cells with l-mimosine resulted in production of H(2)O(2). Addition of FeSO(4) restored IKK activation by TNFalpha as did ectopic expression of catalase or pretreatment of cells with N-aceltyl-l-cysteine, indicating a role for intracellular H(2)O(2) as a mediator of inhibition. Cleavage and degradation of TNF pathway components TNFR1, RIP, and Hsp90 were observed in l-mimosine and H(2)O(2) treated cells indicating a putative mechanism for selective inhibition of TNF but not IL-1beta-induced IKK activation.

Highlights

From the ‡Department of Molecular Microbiology and Immunology, University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine at USC, Los Angeles, California 90033 and ¶Department of Anatomy and Cell Biology, University of Iowa College of Medicine, Iowa City, Iowa 52242
In this study we demonstrate that L-mimosine treatment of cells results in production of H2O2, which selectively inhibits tumor necrosis factor (TNF)- but not IL-1-induced IKK activation in a cell type-specific manner
Treatment of mouse embryonic fibroblasts (MEF) with L-Mimosine Prevents Activation of IKK by TNF␣ but Does Not Prevent IKK Activation by IL-1␤—In a series of experiments originally designed to investigate the regulation of IKK by TNF␣ at different stages of the cell cycle, we observed that pretreatment of a MEF line with L-mimosine for 12–16 h strongly reduced activation of the IKK complex by TNF␣ as indicated by the reduction of kinase activity (Fig. 1A) [31]

Summary

Introduction

From the ‡Department of Molecular Microbiology and Immunology, University of Southern California/Norris Comprehensive Cancer Center, Keck School of Medicine at USC, Los Angeles, California 90033 and ¶Department of Anatomy and Cell Biology, University of Iowa College of Medicine, Iowa City, Iowa 52242. Whether the signaling pathways of these cytokines are regulated differently in various cell types and cellular conditions induced by exposure to different agents are important questions that need to be answered to better understand the mechanism of the inflammatory process. Both of these cytokines activate the I␬B kinase complex (IKK)/NF-␬B and JNK/AP-1 pathways, but TNF␣ . It has been reported that treatment of cells with free radical scavengers or ectopic expression of oxygen radical scavenging enzymes reduced TNFinduced IKK activation, suggesting ROS as second messengers for cytokine-induced signaling for the IKK/NF-␬B pathway [15, 16]

Methods

Results

Conclusion