UMMS Affiliation

Department of Pathology; Program in Immunology and Virology

Date

8-2011

Document Type

Article

Subjects

Animals; Apoptosis; Caspases; Cell Proliferation; Cyclic AMP-Dependent Protein Kinases; Extracellular Signal-Regulated MAP Kinases; Gene Knockdown Techniques; Humans; Imidazoles; Indoles; JNK Mitogen-Activated Protein Kinases; Jurkat Cells; Lymphocyte Activation; Mice; NIH 3T3 Cells; Necrosis; Phosphorylation; Receptor-Interacting Protein Serine-Threonine Kinases; T-Lymphocytes; Tumor Necrosis Factor-alpha

Disciplines

Immunology and Infectious Disease | Life Sciences | Medicine and Health Sciences | Pathology

Abstract

BACKGROUND: Programmed necrosis/necroptosis is an emerging form of cell death that plays important roles in mammalian development and the immune system. The pro-necrotic kinases in the receptor interacting protein (RIP) family are crucial mediators of programmed necrosis. Recent advances in necrosis research have been greatly aided by the identification of chemical inhibitors that block programmed necrosis. Necrostatin-1 (Nec-1) and its derivatives were previously shown to target the pro-necrotic kinase RIP1/RIPK1. The protective effect conferred by Nec-1 and its derivatives in many experimental model systems was often attributed to the inhibition of RIP1 function.

METHODOLOGY/PRINCIPAL FINDINGS: We compared the effect of Nec-1 and siRNA-mediated silencing of RIP1 in the murine fibrosarcoma cell line L929. Treatment of L929 cells with the pan-caspase inhibitor zVAD-fmk or exogenous TNF induces necrosis. Strikingly, we found that siRNA-mediated silencing of RIP1 inhibited zVAD-fmk induced necrosis, but not TNF-induced necrosis. TNF-induced cell death in RIP1 knocked down L929 cells was inhibited by Nec-1, but not the caspase inhibitor zVAD-fmk. We found that PKA-C section sign expression, but not Jnk or Erk activation, was moderately inhibited by Nec-1. Moreover, we found that Nec-1 inhibits proximal T cell receptor signaling independent of RIP1, leading to inhibition of T cell proliferation.

CONCLUSIONS/SIGNIFICANCE: Our results reveal that besides RIP1, Nec-1 also targets other factors crucial for necrosis induction in L929 cells. In addition, high doses of Nec-1 inhibit other signal transduction pathways such as that for T cell receptor activation. These results highlight the importance to independently validate results obtained using Nec-1 with other approaches such as siRNA-mediated gene silencing. We propose that some of the previous published results obtained using Nec-1 should be re-evaluated in light of our findings.

Comments

Citation: Cho Y, McQuade T, Zhang H, Zhang J, Chan FK-M (2011) RIP1-Dependent and Independent Effects of Necrostatin-1 in Necrosis and T Cell Activation. PLoS ONE 6(8): e23209. doi:10.1371/journal.pone.0023209. Link to article on publisher's site

Copyright: © 2011 Cho et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Related Resources

Link to Article in PubMed

Journal Title

PloS one

PubMed ID

21853090

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