RIP1-Dependent and Independent Effects of Necrostatin-1 in Necrosis and T Cell Activation

Youngsik Cho,Jianke Zhang,Francis Ka-Ming Chan,Haibing Zhang,Thomas Mcquade,Jose Alberola-Ila

doi:10.1371/journal.pone.0023209

Abstract

BackgroundProgrammed 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 FindingsWe 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§ 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/SignificanceOur 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.

Highlights

Cell death by necrosis is often associated with inflammation
Nec-1 has been reported to inhibit the expression of ectopically expressed RIP1 in HEK293T cells [6], it did not affect endogenous RIP1 expression in L929 cells (Fig. 1C)
The emerging role of RIP1 and RIP3-dependent necrosis in many disease pathologies have led to the wide use of Nec-1 and its derivatives by many investigators

Summary

Introduction

Cell death by necrosis is often associated with inflammation. necrosis was once thought to be an un-regulated form of cell injury due to trauma, recent evidence indicates that necrosis is a highly regulated process that involves a dedicated molecular circuitry [1,2]. Necrosis is the dominant response when cells are stimulated with TNF-like death cytokines in the presence of caspase inhibitors [1]. This form of necrosis, often referred to as programmed necrosis/necroptosis [3], is characterized by organelle and cell swelling and eventually plasma membrane rupture. The loss of plasma membrane integrity often coincides or precedes the exposure of phosphatidyl serine [4] This morphological signature of necrosis distinguishes it from apoptosis, in which the early exposure of phosphatidyl serine and other ‘‘eat-me’’ signals prompt their clearance by professional phagocytes before membrane leakage and release of inflammatory cellular adjuvants [5]. The protective effect conferred by Nec-1 and its derivatives in many experimental model systems was often attributed to the inhibition of RIP1 function

Methods

Results

Conclusion