RIP1-Dependent and Independent Effects of Necrostatin-1 in Necrosis and T Cell Activation YoungSik Cho1.¤, Thomas McQuade1., Haibing Zhang2, Jianke Zhang2, Francis Ka-Ming Chan1,3* 1 Department of Pathology, Program in Immunology and Virology, The University of Massachusetts Medical School, Worcester, Massachusetts, United States of America, 2 Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America, 3 UMass Diabetes and Endocrinology Research Center, The University of Massachusetts Medical School, Worcester, Massachusetts, United States of America 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-C1 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. 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 Editor: Jose Alberola-Ila, Oklahoma Medical Research Foundation, United States of America Received March 23, 2011; Accepted July 12, 2011; Published August 10, 2011 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. Funding: This work was supported in part by grants from the National Institutes of Health (AI088502, AI083497, and DK32520 to F.K.C. and AI076788 to J.Z.). No additional external funding was received for this study. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Francis.chan@umassmed.edu . These authors contributed equally to this work. ¤ Current address: College of Pharmacy, Keimyung University, Daegu, Korea Introduction Cell death by necrosis is often associated with inflammation. Although 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]. Specifically, 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 character- ized by organelle and cell swelling and eventually plasma membrane rupture. In necrosis, 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]. Thus, necrosis is a more inflammatory form of cell death than apoptosis. Recent work shows that the receptor interacting protein (RIP) kinases RIP1 and RIP3 play essential roles in TNF-induced necrosis [6,7,8]. In addition to TNF-like death cytokines, RIP1/ RIP3-dependent necrosis also contributes to the abortive embry- onic development and impaired T lymphocyte proliferation of FADD2/2 and caspase-82/2 mice [9,10,11,12,13]. Furthermore, RIP1/RIP3-dependent necrosis has been shown to mediate cerulein-induced pancreatitis [7,8], retinal detachment induced photoreceptor necrosis [14], ischemia-induced brain injury [15,16] and myocardial infarction [17]. Necrostatin-1 (Nec-1) is a small molecule inhibitor originally identified in a chemical library screen as a potent inhibitor of TNF-induced necrosis [15]. Subsequent studies show that Nec-1 PLoS ONE | www.plosone.org 1 August 2011 | Volume 6 | Issue 8 | e23209