TAK1 inhibition-induced RIP1-dependent apoptosis in murine macrophages relies on constitutive TNF-α signaling and ROS production

Wang et al. Journal of Biomedical Science (2015) 22:76 DOI 10.1186/s12929-015-0182-7 RESEARCH Open Access TAK1 inhibition-induced RIP1-dependent apoptosis in murine macrophages relies on constitutive TNF-α signaling and ROS production Jang-Shiun Wang1,2†, Dean Wu3†, Duen-Yi Huang1 and Wan-Wan Lin1,2* Abstract Background: Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is a key regulator of signal cascades of TNF-α receptor and TLR4, and can induce NF-κB activation for preventing cell apoptosis and eliciting inflammation response. Results: TAK1 inhibitor (TAKI) can decrease the cell viability of murine bone marrow-derived macrophages (BMDM), RAW264.7 and BV-2 cells, but not dermal microvascular endothelial cells, normal human epidermal keratinocytes, THP-1 monocytes, human retinal pigment epithelial cells, microglia CHME3 cells, and some cancer cell lines (CL1.0, HeLa and HCT116). In BMDM, TAKI-induced caspase activation and cell apoptosis were enhanced by lipopolysaccharide (LPS). Moreover, TAKI treatment increased the cytosolic and mitochondrial reactive oxygen species (ROS) production, and ROS scavengers NAC and BHA can inhibit cell death caused by TAKI. In addition, RIP1 inhibitor (necrostatin-1) can protect cells against TAKI-induced mitochondrial ROS production and cell apoptosis. We also observed the mitochondrial membrane potential loss after TAKI treatment and deterioration of oxygen consumption upon combination with LPS. Notably TNF-α neutralization antibody and inhibitor enbrel can decrease the cell death caused by TAKI. Conclusions: TAKI-induced cytotoxicity is cell context specific, and apoptosis observed in macrophages is dependent on the constitutive autocrine action of TNF-α for RIP1 activation and ROS production. Keywords: TAK1, RIP1, Macrophages, TNF-α, ROS, Apoptosis Background Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is a ubiquitously expressed mitogen-activated protein kinase kinase kinase and plays a key role in regulating inflammation, immunity, cell differentiation and death [1–3]. Accumulating evidence indicates that TAK1 is a key regulator of signal transduction cascades and is activated by various inflammatory mediators and cytokines such as transforming growth factor (TGF)-β, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, CD40 ligand, toll-like receptor (TLR) ligands, T and B cell receptor * Correspondence: † Equal contributors 1 Department of Pharmacology, College of Medicine, National Taiwan University, No 1, Sec 1, Jen-Ai Road, Taipei, Taiwan 2 Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan Full list of author information is available at the end of the article ligands [3, 4]. TAK1 activity is tightly regulated by its binding proteins, TAB1 and TAB2/TAB3, as well as by posttranslational modification including ubiquitination and phosphorylation. In TLR4 signaling, TRAF6 through its E3 ubiquitin ligase activity facilitates the formation of K63 polyubiquitin chains to recruit and activate TAK1 [5]. This activation then transduces signals for activating downstream kinases IKK, p38, and JNK, in turn leading to activate NF-κB and activator protein-1 (AP-1) to produce the proinflammatory and anti-apoptosis proteins [4, 6–8]. Consistently the notion implicating TAK1 as a key intermediary in cell survival is evidenced by observing the embryonic lethality associated with multi-tissue defects in germline deficient of TAK1 gene [4]. Subsequent studies in mice with the conditional ablation of TAK1 in hepatocytes [9], liver parenchymal cells [10], keratinocytes [11], intestinal epithelial cells [12], dendritic cells [13], and monocytes [14], further strengthen the cytoprotection role of TAK1. © 2015 Wang et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Wang et al. Journal of Biomedical Science (2015) 22:76 TNF-α contributes to many physiological and pathological processes and plays an important role in mediating survival signaling, apoptosis and necroptosis depending on the cell types and cellular context [1]. On TNF-α binding, TNF receptor 1 (TNFR1) undergoes a conformational change to form TNFR complex I containing TRADD, receptor-interacting protein (RIP)1, cIAPs, TRAF2 and TRAF5. RIP1 ubiquitylation by cIAP1 or TRAF2 can recruit the TAK1 to initiate the canonical NF-κB survival pathway [1, 4, 10, 15]. Upon inhibition of NF-κB signaling, for example by de-ubiquitylation of RIP1 and loss of cIAPs, TNFR complex II (TRADD, FADD, caspase 8, RIP1 and RIP3) can be formed and execute two distinct types of cell death, apoptosis and necroptosis that compete for each other and is switched by TAK1 and caspases. Normally, caspase 8 triggers apoptosis by cleaving RIP1 to inhibit the pro-survival actions derived from NF-κB signaling, and the pro-apoptotic protein Bid to generate a truncated form (tBid) for inducing cytochrome c release and apoptosome formation. However, when caspase 8 is blocked by pharmacological or genetic interventions, RIP1 can recruit RIP3 to form the RIP1/RIP3 necrosome. Within this necrosome, RIP1 and RIP3 phosphorylate each other, further stabilizing the complex and engaging the effector mechanisms of necroptosis, which is a recently identified programmed cell death [16, 17]. Necroptosis has been shown to rely on mitochondrial reactive oxygen species (ROS) production and disintegration of mitochondrial, lysosomal and plasma membranes [18–20]. Moreover, necroptosis has been shown to involve in the pathogenesis of various diseases, such as ischemic injury, neurodegeneration, viral infection, liver injury, traumatic brain injury, and severe drug reaction [21]. Notably, recent studies further demonstrate the role of TAK1 in controlling the RIP1-dependent apoptosis triggered by TNFR complex II. Besides inducing NFκB-dependent anti-apoptotic mechanisms, an NF-κBindependent cell survival pathway downstream of TAK1 and involving the regulation of ROS-cIAPs-RIP1-caspase pathway has been suggested [2, 22, 23]. Moreover, in mouse embryonic fibroblasts (MEF), TAK1 activity was shown to be a switch between apoptosis and necroptosis following TNF-α stimulation [24]. Hyperactivation of TAK1 possibly through inhibition of caspase-1 leads to necroptosis in dermal fibroblasts which results in the delay in wound healing. In contrast, ablation of TAK1 causes RIP1- and caspase-dependent apoptosis in MEF and monocytes [14, 24]. Since TAK1 i (...truncated)