Role For Receptor-interacting Protein 1 Research Articles

Role for RIP1 in mediating necroptosis in experimental intracerebral hemorrhage model both in vivo and in vitro

Cell death is a hallmark of second brain injury after intracerebral hemorrhage (ICH); however, the mechanism still has not been fully illustrated. In this study, we explored whether necroptosis, a type of regulated necrosis, has an essential role in brain injury after ICH. We found that inhibiting receptor-interacting protein 1 (RIP1) – a core element of the necroptotic pathway – by a specific chemical inhibitor or genetic knockdown attenuated brain injury in a rat model of ICH. Furthermore, necroptosis of cultured neurons could be induced by conditioned medium from microglia stimulated with oxygen hemoglobin, and this effect could be inhibited by TNF-α inhibitor, indicating that TNF-α secreted from activated microglia is an important factor in inducing necroptosis of neurons. Undoubtedly, overexpression of RIP1 increased conditioned medium-induced necroptosis in vitro, but this effect was partially diminished in mutation of serine kinase phosphorylation site of RIP1, showing that phosphorylation of RIP1 is the essential molecular mechanism of necroptosis, which was activated in the in vitro model of ICH. Collectively, our investigation identified that necroptosis is an important mechanism of cell death in brain injury after ICH, and inhibition of necroptosis may be a potential therapeutic intervention of ICH.

Abstract B27: RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis

Abstract In the presence of ample oxygen, cancer cells prefer glycolysis to mitochondrial respiration for energy supply. This metabolic alteration, known as the Warburg effect or aerobic glycolysis, has been shown to contribute to cancer cell proliferation. However, how this glucose metabolism pathway is precisely regulated remains elusive. Receptor-interacting protein 1 (RIP1) is an important cellular signaling molecule that plays a pivotal role in the regulation of cell death, survival and proliferation. However, the role of RIP1 in cell metabolism is unclear. To investigate the function of RIP1 in lung cancer cell proliferation, we established stable RIP1 knockdown lung cancer cell lines using lentivius-mediated shRNA expression. RIP1 knockdown inhibited the proliferation of lung cancer cells, which was associated with DNA damage-induced p53 activation. Compared with control cells, the RIP1 knockdown cells had higher glucose consumption and lactate production, indicating a higher glycolytic rate. The increased glycolysis lowered the cellular level of nicotinamide adenine dinucleotide (NAD+), a cofactor essential for DNA damage repair. By microarray profiling and promoter reporter assays, we determined that RIP1 positively regulates the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a key factor involved in the regulation of mitochondrial function. The downregulation of PGC-1α reduced the expression of components of electron transfer chain that impaired oxidative phosphorylation, which underlays the increased glycolysis in RIP1 knockdown cells. Moreover, RIP1 knockdown cells were more sensitive to both DNA damage anticancer agents and glycolysis inhibitors. Our data suggest that while aerobic glycolysis within a certain range favors cancer cell proliferation, excessive glycolysis may cause cytostasis. Thus, maintenance of glycolysis by RIP1 is critical to cancer cell energy homeostasis and DNA integrity, which may be exploited for anticancer therapy. Citation Format: Wenshu Chen, Qiong Wang, Lang Bai, Weijie Chen, Xia Wang, Carmen S. Tellez, Shuguang Leng, Mabel T. Padilla, Toru Nyunoya, Steven A. Belinsky, Yong Lin. RIP1 maintains DNA integrity and cell proliferation by regulating PGC-1α-mediated mitochondrial oxidative phosphorylation and glycolysis. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B27.

Abstract 2910: Suppressing excessive reactive oxygen species accumulation by RIP1 potentiates BPDE-induced transformation of human bronchial epithelial cells.

Abstract Cell survival signaling is important for the malignant phenotypes of cancer cells. Although the role of receptor-interacting protein 1 (RIP1) in cell survival signaling is well documented, whether RIP1 is directly involved in cancer development has never been studied. In this report, we found that RIP1 expression is substantially increased in human non-small cell lung cancer (NSCLC) tissues and cell lines. RIP1 expression in human bronchial epithelial cells (HBECs) was significantly induced by cigarette smoke extract (CSE) or benzo[a]pyrene diol epoxide (BPDE), the active form of the tobacco-specific carcinogen benzo (a) pyrene (BaP). In RIP1 knockdown HBECs, BPDE-induced cytotoxicity was significantly increased, which was associated with induction of cellular reactive oxygen species (ROS), mainly hydrogen peroxide (H2O2), and activation of mitogen-activated protein kinases (MAPKs) including JNK, ERK and p38. Scavenging ROS suppressed BPDE-induced MAPK activation and inhibiting ROS or MAPKs substantially blocked BPDE-induced cytotoxicity, suggesting ROS-mediated MAPK activation is involved in BPDE-induced cell death. The H2O2-reducing enzyme catalase is destabilized in an ERK- and JNK-dependent manner in RIP1 knockdown HBECs and application of catalase effectively blocked BPDE-induced H2O2 accumulation and cytotoxicity. Importantly, BPDE-induced transformation of HBECs was significantly reduced when RIP1 expression was suppressed. Altogether, these results strongly suggest an oncogenic role for RIP1, which promotes malignant transformation through protecting DNA-damaged cells against carcinogen-induced cytotoxicity associated with excessive ROS production. Citation Format: Qiong Wang, Wenshu Chen, Xiuling Xu, Bilan Li, Weiyang He, Mabel T. Padilla. Suppressing excessive reactive oxygen species accumulation by RIP1 potentiates BPDE-induced transformation of human bronchial epithelial cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2910. doi:10.1158/1538-7445.AM2013-2910

Increasing complexity in TNFR1 signaling

Tumor necrosis factor (TNF) and its receptors, TNF receptor-1 (TNFR1) and TNFR2, have received considerable attention in basic and clinical research for more than two decades. This is because of, on the one hand, the overwhelming importance of TNF-blocking reagents in the treatment of autoimmune diseases and, on the other hand, the fact that practically every cell type responds in some way or another to TNF. The effects of TNF on cells are highly variable and reach from the induction of proliferation or differentiation over the activation of inflammatory processes to cell death. This is mirrored by the fact that TNF stimulates a diverse set of signaling pathways resulting in the activation of apoptotic caspases, inhibitor of jB kinases, MAP3 kinases and sphingomyelinases. It is particularly intriguing that, depending on environmental factors, the same cell-type can respond to TNF with such contrasting effects as cell death and inflammatory cell activation. Research on the signaling pathways ⁄mechanisms mediating these opposing cell fates was therefore in the center of TNF-related research early on, and was formative for the field of cell death and NF-jB research. TNF-related studies, for example, not only resulted in the definition of two novel adapter protein families, the death domain-containing adaptor proteins and the TNF receptor-associated factors (TRAFs), but also led to the identification of the inhibitor of jB kinase complex and a variety of NF-jB regulatory mechanisms. Until a few years ago, the simple picture prevailed that TNFR1-induced apoptosis and necrosis and TNFR1-stimulated activation of NF-jB transcription factors rely on the use of basically linear signaling pathways. Activation of the classical NF-jB pathway by TNFR1 was assigned to the recruitment of receptorinteracting protein-1 (RIP1), TNF receptor-associated death domain (TRADD) and TRAF2, which in a cooperative manner then secondarily recruit and activate the inhibitor of jB kinase complex and thus the classical NF-jB pathway. TNF-induced necrosis was further traced to activation of the kinase activity of RIP1 which, however, is dispensable for NF-jB signaling. TNFR1mediated apoptosis was found to require internalization and sequential action of the TRADD-interacting death domain adapter protein Fas-associated death domain (FADD) and the FADD-interacting protease caspase 8. The balance between these TNFR1-associated pathways and thus the cellular fate in response to TNF was considered to be mainly controlled by pre-existing and NF-jB-induced prosurvival proteins. In recent years, it has turned out that the situation is much more complicated than initially anticipated. Different types of ubiquitylation of several TNF signaling intermediates have been identified not only as pivotal events in TNFR1-mediated NF-jB activation, but also play a crucial role in the control of a novel RIP1-dependent mode of caspase 8 activation. Further, it became evident that most proteins involved in TNF signaling are less obligatory than previously thought, and more than 10 years after their identification as components of the TNFR1 and TNFR2 signaling complexes, the TRAF2-associated proteins cIAP1 and cIAP2 are recognized as important regulators of apoptotic and inflammatory TNFR1 signaling. Moreover, it has been recognized that TNFR2 modulates TNFR1 signaling by regulation of the TNFR1-‘available’ cytoplasmic pool of TRAF2–cIAP complexes. This minireviews series covers these novel and exciting aspects of TNF signaling and tries to address new questions emerging from these developments. The first minireview by Wajant and Scheurich is focused on the mechanisms of TNFR1-induced NF-jB activation and particularly highlights recent details of the regulatory role of nondegrading protein ubiquitylation for this process. The second minireview by O’Donnell and Ting focuses on the role of RIP1 and ubiquitylation in TNFR1-induced cell death. Finally, Naude et al. discuss, in the context of TNFR2, how the aforementioned TNFR1-associated signaling pathways can be modulated by external cues.