PPP1R3G Deletion Blocks RIPK1-Mediated Apoptosis and Necroptosis in Doxorubicin-Induced Cardiotoxicity.

Background. Acute kidney injury (AKI) is an independent risk factor for mortality and morbidity. Inflammation is now believed to play a major role in the pathophysiology of AKI. Receptor‐interacting protein kinase 3 (RIP3) is a member of the receptor‐interacting protein (RIP) family of serine/threonine protein kinases. RIP3 is a regulator of both programmed necrosis/necroptosis, an inflammatory form of cell death observed in pathogen‐induced and sterile inflammation, and TNFα‐induced apoptosis. In the induction of necroptosis, RIP3 is a major component of the tumor necrosis factor (TNF) receptor‐I signaling complex which through additional interactions with (RIP1) and pseudokinase mixed lineage kinase domain‐like protein (MLKL) forms the necrosome. More recently, RIP3 activity was found to promote sepsis‐induced AKI via mitochondrial dysfunction. Since I/R injury triggers numerous pathological changes, including apoptosis, oxidative stress, and inflammation, suggesting a role of mitochondrial function in RIP3‐dependent I/R injury.ObjectiveWe investigated whether RIP3 translocates into mitochondria in response to ischemia/reperfusion (I/R) to interact with Mitofilin and promote mitochondria damage that facilitates mtDNA release into the cytosol. We postulated that release of mtDNA activates cGAS/STING pathway leading to increased nuclear transcription of pro‐inflammatory markers that exacerbates renal I/R injury.Material and methods. C57/6N and RIP3‐/‐ mice as well as HK2 cells were used. Monolateral kidneys were subjected to 30 min of ischemia followed by either 12, 24, or 48 h of reperfusion. Protein levels of RIP3, Mitofilin, cGAS, STING, and p‐p65 were measured using Western and immunofluorescence analysis, while IL‐6, TNF‐α, and ICAM‐1 expressions as well as mtDNA release were assessed by qRT‐PCR, ELISA. Kidney function was measured by blood urea nitrogen and creatinine kits, and the interaction between RIP3 and Mitofilin was measured by Co‐Immunoprecipitation. In WT,Results. We found that renal I/R increased RIP3 levels, and its translocation into mitochondria. We observed that RIP3 interacts with Mitofilin likely promoting its degradation. While renal I/R associated with mitochondria damage, increased mtDNA release, activation of cGAS/STING/p65 pathway and increased transcription of pro‐inflammatory markers including IL‐6, TNF‐α and ICAM‐1, all these effects were decreased in RIP3‐/‐ mice. In HK‐2, RIP3 overexpression or Mitofilin knockdown increased cell death by activating the cGAS‐STING/p65 pathway.Conclusion. We demonstrated that kidney I/R increases RIP3 levels in the cytosol and it translocates into mitochondria, where it interacts with and promote Mitofilin degradation leading to increased mitochondrial structures damage and dysfunction. The subsequent increase in ROS production in mitochondria is postulated to facilitate mtDNA damage and release into the cytosol where it activates the cGAS/STING/p‐p65 pathway leading to amplified nuclear transcription of pro‐inflammatory markers that subsequently increase renal I/R injury. Together, this study point to an important role of RIP3 in the initiation and development of renal I/R injury.

The Ripoptosome: Death Decision in the Cytosol

The receptor interacting protein kinase 1 (RIP1) is a crucial component of the TNFR1 response. In this issue of Cell Death and Differentiation, Wong et al. has set a cat among the pigeons by challenging the commonly accepted model in which RIP1 is essential for TNFR1-induced NF-kB activation. Their new data will force the scientific community to adapt and refine the model of NF-kB activation. Tumor necrosis factor (TNF) is a multifunctional cytokine. Upon binding to TNFR1, it activates distinct pathways with diametrically opposed consequences: killing cells or promoting survival. Its protective effect is achieved mainly by activating the NF-kB pathway, which induces the transcription of a set of pro-survival genes. In most cells, exposure to TNF is lethal only if the NF-kB signaling pathway is inhibited. Because NF-kB has a crucial role in the pathological consequences of TNF action, the mechanism of its activation has attracted the attention of scientists over many years. The data collected so far indicate an important role for RIP1 as a signaling node that contributes to TNFR1’s life and death decisions and have led to the development of a commonly accepted model (reviewed by Wertz and Dixit and Skaug et al.). According to this model, exposure to TNF results in recruitment of a complex consisting of TRADD, RIP1, TRAF2, cIAP1 and cIAP2 at the receptor, allowing cIAP1 and cIAP2 to conjugate RIP1 with K-linked polyubiquitin chains. The addition of these chains to RIP1 has two major consequences. First, it prevents RIP1 from activating cell death signaling pathways, which is dependent on FADD and caspase-8 in apoptosis and on RIP3 in necroptosis. Second, it creates a platform for the recruitment of the protein kinase TAK1 (which acts in concert with the regulatory proteins TAB2 and TAB3) and the IkB kinase complex IKKa–IKKb–NEMO. Both TAB and NEMO were shown to dock at K-linked polyubiquitin chains, and it is believed that the close proximity of TAK1 to the IKK complex on RIP1’s K-polyubiquitin chains is sufficient for TAK1 to activate IKKb by phosphorylation. Once activated, IKKb phosphorylates IkBa, a signal for the K-ubiquitination and proteasomal degradation of IkBa. Releasing the inhibitory effect of IkBa then permits NF-kB dimers to translocate to the nucleus and transactivate pro-survival genes. The de-ubiquitinating enzymes A20 and CYLD have been identified as negative regulators that edit RIP1 K-polyubiquitin chains, thereby limiting the duration of the NF-kB response. In the absence of cIAP1 and cIAP2, RIP1 does not get K-ubiquitinated and TNF exposure induces RIP1-dependent cell death. Therefore, in the present model, RIP1 is crucial for cell survival through activation of the NF-kB pathway. In the current issue from Cell Death and Differentiation, Wong et al. re-examined RIP1’s functions downstream of the TNFR1 using wt and ripk1 / primary and SV40 large T immortalized mouse embryonic fibroblasts (MEFs). When the authors treated the cells with a combination of TNF and the IAP antagonist compound A, they observed that wt MEFs succumbed to the treatment but ripk1 / MEFs did not. These results are consistent with several recently published studies and confirm that RIP1 has a pro-cell death function in the absence of cIAP1 and cIAP2 activity. More interestingly, the authors report that TNF alone has a minor impact on the viability of ripk1 / MEFs, and that a significant difference between the survival of wt and ripk1 / MEFs is observed only when NF-kB action is blocked by the translation inhibitor cycloheximide. These results are remarkable because TNF-induced activation of NF-kB had been shown to protect cells from death. Therefore, although these new data confirm a pro-survival function of RIP1, they seriously question the obligate role of RIP1 in TNFR1-dependent NF-kB activation. These doubts were confirmed when Wong et al. demonstrated that TNF-induced IkBa degradation and recovery, as well as RelA nuclear translocation, occurs normally in both primary and transformed ripk1 / MEFs. The MEF results provided by Wong et al. are at odds with previous studies that report a crucial role for RIP1 in NF-kB activation. For example, Kelliher et al. reported that the nuclear extract from TNF-treated Ripk1 / Abelson virustransformed pre-B cells failed to bind to an NF-kB probe in electrophoretic mobility shift assay. Ea et al. showed that the K-polyubiquitination on lysine 377 of RIP1 is required for NF-kB activation in human Jurkat T cells by serving as a docking site for the recruitment of TAK1 and the IKK complex. These studies might be reconciled by considering a cell-typespecific role for RIP1 in NF-kB activation, perhaps highlighting the pitfalls of not integrating cell specificity in many of the established signaling models. Differences in responses

Introduction. Impairment in cell death pathways represents a general characteristic of most cancer cells. The receptor-interacting protein kinase 3 (RIP3) associates with RIP1 in a necrosome complex that can induce necroptosis, apoptosis, or cell proliferation. The role of RIP3 in necroptosis and inflammation has been extensively studied, but its role in cancer remains poorly understood Methods. We analyzed the expression of RIP1 and RIP3 in CD34+ leukemia cells from a cohort of patients with acute myeloid leukemia (AML) and CD34+ cells from healthy donors. To analyze the potential advantages for myeloid malignant cells due to reduced RIP3 expression, we induced the expression of RIP3 in the DA1-3b mouse leukemia cell line. Results. RIP3 expression was significantly reduced in most AML samples, whereas the expression of RIP1 did not differ significantly. When re-expressed in the mouse DA1-3b leukemia cell line, RIP3 induced apoptosis, and necroptosis in the presence of caspase inhibitors. Surprisingly, the re-expression of a RIP3 mutant with an inactive kinase domain (RIP3-KD) induced significantly more and earlier apoptosis than wild-type RIP3 (RIP3 WT), indicating that the RIP3 kinase domain is an essential regulator of apoptosis/necroptosis in leukemia cells. The induced in vivo expression of RIP3-KD, but not RIP3 WT prolonged the survival of mice injected with leukemia cells. RIP3-KD-induced cell death but not RIP3 WT was significantly antagonized by an IKKβSSEE constitutively active mutant, showing that RIP3-KD-induced apoptosis, but not RIP3 WT-induced apoptosis, was dependent on NF-κB activity. The expression of RIP3-KD induced p65/RelA NF-κB subunit caspase-dependent cleavage, and a non-cleavable p65/RelA D361E mutant rescued cells from apoptosis. The protective effect of the p65/RelA D361E mutant against apoptosis was specific to RIP3-KD-induced cell death because no change in cell death was observed when apoptosis was instead induced by treatment with imatinib or DMSO. The p65/RelA D361E mutant was generated by mutating the INFD putative consensus recognition site for caspase-6. The caspase-6 inhibitor Z-VEID-fmk partially reduced the cell death induced by RIP3-KD and slightly reduced p65/RelA cleavage. p65/RelA cleavage appears to be at least partially mediated by caspase-6. Conclusions. These data indicate that RIP3 silencing in leukemia cells results in suppression of the complex regulation of the apoptosis/necroptosis switch and the modulation of the NF-κB pathway through the caspase-mediated cleavage of p65/RelA. Citation Format: Anne-Lucie Nugues, Hassiba Bouafia, Dominique Hetuin, Celine Berthon, Anne Loyens, Elisabeth Bertrand, Nathalie Jouy, Thierry Idziorek, Bruno Quesnel. RIP3 is downregulated in human myeloid leukemia cells and modulates apoptosis and caspase-mediated p65/RelA cleavage. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1342. doi:10.1158/1538-7445.AM2014-1342

RIPK1 and RIPK3 regulate TNFα-induced β-cell death in concert with caspase activity

Irigenin treatment alleviates doxorubicin (DOX)-induced cardiotoxicity by suppressing apoptosis, inflammation and oxidative stress via the increase of miR-425

Receptor interacting protein kinase 1 (RIPK1) is a cytosolic multidomain protein that controls cell life and death. While RIPK1 promotes cell death through its kinase activity, it also functions as a scaffold protein to promote cell survival by inhibiting FADD-caspase 8-dependent apoptosis and RIPK3-MLKL-dependent necroptosis. This pro-survival function is highlighted by excess cell death and perinatal lethality in Ripk1−/− mice. Recently, loss of function mutation of RIPK1 was found in patients with immunodeficiency and inflammatory bowel diseases. Hematopoietic stem cell transplantation restored not only immunodeficiency but also intestinal inflammatory pathology, indicating that RIPK1 in hematopoietic cells is critical to maintain intestinal immune homeostasis. Here, we generated dendritic cell (DC)-specific Ripk1−/− mice in a genetic background with loss of RIPK1 kinase activity and found that the mice developed spontaneous colonic inflammation characterized by increased neutrophil and Ly6C+ monocytes. In addition, these mice were highly resistant to injury-induced colitis. The increased colonic inflammation and the resistance to colitis were restored by dual inactivation of RIPK3 and FADD, but not by inhibition of RIPK3, MLKL, or ZBP1 alone. Altogether, these results reveal a scaffold activity-dependent role of RIPK1 in DC-mediated maintenance of colonic immune homeostasis.

Background/HypothesisExcessive β‐adrenergic receptor activation causes maladaptive cardiac remodeling in humans with myocardial infarction. Cardiomyocyte apoptosis and necrosis are both considered as the main modes of cell death in β‐adrenergic receptor agonist isoproterenol (ISO)‐induced myocardial injury but the mechanism underlying necrosis remains elusive. Necroptosis is a newly discovered form of regulated cell death that exhibits morphological characteristics of necrosis but is regulated by the receptor interacting protein kinase (RIP) 3‐dependant pathway. Necroptosis has been suggested to play a role in the pathogenesis of common cardiovascular diseases. During necroptosis, the formation of necroptosomes consisting of proteins including RIP3 and RIP1 is critical. More recently, Ca2+/calmodulin‐dependent kinase II (CaMKII), which can be phosphorylated and thereby activated by β‐adrenergic receptor activation, was also reported to be involved in cardiomyocyte necroptosis by directly binding to and being phosphorylated by RIP3. However, whether ISO‐induced cardiomyocyte death involves necroptosis requires further investigation. Hence, we sought to test the hypothesis that RIP3‐depedent cardiomyocyte necroptosis contributes to ISO‐induced cardiac injury.Methods and ResultsYoung adult mixed‐sex wild type (RIP3+/+) or RIP3 global knockout (RIP3−/−) C57BL/6 mice were subject to two consecutive subcutaneous injections of ISO (85mg/kg body weight/injection, 24h interval) or vehicle, myocardial tissues were collected at 24h after the 2nd ISO injection. Evan's blue dye (EBD) was administered (100ug/g body weight, i.p.) at 18h before tissues collection for the EBD‐based detection of increased cardiomyocyte membrane permeability, a hallmark of necrosis. In RIP3+/+ mice, the ISO treatment significantly increased myocardial protein expression of RIP3 (both in the soluble and insoluble fractions), RIP1, RIP3 bund RIP1 (as detected by immunoprecipitation), and phosphorylated CaMKII and resulted in increased EDB uptake by a large number of cardiomyocytes (p<0.01). By contrast, the elevation of RIP1 and the number of EBD‐positive cardiomyocytes, but not phosphorylated CaMKII, were significantly less in the RIP3−/− mice subject to the same ISO treatment (p<0.01).ConclusionInduction of myocardial injury by excessive β‐adrenergic receptor activation involves cardiomyocyte necroptosis via a RIP3‐dependent but CaMKII‐independent pathway.Support or Funding InformationThis work is supported in part by grants from the NIH [HL072166, HL085629, and HL131667] and the National Natural Science Foundation of China [81570278]This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Investigating the role of receptor interacting protein kinase 3 in venous thrombosis

The receptor-interacting protein kinase 3 (RIP3) has been reported to regulate programmed necrosis–necroptosis forms of cell death with important functions in inflammation. We investigated whether RIP3 translocates into mitochondria in response to renal ischemia–reperfusion (I/R) to interact with inner mitochondrial protein (Mitofilin) and promote mtDNA release into the cytosol. We found that release of mtDNA activates the cGAS–STING pathway, leading to increased nuclear transcription of pro-inflammatory markers that exacerbate renal I/R injury. Monolateral C57/6N and RIP3−/− mice kidneys were subjected to 60 min of ischemia followed by either 12, 24, or 48 h of reperfusion. In WT mice, we found that renal I/R injury increased RIP3 levels, as well as its translocation into mitochondria. We observed that RIP3 interacts with Mitofilin, likely promoting its degradation, resulting in increased mitochondria damage and mtDNA release, activation of the cGAS–STING–p65 pathway, and increased transcription of pro-inflammatory markers. All of these effects observed in WT mice were decreased in RIP3−/− mice. In HK-2, RIP3 overexpression or Mitofilin knockdown increased cell death by activating the cGAS–STING–p65 pathway. Together, this study point to an important role of the RIP3–Mitofilin axis in the initiation and development of renal I/R injury.

RIPK3 inhibitor-AZD5423 alleviates acute kidney injury by inhibiting necroptosis and inflammation

Islet amyloid is a hallmark of type 2 diabetes that is associated with β-cell loss. Aggregation of the β-cell secretory product human islet amyloid polypeptide (hIAPP) to form islet amyloid promotes β-cell death 1) via direct effects on the β cell and 2) by stimulating islet immune cell cytokine production. We recently showed that β cells are susceptible to TNFα-induced necroptosis, a form of caspase-independent, receptor interacting protein kinase 3 (RIPK3)-mediated cell death. We hypothesized that RIPK3 also promotes β-cell death in response to islet amyloid formation. To test this hypothesis, we evaluated INS-1, NIT-1 and isolated mouse islet cells treated with synthetic hIAPP (20 μM) +/− the pan-caspase inhibitor zVAD (40 μM). We found synthetic hIAPP increased cell death and caspase activity in INS-1, NIT-1 and mouse islet cells over 48 h. Remarkably, inhibition of caspases with zVAD (as measured by caspase 3/7 activity) failed to reduce synthetic hIAPP-mediated cell death in INS-1 (n=3, p=0.99), NIT-1 (n=3, p=0.99) or mouse islet cells (n=4, p=0.98). To evaluate RIPK3 in hIAPP-induced β-cell death, we examined a small molecule RIPK3 inhibitor (GSK’872, 5 μM), gene-edited NIT-1 Ripk3Δ cells (gRNA targeting exon 6), and intact isolated Ripk3−/− mouse islets. We found GSK’872 failed to protect INS-1 (n=3, p=0.32) or NIT-1 (n=3, p=0.30) cells from hIAPP-induced cell death over 48 h. Similarly, neither NIT-1 Ripk3Δ cells (n=2, hIAPP: p=0.82, hIAPP+zVAD: p=0.97) nor Ripk3−/− islets (n=4, hIAPP: p=0.63, hIAPP+zVAD: p=0.86) were protected from synthetic hIAPP-induced cell death either when caspases were active or inhibited. Our data indicate that caspase 3/7 activity and RIPK3 are dispensable for synthetic hIAPP-induced β-cell death in vitro. However, we found that RIPK3 is upregulated in response to endogenous islet amyloid formation in vitro. Future studies will determine whether RIPK3 promotes β-cell loss in part via effects on endogenous amyloid-induced cytokine signaling. Disclosure N.Mukherjee: None. C.J.Contreras: None. L.Lin: None. E.P.Cai: None. S.E.Kahn: Advisory Panel; Anji Pharmaceuticals, Bayer Inc., Boehringer Ingelheim Inc., Eli Lilly and Company, Merck & Co., Inc., Other Relationship; Novo Nordisk. A.T.Templin: None. Funding U.S. Department of Veterans Affairs (IK2BX004659 to A.T.T.); National Institutes of Health (T32DK064466 to C.J.C.); Indiana University School of Medicine (to N.M.)

Introduction and Objective: Type 1 diabetes (T1D) is characterized by immune-associated destruction of insulin-producing β-cells. Activation of antiviral response programs has been linked to β-cell demise in T1D, and previous studies show that IFNγ + poly I:C (a synthetic double stranded RNA) synergistically elicit β-cell death. However, the mechanisms that underlie this form cytotoxicity are not fully understood. We previously identified receptor interacting protein kinase 1 (RIPK1) as a regulator of β-cell fate. Here, we hypothesized that RIPK1 promotes IFNγ + poly I:C-induced β-cell death. Methods: We treated NIT-1 β-cells with IFNγ + poly I:C for 24 hours and quantified cell death, caspase 3/7 activity, and gene expression. We tested control (CTL, non-targeting gRNA) and Ripk1 gene-edited (Ripk1Δ, gRNA targeting Ripk1 exons 2-3) NIT-1 β-cells and evaluated a small molecule RIPK1 kinase inhibitor (SZM679). Results: Treatment with IFNγ alone or poly I:C alone failed to increase caspase 3/7 activity or cell death in NIT-1 CTL cells, whereas co-treatment led to increases in both of these parameters. Blockade of RIPK1 with SZM679 or in NIT-1 Ripk1Δ cells protected from IFNγ + poly I:C-induced caspase 3/7 activation and cell death. RNAseq analysis revealed that RIPK1-deficient NIT-1 Ripk1Δ cells have decreased expression of nucleic acid sensors including Tlr3, cGas, Sting1, and Ifih1 compared to NIT-1 CTL cells, suggestive of a role for RIPK1 in nucleic acid sensing-related cell death. We also found that Ripk1 expression is increased 5-fold in 20- versus 8-week-old NOD mouse islets, and expression of the nucleic acid sensors Sting1, cGas, Zbp1, and Adar were also increased at this timepoint. Conclusion: Our data indicate that RIPK1 regulates nucleic acid sensor expression and mediates IFNγ + poly I:C-induced NIT-1 β-cell death. Studies are needed to determine the role of RIPK1 in nucleic acid sensor expression and β-cell demise in additional preclinical models of T1D. Disclosure R.C.S. Branco: None. C.J. Contreras: None. N. Mukherjee: None. E. Mather: None. L. Lin: None. K.A. Colglazier: Employee; Eli Lilly and Company. E.P. Cai: None. A.T. Templin: None. Funding U.S. Department of Veterans Affairs (IK2 BX004659 to ATT, I01 BX001060 to SEK); National Institutes of Health (P30 DK097512 to Indiana UniversityCenter for Diabetes and Metabolic Diseases, P30 DK017047 to University of Washington Diabetes Research Center); Ralph W. and Grace M. Showalter Research Trust (080657-00002B to ATT); VA Puget Sound Health Care System and the Richard L. Roudebush VA Medical Center.

RIPK-Dependent Necrosis and Its Regulation by Caspases: A Mystery in Five Acts

Although many studies in recent years have uncovered an important role of receptor-interacting protein kinase 1 (RIP1) in mediating cell survival and death signaling, its potential part in the pathogenesis of cancer remains less understood. Here we report that RIP1 functions as an oncogenic regulator in human melanoma. While the expression of RIP1 was commonly upregulated in melanoma, knockdown of RIP1 inhibited melanoma cell proliferation in vitro, and retarded melanoma growth in a xenograft model. Conversely, despite induction of apoptosis in a small proportion of melanoma cells, overexpression of RIP1 enhanced proliferation in the remaining cells. The promoting effect of RIP1 on melanoma cell proliferation was mediated by activation of NF-κB, as blockade of NF-κB activation eliminated RIP1 overexpression-triggered increase in cell proliferation, whereas hyperactivation of NF-κB abolished inhibition of cell proliferation caused by RIP1 knockdown. In support, RIP1 knockdown led to reduction, whereas its overexpression caused an increase, in NF-κB activation. Strikingly, ectopic expression of RIP1 enhanced melanocyte proliferation and triggered anchorage-independent growth of the cells similarly in a NF-κB-dependent manner. While upregulation of RIP1 was associated with DNA copy number gain in a subset of melanomas, constitutive ubiquitination and subsequent stabilization of the RIP1 protein driven by TNFα autocrine appeared to be another mechanism commonly responsible for upregulation of RIP1 in melanoma cells. Collectively, these results identify RIP1 as an oncogenic regulator in melanoma, and points to the possibility of targeting the NF-κB activating mechanism of RIP1 as a novel approach in the treatment of the disease. Citation Format: Lei Jin, Xiao Ying Liu, Fritz Lai, Xu Guang Yan, Chen Chen Jiang, Su Tang Guo, Chun Yan Wang, Amanda Croft, Hsin-Yi Tseng, James S. Wilmott, Richard A. Scolyer, Xu Dong Zhang. Receptor-Interacting protein kinase 1 functions as an oncogenic regulator in human melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 56. doi:10.1158/1538-7445.AM2015-56