RIPK3 Expression Research Articles

RIPK3 Induces Cardiomyocyte Necroptosis via Inhibition of AMPK-Parkin-Mitophagy in Cardiac Remodelling after Myocardial Infarction.

Receptor-interacting protein 3- (RIPK3-) modulated necroptosis plays a critical role in cardiac remodelling after myocardial infarction (MI). However, the precise regulatory mechanism is not fully elucidated yet. In the present study, we showed that RIPK3 expression was upregulated in myocardial tissue after MI in a mouse model by coronary artery ligation, as well as in the cardiomyocytes following hypoxic injury in vitro. The increase of RIPK3 expression was found to be accompanied by severe cardiac remodelling, cardiac dysfunction, and higher mortality. Elevated RIPK3 expression subsequently abrogated the AMPK pathway that was accompanied by inhibition of Parkin-mediated mitophagy. Loss of mitophagy increased the opening of mitochondrial permeability transition pore (mPTP), which ultimately induced the cardiomyocyte necroptosis. In contrast, genetic ablation of Ripk3 induced the AMPK/Parkin-mitophagy pathway, favouring a prosurvival state that eventually inhibited mPTP opening and induced the necroptosis of cardiomyocytes in the post-MI cardiac remodelling. In conclusion, our results revealed a key mechanism by which necroptosis could be mediated by RIPK3 via the AMPK/Parkin-mitophagy/mPTP opening axis, which provides a potential therapeutic target in the management of heart failure after MI.

Investigation on the expression regulation of RIPK1/RIPK3 in the retinal ganglion cells (RGCs) cultured in high glucose

ABSTRACT Diabetic retinopathy (DR) represents the most typical complication of type 2 diabetes mellitus and one of the most primary oculopathy causing blindness. However, the mechanism of DR remains unknown. RIPK1/RIPK3, as homologous serine/threonine kinases, are key elements in mediating necroptosis and may have functions in DR development. To clarify the relationship between DR and RIPK1/RIPK3, this study established a model of apoptosis using high-glucose induced RGCs, which were treated with 7.5, 19.5, and 35 mM D-glucose for 12, 24, and 48 h, respectively. Subsequently, the expression of RIPK1/RIPK3 was determined and the protective effect of necrostatin-1 on RGCs injury induced by high glucose was explored. The results demonstrated that the expression of RIPK1 and RIPK3 in the cells was increased markedly following 12 h treatment with 19.5 mM D-glucose. Additionally, following an addition of 100 μM necrostatin-1 in 19.5 mM D-glucose medium for RGCs treatment 12 h, the protein expression of RIPK1 and RIPK3 was decreased markedly, and the number of Nissl bodies in cells was increased substantially. The findings of the present study indicated that high glucose could induce the expression of RIPK1/RIPK3, and necrostatin-1 could effectively protect RGCs from D-glucose-induced cell necrosis.

RIPK3-Mediated Necroptosis in Diabetic Cardiomyopathy Requires CaMKII Activation.

Activation of Ca2+/calmodulin-dependent protein kinase (CaMKII) has been proved to play a vital role in cardiovascular diseases. Receptor-interaction protein kinase 3- (RIPK3-) mediated necroptosis has crucially participated in cardiac dysfunction. The study is aimed at investigating the effect as well as the mechanism of CaMKII activation and necroptosis on diabetic cardiomyopathy (DCM). Wild-type (WT) and the RIPK3 gene knockout (RIPK3−/−) mice were intraperitoneally injected with 60 mg/kg/d streptozotocin (STZ) for 5 consecutive days. After 12 w of feeding, 100 μL recombinant adenovirus solution carrying inhibitor 1 of protein phosphatase 1 (I1PP1) gene was injected into the caudal vein of mice. Echocardiography, myocardial injury, CaMKII activity, necroptosis, RIPK1 expression, mixed lineage kinase domain-like protein (MLKL) phosphorylation, and mitochondrial ultrastructure were measured. The results showed that cardiac dysfunction, CaMKII activation, and necroptosis were aggravated in streptozotocin- (STZ-) stimulated mice, as well as in (Lepr) KO/KO (db/db) mice. RIPK3 deficiency alleviated cardiac dysfunction, CaMKII activation, and necroptosis in DCM. Furthermore, I1PP1 overexpression reversed cardiac dysfunction, myocardial injury and necroptosis augment, and CaMKII activity enhancement in WT mice with DCM but not in RIPK3−/− mice with DCM. The present study demonstrated that CaMKII activation and necroptosis augment in DCM via a RIPK3-dependent manner, which may provide therapeutic strategies for DCM.

TNF-mediated alveolar macrophage necroptosis drives disease pathogenesis during respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is the major cause of acute bronchiolitis in infants under 2 years old. Necroptosis has been implicated in the outcomes of respiratory virus infections. We report that RSV infection triggers necroptosis in primary mouse macrophages and human monocytes in a RIPK1-, RIPK3- and MLKL-dependent manner. Moreover, necroptosis pathways are harmful to RSV clearance from alveolar macrophages. Additionally, Ripk3−/− mice were protected from RSV-induced weight loss and presented with reduced viral loads in the lungs.Alveolar macrophage depletion also protected mice from weight loss and decreased lung RSV virus load. Importantly, alveolar macrophage depletion abolished the upregulation of Ripk3 and Mlkl gene expression induced by RSV infection in the lung tissue.Autocrine tumor necrosis factor (TNF)-mediated RSV-triggered macrophage necroptosis and necroptosis pathways were also involved in TNF secretion even when macrophages were committed to cell death, which can worsen lung injury during RSV infection. In line, Tnfr1−/− mice had a marked decrease in Ripk3 and Mlkl gene expression and a sharp reduction in the numbers of necrotic alveolar macrophages in the lungs. Finally, we provide evidence that elevated nasal levels of TNF are associated with disease severity in infants with RSV bronchiolitis.We propose that targeting TNF and/or the necroptotic machinery may be valuable therapeutic approaches to reduce the respiratory morbidity caused by RSV infection in young children.

Ubiquitination of RIPK1 regulates its activation mediated by TNFR1 and TLRs signaling in distinct manners

RIPK1 is a death-domain (DD) containing kinase involved in regulating apoptosis, necroptosis and inflammation. RIPK1 activation is known to be regulated by its DD-mediated interaction and ubiquitination, though underlying mechanisms remain incompletely understood. Here we show that K627 in human RIPK1-DD and its equivalent K612 in murine RIPK1-DD is a key ubiquitination site that regulates the overall ubiquitination pattern of RIPK1 and its DD-mediated interactions with other DD-containing proteins. K627R/K612R mutation inhibits the activation of RIPK1 and blocks both apoptosis and necroptosis mediated by TNFR1 signaling. However, Ripk1K612R/K612R mutation sensitizes cells to necroptosis and caspase-1 activation in response to TLRs signaling. Ripk1K612R/K612R mice are viable, but develop age-dependent reduction of RIPK1 expression, spontaneous intestinal inflammation and splenomegaly, which can be rescued by antibiotic treatment and partially by Ripk3 deficiency. Furthermore, we show that the interaction of RIPK1 with FADD contributes to suppressing the activation of RIPK3 mediated by TLRs signaling. Our study demonstrates the distinct roles of K612 ubiquitination in mRIPK1/K627 ubiquitination in hRIPK1 in regulating its pro-death kinase activity in response to TNFα and pro-survival activity in response to TLRs signaling.

Selenium ameliorates aflatoxin B1-induced uterine injury in female mice and necrosis of human endometrial microvascular endothelial cells.

This study aimed to observe the effects of Selenium (Se) on aflatoxin B1 (AFB1)-induced uterine injury in female mice and necrosis of human endometrial microvascular endothelial cells (HEMECs). Fifty female mice were randomly divided into control group; AFB1 group; Se group (0.2 mg/kg each day); AFB1 + Se group; and positive control group. After continuous treatment for 30 days, uterine tissues were harvested, which were used for hematoxylin and eosin (H&E) staining. Necrosis-related proteins including RIPK1, RIPK3, and MLKL were examined in uterine tissues using western blot and immunohistochemistry. HEMECs were treated with different concentrations of AFB1 or Se, which were used for selecting the optimal concentrations. RIPK1, RIPK3 and MLKL expression was detected in HEMECs exposed to AFB1 and/or Se via western blot and immunofluorescence. H&E staining results showed that AFB1 induced uterine injury of female male, which was ameliorated by Se treatment. According to western blot and immunohistochemistry, RIPK1, RIPK3, and MLKL expression was distinctly increased in uterine tissues of AFB1-treated mice, which was decreased by Se treatment. Cell viability of HEMECs was gradually lowered as the concentrations of AFB1and Se increased. A 10-μM AFB1 exposure significantly increased RIPK1, RIPK3, and MLKL expression in HEMECs, which was improved following co-treatment with 5-μM Se. Thus, our findings revealed that Se may ameliorate AFB1-induced uterine injury in female mice and necrosis of HEMECs.

Correlation of NK cell RIPK1 with Prognosis of Acute Myeloid Leukemia Patients with FLT3-ITD Mutation

To investigate the correlation of NK cell receptor-interacting serine/threonine kinase 1（RIPK1） activity and expression with prognosis of acute myeloid leukemia (AML) patients with FLT3-ITD mutation. A total of 132 AML patients with FLT3-ITD mutation and 136 AML patients with FLT3-WT were selected. Clinical data and the number, length and rearrangement ratio of FLT3-ITD mutations were collected. The ratio of CD4+ T cells, regulatory T cells（Tregs）, CD8+ T cells, B cells, natural killer cells（NK cells） and macrophage in peripheral blood（PB） were analyzed by flow cytometry. Correlation of NK cell ratio with FLT3-ITD mutation number, length and rearrangement ratio was analyzed by Pearson correlation analysis. Western blot and RT-qPCR were used to detect RIPK1 protein and mRNA levels in NK cells, respectively. Plasma RIPK activity was detected by ELISA, and Pearson corre-lation analysis was performed for the correlation of RIPK with FLT3-ITD mutation number, length and rearrangement ratio. Kaplan-Meier curve was used to analyze the survival rate of AML patients with FLT3-ITD mutation. Compared with AML patients with FLT3-WT, the white blood cell count in patients with FLT3-ITD mutation significantly increased, PB and BM blasts significantly decreased. There was no significant change in PB CD3+ T cells, Tregs, CD8+ T cells, B cells, M1 and M2 type macrophage of AML patients with FLT3-WT and FLT3-ITD mutation；Compared with AML patients with FLT3-WT, CD4+ T cells significantly decreased, NK cells significantly increased in AML patients wtih FLT3-ITD mutation. NK cells ratio in AML patients with FLT3-ITD mutation significantly positively correlated with the number of FLT3-ITD mutations and rearrangement bases. Plasma RIPK1 activity, RIPK1 protein and mRNA levels in NK cells of AML patients with FLT3-ITD were significantly lower than those of AML patients with FLT3-WT, and negatively correlated with the number of FLT3-ITD mutations, the length of rearranged bases (≥52 bp) and the ratio of rearranged bases. The survival rate of AML patients with FLT3-ITD mutation in low RIPK1 activity after Rydapt treatment was significantly higher than that in high RIPK1 activity. The prognosis of AML patients with FLT3-ITD mutation closely relates with plasma RIPK1 activity and RIPK1 expression in NK cells.

Expression of Protein Kinases RIPK-1 and RIPK-3 in Mouse and Human Hair Follicle.

Expression of cell death regulators RIPK-1 and RIPK-3 in mouse and human hair follicle structures was studied by immunohistochemistry. At anagen and catagen stages of mouse hair follicle, RIPK-1+ cells were located in the inner root sheath, whereas RIPK-3+ cells were found in the inner and outer root sheath, dermal papilla, and interfollicular epidermis. RIPK-1 expression intensity was low in the early anagen and increased as mature anagen and catagen approached. RIPK-1+ and RIPK-3+ cells were also found in human hair follicle. It is assumed that the role of necroptosis markers in hair follicle life activity is independent of programmed cell death and that they may have yet unknown functions and take part in noncanonical signal cascades.

Functional suppression of Ripk1 blocks the NF-κB signaling pathway and induces neuron autophagy after traumatic brain injury.

Traumatic brain injury (TBI), known as intracranial injury, has been a serious threat to human health. Evidence exists indicating that autophagy and inflammatory responses contribute to secondary brain injury after TBI. Notably, receptor-interacting protein kinase 1 (Ripk1) exerts an important role in cell autophagy. Therefore, this study aims to explore the effect of Ripk1 on neuron autophagy and apoptosis in TBI. Initially, blood samples of patients with TBI and healthy persons were collected to detect the expression of Ripk1, nuclear factor-kappa B (NF-κB), and NF-kB inhibitor α (IKBα). Then rat models with TBI were successfully established and, respectively, treated with shRNA targeting Ripk1 (sh-Ripk1), Ripk1 overexpression plasmid (oe-Ripk1), or IKKα inhibitor (BAY 11-7082). Subsequently, reverse transcription quantitative polymerase chain reaction and Western blot analysis were conducted to detect the expression of Ripk1, IKBα, NF-κB signaling pathway-, and apoptosis-related factors. Enzyme-linked immunosorbent assay was used to detect the expression of inflammatory cytokines. Compared with healthy persons, the expression of Ripk1, NF-κB and IKBα in blood of TBI patients was significantly upregulated. After silencing of Ripk1 or inhibition of the NF-κB signaling pathway, the expression of IL-1β, IL-6, TNF-α, Bax, and cleaved-caspase-3 was downregulated, and the expression of Bcl-2, ATG5, and LC3II/LC3I was upregulated. Furthermore, neuron injury and apoptosis were notably reduced and neuron autophagy increased significantly by Ripk1 downregulation or IKKα inhibitor. Ripk1 overexpression contributed to activation of NF-κB signaling pathway, whereby aggravating TBI-induced damage. Silencing Ripk1 suppresses TBI by inhibiting inflammation and promoting autophagy of neurons via inhibition of NF-κB signaling pathway.

Functional role of microRNA-500a-3P-loaded liposomes in the treatment of cisplatin-induced AKI.

Cisplatin treatment results in acute kidney injury (AKI) by the phosphorylation of mixed lineage kinase domain-like protein (MLKL). The knockout of MLKL, which is a principle mediator of necroptosis, is believed to alleviate the AKI symptoms. The present study was aimed to improve the therapeutic efficacy in AKI. For this purpose, miR-500a-3P was identified as appropriate miRNA therapeutics and loaded in liposome delivery carrier. The authors have showed that the miR-LIP directly controls the expression of RIPK3 and MLKL - a modulator of necroptosis and thereby reduces the severity of kidney injury. The miR-LIP significantly controlled the phosphorylation of MLKL compared to that of CDDP-treated HK2 cells. Similar results are observed with RIPK3. The miR-LIP has also been demonstrated to control the inflammatory response in tubular cells. Western blot analysis further revealed that the phosphorylation of P-65 was mainly responsible for the inflammatory response and miR-LIP significantly decreased the CDDP-induced NF-kB phosphorylation. Overall, the present study explored the molecular mechanism behind the necroptosis in AKI and potential of miRNA in targeting MLKL pathways. Study further highlights the potential advantage of liposome as a delivery carrier for miRNA therapeutics.

Prognostic Significance of CHIP and RIPK3 in Non-Small Cell Lung Cancer.

RIPK3 is a key regulator of necroptosis, which plays a double-edged sword role in tumor progression. CHIP is an E3 ubiquitin ligase that regulates necroptosis by degrading RIPK3. Here, we investigated the prognostic value of RIPK3 and CHIP expression in 404 patients with non-small cell lung cancer (NSCLC). Expressions of CHIP and RIPK3 showed opposite correlations with survival. CHIP expression was associated with the longer overall survival (OS), whereas RIPK3 expression was associated with the shorter OS. RIPK3 positivity showed marginal association with shorter OS and disease-free survival (DFS) in adjuvant radiotherapy recipients but not in non-recipients, suggesting that necroptosis may induce radioresistance. In multivariate analysis, CHIP expression was associated with longer OS. Compared with other patients, CHIP(−)/RIPK3(+) patients had shorter OS and DFS. In summary, in patients with NSCLC, the expression of CHIP was an independent favorable prognostic factor while that of RIPK3 was an adverse prognostic factor.

Apoptosis and necroptosis occur in the different brain regions of hippocampus in a rat model of hypoxia asphyxia

Aim of the study Hypoxic-ischemic encephalopathy (HIE) is a major cause of newborn brain injury. Apoptosis and necroptosis are two forms of cell death which may occur in HIE but reported data are yet limited. This study investigates the expression of receptor interacting protein kinase (RIPK) 1 and 3, and caspase3, the key modulators of necroptosis and apoptosis, respectively, in a model of HIE to determine whether both forms of cell death occur in the corresponding brain regions. Materials and methods Postneonatal day 7 Sprague-Dawley rats were subjected to right carotid artery ligation followed by hypoxia or subjected to skin incision under surgical anesthesia without ligation and hypoxia. Neuroglioma (H4) cell was cultured and subjected to 24 h hypoxic insults. Necrostatin-1, a RIPK1 inhibitor, was administered in both in vivo and in vitro settings before insult. Results After hypoxic-ischemic insults, both RIPK1 and RIPK3 expression were significantly increased in the region of hippocampal dentate gyrus in the injurious hemisphere. However, cleaved caspase3 was significantly increased in the hippocampal cornu ammonis 1 region in the injurious hemisphere. After hypoxic insults, RIPK1 and RIPK3 expression was also found in H4 cells. In addition, it was identified that the increased RIPK1 and RIPK3 can be inhibited by necrostatin-1 in both in vivo and in vitro. Conclusions These data indicated that apoptosis and necroptosis occur in different brain regions of hippocampus in a model of HIE which may suggest that strategies to prevent each form of neuronal death is valuable to be developed.

Inhibition of RIPK1/RIPK3 ameliorates osteoclastogenesis through regulating NLRP3-dependent NF-κB and MAPKs signaling pathways

Osteoblast-induced bone formation and osteoclast-regulated bone resorption are the essential events contributing to bone homeostasis. It is critical to investigate the underlying molecular mechanisms. In this study, we explored the effects of receptor-interacting serine-threonine kinases (RIPKs) on osteoclastogenesis and bone loss in vitro and in vivo. We found that both RIPK1 and RIPK3 expression levels were highly up-regulated during osteoclastogenesis. Inhibiting RIPK1 and RIPK3 by their inhibitors Necrostatin-1 (Nec-1) and GSK-872, respectively, showed effective activities against osteoclast differentiation and bone resorption induced by receptor activator of nuclear factor-κB ligand (Rankl). Osteoclast-specific gene expression levels were also impeded by RIPK1/RIPK3 blockage in a time-dependent manner. Subsequently, we found that the pyrin domain-containing protein 3 (NLRP3) inflammasome stimulated by Rankl during osteoclastogenesis was greatly inhibited by Nec-1 and GSK-872. Additionally, reducing RIPK1/RIPK3 overtly reduced the activation of NF-κB (p65) and mitogen-activated protein kinases (MAPKs) signaling during Rankl-induced osteoclast formation. Notably, adenovirus-regulated NLRP3 over-expression significantly abrogated the inhibitory effects of Nec-1 and GSK-872 on NF-κB and MAPKs signaling pathways, as well as the osteoclastogenesis. Finally, the in vivo studies indicated that suppressing RIPK1/RIPK3 could effectively ameliorate ovariectomy (OVX)-induced bone loss in mice through repressing osteoclastogenesis, as proved by the clearly down-regulated number of osteoclasts via histological staining. In conclusion, our study elucidated that restraining RIPK1/RIPK3 could hinder osteoclastogenesis and attenuate bone loss through suppressing NLRP3-dependent NF-κB and MAPKs signaling pathways. Therefore, targeting RIPK1/RIPK3 signaling might be a potential therapeutic strategy to develop effective treatments against osteoclast-related bone lytic diseases.

Mitochondrial Damage and Necroptosis in Aging Cochlea.

Age-related hearing loss (ARHL) is an irreversible, progressive neurodegenerative disorder and is presently untreatable. Previous studies using animal models have suggested mitochondrial damage and programmed cell death to be involved with ARHL. Thus, we further investigated the pathophysiologic role of mitochondria and necroptosis in aged C57BL/6J male mice. Aged mice (20 months old) exhibited a significant loss of hearing, number of hair cells, neuronal fibers, and synaptic ribbons compared to young mice. Ultrastructural analysis of aged cochleae revealed damaged mitochondria with absent or disorganized cristae. Aged mice also showed significant decrease in cochlear blood flow, and exhibited increase in gene expression of proinflammatory cytokines (IL-1β, IL-6, and TNF-α), receptor-interacting serine/threonine-protein kinase 1 and 3 (RIPK1 and RIPK3) and the pseudokinase mixed-lineage kinase domain-like (MLKL). Immunofluorescence (IF) assays of cytochrome C oxidase I (COX1) confirmed mitochondrial dysfunction in aged cochleae, which correlated with the degree of mitochondrial morphological damage. IF assays also revealed localization and increased expression of RIPK3 in sensorineural tissues that underwent significant necroptosis (inner and outer hair cells and stria vascularis). Together, our data shows that the aging cochlea exhibits damaged mitochondria, enhanced synthesis of proinflammatory cytokines, and provides new evidence of necroptosis in the aging cochlea in in vivo.

Gut epithelial TSC1/mTOR controls RIPK3-dependent necroptosis in intestinal inflammation and cancer.

Although Western diet and dysbiosis are the most prominent environmental factors associated with inflammatory bowel diseases (IBDs), the corresponding host factors and cellular mechanisms remain poorly defined. Here we report that the TSC1/mTOR pathway in the gut epithelium represents a metabolic and innate immune checkpoint for intestinal dysfunction and inflammation. mTOR hyperactivation triggered by Western diet or Tsc1 ablation led to epithelium necroptosis, barrier disruption, and predisposition to dextran sulfate sodium-induced colitis and inflammation-associated colon cancer. Mechanistically, our results uncovered a critical role for TSC1/mTOR in restraining the expression and activation of RIPK3 in the gut epithelium through TRIM11-mediated ubiquitination and autophagy-dependent degradation. Notably, microbiota depletion by antibiotics or gnotobiotics attenuated RIPK3 expression and activation, thereby alleviating epithelial necroptosis and colitis driven by mTOR hyperactivation. mTOR primarily impinged on RIPK3 to potentiate necroptosis induced by TNF and by microbial pathogen-associated molecular patterns (PAMPs), and hyperactive mTOR and aberrant necroptosis were intertwined in human IBDs. Together, our data reveal a previously unsuspected link between the Western diet, microbiota, and necroptosis and identify the mTOR/RIPK3/necroptosis axis as a driving force for intestinal inflammation and cancer.

Dose- and Time-Dependent Cytotoxicity of Carteolol in Corneal Endothelial Cells and the Underlying Mechanisms.

Carteolol is a non-selective β-adrenoceptor antagonist used for the treatment of glaucoma, and its abuse might be cytotoxic to the cornea. However, its cytotoxicity and underlying mechanisms need to be elucidated. Herein, we used an in vivo model of feline corneas and an in vitro model of human corneal endothelial cells (HCECs), respectively. In vivo results displayed that 2% carteolol (clinical dosage) could induce monolayer density decline and breaking away of feline corneal endothelial (FCE) cells. An in vitro model of HCECs that were treated dose-dependently (0.015625–2%) with carteolol for 2–28 h, resulted in morphological abnormalities, declining in cell viability and elevating plasma membrane (PM) permeability in a dose- and time- dependent manner. High-dose (0.5–2%) carteolol treatment induced necrotic characteristics with uneven distribution of chromatin, marginalization and dispersed DNA degradation, inactivated caspase-2/-8, and increased RIPK1, RIPK3, MLKL, and pMLKL expression. The results suggested that high-dose carteolol could induce necroptosis via the RIPK/MLKL pathway. While low-dose (0.015625–0.25%) carteolol induced apoptotic characteristics with chromatin condensation, typical intranucleosomal DNA laddering patterns, G1 cell-cycle arrest, phosphatidylserine (PS) externalization, and apoptotic body formation in HCECs. Meanwhile, 0.25% carteolol treatment resulted in activated caspase-2, -3, -8, and -9, downregulation of Bcl-2 and Bcl-xL, upregulation of Bax and Bad, ΔΨm disruption, and release of cytoplasmic cytochrome c (Cyt.c) and AIF into the cytoplasm. These observations suggested that low-dose carteolol could induce apoptosis via a caspase activated and mitochondrial-dependent pathway. These results suggested that carteolol should be used carefully, as low as 0.015625% cartelol caused apoptotic cell death in HCECs in vitro.

Up-regulated microRNA-411 or declined RIPK1 inhibits proliferation and promotes apoptosis of synoviocytes in rheumatoid arthritis mice via decreased NF-κB pathway

ABSTRACT Rheumatoid arthritis (RA) is a chronic and inflammatory synovitis systemic disease. Due to the unknown pathogenesis, this study was to investigate the effect of microRNA (miR)-411 on apoptosis and joint function of synoviocytes in RA mice via RIPK1-mediated NF-κB signaling pathway. The collagen-induced arthritis model mice were induced via collagen type II and Freund’s adjuvant. The mice were injected with miR-411 mimics, si-RIPK1 or miR-411 mimics + oe-RIPK1 to figure out their roles in cell apoptosis and inflammation of synovial tissues. Synoviocytes were grouped as in animal experiments. Proliferation and apoptosis of synoviocytes were detected upon treatment with overexpressed miR-411 and silenced RIPK1. The expression of miR-411, RIPK1 and NF-κB in synovial tissues and synoviocytes of RA mice was detected by RT-qPCR and Western blot analysis. Poorly expressed miR-411, and highly expressed NF-κB and RIPK1 existed in synovial tissue and synoviocytes of RA. Additionally, it was found that si-RIPK1 decreased NF-κB expression, and miR-411 mimics decreased both RIPK1 and NF-κB. MiR-411 had a targeted relationship with RIPK1. si-RIPK1 or miR-411 mimics promoted cell apoptosis and strained inflammation in synovial tissues of mice with RA. Overexpressed miR-411 or silencing RIPK1 inhibited the proliferation and promoted apoptosis of synoviocytes of RA mice. Up-regulation of miR-411 or down-regulation of RIPK1 had a certain inhibitory effect on RA. This study suggests that up-regulated miR-411 or down-regulated RIPK1 promoted apoptosis and inhibited proliferation of synoviocytes of RA mice, which may be related to the inhibition of NF-κB activation.

Coxsackievirus A6 Induces Necroptosis for Viral Production.

Hand, foot, and mouth disease (HFMD) is a febrile exanthematous disease with typical or atypical symptoms. Typical HFMD is usually caused by enterovirus 71 (EV71) or coxsackievirus A16, while atypical HFMD is usually caused by coxsackievirus A6 (CA6). In recent years, worldwide outbreaks of CA6-associated HFMD have dramatically increased, although the pathogenic mechanism of CA6 is still unclear. EV71 has been established to induce caspase-dependent apoptosis, but in this study, we demonstrate that CA6 infection promotes a distinct pathway of cell death that involves loss of cell membrane integrity. Necrostatin-1, an inhibitor of necroptosis, blocks the cell death induced by CA6 infection, but Z-DEVD-FMK, an inhibitor of caspase-3, has no effect on CA6-induced cell death. Furthermore, CA6 infection up-regulates the expression of the necroptosis signaling molecule RIPK3. Importantly, necrostatin-1 inhibits CA6 viral production, as assessed by its ability to inhibit levels of VP1 protein and genomic RNA and infectious particles. CA6-induced necroptosis is not dependent on the generation of reactive oxygen species; however, viral 3D protein can directly bind RIPK3, which is suggestive of a direct mechanism of necroptosis induction. Therefore, these results indicate that CA6 induces a mechanism of RIPK3-dependent necroptosis for viral production that is distinct from the mechanism of apoptosis induced by typical HFMD viruses.

RIPK3 upregulation confers robust proliferation and collateral cystine-dependence on breast cancer recurrence.

The molecular and genetic basis of tumor recurrence is complex and poorly understood. RIPK3 is a key effector in programmed necrotic cell death and, therefore, its expression is frequently suppressed in primary tumors. In a transcriptome profiling between primary and recurrent breast tumor cells from a murine model of breast cancer recurrence, we found that RIPK3, while absent in primary tumor cells, is dramatically reexpressed in recurrent breast tumor cells by an epigenetic mechanism. Unexpectedly, we found that RIPK3 knockdown in recurrent tumor cells reduced clonogenic growth, causing cytokinesis failure, p53 stabilization, and repressed the activities of YAP/TAZ. These data uncover a surprising role of the pro-necroptotic RIPK3 kinase in enabling productive cell cycle during tumor recurrence. Remarkably, high RIPK3 expression also rendered recurrent tumor cells exquisitely dependent on extracellular cystine and undergo necroptosis upon cystine deprivation. The induction of RIPK3 in recurrent tumors unravels an unexpected mechanism that paradoxically confers on tumors both growth advantage and necrotic vulnerability, providing potential strategies to eradicate recurrent tumors.

Crucial role of the terminal complement complex in chondrocyte death and hypertrophy after cartilage trauma

Innate immune response and particularly terminal complement complex (TCC) deposition are thought to be involved in the pathogenesis of posttraumatic osteoarthritis. However, the possible role of TCC in regulated cell death as well as chondrocyte hypertrophy and senescence has not been unraveled so far and was first addressed using an ex vivo human cartilage trauma-model. Cartilage explants were subjected to blunt impact (0.59J) and exposed to human serum (HS) and cartilage homogenate (HG) with or without different potential therapeutics: RIPK1-inhibitor Necrostatin-1 (Nec), caspase-inhibitor zVAD, antioxidant N-acetyl cysteine (NAC) and TCC-inhibitors aurintricarboxylic acid (ATA) and clusterin (CLU). Cell death and hypertrophy/senescence-associated markers were evaluated on mRNA and protein level. Addition of HS resulted in significantly enhanced TCC deposition on chondrocytes and decrease of cell viability after trauma. This effect was potentiated by HG and was associated with expression of RIPK3, MLKL and CASP8. Cytotoxicity of HS could be prevented by heat-inactivation or specific inhibitors, whereby combination of Nec and zVAD as well as ATA exhibited highest cell protection. Moreover, HS+HG exposition enhanced the gene expression of CXCL1, IL-8, RUNX2 and VEGFA as well as secretion of IL-6 after cartilage trauma. Our findings imply crucial involvement of the complement system and primarily TCC in regulated cell death and phenotypic changes of chondrocytes after cartilage trauma. Inhibition of TCC formation or downstream signaling largely modified serum-induced pathophysiologic effects and might therefore represent a therapeutic target to maintain the survival and chondrogenic character of cartilage cells.