Receptor-interacting Protein Kinase Research Articles

Divergent roles of RIPK3 and MLKL in high-fat diet-induced obesity and MAFLD in mice.

Cell death frequently occurs in the pathogenesis of obesity and metabolic dysfunction-associated fatty liver disease (MAFLD). However, the exact contribution of core cell death machinery to disease manifestations remains ill-defined. Here, we show via the direct comparison of mice genetically deficient in the essential necroptotic regulators, receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL), as well as mice lacking apoptotic caspase-8 in myeloid cells combined with RIPK3 loss, that RIPK3/caspase-8 signaling regulates macrophage inflammatory responses and drives adipose tissue inflammation and MAFLD upon high-fat diet feeding. In contrast, MLKL, divergent to RIPK3, contributes to both obesity and MAFLD in a manner largely independent of inflammation. We also uncover that MLKL regulates the expression of molecules involved in lipid uptake, transport, and metabolism, and congruent with this, we discover a shift in the hepatic lipidome upon MLKL deletion. Collectively, these findings highlight MLKL as an attractive therapeutic target to combat the growing obesity pandemic and metabolic disease.

M6A-modified circCacna1c regulates necroptosis and ischemic myocardial injury by inhibiting Hnrnpf entry into the nucleus.

Circular RNAs (circRNAs) are differentially expressed in various cardiovascular diseases, including myocardial infarction (MI) injury. However, their functional role in necroptosis-induced loss of cardiomyocytes remains unclear. We identified a cardiac necroptosis-associated circRNA transcribed from the Cacna1c gene (circCacna1c) to investigate the involvement of circRNAs in cardiomyocyte necroptosis. To investigate the role of circCacna1c during oxidative stress, H9c2 cells and neonatal rat cardiomyocytes were treated with hydrogen peroxide (H2O2) to induce reactive oxygen species (ROS)-induced cardiomyocyte death. The N6-methyladenosine (m6A) modification level of circCacna1c was determined by methylated RNA immunoprecipitation quantitative polymerase chain reaction (MeRIP-qPCR) analysis. Additionally, an RNA pull-down assay was performed to identify interacting proteins of circCacna1c in cardiomyocytes, and the regulatory role of circCacna1c in target protein expression was tested using a western blotting assay. Furthermore, theMI mouse model was constructed to analyze the effect of circCacna1c on heart function and cardiomyocyte necroptosis. The expression of circCacna1c was found to be reduced in cardiomyocytes exposed to oxidative stress and in mouse hearts injured by MI. Overexpression of circCacna1c inhibited necroptosis of cardiomyocytes induced by hydrogen peroxide and MI injury, resulting in a significant reduction in myocardial infarction size and improved cardiac function. Mechanistically, circCacna1c directly interacts with heterogeneous nuclear ribonucleoprotein F (Hnrnpf) in the cytoplasm, preventing its nuclear translocation and leading to reduced Hnrnpf levels within the nucleus. This subsequently suppresses Hnrnpf-dependent receptor-interacting protein kinase 1 (RIPK1) expression. Furthermore, fat mass and obesity-associated protein (FTO) mediates demethylation of m6A modification on circCacna1c during necrosis and facilitates degradation of circCacna1c. Our study demonstrates that circCacna1c can improve cardiac function following MI-induced heart injury by inhibiting the Hnrnpf/RIPK1-mediated cardiomyocyte necroptosis. Therefore, the FTO/circCacna1c/Hnrnpf/RIPK1 axis holds great potential as an effective target for attenuating cardiac injury caused by necroptosis in ischemic heart disease.

Mechanistic Insights into Influenza A Virus-Induced Cell Death and Emerging Treatment Strategies

Influenza A virus (IAV) infection initiates a complex interplay of cell death modalities, including apoptosis, necroptosis, pyroptosis, and their integration, known as PANoptosis, which significantly impacts host immune responses and tissue integrity. These pathways are intricately regulated by viral proteins and host factors, contributing to both viral clearance and pathogenesis-related tissue damage. This review comprehensively explores the molecular mechanisms underlying these cell death processes in influenza infection. We highlight the roles of key regulatory proteins, such as ZBP1 (Z-DNA binding protein 1) and RIPK3 (receptor-interacting protein kinase 3), in orchestrating these responses, emphasizing the dual roles of cell death in both antiviral defense and tissue injury. Furthermore, we discuss emerging therapeutic strategies targeting these pathways, aiming to enhance antiviral efficacy while minimizing collateral tissue damage. Future research should focus on targeted approaches to modulate cell death mechanisms, aiming to reduce tissue damage and improve clinical outcomes for patients with severe influenza.

Deficiency of muscle-generated brain-derived neurotrophic factor causes inflammatory myopathy through reactive oxygen species-mediated necroptosis and pyroptosis

Idiopathic inflammatory myopathy (commonly known as myositis) is a group of immune-related diseases characterized by muscle damage, weakness, and fatigue with unknown causes. Although overactivated innate immunity is a widely believed cause of myositis onset, the mechanism that provokes and maintains a high immune response in myositis patients is still unclear. This study aims to test if brain-derived neurotrophic factor (BDNF) deficiency per se is sufficient to cause myositis and determine its underlying mechanism. We found that ablating BDNF production in skeletal muscle is sufficient to trigger myositis development in mice. Muscle-specific Bdnf knockout (MBKO) mice displayed extensive myocyte necrosis, mononuclear cell infiltration, and myophagocytosis. In association with these damages, elevated production of pro-inflammatory cytokines such as interleukin (IL) 23, IL-1β, IL-18, and tumor necrosis factor α (TNFα) was found in the muscle of MBKO mice. Disruption of sarcolemma integrity was also detected in MBKO mice, which is a result of necroptosis executioner Mixed lineage kinase domain-like protein (MLKL) and pyroptosis executioner Gasdermin D (GSDMD) activation. Mechanistically, diminishing BDNF synthesis in myotubes enhances the accumulation of mitochondrial reactive oxygen species (mtROS), which sensitizes the cells towards TNFα-induced receptor-interacting protein kinase (RIPs) activation and promotes the formation of NLR family pyrin domain containing 3 (NLRP3)-containing inflammasome. BDNF deficiency-induced cell death could be alleviated by scavenging mtROS, suppressing the activity of GSDMD, or inhibiting receptor-interacting kinase 3 (RIP3). Similarly, supplementation of BDNF mimetics, suppression of RIP3 activity, increasing the intramyocellular antioxidant, or enhancing mitophagy ameliorated the myopathies of MBKO mice and improved their muscle strength. Together, our study demonstrates that insufficient BDNF production in mouse muscle causes the development of pathological features of myositis via enhancing oxidative stress, necroptosis, and pyroptosis in myofibers.

Receptor-Interacting Protein Kinase 3 as a Serological Biomarker in Relation to Disease Severity and Delirium After Acute Pancreatitis: A Prospective Cohort Study

Receptor-Interacting Protein Kinase 3 as a Serological Biomarker in Relation to Disease Severity and Delirium After Acute Pancreatitis: A Prospective Cohort Study

Mechanisms Underlying Sensory Nerve-Predominant Damage by Methylmercury in the Peripheral Nervous System.

Sensory disturbances and central nervous system symptoms are important in patients with Minamata disease. In the peripheral nervous system of these patients, motor nerves are not strongly injured, whereas sensory nerves are predominantly affected. In this study, we investigated the mechanisms underlying the sensory-predominant impairment of the peripheral nervous system caused by methylmercury. We found that the types of cell death in rat dorsal root ganglion (DRG) neurons caused by methylmercury included apoptosis, necrosis, and necroptosis. Methylmercury induced apoptosis in cultured rat DRG neurons but not in anterior horn neurons or Schwann cells. Additionally, methylmercury activated both caspase 8 and caspase 3 in DRG neurons. It increased the expression of tumor necrosis factor (TNF) receptor-1 and the phosphorylation of receptor-interacting protein kinase 3 (RIP3) and mixed-lineage kinase domain-like pseudokinase (MLKL). The expression of TNF-α was increased in macrophage-like RAW264.7 cells by methylmercury. The increase was suggested to be mediated by the NF-κB pathway. Moreover, methylmercury induced neurological symptoms, evaluated by a hindlimb extension response, were significantly less severe in TNF-α knockout mice. Based on these results and our previous studies, we propose the following hypothesis regarding the pathogenesis of sensory nerve-predominant damage by methylmercury: First, methylmercury accumulates within sensory nerve neurons and initiates cell death mechanisms, such as apoptosis, on a small scale. Second, cell death triggers the infiltration of macrophages into the sensory fibers. Third, the macrophages are stimulated by methylmercury and secrete TNF-α through the NF-κB pathway. Fourth, TNF-α induces cell death mechanisms, including necrosis, apoptosis through the caspase 8/3 pathway, and necroptosis through the TNFR1-RIP1-RIP3-MLKL pathway, activated by methylmercury in sensory neurons. Consequently, methylmercury exhibits potent cytotoxicity specific to the DRG/sensory nerve cells in the peripheral nervous system. This chain of events caused by methylmercury may contribute to sensory disturbances in patients with Minamata disease.

Suppression of ZBP1-mediated NLRP3 inflammasome by the tegument protein VP22 facilitates pseudorabies virus infection.

Z-DNA binding protein 1 (ZBP1) functions as a pivotal innate immune sensor that regulates inflammatory cell death during viral infections. However, its role in pseudorabies virus (PRV) infection remains unknown. Here, we demonstrate that ZBP1 serves as a restrictive factor by triggering the activation of the NLR family pyrin domain-containing 3 inflammasome, a process counteracted by PRV-encoded protein VP22. Furthermore, VP22 interferes with the interaction between ZBP1 and receptor-interacting protein kinase 3/Caspase-8, particularly through its N-terminal 1-50 amino acids. Importantly, deficiency in ZBP1 enhances the replication and virulence of recombinant viruses lacking VP22 or its N-terminal 1-50 amino acids. These findings reveal how PRV escapes ZBP1-mediated inflammatory responses during infection, potentially informing the rational design of therapeutic interventions.

4-Anilinoquinazoline Derivatives as the First Potent NOD1-RIPK2 Signaling Pathway Inhibitors at the Nanomolar Range.

Inflammation is a defense mechanism that restores tissue damage and eliminates pathogens. Among the pattern recognition receptors that recognize danger or pathogenic signals, nucleotide oligomerization domains 1 and 2 (NOD1/2) have been identified to play an important role in innate immunity responses, and inhibition of NOD1 could be interesting to treat severe infections and inflammatory diseases. In this work, we identified the first selective NOD1 versus NOD2 pathway inhibitors at the nanomolar range based on a 4-anilinoquinazoline scaffold. We demonstrated that NOD1 inhibition occurs through the inhibition of receptor interacting protein kinase 2 (RIPK2), which is involved in its downstream signaling pathways. Compound 37 demonstrates no cytotoxicity, a selectivity for RIPK2 over epithelial and vascular endothelial growth factor receptors (EGFR/VEGFR), and a capacity to reduce pro-inflammatory cytokine IL-8 secretion. The structure of the RIPK2-compound 37 complex was resolved by crystallography. The 4-anilinoquinazoline scaffold offers novel perspectives to design NOD1-RIPK2 signaling inhibitors.

Extracellular RIPK3 Acts as a Danger-Associated Molecular Pattern to Exaggerate Cardiac Ischemia/Reperfusion Injury.

Cardiac ischemia/reperfusion (I/R) injury has emerged as an important therapeutic target for ischemic heart disease. Currently, there is no effective therapy for reducing cardiac I/R injury. Damage-associated molecular patterns are endogenous molecules released after cellular damage to exaggerate tissue inflammation and injury. RIPK3 (receptor-interacting protein kinase 3), a well-established intracellular mediator of cell necroptosis and inflammation, serves as a circulating biomarker of multiple diseases. However, whether extracellular RIPK3 also exerts biological functions in cardiac I/R injury remains totally unknown. Patients with acute myocardial infarction receiving percutaneous coronary intervention (PCI) were recruited independently in the discovery cohort (103 patients) and validation cohort (334 patients), and major adverse cardiovascular events were recorded. Plasma samples were collected before and after PCI (6 and 24 h) for RIPK3 concentration measurement. Cultured neonatal rat ventricular myocytes, macrophages and endothelial cells, and in vivo mouse models with myocardial injury induced by I/R (or hypoxia/reoxygenation) were used to investigate the role and mechanisms of extracellular RIPK3. Another cohort including patients with acute myocardial infarction receiving PCI and healthy volunteers was recruited to further explore the mechanisms of extracellular RIPK3. In the discovery cohort, elevated plasma RIPK3 levels after PCI are associated with poorer short- and long-term outcomes in patients with acute myocardial infarction, as confirmed in the validation cohort. In both cultured cells and in vivo mouse models, recombinant RIPK3 protein exaggerated myocardial I/R (or hypoxia/reoxygenation) injury, which was alleviated by the RIPK3 antibody. Mechanistically, RIPK3 acted as a damage-associated molecular pattern and bound with RAGE (receptor of advanced glycation end-products), subsequently activating CaMKII (Ca2+/calmodulin-dependent kinase II) to elicit the detrimental effects. The positive correlation between plasma RIPK3 concentrations and CaMKII phosphorylation in human peripheral blood mononuclear cells was confirmed. We identified the positive relationship between plasma RIPK3 concentrations and the risk of major adverse cardiovascular events in patients with acute myocardial infarction receiving PCI. As a damage-associated molecular pattern, extracellular RIPK3 plays a causal role in multiple pathological conditions during cardiac I/R injury through RAGE/CaMKII signaling. These findings expand our understanding of the physiological and pathological roles of RIPK3, and also provide a promising therapeutic target for myocardial I/R injury and the associated complications.

Targeted Degradation of Receptor-Interacting Protein Kinase 1 to Modulate the Necroptosis Pathway.

Necroptosis is a highly regulated form of necrotic cell death that plays an essential role in pathogen defense and tissue homeostasis. Abnormal regulation of the necroptotic pathway has been implicated in the pathogenesis of various human diseases, including cancer, inflammatory, and neurodegenerative diseases. Receptor-interacting protein kinase 1 (RIPK1) serves as a crucial regulator of the necroptotic signaling pathway and has been identified as a potential therapeutic target. Mechanistically, RIPK1 serves as both a protein kinase and a scaffolding protein, fulfilling its dual function through a combination of kinase activity-dependent and kinase activity-independent mechanisms. Thus, employing a targeted RIPK1 knockdown strategy is a highly effective means of inhibiting RIPK1 functions. To achieve a targeted RIPK1 knockdown, we generated a RIPK1-PROTAC, MS2031, by connecting the ZB-R-55 RIPK1 binder to the VHL ligand, thereby recruiting the CUL2-RING-VHL (CRL2VHL) E3 ubiquitin ligase complex for targeted degradation of RIPK1 through the 26S proteasome. Notably, MS2031 treatment effectively reduced the abundance of RIPK1 protein in the nanomolar range in various cell lines we examined, including HT-29 and T47D cells, and modulated the necroptosis signaling pathway. These results suggest that MS2031 may hold potential for the treatment of human diseases resulting from aberrant regulation of RIPK1.

Role of Z-DNA Binding Protein 1 Sensing Mitochondrial Z-DNA and Triggering Necroptosis in Oxalate-Induced Acute Kidney Injury.

Calcium oxalate-induced acute kidney injury is a severe condition in which the kidneys suffer rapid damage due to the deposition of oxalate crystals. Known factors contributing to cell death induced by calcium oxalate include receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) protein dependent necroptosis, as well as necrosis involving peptidylprolyl isomerase F (PPIF) mediated mitochondrial permeability transition. However, the detailed molecular mechanisms linking mitochondrial dysfunction to RIPK3 activation are not fully understood. Mice with gene knock-out of Zbp1, Ripk3, or Mlkl and mice with mutations in the Z-nucleic acid sensing domain of ZBP1 or deletion of Zα1 were used in an oxalate-induced AKI model. Proximal renal tubule cells were isolated and cultured for further investigation. Human oxalate nephropathy biopsy samples were analyzed. Specific gene deletions of Zbp1, Ripk3, or Mlkl in proximal renal tubules significantly reduced the severity of oxalate-induced AKI by preventing necroptosis and subsequent inflammation. Notably, mice with mutations in the Z-nucleic acid sensing domain of ZBP1 or deletion of Zα1 were protected from AKI. In cultured proximal tubular cells, calcium oxalate damaged mitochondria, accompanied by an increase in Bax and a decrease in BCL2 and TAFM, leading to the release of mitochondrial Z-DNA. ZBP1 sensed this mitochondrial Z-DNA and then recruited RIPK3 via the RIP homotypic interaction motifs (RHIM), which in turn activated MLKL through RIPK3 phosphorylation, leading to necroptosis and contributing to AKI. ZBP1 plays a critical role in sensing mitochondrial Z-DNA and initiating RIPK3/MLKL-mediated necroptosis, contributing to the development of oxalate-induced AKI.

Induced Necroptosis and Its Role in Cancer Immunotherapy

Necroptosis is a type of regulated cell death (RCD) that is triggered by changes in the extracellular or intracellular milieu that are picked up by certain death receptors. Thanks to its potent capacity to induce immunological responses and overcome apoptotic resistance, it has garnered significant attention as a potential cancer treatment. Basic information for the creation of nano-biomedical treatments is provided by studies on the mechanisms underlying tumor necroptosis. Receptor-interacting protein kinase 1 (RIPK1)–RIPK3-mediated necroptosis, Toll-like receptor domain-containing adapter-inducing interferon (IFN)-β (TRIF)–RIPK3-mediated necroptosis, Z-DNA-binding protein 1 (ZBP1)–RIPK3-mediated necroptosis, and IFNR-mediated necroptosis are the four signaling pathways that collectively account for triggered necroptosis in this review. Necroptosis has garnered significant interest as a possible cancer treatment strategy because, in contrast to apoptosis, it elicits immunological responses that are relevant to therapy. Thus, a thorough discussion is held on the connections between tumor cell necroptosis and the immune environment, cancer immunosurveillance, and cells such as dendritic cells (DCs), cytotoxic T cells, natural killer (NK) cells, natural killer T (NKT) cells, and their respective cytokines. Lastly, a summary of the most recent nanomedicines that cause necroptosis in order to cause immunogenic cell death is provided in order to emphasize their promise for cancer immunotherapy.

Genomic and Transcriptional Analysis of the Necroptosis Pathway Elements RIPK and MLKL in Sea Cucumber, Holothuria leucospilota.

Background: Receptor-interacting protein kinases (RIPKs) and mixed-lineage kinase domain-like protein (MLKL) are crucial in regulating innate immune responses and cell death signaling (necroptosis and apoptosis), and are potential candidates for genetic improvement in breeding programs. Knowledge about the RIPK family and MLKL in sea cucumber remains limited. Methods: We searched the genomes of sea cucumber Holothuria leucospilota for genes encoding RIPKs and MLKL, performed phylogenetic tree, motif and functional domain analyses, and examined tissue distribution and embryonic development patterns using qPCR. Results: RIPK5 (Hl-RIPK5), RIPK7 (Hl-RIPK7) and MLKL (Hl-MLKL) were identified in sea cucumber H. leucospilota. Hl-RIPK5 and Hl-RIPK7 were mainly expressed in coelomocytes, suggesting that they play a role in innate immunity, whereas Hl-MLKL exhibited relatively low expression across tissues. During embryonic development, Hl-MLKL was highly expressed from the 2-cell stage to the morula stage, while Hl-RIPK5 and Hl-RIPK7 were primarily expressed after the morula stage, indicating different roles in embryonic development. In primary coelomocytes, Hl-RIPK5 transcriptional activity was significantly depressed by LPS, poly(I:C), or pathogen Vibrio harveyi. Hl-RIPK7 expression levels were unchanged following the same challenges. Hl-MLKL mRNA levels were significantly decreased with poly(I:C) or V. harveyi, but did not change with LPS. Conclusions: These findings provide valuable insights into the evolutionary tree and characterization of RIPK and MLKL genes in sea cucumber, contributing to the broader understanding of the RIPK gene family and MLKL in ancient echinoderms.

Local receptor-interacting protein kinase 2 inhibition mitigates house dust mite-induced asthma.

House dust mite is the most frequent trigger of allergic asthma, with innate and adaptive immune mechanisms playing critical roles in outcomes. We recently identified the nucleotide-binding oligomerisation domain 1 (NOD1)/receptor-interacting serine/threonine protein kinase 2 (RIPK2) signalling pathway as a relevant contributor to murine house dust mite-induced asthma. This study aimed to evaluate the effectiveness of a pharmacological RIPK2 inhibitor administered locally as a preventive and therapeutic approach using a house dust mite-induced asthma model in wild-type and humanised NOD1 mice harbouring an asthma-associated risk allele, and its relevance using air-liquid interface epithelial cultures from asthma patients. A RIPK2 inhibitor was administered intranasally either preventively or therapeutically in a murine house dust mite-induced asthma model. Airway hyperresponsiveness, bronchoalveolar lavage composition, cytokine/chemokine expression and mucus production were evaluated, as well as the effect of the inhibitor on precision-cut lung slices. Furthermore, the inhibitor was tested on air-liquid interface epithelial cultures from asthma patients and controls. While local preventive administration of the RIPK2 inhibitor reduced airway hyperresponsiveness, eosinophilia, mucus production, T-helper type 2 cytokines and interleukin 33 (IL-33) in wild-type mice, its therapeutic administration failed to reduce the above parameters, except IL-33. By contrast, therapeutic RIPK2 inhibition mitigated all asthma features in humanised NOD1 mice. Results in precision-cut lung slices emphasised an early role of thymic stromal lymphopoietin and IL-33 in the NOD1-dependent response to house dust mite, and a late effect of NOD1 signalling on IL-13 effector response. RIPK2 inhibitor downregulated thymic stromal lymphopoietin and chemokines in house dust mite-stimulated epithelial cultures from asthma patients. These data support that local interference of the NOD1 signalling pathway through RIPK2 inhibition may represent a new therapeutic approach in house dust mite-induced asthma.

A Novel Approach for Apical Periodontitis Prevention Through RIPK3 Inhibition Using Organic Flavonoids

Background: Apical periodontitis (AP) entails inflammatory bone resorption facilitated by the induction of necroptosis through receptor-interacting protein kinase 3 (RIPK3). Consequently, the inhibition of RIPK3 holds the potential for favorable effects in addressing AP. A comprehensive investigation, encompassing 44 flavonoids, was undertaken to identify potential inhibitors of RIPK3. Methods: The crystal structure of the RIPK3 catalytic domain (PDB ID: 7MON) underwent meticulous preparation through the removal of ligands and energy optimization. Overall, 44 flavonoids were examined in this study. Utilizing AutoDock 4.0, molecular docking analyses were performed to evaluate the binding energies within the active site of RIPK3 concerning the examined herbal isolates, with subsequent comparison to the results obtained from a positive control inhibitor. Furthermore, the BIOVIA Discovery Studio Visualizer played a crucial role in elucidating the interactions between the top-ranked flavonoids and the residues of RIPK3. Results: Five distinct flavonoids—orientin, rutin, vicenin-2, amentoflavone, and nicotiflorin—manifested notable inhibitory effects on RIPK3, boasting inhibition constant values at either the femtomolar or picomolar scale. Notably, the first three members of this set showcased superior binding affinity to the active site of RIPK3 compared to the reference compound. Conclusion: The proposition arises that flavonoid, particularly orientin, rutin, vicenin-2, amentoflavone, and nicotiflorin, stand as promising herbal isolates for the therapeutic management of AP.

RIPK4 promotes oxidative stress and ferroptotic death through the downregulation of ACSM1

One of the most critical axes for cell fate determination is how cells respond to excessive reactive oxygen species (ROS)-oxidative stress. Extensive lipid peroxidation commits cells to death via a distinct cell death paradigm termed ferroptosis. However, the molecular mechanism regulating cellular fates to distinct ROS remains incompletely understood. Through siRNA against human receptor-interacting protein kinase (RIPK) family members, we found that RIPK4 is crucial for oxidative stress and ferroptotic death. Upon ROS induction, RIPK4 is rapidly activated, and the kinase activity of RIPK4 is indispensable to induce cell death. Specific ablation of RIPK4 in kidney proximal tubules protects mice from acute kidney injury induced by cisplatin and renal ischemia/reperfusion. RNA sequencing revealed the dramatically decreased expression of acyl-CoA synthetase medium-chain (ACSM) family members induced by cisplatin treatment which is compromised in RIPK4-deficient mice. Among these ACSM family members, suppression of ACSM1 strongly augments oxidative stress and ferroptotic cell death with induced expression of ACS long-chain family member 4, an important component for ferroptosis execution. Our lipidome analysis revealed that overexpression of ACSM1 leads to the accumulation of monounsaturated fatty acids, attenuation of polyunsaturated fatty acid (PUFAs) production, and thereby cellular resistance to ferroptosis. Hence, knockdown of ACSM1 resensitizes RIPK4 KO cells to oxidative stress and ferroptotic death. In conclusion, RIPK4 is a key player involved in oxidative stress and ferroptotic death, which is potentially important for a broad spectrum of human pathologies. The link between the RIPK4-ASCM1 axis to PUFAs and ferroptosis reveals a unique mechanism to oxidative stress-induced necrosis and ferroptosis.

Exploring the role of macromolecular crowding and TNFR1 in cell volume control

The excessive cosolute densities in the intracellular fluid create a physicochemical condition called macromolecular crowding (MMC). Intracellular MMC entropically maintains the biochemical thermodynamic equilibria by favouring associative reactions while hindering transport processes. Rapid cell volume shrinkage during extracellular hypertonicity elevates the MMC and disrupts the equilibria, potentially ushering cell death. Consequently, cells actively counter the hypertonic stress through regulatory volume increase (RVI) and restore the MMC homeostasis. Here, we establish fluorescence anisotropy of EGFP as a reliable tool for studying cellular MMC and explore the spatiotemporal dynamics of MMC during cell volume instabilities under multiple conditions. Our studies reveal that the actin cytoskeleton enforces spatially varying MMC levels inside adhered cells. Within cell populations, MMC is uncorrelated with nuclear DNA content but anti-correlated with the cell spread area. Although different cell lines have statistically similar MMC distributions, their responses to extracellular hypertonicity vary. The intensity of the extracellular hypertonicity determines a cell's ability for RVI, which correlates with Nuclear Factor Kappa Beta (NFkB) activation. Pharmacological inhibition and knockdown experiments reveal that Tumour Necrosis Factor Receptor 1 (TNFR1) initiates the hypertonicity induced NFkB signalling and RVI. At severe hypertonicities, the elevated MMC amplifies cytoplasmic microviscosity and hinders Receptor Interacting Protein Kinase 1 (RIPK1) recruitment at the TNFR1 complex, incapacitating the TNFR1-NFkB signalling and consequently, RVI. Together, our studies unveil the involvement of TNFR1-NFkB signalling in modulating RVI and demonstrate the pivotal role of MMC in determining cellular osmoadaptability.

Prognostic significance and response to immune checkpoint inhibitors of RIPK3, MLKL and necroptosis in non-small cell lung cancer

Lung cancer remains the leading cause of cancer death. Treatment with immune checkpoint inhibitor (ICI) alone or combination with chemotherapy served as first-line therapy in non-small cell lung cancer (NSCLC). However, only 20–50% of NSCLC patients respond to ICI. Necroptosis, an inflammatory form of cell death plays an important role in the regulation of tumor immune microenvironment which may affect prognosis and ICI response but its clinical significance in NSCLC patients has remained largely unknown. Therefore, we aimed to analyze the correlation between key necroptotic proteins and necroptosis and clinical outcomes, the status of tumor-infiltrating immune cells, and response to ICI in NSCLC patients. The expression of receptor-interacting protein kinase 3 (RIPK3), mixed lineage kinase domain-like protein (MLKL) and phosphorylated MLKL (pMLKL) were immunolocalized in 125 surgically resected NSCLC patients and 23 NSCLC patients administered with ICI therapy. CD8 + and FOXp3 + T cells and CD163 + M2 macrophages were also immunolocalized. High RIPK3 status was positively correlated with survival of the patients and RIPK3 turned out an independent favorable prognostic factor of the patients. RIPK3 was negatively correlated with CD8 + T cells, while MLKL positively correlated with CD163 + M2 macrophages, suggesting the possible involvement of RIPK3 and MLKL in formulating immunosuppressive microenvironment. In addition, high RIPK3 status tended to be associated with clinical resistance to ICI therapy (P-value = 0.057). Furthermore, NSCLC cells-expressing RIPK3 suppressed T cells response to ICI therapy in vitro. Therefore, RIPK3 and MLKL could induce an immunosuppressive microenvironment, resulting in low response to ICI therapy in NSCLC.

Zinc finger domain of p62/SQSTM1 is involved in the necroptosis of human cisplatin‑resistant ovarian cancer cells treated with sulfasalazine.

Cisplatin resistance in ovarian cancer cells is mainly apoptosis resistant. Although other types of programmed cell death are highly involved in chemoresistance, which type can overcome cisplatin resistance remains unclear. The present study observed that cisplatin-sensitive SKOV3 cells and cisplatin-resistant SKOV3/DDP cells had different levels of sensitivity to sulfasalazine (SAS). The present study aimed to investigate the effect of SAS on necroptosis under the same inhibition rate in these two types of cells. Necroptosis inhibitor Necrostatin-1 (Nec-1) attenuated SAS-induced SKOV3/DDP cytotoxicity. SAS decreased SKOV3/DDP cells survival rate, accompanied by decreased cell adhesion and spreading. SAS treatment activated necrosome formation in SKOV3/DDP cells, suggesting the possibility of necroptosis. p62/sequestosome-1 (SQSTM1) protein expression levels were also increased over the same time period. The transfection of small interfering (si)-p62 could decrease the ratios of phosphorylated (p)-receptor-interacting serine/threonine kinase 1 (RIP1)/RIP1, p-receptor-interacting serine/threonine kinase 3 (RIP3)/RIP3 and p-mixed lineage kinase domain-like protein (MLKL)/MLKL proteins in SKOV3/DDP cells. Under the si-p62 condition, there was no increase in the rate of cell survival in Nec-1 and SAS combination group compared with SAS. The zinc finger domain deletion of p62/SQSTM1 effectively decreased the expression levels of necroptosis-related p-proteins. Collectively, certain drugs were able to induce necroptosis in SKOV3/DDP, while p62/RIP1/RIP3/MLKL was associated with the induction of necroptosis and with increasing the sensitivity of cisplatin-resistant ovarian cancer cells, which provided evidence for potential as a therapeutic target for overcoming resistance.

Mechanisms of retinal photoreceptor loss in spontaneously hypertensive rats

Retinal neurodegenerative diseases, including hypertensive retinopathy, involve progressive damage to retinal neurons, leading to visual impairment. In this study, we investigated the pathological mechanisms underlying retinal neurodegeneration in spontaneously hypertensive rats (SHR), using Wistar Kyoto (WKY) rats as normotensive controls. We observed that SHR exhibited significantly higher blood pressure and decreased retinal thickness, indicating retinal neurodegeneration. Molecular tests including quantitative real-time polymerase chain reaction, immunoblot, and immunofluorescent staining showed elevated levels of the pro-inflammatory cytokine tumor necrosis factor-α, apoptotic markers (Fas, FasL, caspase-8, active caspase-3, and cleaved poly (ADP-ribose) polymerase), and necroptotic markers (receptor-interacting protein kinase-1 and -3) in SHR retinas. Additionally, we found elevated transforming growth factor-β (TGF-β) levels in the retinal pigment epithelium (RPE) of SHR, with a decrease in lecithin retinol acyltransferase (LRAT), which regulates retinoid metabolism and photoreceptor health. In human RPE cells (ARPE-19), TGF-β administration suppressed mRNA and protein levels of LRAT; and vactosertib, a selective inhibitor of TGF-β receptor kinase type 1, reversed the effect of TGF-β. These findings suggest that hypertension-induced retinal neurodegeneration involves inflammation, apoptosis, necroptosis, and disrupted retinoid metabolism, providing potential therapeutic targets for hypertensive retinopathy.