Mediators of necroptosis: from cell death to metabolic regulation.

Necroptosis, a programmed cell death mechanism distinct from apoptosis, has garnered attention for its role in various pathological conditions. While initially recognized for its involvement in cell death, recent research has revealed that key necroptotic mediators, including receptor-interacting protein kinases (RIPKs) and mixed lineage kinase domain-like protein (MLKL), possess additional functions that go beyond inducing cell demise. These functions encompass influencing critical aspects of metabolic regulation, such as energy metabolism, glucose homeostasis, and lipid metabolism. Dysregulated necroptosis has been implicated in metabolic diseases, including obesity, diabetes, metabolic dysfunction-associated steatotic liver disease (MASLD) and alcohol-associated liver disease (ALD), contributing to chronic inflammation and tissue damage. This review provides insight into the multifaceted role of necroptosis, encompassing both cell death and these extra-necroptotic functions, in the context of metabolic diseases. Understanding this intricate interplay is crucial for developing targeted therapeutic strategies in diseases that currently lack effective treatments.

Receptor-interacting protein 1 and 3 kinase activity are required for high-fat diet induced liver injury in mice.

Background: The RIP1-RIP3-MLKL-mediated cell death pathway is associated with progression of non-alcohol-associated fatty liver/steatohepatitis (NAFL/NASH). Previous work identified a critical role for MLKL, the key effector regulating necroptosis, but not RIP3, in mediating high fat diet-induced liver injury in mice. RIP1 and RIP3 have active N-terminus kinase domains essential for activation of MLKL and subsequent necroptosis. However, little is known regarding domain-specific roles of RIP1/RIP3 kinase in liver diseases. Here, we hypothesized that RIP1/RIP3 kinase activity are required for the development of high fat diet-induced liver injury. Methods: Rip1K45A/K45A and Rip3K51A/K51A kinase-dead mice on a C57BL/6J background and their littermate controls (WT) were allowed free access to a diet high in fat, fructose and cholesterol (FFC diet) or chow diet. Results: Both Rip1K45A/K45A and Rip3K51A/K51A mice were protected against FFC diet-induced steatosis, hepatocyte injury and expression of hepatic inflammatory cytokines and chemokines. FFC diet increased phosphorylation and oligomerization of MLKL and hepatocyte death in livers of WT, but not in Rip3K51A/K51A, mice. Consistent with in vivo data, RIP3 kinase deficiency in primary hepatocytes prevented palmitic acid-induced translocation of MLKL to the cell surface and cytotoxicity. Additionally, loss of Rip1 or Rip3 kinase suppressed FFC diet-mediated formation of crown-like structures (indicators of dead adipocytes) and expression of mRNA for inflammatory response genes in epididymal adipose tissue. Moreover, FFC diet increased expression of multiple adipokines, including leptin and plasminogen activator inhibitor 1, in WT mice, which was abrogated by Rip3 kinase deficiency. Discussion: The current data indicate that both RIP1 and RIP3 kinase activity contribute to FFC diet-induced liver injury. This effect of RIP1 and RIP3 kinase deficiency on injury is consistent with the protection of Mlkl-/- mice from high fat diet-induced liver injury, but not the reported lack of protection in Rip3-/- mice. Taken together with previous reports, our data suggest that other domains of RIP3 likely counteract the effect of RIP3 kinase in response to high fat diets.

Necroptosis in CNS diseases: Focus on astrocytes.

In the last few years, necroptosis, a recently described type of cell death, has been reported to play an important role in the development of various brain pathologies. Necroptosis is a cell death mechanism that has morphological characteristics similar to necrosis but is mediated by fundamentally different molecular pathways. Necroptosis is initiated by signaling through the interaction of RIP1/RIP3/MLKL proteins (receptor-interacting protein kinase 1/receptor-interacting protein kinase 3/mixed lineage kinase domain-like protein). RIPK1 kinase is usually inactive under physiological conditions. It is activated by stimulation of death receptors (TNFR1, TNFR2, TLR3, and 4, Fas-ligand) by external signals. Phosphorylation of RIPK1 results in the formation of its complex with death receptors. Further, complexes with the second member of the RIP3 and MLKL cascade appear, and the necroptosome is formed. There is enough evidence that necroptosis plays an important role in the pathogenesis of brain ischemia and neurodegenerative diseases. In recent years, a point of view that both neurons and glial cells can play a key role in the development of the central nervous system (CNS) pathologies finds more and more confirmation. Astrocytes play complex roles during neurodegeneration and ischemic brain damage initiating both impair and protective processes. However, the cellular and molecular mechanisms that induce pathogenic activity of astrocytes remain veiled. In this review, we consider these processes in terms of the initiation of necroptosis. On the other hand, it is important to remember that like other types of programmed cell death, necroptosis plays an important role for the organism, as it induces a strong immune response and is involved in the control of cancerogenesis. In this review, we provide an overview of the complex role of necroptosis as an important pathogenetic component of neuronal and astrocyte death in neurodegenerative diseases, epileptogenesis, and ischemic brain damage.

The Key Regulator of Necroptosis, RIP1 Kinase, Contributes to the Formation of Astrogliosis and Glial Scar in Ischemic Stroke.

Necroptosis initiation relies on the receptor-interacting protein 1 kinase (RIP1K). We recently reported that genetic and pharmacological inhibition of RIP1K produces protection against ischemic stroke-induced astrocytic injury. However, the role of RIP1K in ischemic stroke-induced formation of astrogliosis and glial scar remains unknown. Here, in a transient middle cerebral artery occlusion (tMCAO) rat model and an oxygen and glucose deprivation and reoxygenation (OGD/Re)-induced astrocytic injury model, we show that RIP1K was significantly elevated in the reactive astrocytes. Knockdown of RIP1K or delayed administration of RIP1K inhibitor Nec-1 down-regulated the glial scar markers, improved ischemic stroke-induced necrotic morphology and neurologic deficits, and reduced the volume of brain atrophy. Moreover, knockdown of RIP1K attenuated astrocytic cell death and proliferation and promoted neuronal axonal generation in a neuron and astrocyte co-culture system. Both vascular endothelial growth factor D (VEGF-D) and its receptor VEGFR-3 were elevated in the reactive astrocytes; simultaneously, VEGF-D was increased in the medium of astrocytes exposed to OGD/Re. Knockdown of RIP1K down-regulated VEGF-D gene and protein levels in the reactive astrocytes. Treatment with 400 ng/ml recombinant VEGF-D induced the formation of glial scar; conversely, the inhibitor of VEGFR-3 suppressed OGD/Re-induced glial scar formation. RIP3K and MLKL may be involved in glial scar formation. Taken together, these results suggest that RIP1K participates in the formation of astrogliosis and glial scar via impairment of normal astrocyte responses and enhancing the astrocytic VEGF-D/VEGFR-3 signaling pathways. Inhibition of RIP1K promotes the brain functional recovery partially via suppressing the formation of astrogliosis and glial scar.

Inhibitors of RIP1 kinase: a patent review (2016-present).

Introduction: RIP1 kinase is a serine/threonine-protein kinase that has recently emerged as a central regulator of TNF-α dependent programmed necrosis (necroptosis), an inflammatory form of cell death, with important roles in inflammation and neurodegeneration. Small molecule RIP1 kinase inhibitors may provide new opportunities for treating a variety of autoimmune, inflammatory, and neurodegenerative diseases, among others, and thus have attracted widespread drug development efforts and a corresponding large amount of patent activity in recent years. Areas covered: This review focuses on the patent literature covering small molecule inhibitors of RIP1 kinase from 2016-present. Expert opinion: Inhibition of programmed necrosis (necroptosis) by RIP1 kinase inhibitors is a new field that has attracted widespread recent interest as a possible therapeutic means to treat a number of diseases in the inflammatory, neurodegenerative, and oncology areas. The interest in the therapeutic potential of RIP1kinase is evidenced by more than 40 small molecule patent applications published since 2016. To date, only a few RIP1 kinase inhibitors have entered the clinic. An understanding of the optimal clinical setting, in terms of dosing and disease indications for RIP1 inhibition, will require further clinical readouts as the current inhibitors progress and additional molecules enter into full development.

RIP1, RIP3, and MLKL Contribute to Cell Death Caused by Clostridium perfringens Enterotoxin.

Clostridium perfringens type F strains cause gastrointestinal disease when they produce a pore-forming toxin named C. perfringens enterotoxin (CPE). In human enterocyte-like Caco-2 cells, low CPE concentrations cause caspase-3-dependent apoptosis, while high CPE concentrations cause necrosis. Since necrosis or apoptosis sometimes involves receptor-interacting serine/threonine-protein kinase-1 or 3 (RIP1 or RIP3), this study examined whether those kinases are important for CPE-induced apoptosis or necrosis. Highly specific RIP1 or RIP3 inhibitors reduced both CPE-induced apoptosis and necrosis in Caco-2 cells. Those findings suggested that the form of necrosis induced by treating Caco-2 cells with high CPE concentrations involves necroptosis, which was confirmed when high, but not low, CPE concentrations were shown to induce oligomerization of mixed-lineage kinase domain-like pseudokinase (MLKL), a key late step in necroptosis. Furthermore, an MLKL oligomerization inhibitor reduced cell death caused by high, but not low, CPE concentrations. Supporting RIP1 and RIP3 involvement in CPE-induced necroptosis, inhibitors of those kinases also reduced MLKL oligomerization during treatment with high CPE concentrations. Calpain inhibitors similarly blocked MLKL oligomerization induced by high CPE concentrations, implicating calpain activation as a key intermediate in initiating CPE-induced necroptosis. In two other CPE-sensitive cell lines, i.e., Vero cells and human enterocyte-like T84 cells, low CPE concentrations also caused primarily apoptosis/late apoptosis, while high CPE concentrations mainly induced necroptosis. Collectively, these results establish that high, but not low, CPE concentrations cause necroptosis and suggest that RIP1, RIP3, MLKL, or calpain inhibitors can be explored as potential therapeutics against CPE effects in vivoIMPORTANCEC. perfringens type F strains are a common cause of food poisoning and antibiotic-associated diarrhea. Type F strain virulence requires production of C. perfringens enterotoxin (CPE). In Caco-2 cells, high CPE concentrations cause necrosis while low enterotoxin concentrations induce apoptosis. The current study determined that receptor-interacting serine/threonine-protein kinases 1 and 3 are involved in both CPE-induced apoptosis and necrosis in Caco-2 cells, while mixed-lineage kinase domain-like pseudokinase (MLKL) oligomerization is involved in CPE-induced necrosis, thereby indicating that this form of CPE-induced cell death involves necroptosis. High CPE concentrations also caused necroptosis in T84 and Vero cells. Calpain activation was identified as a key intermediate for CPE-induced necroptosis. These results suggest inhibitors of RIP1, RIP3, MLKL oligomerization, or calpain are useful therapeutics against CPE-mediated diseases.

Extracts derived from a traditional Chinese herbal formula triggers necroptosis in ectocervical Ect1/E6E7 cells through activation of RIP1 kinase.

ETHNOPHARMACOLOGICAL RELEVANCE: As one of the most common female malignant tumors mainly infected by human papillomavirus (HPV) worldwide, cervical cancer is widely distributed in about 90% developing countries. An in-hospital preparation derived from a traditional Chinese herbal formula, Youdujing (YDJ), has been developed and clinically used for more than 20 years in our hospital for treating multiple diseases caused by HPV infection, such as cervical precancerous lesions, recurrent condyloma acuminata, fla t warts, etc. However, few investigations on the effect and mechanism of YDJ extract on treating and preventing HPV infection induced cervical cancer have been reported. AIM OF THE STUDY: Previous reports showed that YDJ extract is effective in triggering human cervical cancer cells (ectocervical Ect1/E6E7) death in a necrotic manner. Herein, we aim to investigate the anti-proliferation effects and potential mechanisms of YDJ extract in inducing necroptosis in ectocervical Ect1/E6E7 cells. MATERIALS AND METHODS: The high-performance liquid chromatography (HPLC) fingerprint method was firstly used for better quality control of the chemical components in YDJ extract. MTT assay and flow cytometer were applied for evaluating cytotoxicity and necroptosis induced by YDJ extract in ectocervical Ect1/E6E7 cells. Besides, Western blotting, receptor-interacting protein serine-threonine kinase 1 (RIP1) inhibitor (necrostatin-1), and RIP1 shRNA and pCDNA transfection assays were employed for investigation on the underlying mechanisms and validation the role of RIP1 in YDJ extract induced necroptosis. RESULTS: YDJ extract induced necroptosis in ectocervical Ect1/E6E7 cells both in time- and concentration-dependent manners, without affecting activation of caspases and elevation of intracellular reactive oxygen species (ROS) level. Moreover, a selective increasing in RIP1expression was observed in YDJ extract treated ectocervical Ect1/E6E7 cells. The induction effect of necroptosis by YDJ extract was partially blocked by the addition of RIP1 inhibitor (necrostatin-1). Co-immunoprecipitation assay demonstrated that the treatment of YDJ extract in ectocervical Ect1/E6E7 cells promoted the combination of RIP1 with RIP3 and MLKL to form necrosome, which facilitates the process of necroptosis. CONCLUSIONS: Taken together, YDJ preparation displays an effective ability of inducing necroptosis in cervical cancer cells through activation of RIP1 kinase. However, although the treatment efficacy and potential mechanisms of YDJ extract in vivo remain unclear and need further investigation, it is believed that YDJ extract has the great potential to be used as a starting point to develop more potent agent for treating or preventing cervical cancer and other proliferative diseases.

RIP1 kinase inhibitor halts the progression of an immune-induced demyelination disease at the stage of monocyte elevation.

Demyelination in the central nervous system (CNS) underlies many human diseases, including multiple sclerosis (MS). We report here the findings of our study of the CNS demyelination process using immune-induced [experimental autoimmune encephalomyelitis (EAE)] and chemical-induced [cuprizone (CPZ)] mouse models of demyelination. We found that necroptosis, a receptor-interacting protein 3 (RIP3) kinase and its substrate mixed lineage kinase domain-like protein (MLKL)-dependent cell death program, played no role in the demyelination process, whereas the MLKL-dependent, RIP3-independent function of MLKL in the demyelination process initially discovered in the peripheral nervous system in response to nerve injury, also functions in demyelination in the CNS in these models. Moreover, a receptor-interacting protein 1 (RIP1) kinase inhibitor, RIPA-56, blocked disease progression in the EAE-induced model but showed no effect in the CPZ-induced model. It does so most likely at a step of monocyte elevation downstream of T cell activation and myelin-specific antibody generation, although upstream of breakdown of the blood-brain barrier. RIP1-kinase dead knock-in mice shared a similar result as mice treated with the RIP1 inhibitor. These results indicate that RIP1 kinase inhibitor is a potential therapeutic agent for immune-mediated demyelination diseases that works by prevention of monocyte elevation, a function previously unknown for RIP1 kinase.

Use of RIP1 Kinase Small-Molecule Inhibitors in Studying Necroptosis.

RIP1 kinase plays a key role in regulating signaling pathways downstream of a number of innate immune receptors such as TNFRI and TLRs. The discovery of Necrostatin-1 (Nec-1) as a small-molecule inhibitor of RIP1 kinase has been very instrumental in defining the necroptotic and other signalling pathways regulated by RIP1, but certain characteristics of Nec-1 limits its utility in experimental systems. Next generation RIP1 kinase inhibitors have been identified and the use of these tool inhibitors along with Nec-1 has revealed that RIP1 is emerging as a key driver of inflammation and tissue injury in the pathogenesis of various diseases. Further studying the role of RIP1 to carefully unravel the complex biology requires the selection of the correct tool small-molecule inhibitors. In addition, it is important to consider the proper application of current tool inhibitors and understand the current limitiations. Here we will discuss key parameters that need to be considered when selecting and applying tool inhibitors to novel biological assays and systems. General protocols to explore the in vitro and in vivo potency, cellular selectivity, and pharmacokinetic properties of current small-molecule inhibitors of RIP1 kinase are provided.

TNF-α induced RIP1-dependent apoptosis in L929-A cells by interrupting mitochondrial respiratory chain complex Ⅲ / 军事医学

Objective To explore the mechanism by which tumor necrosis factor alpha(TNF-α) induces RIP1 kinase-dependented apoptosis in L929-A fibroblastoma cells.Methods The sub-mitochondrial localization of receptor-interacting protein 1(RIP1),caspase-8 and Bid proteins was detected by dose-gradient trypsin digestion and Western blotting.The levels of reactive oxygen species (ROS),intracellular calcium concentration,mitochondrial membrane potential (MMP),and cellular adenosine triphosphate(ATP) content were determined by fluorescent probe labeling and flow cytometry assay.The mitochondrial respiratory chain complex Ⅰ and Ⅲ activities were detected by commercial kits.Nec-1,A RIP1 kinase specific inhibitor,and RIP1-/-or Bid-/-L929-A cells were used to detect the roles of RIP1 kinase and Bid protein in cell death.Results RIP1,caspase-8 and Bid proteins were co-located in the outer membrane of mitochondrial.TNF-α exposure for 3 h could induce Bid cleavage,inhibit mitochondrial respiratory chain complex Ⅲ activity and reduce MMP.Following these changes and after TNF-α exposure for 6-12 h,the intracellular calcium concentration and ROS were increased,whereas the ATP concentration was decreased,and the cells were killed.Inhibiting RIP1 kinase or knockdown RIP1 or Bid protein could suppress all the cytotoxic effects of TNF-α.Conclusion TNF-α treatment can result in RIP1 kinase-mediated Bid cleavage and inhibit mitochondrial respiratory chains and cell energy metabolism,which ultimately leads to the death of L929-A cells.

Targeting Cell Death Pathways for Therapeutic Intervention in Kidney Diseases.

Precise regulation of cell death and survival is essential for proper maintenance of organismal homeostasis, development, and the immune system. Deregulated cell death can lead to developmental defects, neuropathies, infections, and cancer. Kidney diseases, especially acute pathologies linked to ischemia-reperfusion injury, are among illnesses that profoundly are affected by improper regulation or execution of cell death pathways. Attempts to develop medicines for kidney diseases have been impacted by the complexity of these pathologies given the heterogeneous patient population and diverse etiologies. By analyzing cell death pathways activated in kidney diseases, we attempt to differentiate their importance for these pathologies with a goal of identifying those that have more profound impact and the best therapeutic potential. Although classic apoptosis still might be important, regulated necrosis pathways including necroptosis, ferroptosis, parthanatos, and mitochondrial permeability transition-associated cell death play a significantly role in kidney diseases, especially in acute kidney pathologies. Although targeting receptor-interacting protein 1 kinase appears to be the best therapeutic strategy, combination with inhibitors of other cell death pathways is likely to bring superior benefit and possible cure to patients suffering from kidney diseases.