Sublethal necroptosis signaling promotes inflammation and liver cancer.

It is currently not well known how necroptosis and necroptosis responses manifest in vivo. Here, we uncovered a molecular switch facilitating reprogramming between two alternative modes of necroptosis signaling in hepatocytes, fundamentally affecting immune responses and hepatocarcinogenesis. Concomitant necrosome and NF-κB activation in hepatocytes, which physiologically express low concentrations of receptor-interacting kinase 3 (RIPK3), did not lead to immediate cell death but forced them into a prolonged "sublethal" state with leaky membranes, functioning as secretory cells that released specific chemokines including CCL20 and MCP-1. This triggered hepatic cell proliferation as well as activation of procarcinogenic monocyte-derived macrophage cell clusters, contributing to hepatocarcinogenesis. In contrast, necrosome activation in hepatocytes with inactive NF-κB-signaling caused an accelerated execution of necroptosis, limiting alarmin release, and thereby preventing inflammation and hepatocarcinogenesis. Consistently, intratumoral NF-κB-necroptosis signatures were associated with poor prognosis in human hepatocarcinogenesis. Therefore, pharmacological reprogramming between these distinct forms of necroptosis may represent a promising strategy against hepatocellular carcinoma.

Excessive apoptosis of Rip1-deficient T cells leads to premature aging.

In mammals, the most remarkable T cell variations with aging are the shrinking of the naïve T cell pool and the enlargement of the memory T cell pool, which are partially caused by thymic involution. However, the mechanism underlying the relationship between T-cell changes and aging remains unclear. In this study, we find that T-cell-specific Rip1 KO mice show similar age-related T cell changes and exhibit signs of accelerated aging-like phenotypes, including inflammation, multiple age-related diseases, and a shorter lifespan. Mechanistically, Rip1-deficient T cells undergo excessive apoptosis and promote chronic inflammation. Consistent with this, blocking apoptosis by co-deletion of Fadd in Rip1-deficient T cells significantly rescues lymphopenia, the imbalance between naïve and memory T cells, and aging-like phenotypes, and prolongs life span in T-cell-specific Rip1 KO mice. These results suggest that the reduction and hyperactivation of T cells can have a significant impact on organismal health and lifespan, underscoring the importance of maintaining T cell homeostasis for healthy aging and prevention or treatment of age-related diseases.

CLDN6 inhibited cellular biological function of nonsmall cell lung cancer cells through suppressing aerobic glycolysis via the RIP1/ASK1/JNK axis.

Claudin-6 (CLDN6) has been extensively studied in different tumors to date. However, in the case of nonsmall cell lung cancer (NSCLC), CLDN6 has a largely unknown role and molecular mechanism. We detected the expression of CLDN6 in NSCLC tissues and cells using reverse transcription-quantitative polymerase chain reaction (PCR) and western blot assays. A gain-of-function experiment was performed to evaluate the biological effects of CLDN6 on NSCLC cell behaviors. Methylation-specific PCR was utilized to detect the DNA methylation of CLDN6 gene promoter region. The interaction of CLDN6 and receptor interacting protein 1 (RIP1) was determined by coimmunoprecipitation assay. Furthermore, the modulation of CLDN6 on RIP1/apoptosis signal-regulating kinase 1 (ASK1)/c-Jun N-terminal kinase (JNK) axis was confirmed. The results showed that in NSCLC tissues and cells, CLDN6 expression level was declined, and was associated with a high level of DNA methylation. CLDN6 overexpression suppressed the viability, invasion, migration, and promoted cell apoptosis. Besides, the enhanced expression of CLDN6 reduced the glycolysis and the dysfunction of mitochondrial respiration of NSCLC cells. Mechanistic investigation confirmed that CLDN6 interacted with RIP1 and inhibited cellular biological function of NSCLC cells via RIP1/ASK1/JNK axis. Besides, CLDN6 overexpression inhibited tumor growth in vivo. In conclusion, CLDN6 inhibited NSCLC cell proliferation through inactivating aerobic glycolysis via the RIP1/ASK1/JNK axis.

RIP1/3-dependent programmed necrosis induces intestinal injury in septic rats.

The regulation of various types of cell death may help to restore the normal physiological function of cells and play a protective role in sepsis. In the current study, we explore the role of programmed cell necrosis in sepsis and the underlying mechanisms. The septic rat model is established by Cecal-ligation and perforation (CLP), and the in vitro model is established by LPS in IEC-6 cells. Our results demonstrate that receptor-interacting protein 1 (RIP1) is significantly upregulated in the ileum of septic rats and LPS-treated IEC-6 cells at both the mRNA and protein levels. Nec-1, an inhibitor of RIP1, reduces the protein levels of RIP1, p-RIP3, and phosphorylated mixed-lineage kinase domain-like (MLKL) (serine 358) and relieves intestinal injury in CLP-induced septic rats with decreased IL-6 and TNF-α levels. The in vitro experiments further reveal that LPS induces the colocalization of RIP1 and RIP3, resulting in the phosphorylation and translocation of MLKL to the plasma membrane in IEC-6 cells. LPS also facilitates ROS production in IEC-6 cells, but this effect is further reversed by Nec-1, si-RIP1 and si-RIP3. Furthermore, LPS-induced necrosis in IEC-6 cells is counteracted by NAC. Thus, we conclude that RIP1/RIP3-dependent programmed cell necrosis participates in intestinal injury in sepsis and may be associated with RIP1/RIP3-mediated ROS.

Downregulation of Mitochondrial Fusion Protein Expression Affords Protection from Canonical Necroptosis in H9c2 Cardiomyoblasts.

Necroptosis, a form of necrosis, and alterations in mitochondrial dynamics, a coordinated process of mitochondrial fission and fusion, have been implicated in the pathogenesis of cardiovascular diseases. This study aimed to determine the role of mitochondrial morphology in canonical necroptosis induced by a combination of TNFα and zVAD (TNF/zVAD) in H9c2 cells, rat cardiomyoblasts. Time-course analyses of mitochondrial morphology showed that mitochondria were initially shortened after the addition of TNF/zVAD and then their length was restored, and the proportion of cells with elongated mitochondria at 12 h was larger in TNF/zVAD-treated cells than in non-treated cells (16.3 ± 0.9% vs. 8.0 ± 1.2%). The knockdown of dynamin-related protein 1 (Drp1) and fission 1, fission promoters, and treatment with Mdivi-1, a Drp-1 inhibitor, had no effect on TNF/zVAD-induced necroptosis. In contrast, TNF/zVAD-induced necroptosis was attenuated by the knockdown of mitofusin 1/2 (Mfn1/2) and optic atrophy-1 (Opa1), proteins that are indispensable for mitochondrial fusion, and the attenuation of necroptosis was not canceled by treatment with Mdivi-1. The expression of TGFß-activated kinase (TAK1), a negative regulator of RIP1 activity, was upregulated and the TNF/zVAD-induced RIP1-Ser166 phosphorylation, an index of RIP1 activity, was mitigated by the knockdown of Mfn1/2 or Opa1. Pharmacological TAK1 inhibition attenuated the protection afforded by Mfn1/2 and Opa1 knockdown. In conclusion, the inhibition of mitochondrial fusion increases TAK1 expression, leading to the attenuation of canonical necroptosis through the suppression of RIP1 activity.

Acacetin induces sustained ERK1/2 activation and RIP1-dependent necroptotic death in breast cancer cells.

Acacetin (AC), a naturally occurring flavonoid has shown anticancer potential. Herein, we studied the mechanisms of cell death and growth inhibition by AC in breast carcinoma T-47D and MDA-MB-231 cells. AC (10-40 µM) significantly decreased the levels of G2/M phase cyclins and CDKs, simultaneously increasing the expression of CDK inhibitors including Cip1/p21. A concentration-dependent increase in cell death was noted in both breast cancer cell lines with no such considerable effects on MCF-10A non-tumorigenic breast cells. The cell death-inducing potential of AC was further confirmed using confocal microscopy and flow cytometry analysis. AC resulted in mitochondrial superoxide generation, DNA damage, and ROS generation. N-acetyl cysteine (NAC) pre-treatment inhibited ROS generation and partially reversed ERK1/2 activation as well as cell death by AC. Further, AC enhanced the expression of RIP1 and RIP3, which mediate necroptosis. RIP1-specific inhibitor Necrostatin-1 (NS-1) reversed the AC-induced DNA damage and cell death. Collectively, these findings, for the first time, suggested that AC exerts its antitumor potential through ROS induction and RIP1-dependent necroptosis in breast carcinoma cells.

Nuclear translocation of MLKL enhances necroptosis by a RIP1/RIP3-independent mechanism in H9c2 cardiomyoblasts.

Accumulating evidence suggests that necroptosis of cardiomyocytes contributes to cardiovascular diseases. Lethal disruption of the plasma membrane in necroptosis is induced by oligomers of mixed lineage kinase domain-like (MLKL) that is translocated to the membrane from the cytosol. However, the role played by cytoplasmic-nuclear shuttling of MLKL is unclear. Here, we tested the hypothesis that translocation of MLKL to the nucleus promotes the necroptosis of cardiomyocytes. Activation of the canonical necroptotic signaling pathway by a combination of TNF-α and zVAD (TNF/zVAD) increased nuclear MLKL levels in a RIP1-activity-dependent manner in H9c2 cells, a rat cardiomyoblast cell line. By use of site-directed mutagenesis, we found a nuclear export signal sequence in MLKL and prepared its mutant (MLKL-L280/283/284A), though a search for a nuclear import signal was unsuccessful. MLKL-L280/283/284A localized to both the cytosol and the nucleus. Expression of MLKL-L280/283/284A induced necroptotic cell death, which was attenuated by GppNHp, an inhibitor of Ran-mediated nuclear import, but not by inhibition of RIP1 activity or knockdown of RIP3 expression. GppNHp partly suppressed H9c2 cell death induced by TNF/zVAD treatment. These results suggest that MLKL that is translocated to the nucleus via RIP1-mediated necroptotic signaling enhances the necroptosis of cardiomyocytes through a RIP1-/RIP3-independent mechanism.

Retinoic acid-inducible gene 1 (RIG-1) and IFN-ß promoter stimulator-1 (IPS-1) significantly down-regulated in the severe coronavirus disease 2019 (COVID-19).

INTRODUCTION: Retinoic acid-inducible gene 1 (RIG-1) and melanoma differentiation-associated protein 5 (MDA5) are the well-known cytoplasmic sensors that recognize microbial DNA or RNA and active down-stream molecules, including IFN-ß promoter stimulator-1 (IPS-1) and receptor interacting protein 1 (RIP1). The roles played by the networked molecules on the infection with SARS-CoV-2 needs more investigations. MATERIAL AND METHOD: In this project MDA5, RIG-1, IPS-1 and RIP1 mRNA levels were evaluated in 45 hospitalized patients suffering from coronavirus disease of 2019 (COVID-19) and 45 healthy subjects using Real Time-qPCR technique. RESULT: The results showed significant decreased RIG-1 and IPS-1 in the SARS-CoV-2 infected patients when compared to healthy cases. MDA5 and RIP1 did not change when compared two groups. Male patients had similar expression of MDA5, RIG-1, IPS-1 and RIP1 when compared to female patients. CONCLUSION: Based on the results, it seems that RIG-1 and its signaling molecule, IPS-1, play key roles in the peripheral blood immune cells against SARS-CoV-2 and, their down-regulation may be induced by the virus to escape from immune responses.

Brazilin inhibits bladder cancer by promoting cell necroptosis.

Brazilin possesses anticancer effects, but the mechanisms are poorly understood. This study investigated the mechanisms of brazilin-induced cell death in the T24 human bladder cancer cell line. Low serum cell culture and the lactate dehydrogenase assay were used to confirm the antitumor effect of brazilin. Annexin V and propidium iodide double staining, transmission electron microscopy, fluo-3-AM assay for Ca2+ mobilization and caspase activity assay were performed to identify the type of cell death after brazilin treatment. Mitochondria membrane potentials were measured using JC-1. Quantitative real-time polymerase chain reaction and western blot analyses were performed to verify the expression of the necroptosis-related genes and proteins receptor interacting protein 1 (RIP1), RIP3 and mixed lineage kinase domain-like (MLKL). The results showed that brazilin induced necrosis in T24 cells and upregulated the mRNA and protein levels of RIP1, RIP3 and MLKL and Ca2+ influx. The necroptosis-mediated cell death was rescued by the necroptosis inhibitor necrostatin-1 (Nec-1), but not by the apoptosis inhibitor z-VAD-fmk. Brazilin repressed caspase 8 expression and decreased the mitochondrial membrane potentials; both effects were partially reversed by Nec-1. Brazilin induced physiological and morphological changes in T24 cells and RIP1/RIP3/MLKL-mediated necroptosis might be involved. In conclusion, the results confirm the involvement of necroptosis in brazilin-induced cell death and suggest that brazilin could be explored as an anticancer agent against bladder cancer.

RIP1 post-translational modifications.

Receptor interacting protein 1 (RIP1) kinase is a critical regulator of inflammation and cell death signaling, and plays a crucial role in maintaining immune responses and proper tissue homeostasis. Mounting evidence argues for the importance of RIP1 post-translational modifications in control of its function. Ubiquitination by E3 ligases, such as inhibitors of apoptosis (IAP) proteins and LUBAC, as well as the reversal of these modifications by deubiquitinating enzymes, such as A20 and CYLD, can greatly influence RIP1 mediated signaling. In addition, cleavage by caspase-8, RIP1 autophosphorylation, and phosphorylation by a number of signaling kinases can greatly impact cellular fate. Disruption of the tightly regulated RIP1 modifications can lead to signaling disbalance in TNF and/or TLR controlled and other inflammatory pathways, and result in severe human pathologies. This review will focus on RIP1 and its many modifications with an emphasis on ubiquitination, phosphorylation, and cleavage, and their functional impact on the RIP1's role in signaling pathways.