Receptor-interacting Protein Kinase 3 Expression Research Articles

RIPK1-Induced A1 Reactive Astrocytes in Brain in MPTP-Treated Murine Model of Parkinson's Disease.

Neuroinflammation is one of the hallmarks of Parkinson's disease, including the massive activation of microglia and astrocytes and the release of inflammatory factors. Receptor-interacting protein kinase 1 (RIPK1) is reported to mediate cell death and inflammatory signaling, and is markedly elevated in the brain in PD mouse models. Here, we aim to explore the role of RIPK1 in regulating the neuroinflammation of PD. C57BL/6J mice were intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 20 mg/kg four times/day), followed by necrostatin-1 treatment (Nec-1, RIPK1 inhibitor; 1.65 mg/kg once daily for seven days. Notably, the first Nec-1 was given 12 h before MPTP modeling). Behavioral tests indicated that inhibition of RIPK1 greatly relieved motor dysfunction and anxiety-like behaviors of PD mice. It also increased striatal TH expression, rescue the loss of dopaminergic neurons, and reduce activation of astrocytes in the striatum of PD mice. Furthermore, inhibition of RIPK1 expression reduced A1 astrocytes' relative gene expression (CFB, H2-T23) and inflammatory cytokine or chemokine production (CCL2, TNF-α, IL-1β) in the striatum of PD mice. Collectively, inhibition of RIPK1 expression can provide neuroprotection to PD mice, probably through inhibition of the astrocyte A1 phenotype, and thus RIPK1 might be an important target in PD treatment.

Encephalitis and poor neuronal death-mediated control of herpes simplex virus in human inherited RIPK3 deficiency.

Inborn errors of TLR3-dependent type I IFN immunity in cortical neurons underlie forebrain herpes simplex virus-1 (HSV-1) encephalitis (HSE) due to uncontrolled viral growth and subsequent cell death. We report an otherwise healthy patient with HSE who was compound heterozygous for nonsense (R422*) and frameshift (P493fs9*) RIPK3 variants. Receptor-interacting protein kinase 3 (RIPK3) is a ubiquitous cytoplasmic kinase regulating cell death outcomes, including apoptosis and necroptosis. In vitro, the R422* and P493fs9* RIPK3 proteins impaired cellular apoptosis and necroptosis upon TLR3, TLR4, or TNFR1 stimulation and ZBP1/DAI-mediated necroptotic cell death after HSV-1 infection. The patient's fibroblasts displayed no detectable RIPK3 expression. After TNFR1 or TLR3 stimulation, the patient's cells did not undergo apoptosis or necroptosis. After HSV-1 infection, the cells supported excessive viral growth despite normal induction of antiviral IFN-β and IFN-stimulated genes (ISGs). This phenotype was, nevertheless, rescued by application of exogenous type I IFN. The patient's human pluripotent stem cell (hPSC)-derived cortical neurons displayed impaired cell death and enhanced viral growth after HSV-1 infection, as did isogenic RIPK3-knockout hPSC-derived cortical neurons. Inherited RIPK3 deficiency therefore confers a predisposition to HSE by impairing the cell death-dependent control of HSV-1 in cortical neurons but not their production of or response to type I IFNs.

Abstract 2525: Analyzing the role of MDMX for regulating cell death in prostate cancer cells

Abstract Prostate cancer (PCa) is the most prominent cancer type and the second leading cause of cancer death among men in the USA. The five-year survival rate for localized PCa is 98% but drops to 31% when PCa spreads to different parts of the body. Various treatment strategies that can induce cell death through different mechanisms are used while treating cancers. In recent years, necroptosis has been established as one of the effective mechanisms of inducing cell death in cancers. Necroptosis can be identified as a regulated form of cell death that can occur independently of executioner caspases. Despite numerous studies on the effects of necroptosis in cancer, it’s role on PCa has not been fully elucidated. In this study, we have evaluated the role of MDMX, a proto-oncogene involved in the negative regulation of p53, in causing cell death through p53-independent mechanisms. We have observed that inhibition of MDMX decreased the cell viability of LNCaP cells in response to 20 µM of NSC (NSC-207895), a MDMX specific inhibitor treatment, in a concentration-dependent manner. MDMX inhibition appears to induce necroptosis in LNCaP prostate cancer cells by elevating the expression of receptor-interacting protein kinase 1 (RIP1), which is a key element of the necroptotic pathway. In general, the necroptosis pathway can trigger two opposing mechanisms in cancer where it can promote tumor migration and metastasis on one hand and can also act as a defensive mechanism on the other hand when apoptosis is disrupted. To further assess the effect of MDMX on cell migration, the LNCaP cells were treated with 20 µM of NSC, which effectively blocked the cancer cell migration for up to 48 h, which was confirmed using the scratch assay. Interestingly, NSC treatment also reduced the expression of Vascular Endothelial Growth Factor A (VEGFA), which is one of the main markers for angiogenesis. Our results show that the p53-independent mechanisms that are activated following MDMX inhibition can regulate both cancer cell migration and angiogenesis. MDMX also seemed to be involved in regulating PCa cell survival through necroptosis. The outcomes of our experiments provide additional knowledge and strategies for making an accurate prognosis and choosing the most suitable therapeutic strategies for offering effective treatments for MDMX-positive PCa. (This research was supported by the Royal Dames of Cancer Research Inc. Ft. Lauderdale, Florida) Citation Format: Ahmed Alsarrani, Umamaheswari Natarajan, Samia Alsubhi, Amal Alzahrani, Appu Rathinavelu. Analyzing the role of MDMX for regulating cell death in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2525.

P116 RIPK3 - a new marker for assessment of ulcerative colitis severity

Abstract Background Necroptosis is a novel form of regulated necrotic cell death that is dependent on Receptor interacting protein kinase-3 (RIPK3). It leads to cell lysis and directly triggers inflammation. Several clinical and preclinical studies have implicated necroptosis in the etiopathogenesis of inflammatory bowel disease. Methods The aim of the study was to evaluate the expression of RIPK3 according to the activity and severity of ulcerative colitis (UC). Tissue expression of RIPK3 was determined immunohistochemically in intestinal biopsies from 85 patients with ulcerative colitis. Тo evaluate the severity and activity of UC were used the Montreal classification for disease severity (S), full Mayo score (MS) and Mayo endoscopic score (MES). Results A moderate direct correlation was found between disease severity and RIPK3 expression (r=0.354; p=0.001), with severe disease (S3) being characterized by overexpression of the marker (199.67). A moderate right-proportional relationship was also found in the analysis of RIPK3 expression and UC activity, measured by the full Mayo score (r=0.481; p<0.001). RIPK3 expression increased with increasing disease activity (p<0.001), and patients with severe disease activity (MS =11-12) had overexpression of the marker (211.71). A moderate correlation between RIPK3 expression and the degree of endoscopic activity was also found (r=0.363; p=0.001), with patients with severe endoscopic activity (MES=3) having high expression of the marker (198.27). Conclusion High expression of RIPK3 in UC patients was associated with severe disease activity assessed by the Montreal classification and full Mayo score and marked endoscopic activity.

Effects of drugs commonly used in Sars-CoV-2 infection on renal tissue in rats

Aim: In December 2019, the coronavirus disease was caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Some drugs were repurposed for this disease treatment. Hydroxychloroquine (HCQ), favipiravir (FAV), molnupiravir (MOL), and dexamethasone (DEX) were widely used for the treatment of the disease. To increase the success of the treatment of coronavirus disease, there was used some of these drugs in combination. On the other hand, limited studies report these drugs’ side effects. Therefore, this study was designed to evaluate the possible effects of these drugs and their combinations on the kidney tissues of rats without viral load. Materials and Methods: Male Wistar albino rats were divided into control, HCQ, FAV, HCQ+FAV, HCQ+FAV+DEX, MOL, and MOL+DEX groups. At the end of the experiment, the serum kidney tissue samples were taken. Serum samples were analyzed for urea and creatinine. Kidney tissue samples were assessed as histopathological and immunohistochemical for heat shock protein 60 (HSP60), caspase-3, and receptor-interacting protein kinase-3 (RIPK3). Results: Urea and creatinine levels were within the normal range in all groups. Histopathologically, all drugs and their combinations caused tubular degeneration. On the other hand, histopathological alterations were more prominent in the HCQ group. The oxidative stress marker HSP60 was significantly increased in FAV, MOL, and MOL+DEX groups, while it was similar to the control group in the HCQ groups. Apoptosis marker caspase-3 expression was found to be prominently higher in other drug groups except the FAV group. Expression of RIPK3, a marker of necroptosis, was significantly increased in all drug groups. Conclusion: Taken together, the data of our study show that the administration of all drugs alone and in combination may cause structural damage to the kidney. Furthermore, our results indicate that HCQ can exhibit more damaging effects compared to other drugs.

RIPK1 kinase-dependent inflammation and cell death contribute to the pathogenesis of COPD.

Receptor-interacting protein kinase 1 (RIPK1) is a key mediator of regulated cell death (including apoptosis and necroptosis) and inflammation, both drivers of COPD pathogenesis. We aimed to define the contribution of RIPK1 kinase-dependent cell death and inflammation in the pathogenesis of COPD. We assessed RIPK1 expression in single-cell RNA sequencing (RNA-seq) data from human and mouse lungs, and validated RIPK1 levels in lung tissue of COPD patients via immunohistochemistry. Next, we assessed the consequences of genetic and pharmacological inhibition of RIPK1 kinase activity in experimental COPD, using Ripk1 S25D/S25D kinase-deficient mice and the RIPK1 kinase inhibitor GSK'547. RIPK1 expression increased in alveolar type 1 (AT1), AT2, ciliated and neuroendocrine cells in human COPD. RIPK1 protein levels were significantly increased in airway epithelium of COPD patients compared with never-smokers and smokers without airflow limitation. In mice, exposure to cigarette smoke (CS) increased Ripk1 expression similarly in AT2 cells, and further in alveolar macrophages and T-cells. Genetic and/or pharmacological inhibition of RIPK1 kinase activity significantly attenuated airway inflammation upon acute and subacute CS exposure, as well as airway remodelling, emphysema, and apoptotic and necroptotic cell death upon chronic CS exposure. Similarly, pharmacological RIPK1 kinase inhibition significantly attenuated elastase-induced emphysema and lung function decline. Finally, RNA-seq on lung tissue of CS-exposed mice revealed downregulation of cell death and inflammatory pathways upon pharmacological RIPK1 kinase inhibition. RIPK1 kinase inhibition is protective in experimental models of COPD and may represent a novel promising therapeutic approach.

Rapamycin Inhibits Light-Induced Necrosome Activation Occurring in Wild-Type, but not RPE65-Null, Mouse Retina.

Both photodamage and aberrant visual cycle contribute to disease progress of many retinal degenerative disorders, whereas the signaling pathways causing photoreceptor death remain unclear. Here we investigated the effects of intense photo-stress on (1) necrosome activation in wild-type and RPE65-null mice, (2) interaction of p62/Sequestosome-1 with the necrosome proteins, and (3) the effects of rapamycin on photodamage-induced necrosome activation and retinal degeneration in wild-type mice. Dark-adapted rd12 mice and 129S2/Sv mice with or without rapamycin treatment were exposed to 15,000 lux light for different times. Expression levels and subcellular localization of proteins were determined through immunoblot and immunohistochemical analyses. Cone sheaths were stained with peanut agglutinin. Correlation between photoreceptor degeneration and receptor-interacting protein kinase-1 (RIPK1) expression was assessed with Spearman's correlation analysis. Protein-protein interaction was analyzed by immunoprecipitation. Intense light caused rod and cone degeneration accompanied by a significant increase in RIPK1-RIPK3 expressions, mixed lineage kinase domain-like protein phosphorylation, damage-associated molecular patterns protein release, and inflammatory responses in wild-type mouse retina. The same intense light did not induce the necrosome activation in rd12 retina, but it did in rd12 mice that received 9-cis-retinal supply. RIPK1 expression levels are positively correlated with the degrees of rod and cone degeneration. Photodamage upregulated expression and interaction of the p62 autophagosome cargo protein with the necrosome proteins, whereas rapamycin treatment attenuated the light-induced necrosome activation and photoreceptor degeneration. Necrosome activation contributed to photodamage-induced rod and cone degeneration. The visual cycle and autophagy are the important therapeutic targets to alleviate light-induced retinal necroptosis.

Caspase-8 and FADD prevent spontaneous ZBP1 expression and necroptosis

The absence of Caspase-8 or its adapter, Fas-associated death domain (FADD), results in activation of receptor interacting protein kinase-3 (RIPK3)- and mixed-lineage kinase-like (MLKL)-dependent necroptosis invivo. Here, we show that spontaneous activation of RIPK3, phosphorylation of MLKL, and necroptosis in Caspase-8- or FADD-deficient cells was dependent on the nucleic acid sensor, Z-DNA binding protein-1 (ZBP1). We genetically engineered a mouse model by a single insertion of FLAG tag onto the N terminus of endogenous MLKL (MlklFLAG/FLAG), creating an inactive form of MLKL that permits monitoring of phosphorylated MLKL without activating necroptotic cell death. Casp8-/-MlklFLAG/FLAG mice were viable and displayed phosphorylated MLKL in a variety of tissues, together with dramatically increased expression of ZBP1 compared to Casp8+/+ mice. Studies invitro revealed an increased expression of ZBP1 in cells lacking FADD or Caspase-8, which was suppressed by reconstitution of Caspase-8 or FADD. Ablation of ZBP1 in Casp8-/-MlklFLAG/FLAG mice suppressed spontaneous MLKL phosphorylation invivo. ZBP1 expression and downstream activation of RIPK3 and MLKL in cells lacking Caspase-8 or FADD relied on a positive feedback mechanism requiring the nucleic acid sensors cyclic GMP-AMP synthase (cGAS), stimulator of interferon genes (STING), and TBK1 signaling pathways. Our study identifies a molecular mechanism whereby Caspase-8 and FADD suppress spontaneous necroptotic cell death.

LCK-Mediated RIPK3 Activation Controls Double-Positive Thymocyte Proliferation and Restrains Thymic Lymphoma by Regulating the PP2A-ERK Axis.

Receptor-interacting protein kinase 3 (RIPK3) is the primary regulator of necroptotic cell death. RIPK3 expression is often silenced in various cancer cells, which suggests that it may have tumor suppressor properties. However, the exact mechanism by which RIPK3 negatively regulates cancer development and progression remains unclear. This report indicates that RIPK3 acts as a potent regulator of the homeostatic proliferation of CD4+ CD8+ double-positive (DP) thymocytes. Abnormal proliferation of RIPK3-deficient DP thymocytes occurs independently of the well-known role for RIPK3 in necroptosis (upstream of MLKL activation), and is associated with an incidental thymic mass, likely thymic hyperplasia. In addition, Ripk3-null mice develop increased thymic tumor formation accompanied by reduced host survival in the context of an N-ethyl-N-nitrosourea (ENU)-induced tumor model. Moreover, RIPK3 deficiency in p53-null mice promotes thymic lymphoma development via upregulated extracellular signal-regulated kinase (ERK) signaling, which correlates with markedly reduced survival rates. Mechanistically, lymphocyte-specific protein tyrosine kinase (LCK) activates RIPK3, which in turn leads to increases in the phosphatase activity of protein phosphatase 2 (PP2A), thereby suppressing hyper-activation of ERK in DP thymocytes. Overall, these findings suggest that a RIPK3-PP2A-ERK signaling axis regulates DP thymocyte homeostasis and may provide a potential therapeutic target to improve thymic lymphoma therapies.

Zearalenone Induces MLKL-Dependent Necroptosis in Goat Endometrial Stromal Cells via the Calcium Overload/ROS Pathway.

Zearalenone (ZEA) is a fungal mycotoxin known to exert strong reproductive toxicity in animals. As a newly identified type of programmed cell death, necroptosis is regulated by receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), and mixed-lineage kinase domain-like pseudokinase (MLKL). However, the role and mechanism of necroptosis in ZEA toxicity remain unclear. In this study, we confirmed the involvement of necroptosis in ZEA-induced cell death in goat endometrial stromal cells (gESCs). The release of lactate dehydrogenase (LDH) and the production of PI-positive cells markedly increased. At the same time, the expression of RIPK1 and RIPK3 mRNAs and P-RIPK3 and P-MLKL proteins were significantly upregulated in ZEA-treated gESCs. Importantly, the MLKL inhibitor necrosulfonamide (NSA) dramatically attenuated gESCs necroptosis and powerfully blocked ZEA-induced reactive oxygen species (ROS) generation and mitochondrial dysfunction. The reactive oxygen species (ROS) scavengers and N-acetylcysteine (NAC) inhibited ZEA-induced cell death. In addition, the inhibition of MLKL alleviated the intracellular Ca2+ overload caused by ZEA. The calcium chelator BAPTA-AM markedly suppressed ROS production and mitochondrial damage, thus inhibiting ZEA-induced necroptosis. Therefore, our results revealed the mechanism by which ZEA triggers gESCs necroptosis, which may provide a new therapeutic strategy for ZEA poisoning.

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

ObjectiveType 1 diabetes (T1D) is characterized by autoimmune-associated β-cell loss, insulin insufficiency, and hyperglycemia. Although TNFα signaling is associated with β-cell loss and hyperglycemia in non-obese diabetic mice and human T1D, the molecular mechanisms of β-cell TNF receptor signaling have not been fully characterized. Based on work in other cell types, we hypothesized that receptor interacting protein kinase 1 (RIPK1) and receptor interacting protein kinase 3 (RIPK3) regulate TNFα-induced β-cell death in concert with caspase activity. MethodsWe evaluated TNFα-induced cell death, caspase activity, and TNF receptor pathway molecule expression in immortalized NIT-1 and INS-1 β-cell lines and primary mouse islet cells in vitro. Our studies utilized genetic and small molecule approaches to alter RIPK1 and RIPK3 expression and caspase activity to interrogate mechanisms of TNFα-induced β-cell death. We used the β-cell toxin streptozotocin (STZ) to determine the susceptibility of Ripk3+/+ and Ripk3−/− mice to hyperglycemia in vivo. ResultsExpression of TNF receptor signaling molecules including RIPK1 and RIPK3 was identified in NIT-1 and INS-1 β cells and isolated mouse islets at the mRNA and protein levels. TNFα treatment increased NIT-1 and INS-1 cell death and caspase activity after 24–48 h, and BV6, a small molecule inhibitor of inhibitor of apoptosis proteins (IAPs) amplified this TNFα-induced cell death. RIPK1 deficient NIT-1 cells were protected from TNFα- and BV6-induced cell death and caspase activation. Interestingly, small molecule inhibition of caspases with zVAD-fmk (zVAD) did not prevent TNFα-induced cell death in either NIT-1 or INS-1 cells. This caspase-independent cell death was increased by BV6 treatment and decreased in RIPK1 deficient NIT-1 cells. RIPK3 deficient NIT-1 cells and RIPK3 kinase inhibitor treated INS-1 cells were protected from TNFα+zVAD-induced cell death, whereas RIPK3 overexpression increased INS-1 cell death and promoted RIPK3 and MLKL interaction under TNFα+zVAD treatment. In mouse islet cells, BV6 or zVAD treatment promoted TNFα-induced cell death, and TNFα+zVAD-induced cell death was blocked by RIPK3 inhibition and in Ripk3−/− islet cells in vitro. Ripk3−/− mice were also protected from STZ-induced hyperglycemia and glucose intolerance in vivo. ConclusionsRIPK1 and RIPK3 regulate TNFα-induced β-cell death in concert with caspase activity in immortalized and primary islet β cells. TNF receptor signaling molecules such as RIPK1 and RIPK3 may represent novel therapeutic targets to promote β-cell survival and glucose homeostasis in T1D.

Anti-cancer Properties of Myricetin Against HT-29 Colon Cancer Cells Through Regulation of RIPK1/RIPK3 Signaling Pathway

Background: Myricetin (MCN), a dietary flavonoid, is present in walnuts, fruits, vegetables, tea, and berries and has been suggested as an anti-inflammatory, antiplatelet, antimicrobial, antioxidant, and antiviral drug. Objectives: This study aimed to investigate whether the necroptosis pathway involves the toxic impacts of myricetin (MCN) on the colon adenoma-carcinoma HT-29 cells. Methods: For 48 hours, HT-29 cells were exposed to 50 µM MCN or three mM Necrosatine-1 (Nec-1), a necroptosis preventive. Apoptosis, cell viability, and expression of necroptosis-related genes, including mixed lineage kinase domain-like protein (MLKL), receptor-interacting protein kinase-1 (RIPK1), and receptor-interacting protein kinase-3 (RIPK3), were evaluated. Results: MCN caused a noticeable decline in the survival of HT-29 cells. In contrast to the slight change in the apoptosis index in HT-29 cells, MCN considerably increased the necrosis index. MCN could enhance the expression of MLKL, RIPK1, and RIPK3 in the HT-29 cells. The co-treatment of MCN with Nec-1 resulted in a significant elevation in HT-29 cell survival. Nec-1 could decrease the necrosis index and expression of necroptosis-related proteins in the MCN-exposed HT-29 cells. Conclusions: These findings demonstrated that MCN effectively induced cell death in HT-29 cells by activating the necroptosis pathway.

RIPK3-MLKL signaling activates mitochondrial CaMKII and drives intrarenal extracellular matrix production during CKD

Intrarenal extracellular matrix production or kidney fibrosis is a prevalent feature of all forms of chronic kidney disease (CKD). The transforming growth factor-beta (TGFβ) is believed to be a major driver of extracellular matrix production. Nevertheless, anti-TGFβ therapies have consistently failed to reduce extracellular matrix production in CKD patients indicating the need for novel therapeutic strategies. We have previously shown that necroinflammation contributes to acute kidney injury. Here, we show that chronic/persistent necroinflammation drives intrarenal extracellular matrix production during CKD. We found that renal expression of receptor-interacting protein kinase-1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) increases with the production of intrarenal extracellular matrix and declined kidney function in both humans and mice. Furthermore, we found that TGFβ exposure induces the translocation of RIPK3 and MLKL to mitochondria resulting in mitochondrial dysfunction and ROS production. Mitochondrial ROS activates the serine-threonine kinase calcium/calmodulin-dependent protein kinases-II (CaMKII) that increases phosphorylation of Smad2/3 and subsequent production of alpha-smooth muscle actin (αSMA), collagen (Col) 1α1, etc. in response to TGFβ during the intrarenal extracellular matrix production. Consistent with this, deficiency or knockdown of RIPK3 or MLKL as well as pharmacological inhibition of RIPK1, RIPK3, and CaMKII prevents the intrarenal extracellular matrix production in oxalate-induced CKD and unilateral ureteral obstruction (UUO). Together, RIPK1, RIPK3, MLKL, CaMKII, and Smad2/3 are molecular targets to inhibit intrarenal extracellular matrix production and preserve kidney function during CKD.

Investigation on the cellular mechanism of Prunetin evidenced through next generation sequencing and bioinformatic approaches against gastric cancer

Gastric cancer is the common type of malignancy positioned at second in mortality rate causing burden worldwide with increasing treatment options. More accurate and reliable diagnostic methods/biomarkers are urgently needed. The application of transcriptomics technologies possesses the high efficiency of identifying key metabolic pathways and functional genes in cancer research. In this study, we performed a transcriptome analysis on Prunetin treated AGS cells. A total of 1,118 differentially expressed (DE) genes on Prunetin treated AGS cancer cells, among which 463 were up-regulated and 655 were down-regulated. Notably, around 40 genes were found to be related with necroptosis, among which 16 genes were found to be in close association with Receptor Interacting Protein Kinase (RIPK) family. Validation of the RIPK genes through GEPIA identified 8 genes (NRP1, MNX1, SSRP1, PRDX2, PLRG1, LGALS4, SNX5 and FXYD3) which are highly expressed in stomach cancer were significantly down-regulated in PRU treated samples. In conclusion, the sequencing data explores the expression of RIPK mediated genes through necroptosis signaling network in treating gastric cancer. The futuristic validations on the 8 genes as candidate biomarkers will offer a treatment approach against gastric cancer using PRU.

Ca2+/Calmodulin-Dependent Protein Kinase II Regulation by Inhibitor of Receptor Interacting Protein Kinase 3 Alleviates Necroptosis in Glycation End Products-Induced Cardiomyocytes Injury.

Necroptosisis a regulatory programmed form of necrosis. Receptor interacting protein kinase 3 (RIPK3) is a robust indicator of necroptosis. RIPK3 mediates myocardial necroptosis through activation of calcium/calmodulin-dependent protein kinase II (CaMKII) in cardiac ischemia-reperfusion (I/R) injury and heart failure. However, the exact mechanism of RIPK3 in advanced glycation end products (AGEs)-induced cardiomyocytes necroptosis is not clear. In this study, cardiomyocytes were subjected to AGEs stimulation for 24 h. RIPK3 expression, CaMKII expression, and necroptosis were determined in cardiomyocytes after AGEs stimulation. Then, cardiomyocytes were transfected with RIPK3 siRNA to downregulate RIPK3 followed by AGEs stimulation for 24 h. CaMKIIδ alternative splicing, CaMKII activity, oxidative stress, necroptosis, and cell damage were detected again. Next, cardiomyocytes were pretreated with GSK′872, a specific RIPK3 inhibitor to assess whether it could protect cardiomyocytes against AGEs stimulation. We found that AGEs increased the expression of RIPK3, aggravated the disorder of CaMKII δ alternative splicing, promoted CaMKII activation, enhanced oxidative stress, induced necroptosis, and damaged cardiomyocytes. RIPK3 downregulation or RIPK3 inhibitor GSK′872 corrected CaMKIIδ alternative splicing disorder, inhibited CaMKII activation, reduced oxidative stress, attenuated necroptosis, and improved cell damage in cardiomyocytes.

Hydrogen Sulfide Suppresses Skin Fibroblast Proliferation via Oxidative Stress Alleviation and Necroptosis Inhibition.

Keloid is a common dermatofibrotic disease with excessive skin fibroblast proliferation. Hydrogen sulfide (H2S) as the third gasotransmitter improves fibrosis of various organs and tissues. Our study is aimed at investigating the effects and possible mechanisms of H2S on skin fibroblast proliferation. Scar tissues from six patients with keloid and discarded skin tissue from six normal control patients were collected after surgery, respectively. Plasma H2S content and skin H2S production in patients with keloid were measured. Keloid fibroblasts and transforming growth factor-β1- (TGF-β1, 10 ng/mL) stimulated normal skin fibroblasts were pretreated with H2S donor as NaHS (50 μM) for 4 h. Cell migration after scratch was assessed. The expressions of α-smooth muscle actin (α-SMA), proliferating cell nuclear antigen (PCNA), collagen I, and collagen III were detected by immunofluorescence, real-time PCR, and/or Western blot. Intracellular superoxide anion and mitochondrial superoxide were evaluated by dihydroethidium (DHE) and MitoSOX staining, respectively. Mitochondrial membrane potential was detected by JC-1 staining. Apoptotic cells were detected by TDT-mediated dUTP nick end labeling (TUNEL). The expressions of receptor interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) were measured by Western blot. We found that H2S production was impaired in both the plasma and skin of patients with keloid. In keloid fibroblasts and TGF-β1-stimulated normal skin fibroblasts, exogenous H2S supplementation suppressed the expressions of α-SMA, PCNA, collagen I, and collagen III, reduced intracellular superoxide anion and mitochondrial superoxide, improved the mitochondrial membrane potential, decreased the positive rate of TUNEL staining, and inhibited RIPK1 and RIPK3 expression as well as MLKL phosphorylation. Overall, H2S suppressed skin fibroblast proliferation via oxidative stress alleviation and necroptosis inhibition.

The Regulatory Mechanism and Effect of Receptor-Interacting Protein Kinase 3 on Phenylephrine-Induced Cardiomyocyte Hypertrophy.

As a critical regulatory molecule, receptor-interacting protein kinase 3 (RIPK3) can mediate the signaling pathway of programmed necrosis. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been proved as a new substrate for RIPK3-induced necroptosis. In this study, we aimed to investigate the regulatory mechanism of RIPK3 on phenylephrine (PE)-induced cardiomyocyte hypertrophy. Cardiomyocyte hypertrophy was induced by exposure to PE (100 μM) for 48 hours. Primary cardiomyocytes were pretreated with RIPK3 inhibitor GSK'872 (10 μM), and RIPK3 siRNA was used to deplete the intracellular expression of RIPK3. The indexes related to myocardial hypertrophy, cell injury, necroptosis, CaMKII activation, gene expression, oxidative stress, and mitochondrial membrane potential were measured. We found that after cardiomyocytes were stimulated by PE, the expressions of hypertrophy markers, atrial and brain natriuretic peptides (ANP and BNP), were increased, the release of lactate dehydrogenase was increased, the level of adenosine triphosphate (ATP) was decreased, the oxidation and phosphorylation levels of CaMKII were increased, and CaMKIIδ alternative splicing was disturbed. However, both GSK'872 and depletion of RIPK3 could reduce myocardial dysfunction, inhibit CaMKII activation and necroptosis, and finally alleviate myocardial hypertrophy. In addition, the pretreatment of RIPK3 could also lessen the accumulation of reactive oxygen species (ROS) induced by PE and stabilize the membrane potential of mitochondria. These results indicated that targeted inhibition of RIPK3 could suppress the activation of CaMKII and reduce necroptosis and oxidative stress, leading to alleviated myocardial hypertrophy. Collectively, our findings provided valuable insights into the clinical treatment of hypertrophic cardiomyopathy.

Ca2+/Calmodulin-Dependent Protein Kinase II Regulation by RIPK3 Alleviates Necroptosis in Transverse Arch Constriction-Induced Heart Failure.

Some studies have reported that the activation of Ca2+/calmodulin dependent protein kinase (CaMKII) plays a vital role in the pathogenesis of cardiovascular disease. Moreover, receptor interacting protein kinase 3 (RIPK3)-mediated necroptosis is also involved in the pathological process of various heart diseases. In the present study, we aimed to investigate the effect of RIPK3-regulated CaMKII on necroptosis in heart failure (HF) and its underlying mechanism. Wild type (WT) and RIPK3-depleted (RIPK3–/–) mice were treated with transverse arch constriction (TAC). After 6 weeks, echocardiography, myocardial injury, CaMKII activity, necroptosis, RIPK3 expression, mixed lineage kinase domain-like protein (MLKL) phosphorylation, and mitochondrial ultrastructure were measured. The results showed that TAC aggravated cardiac dysfunction, CaMKII activation, and necroptosis in WT mice. However, depletion of RIPK3 alleviated cardiac insufficiency, CaMKII activation, and necroptosis in TAC-treated mice. To verify the experimental results, WT mice were transfected with AAV-vector and AAV-RIPK3 shRNA, followed by TAC operation. The findings were consistent with the expected results. Collectively, our current data indicated that the activation of CaMKII, MLKL and necroptosis in HF mice were increased in a RIPK3-dependent manner, providing valuable insights into the pathogenesis and treatment strategy of HF.

MicroRNA-4287 alleviates inflammatory response via targeting RIPK1 in osteoarthritis

Studies have confirmed the regulatory effects of microRNAs (miRNAs) in osteoarthritis (OA) progression. MiR-4287 has been identified by a previous study as a downregulated miRNA in chondrocytes treated with IL-1β and TNF-α. However, the function of the underlying mechanism of miR-4287 in OA is elusive. IL-1β-treated chondrocytes were used as OA cell models. RNA expression was accessed using RT-qPCR. Cell Counting Kit-8 (CCK-8) assay was used to determine the chondrocytes' viability and proliferation. The protein levels of inflammation factors (IL-8, IL-6, and TNF-α), matrix metalloproteinases (MMP 1, MMP3, MMP13), and chondrogenic genes (COL2A1, SOX9, and Aggrecan) were detected using western blot analysis. Luciferase reporter assays were performed for interaction exploration. HE staining and Safranin O/Fast Green staining was used to access the pathological changes in OA mouse tissues and cartilage degeneration in OA mouse. MiR-4287 was downregulated in chondrocytes treated with IL-1β and OA mouse models. MiR-4287 overexpression promoted the viability, and proliferation and attenuated the inflammation response and destruction of cartilage in IL-1β-stimulated chondrocytes. Receptor-interacting protein kinase 1 (RIPK1) was a target gene of miR-4287 in chondrocytes. MiR-4287 negatively regulated RIPK1 expression. RIPK1 overexpression was revealed to reverse the miR-4287-mediated effects on proliferation and inflammatory response in IL-1β-stimulated chondrocytes. Moreover, miR-4287 was demonstrated to inhibit the pathological changes, cartilage degeneration and inflammation response in OA mice models. In conclusion, miR-4287 is a critical molecule in OA development, which attenuates inflammatory response in vivo and in vitro by targeting RIPK1.

Piperlongumine increases the sensitivity of bladder cancer to cisplatin by mitochondrial ROS.

BackgroundThe development of cisplatin resistance often results in cisplatin inefficacy in advanced or recurrent bladder cancer. However, effective treatment strategies for cisplatin resistance have not been well established.MethodsGene expression was measured by qRT‐PCR and Western blotting. CCK‐8 assay was performed to detect cell survival. The number of apoptotic cells was determined using the Annexin V‐PI double‐staining assay. The level of reactive oxygen species (ROS) was measured using 2’,7'‐dichlorodihydrofluorescein diacetate fluorescent dye, and the ATP level was detected using an ATP measurement kit.ResultsThe expression of receptor‐interacting protein kinase 1 (RIPK1), a key regulator of necroptosis, gradually decreased during cisplatin resistance. We first used piperlongumine (PL) in combination with cisplatin to act on cisplatin‐resistant BC cells and found that PL‐induced activation of RIPK1 increased the sensitivity of T24 resistant cells to cisplatin treatment. Furthermore, we revealed that PL killed T24 cisplatin‐resistant cells by triggering necroptosis, because cell death could be rescued by the mixed lineage kinase domain‐like (MLKL) protein inhibitor necrotic sulfonamide or MLKL siRNA, but could not be suppressed by the apoptosis inhibitor z‐VAD. We further explored the specific mechanism and found that PL activated RIPK1 to induce necroptosis in cisplatin‐resistant cells by stimulating mitochondrial fission to produce excessive ROS.ConclusionsOur results demonstrated the role of RIPK1 in cisplatin‐resistant cells and the sensitization effect of the natural drug PL on bladder cancer. These may provide a new treatment strategy for overcoming cisplatin resistance in bladder cancer.