Receptor-interacting Protein Kinase-3 Inhibitors Research Articles

Targeting RIP3 inhibits osteoarthritis development by restoring anabolic-catabolic balance in the bone-cartilage unit

BackgroundOsteoarthritis (OA) is a debilitating joint disorder characterized by progressive cartilage degeneration. During OA, subchondral bone undergoes microstructural and molecular changes that precede cartilage degradation. However, specific mechanisms underlying metabolic dysregulation of the bone-cartilage unit remain unclear. This study aims to investigate the role of receptor-interacting protein kinase-3 (RIP3) in OA progression, focusing on bone-cartilage metabolic homeostasis. MethodsRIP3-mediated pathological and metabolic alterations in chondrocytes, osteoblasts, and bone marrow-derived macrophages (BMMs) were evaluated. RIP3-mediated OA manifestations in cartilage and, more importantly, subchondral bone were determined by intra-articular overexpression of RIP3 in rats. The protective effect of RIP3 deficiency on the bone-cartilage unit during OA was systematically investigated using Rip3 knockout mice. The CMap database was used to screen for compounds that abrogate RIP3-induced OA pathological changes. ResultsRIP3 was upregulated in the cartilage and subchondral bone of OA patients and post-traumatic OA mouse model. RIP3 overexpression not only inhibited extracellular matrix (ECM) anabolism in chondrocytes but also attenuated osteoblast differentiation, whereas RIP3 deficiency blunted receptor activator of NF-kappaB ligand-mediated osteoclastogenesis of BMMs. Intra-articular RIP3 overexpression induced the imbalance of SP7+ osteoblasts/tartrate-resistant acid phosphatase (TRAP)+ osteoclasts within the subchondral bone in addition to cartilage degeneration in rats, while Rip3 deletion significantly improved structural outcomes of the bone-cartilage unit, and achieved pain relief as well as functional improvement in surgery-induced and spontaneous OA mouse models. Mechanistically, RIP3 initiates OA by perturbing critical events, including cartilage metabolism, inflammatory responses, senescence, and osteoclast differentiation. Clofibrate, a hypolipidemic drug, was identified as a novel RIP3 inhibitor that reverses ECM catabolism in OA. ConclusionsRIP3 is an essential governor of whole joint metabolic homeostasis by regulating both cartilage metabolism and subchondral bone remodeling. Reconstruction of the bone-cartilage unit by targeting RIP3 might provide a two-birds-one-stone approach for the development of future OA therapies.

Necroptosis blockade prevents lung injury in severe influenza.

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.

Enhancing Fat Transplantation Efficiency in a Mouse Model through Pretreatment of Adipose-Derived Stem Cells with RIP3 Inhibitors.

Autologous fat transplantation, widely used in cosmetic and reparative surgery for volumetric enhancements, faces challenges with its inconsistent long-term survival rates. The technique's efficacy, crucial for its development, is hindered by unpredictable outcomes. Enriching fat grafts with adipose-derived stem cells (ADSCs) shows promise in improving survival efficiency. This study aimed to explore the potential of receptor-interacting protein kinase 3 (RIP3) kinase inhibitors as a pretreatment for ADSCs in enhancing autologous fat graft retention over a long term. ADSCs were isolated, cultured under normal or oxygen-glucose deprivation conditions, and mixed with particulate fat grafts to form distinct experimental groups in female nude mice. Fat graft mass and volume, along with underlying mechanisms, were evaluated using quantitative reverse transcription polymerase chain reaction (RT-qPCR), immunohistochemistry, and Western blot analysis. The experimental group, pretreated with RIP3 kinase inhibitors, had higher graft mass and volume, greater adipocyte integrity, and increased peroxisome proliferator-activated receptor gamma (PPARγ) mRNA levels than control groups. Furthermore, the experimental group demonstrated lower expression of necroptosis pathway proteins in the short term and an ameliorated inflammatory response as indicated by interleukin-1 beta (IL-1β), interleukin-10 (IL-10) mRNA levels, and histological analyses. Notably, enhanced neovascularization was evident in the experimental group. These findings suggest that RIP3 kinase inhibitor pretreatment of ADSCs can improve fat graft survival, promote adipocyte integrity, potentially decrease inflammation, and enhance neovascularization. This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Regulatory mechanism of CaMKII δ mediated by RIPK3 on myocardial fibrosis and reversal effects of RIPK3 inhibitor GSK'872

BackgroundMyocardial fibrosis (MF) remains a prominent challenge in heart disease. The role of receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis is evident in the pathogenesis of numerous heart diseases. Concurrently, the activation of Ca2+/calmodulin-dependent protein kinase (CaMKII) is pivotal in cardiovascular disease (CVD). This study aimed to evaluate the impact and underlying mechanisms of RIPK3 on myocardial injury in MF and to elucidate the potential involvement of CaMKII. MethodsBuilding upon our previous research methods [1], wild-type (WT) mice and RIPK3 knockout (RIPK3 -/-) mice underwent random assignment for transverse aortic constriction (TAC) in vivo. Four weeks post-procedure, the MF model was effectively established. Parameters such as the extent of MF, myocardial injury, RIPK3 expression, necroptosis, CaMKII activity, phosphorylation of mixed lineage kinase domain-like protein (MLKL), mitochondrial ultrastructural details, and oxidative stress levels were examined. Cardiomyocyte fibrosis was simulated in vitro using angiotensin II on cardiac fibroblasts. ResultsTAC reliably produced MF, myocardial injury, CaMKII activation, and necroptosis in mice. RIPK3 depletion ameliorated these conditions. The RIPK3 inhibitor, GSK'872, suppressed the expression of RIPK3 in myocardial fibroblasts, leading to improved fibrosis and inflammation, diminished CaMKII oxidation and phosphorylation levels, and the rectification of CaMKIIδ alternative splicing anomalies. Furthermore, GSK'872 downregulated the expressions of RIPK1, RIPK3, and MLKL phosphorylation, attenuated necroptosis, and bolstered the oxidative stress response. ConclusionsOur data suggested that in MF mice, necroptosis was augmented in a RIPK3-dependent fashion. There seemed to be a positive correlation between CaMKII activation and RIPK3 expression. The adverse effects on myocardial fibrosis mediated by CaMKII δ through RIPK3 could potentially be mitigated by the RIPK3 inhibitor, GSK'872. This offered a fresh perspective on the amelioration and treatment of MF and myocardial injury.

RIPK3 activation promotes DAXX-dependent neuronal necroptosis after intracerebral hemorrhage in mice.

Necroptosis induced by receptor-interacting protein kinase 3 (RIPK3) is engaged in intracerebral hemorrhage (ICH) pathology. In this study, we explored the impact of RIPK3 activation on neuronal necroptosis and the mechanism of the death domain-associated protein (DAXX)-mediated nuclear necroptosis pathway after ICH. Potential molecules linked to the progression of ICH were discovered using RNA sequencing. The level of DAXX was assessed by quantitative real-time PCR, ELISA, and western blotting. DAXX localization was determined by immunofluorescence and immunoprecipitation assays. The RIPK3 inhibitor GSK872 and DAXX knockdown with shRNA-DAXX were used to examine the nuclear necroptosis pathway associated with ICH. Neurobehavioral deficit assessments were performed. DAXX was increased in patients and mice after ICH. In an ICH mouse model, shRNA-DAXX reduced brain water content and alleviated neurologic impairments. GSK872 administration reduced the expression of DAXX. shRNA-DAXX inhibited the expression of p-MLKL. Immunofluorescence and immunoprecipitation assays showed that RIPK3 and AIF translocated into the nucleus and then bound with nuclear DAXX. RIPK3 revitalization promoted neuronal necroptosis in ICH mice, partially through the DAXX signaling pathway. RIPK3 and AIF interacted with nuclear DAXX to aggravate ICH injury.

HSP70 attenuates neuronal necroptosis through the HSP90α-RIPK3 pathway following neuronal trauma.

Necroptosis, a newly defined regulatable necrosis with membrane disruption, has been demonstrated to participate in trauma brain injury (TBI) related neuronal cell death. Heat shock protein 70 (HSP70) is a stress protein with neuroprotective activity, but the potential protective mechanisms are not fully understood. Here, we investigated the effects of HSP70 regulators in a cellular TBI model induced by traumatic neuronal injury (TNI) and glutamate treatment. We found that necroptosis occurred in cortical neurons after TNI and glutamate treatment. Neuronal trauma markedly upregulated HSP70 protein expression within 24h. The results of immunostaining and lactate dehydrogenase release assay showed that necroptosis following neuronal trauma was inhibited by HSP70 activator TRC051384 (TRC), but promoted by the HSP70 inhibitor 2-phenylethyenesulfonamide (PES). In congruent, the expression and phosphorylation of receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL) were differently regulated by HSP70. Furthermore, the expression of HSP90α induced by neuronal trauma was further promoted by PES but decreased by TRC. The data obtained from western blot showed that the phosphorylation of RIPK3 and MLKL induced by HSP70 inhibition were reduced by RIPK3 inhibitor GSK-872 and HSP90α inhibitor geldanamycin (GA). Similarly, inhibition of HSP90α with GA could partially prevented the increased necroptosis induced by PES. Taken together, HSP70 activation exerted protective effects against neuronal trauma via inhibition of necroptosis. Mechanistically, the HSP90α-mediated activation of RIPK3 and MLKL is involved in these effects.

Compound-42 alleviates acute kidney injury by targeting RIPK3-mediated necroptosis.

Necroptosis plays an essential role in acute kidney injury and is mediated by receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), and mixed lineage kinase domain-like pseudokinase (MLKL). A novel RIPK3 inhibitor, compound 42 (Cpd-42) alleviates the systemic inflammatory response. The current study was designed to investigate whether Cpd-42 exhibits protective effects on acute kidney injury and reveal the underlying mechanisms. The effects of Cpd-42 were determined in vivo through cisplatin- and ischaemia/reperfusion (I/R)-induced acute kidney injury and in vitro through cisplatin- and hypoxia/re-oxygenation (H/R)-induced cell damage. Transmission electron microscopy and periodic acid-Schiff staining were used to identify renal pathology. Cellular thermal shift assay and RIPK3-knockout mouse renal tubule epithelial cells were used to explore the relationship between Cpd-42 and RIPK3. Molecular docking and site-directed mutagenesis were used to determine the binding site of RIPK3 with Cpd-42. Cpd-42 reduced human proximal tubule epithelial cell line (HK-2) cell damage, necroptosis and inflammatory responses in vitro. Furthermore, in vivo, cisplatin- and I/R-induced acute kidney injury was alleviated by Cpd-42 treatment. Cpd-42 inhibited necroptosis by interacting with two key hydrogen bonds of RIPK3 at Thr94 and Ser146, which further blocked the phosphorylation of RIPK3 and mitigated acute kidney injury. Acting as a novel RIPK3 inhibitor, Cpd-42 reduced kidney damage, inflammatory response and necroptosis in acute kidney injury by binding to sites Thr94 and Ser146 on RIPK3. Cpd-42 could be a promising treatment for acute kidney injury.

Cadmium (Cd) and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) co-exposure induces acute kidney injury through oxidative stress and RIPK3-dependent necroptosis.

Environmental pollution is complex, and co-exposure can accurately reflect the true environmental conditions that are important for assessment of human health. Cadmium (Cd) is a widespread toxicant that can cause acute kidney injury (AKI), while its combined effect with 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) is not fully understood. Thus, we used an in vivo model where C57BL/6J mice were treated with low dietary intake of Cd (5 mg/kg/day) and/or BDE-47 (1 mg/kg/day) for 28 days to examine AKI, and in vitro experiments to investigate the possible mechanism. Results showed that Cd or BDE-47 caused pathological kidney damage, accompanied by elevated urea nitrogen (BUN) and urinary creatinine, as well as increased interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), and reduced IL-10 in kidney tissues. In vitro Cd or BDE-47 exposure decreased cell viability and induced cell swelling and blebbing of human embryonic kidney 293 (HEK-293) and renal tubular epithelial cell lines (HKCs), and changes in co-exposure was larger than that in Cd and BDE-47 treatment. Oxidative stress indicators of the reactive oxygen species (ROS) and malondialdehyde (MDA) were elevated, while the antioxidant superoxide dismutase (SOD) was decreased. Necrosis occurred with increased lactate dehydrogenase (LDH) release and propidium iodide (PI) staining, which was attenuated by the ROS scavenger N-acetyl-L-cysteine (NAC). Furthermore, necroptotic genes of receptor-interacting protein kinase-3 (RIPK3), classical mixed lineage kinase domain-like protein-dependent (MLKL), IL-1β and TNF-α were up-regulated, whereas RIPK1 was down-regulated, which was attenuated by the RIPK3 inhibitor GSK872. These findings demonstrate that Cd or BDE-47 alone produces kidney toxicities, and co-exposure poses an additive effect, resulting in AKI via inducing oxidative stress and regulating RIPK3-dependent necroptosis, which offers a further mechanistic understanding for kidney damage, and the combined effect of environmental pollutants should be noticed.

1724-P: Caspase 3/7 Activation and RIPK3 Are Dispensable for Synthetic hIAPP-Induced ß-Cell Death

Islet amyloid is a hallmark of type 2 diabetes that is associated with β-cell loss. Aggregation of the β-cell secretory product human islet amyloid polypeptide (hIAPP) to form islet amyloid promotes β-cell death 1) via direct effects on the β cell and 2) by stimulating islet immune cell cytokine production. We recently showed that β cells are susceptible to TNFα-induced necroptosis, a form of caspase-independent, receptor interacting protein kinase 3 (RIPK3)-mediated cell death. We hypothesized that RIPK3 also promotes β-cell death in response to islet amyloid formation. To test this hypothesis, we evaluated INS-1, NIT-1 and isolated mouse islet cells treated with synthetic hIAPP (20 μM) +/− the pan-caspase inhibitor zVAD (40 μM). We found synthetic hIAPP increased cell death and caspase activity in INS-1, NIT-1 and mouse islet cells over 48 h. Remarkably, inhibition of caspases with zVAD (as measured by caspase 3/7 activity) failed to reduce synthetic hIAPP-mediated cell death in INS-1 (n=3, p=0.99), NIT-1 (n=3, p=0.99) or mouse islet cells (n=4, p=0.98). To evaluate RIPK3 in hIAPP-induced β-cell death, we examined a small molecule RIPK3 inhibitor (GSK’872, 5 μM), gene-edited NIT-1 Ripk3Δ cells (gRNA targeting exon 6), and intact isolated Ripk3−/− mouse islets. We found GSK’872 failed to protect INS-1 (n=3, p=0.32) or NIT-1 (n=3, p=0.30) cells from hIAPP-induced cell death over 48 h. Similarly, neither NIT-1 Ripk3Δ cells (n=2, hIAPP: p=0.82, hIAPP+zVAD: p=0.97) nor Ripk3−/− islets (n=4, hIAPP: p=0.63, hIAPP+zVAD: p=0.86) were protected from synthetic hIAPP-induced cell death either when caspases were active or inhibited. Our data indicate that caspase 3/7 activity and RIPK3 are dispensable for synthetic hIAPP-induced β-cell death in vitro. However, we found that RIPK3 is upregulated in response to endogenous islet amyloid formation in vitro. Future studies will determine whether RIPK3 promotes β-cell loss in part via effects on endogenous amyloid-induced cytokine signaling. Disclosure N.Mukherjee: None. C.J.Contreras: None. L.Lin: None. E.P.Cai: None. S.E.Kahn: Advisory Panel; Anji Pharmaceuticals, Bayer Inc., Boehringer Ingelheim Inc., Eli Lilly and Company, Merck & Co., Inc., Other Relationship; Novo Nordisk. A.T.Templin: None. Funding U.S. Department of Veterans Affairs (IK2BX004659 to A.T.T.); National Institutes of Health (T32DK064466 to C.J.C.); Indiana University School of Medicine (to N.M.)

Z-DNA/RNA Binding Protein 1 Senses Mitochondrial DNA to Induce Receptor-Interacting Protein Kinase-3/Mixed Lineage Kinase Domain-Like-Driven Necroptosis in Developmental Sevoflurane Neurotoxicity

Developmental sevoflurane exposure leads to widespread neuronal cell death known as sevoflurane-induced neurotoxicity (SIN). Receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL)-driven necroptosis plays an important role in cell fate. Previous research has shown that inhibition of RIPK1 activity alone did not attenuate SIN. Since RIPK3/MLKL signaling could also be activated by Z-DNA/RNA binding protein 1 (ZBP1), the present study was designed to investigate whether ZBP1-mediated and RIPK3/MLKL-driven necroptosis is involved in SIN through in vitro and in vivo experiments. We found that sevoflurane priming triggers neuronal cell death and LDH release in a time-dependent manner. The expression levels of RIPK1, RIPK3, ZBP1 and membrane phosphorylated MLKL were also dramatically enhanced in SIN. Intriguingly, knockdown of RIPK3, but not RIPK1, abolished MLKL-mediated neuronal necroptosis in SIN. Additionally, inhibition of RIPK3-mediated necroptosis with GSK’872, rather than inhibition of apoptosis with zVAD, significantly ameliorated SIN. Further investigation showed that sevoflurane treatment causes mitochondrial DNA (mtDNA) release into the cytosol. Accordingly, ZBP1 senses cytosolic mtDNA and consequently activates RIPK3/MLKL signaling. This conclusion was reinforced by the evidence that knockdown of ZBP1 or depleting mtDNA with ethidium bromide remarkably improved SIN. Finally, the administration of the RIPK3 inhibitor GSK'872 relieved sevoflurane-induced spatial and emotional disorders without influence on locomotor activity. Altogether, these results illustrate that ZBP1 senses cytosolic mtDNA to induce RIPK3/MLKL-driven necroptosis in SIN. Elucidating the role of necroptosis in SIN will provide new insights into understanding the mechanism of anesthetic exposure in the developing brain.

Cpd-42 protects against calcium oxalate nephrocalcinosis-induced renal injury and inflammation by targeting RIPK3-mediated necroptosis.

Calcium oxalate (CaOx) crystals, as the predominant component of human kidney stones, can trigger excessive cell death and inflammation of renal tubular epithelial cells, involved in the pathogenesis of nephrocalcinosis. Necroptosis mediated by receptor-interacting protein kinase 3 (RIPK3) serves a critical role in the cytotoxicity of CaOx crystals. Here, we assessed the therapeutic potential of a novel RIPK3 inhibitor, compound 42 (Cpd-42), for CaOx nephrocalcinosis by comparison with dabrafenib, a classic RIPK3 inhibitor. Our results demonstrated that Cpd-42 pretreatment attenuated CaOx crystals-induced renal tubular epithelial cell (TEC) injury by inhibiting necroptosis and inflammation in vitro and in vivo. Furthermore, in an established mouse model of CaOx nephrocalcinosis, Cpd-42 also reduced renal injury while improving the impaired kidney function and intrarenal crystal deposition. Consistent with this finding, Cpd-42 was confirmed to exhibit superior inhibition of necroptosis and protection against renal TEC injury compared to the classic RIPK3 inhibitor dabrafenib in vitro and in vivo. Mechanistically, RIPK3 knockout (KO) tubular epithelial cells pretreated with Cpd-42 did not show further enhancement of the protective effect on crystals-induced cell injury and inflammation. We confirmed that Cpd-42 exerted protective effects by specifically targeting and inhibiting RIPK3-mediated necroptosis to block the formation of the RIPK1-RIPK3 necrosome. Taken together, targeted inhibition of RIPK3-mediated necroptosis with Cpd-42 may provide a potential therapeutic approach for CaOx nephrocalcinosis.

RIPK3 inhibitor-AZD5423 alleviates acute kidney injury by inhibiting necroptosis and inflammation

Acute kidney injury (AKI) is a clinical syndrome that is defined as a sudden decline in renal function and characterized by inflammation and programmed cell death of renal tubular epithelial cells. Necroptosis is a form of regulated cell death that requires activation of receptor interacting protein kinase 3 (RIPK3) and its phosphorylation of the substrate MLKL. RIPK3 plays an important role in acute kidney injury, and hence developing its inhibitors is considered as one of the promising strategies aimed at prevention and treatment of AKI. Recently, we discovered AZD5423 as a novel potent RIPK3 inhibitor using a computer-aided hybrid virtual screening strategy according to three-dimensional structure of RIPK3. Our findings revealed that AZD5423 strongly inhibits activation of RIPK3, and MLKL phosphorylation upon cisplatin-, hypoxia/reoxygenation (H/R)- and TNF-α stimuli as compared with GSK872, which is a previously identified RIPK3 inhibitor. Importantly, AZD5423 exerts effective protection against cisplatin- and ischemia/reperfusion (I/R)-induced AKI mouse model. The results of cellular thermal shift assay and experiments in RIPK3 knockout cells indicated that AZD5423 could directly target RIPK3 to inhibit RIPK3 kinase activity. Mechanistically, the docking of AZD5423 and RIPK3 suggested that the kinase domain of RIPK3 for Lys50, Arg313, Lys29, Arg37 might form hydrogen bonds with AZD5423. Site-directed mutagenesis further revealed that AZD5423 reduces injury response via interacting with the key RIPK3 amino acid residues of Lys50 and Arg313. In conclusion, our study has demonstrated that AZD5423 may serve as a potent inhibitor of RIPK3 kinase and a promising clinical candidate for AKI treatment.

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.

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.

Receptor-Interacting Protein Kinase 3 Inhibition Relieves Mechanical Allodynia and Suppresses NLRP3 Inflammasome and NF-κB in a Rat Model of Spinal Cord Injury.

BackgroundNeuroinflammation is critical in developing and maintaining neuropathic pain after spinal cord injury (SCI). The receptor-interacting protein kinase 3 (RIPK3) has been shown to promote inflammatory response by exerting its non-necroptotic functions. In this study, we explored the involvement of RIPK3 in neuropathic pain after SCI.MethodsThoracic (T10) SCI rat model was conducted, and the mechanical threshold in rats was measured. The expressions of RIPK3, nod-like receptor family pyrin domain-containing protein 3 (NLRP3), caspase-1, and nuclear factor-κB (NF-κB) were measured with western blotting analysis or quantitative real-time polymerase chain reaction (qRT-PCR). Double immunofluorescence staining was used to explore the colabeled NLRP3 with NeuN, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adapter molecule 1 (IBA1). In addition, enzyme-linked immunosorbent assay (ELISA) was applied to analyze the levels of proinflammatory factors interleukin 1 beta (IL-1β), interleukin 18 (IL-18), and tumor necrosis factor alpha (TNF-α).ResultsThe expression of RIPK3 was elevated from postoperative days 7–21, which was consistent with the development of mechanical allodynia. Intrathecal administration of RIPK3 inhibitor GSK872 could alleviate the mechanical allodynia in SCI rats and reduce the expression levels of RIPK3. The activation of NLRP3 inflammasome and NF-κB was attenuated by GSK872 treatment. Furthermore, immunofluorescence suggested that NLRP3 had colocalization with glial cells and neurons in the L4–L6 spinal dorsal horns. In addition, GSK872 treatment reduced the production of inflammatory cytokines.ConclusionOur findings indicated that RIPK3 was an important facilitated factor for SCI-induced mechanical allodynia. RIPK3 inhibition might relieve mechanical allodynia by inhibiting NLRP3 inflammasome, NF-κB, and the associated inflammation.

Investigating the role of receptor interacting protein kinase 3 in venous thrombosis

ObjectiveVenous thromboembolism is a disease that encompasses both deep vein thrombosis and pulmonary embolism. Recent investigations have shown that receptor interacting protein kinase 3 (RIPK3), a protein known for its role in the programmed form of cell death necroptosis, may play a role in thrombosis. Specifically, RIPK3 has been shown to promote platelet activation in arterial thrombosis and mixed lineage kinase domain-like pseudokinase (MLKL), a protein downstream of RIPK3 in the necroptosis pathway, has been shown to promote neutrophil extracellular trap formation in deep vein thrombosis. This investigation sought to comprehensively investigate the role of RIPK3 in deep vein thrombogenesis. MethodsThe inferior vena cava ligation and stenosis models of deep vein thrombosis were used in C57BL/6J, RIPK3 wild-type (Ripk3+/+) and RIPK3-deficient (Ripk3–/–) mice. Downstream tissue analyses included measurement of thrombus weight and histological and Western blot analysis of tissues for markers of necroptosis and cell death. A subset of C57BL/6J mice were treated with a RIPK3 inhibitor to determine the effect on venous thrombosis. ResultsC57BL/6J mice showed significant increases in thrombus weight from 6 to 48 hours. During the same time frame, RIPK3 progressively accumulated in the vein wall (a 35-fold increase from 0 to 48 hours). RIPK3 was present in the thrombus; however, it decreased with time. Although present in the thrombus, MLKL was nearly undetectable in the vein wall by Western blot at any timepoint. Immunostaining confirmed the high accumulation of RIPK3 in the vein wall, primarily colocalized to endothelial and smooth muscle cells. Phosphorylated MLKL, the active form of MLKL and executioner of necroptotic cell death, was detectable by immunostaining in the thrombus, but was present at low to undetectable levels in the vein wall. Propidium iodide and terminal deoxynucleotidyl transferase dUTP nick end labeling staining revealed a high burden of necrotic and apoptotic cells within the thrombus at 48 hours, but a relatively lower burden within the vein wall. Despite robust accumulation of RIPK3 within the vessel wall and the thrombus, knockout and inhibition of RIPK3 failed to impact thrombus incident or weight at 48 hours after inferior vena cava ligation. Neutrophil extracellular trap burden did not differ between Ripk3+/+ and Ripk3–/– mice. ConclusionsIn mice, the vein wall responded to deep vein thrombosis induction with elevation of RIPK3 without showing markers of necroptosis and apoptosis. Studies using genetic or pharmacological inhibition of RIPK3 suggest that this cell death mediator may not have a major role in the acute phase of venous thrombogenesis. Further investigation is needed to determine if RIPK3 plays a potentially non-necroptotic role within the vein wall during later stages of thrombus resolution and vein wall remodeling.

RIPK3-Dependent Necroptosis Activates MCP-1-Mediated Inflammation in Mice after Intracerebral Hemorrhage

BackgroundRecent studies have reported that receptor-interacting protein kinase 3 (RIPK3)-dependent necroptosis is related to the pathological process of intracerebral hemorrhage (ICH). Some studies support the view that inhibiting necroptosis is a key mechanism preventing inflammation. Inflammation is a crucial factor contributing to neurological injuries and unfavorable outcomes after ICH. The aim of this study was to clarify the association between necroptosis and monocyte chemoattractant protein-1 (MCP-1)-mediated inflammation and identify a new target for the treatment of ICH. Methods: An ICH model was established in C57BL/6 mice by injecting collagenase IV into the right basal ganglia. The RIPK3 inhibitor GSK872 was administered through intraventricular injection. Then, we assessed brain edema and neurobehavioral function. Western blotting was employed to detect changes in RIPK3, phospho-mixed lineage kinase domain-like protein (p-MLKL), MCP-1, phospho-c-Jun N-terminal kinase (p-JNK) and interleukin 6 (IL-6) levels in the brain tissue. The localization of RIPK3 and MCP-1 was observed using immunofluorescence staining. Co-immunoprecipitation was performed to determine the interaction between RIPK3 and MCP-1. Results: Compared with the sham group, the levels of RIPK3, p-MLKL, MCP-1, p-JNK and IL-6 were increased post-ICH. GSK872 pretreatment significantly reduced RIPK3, p-MLKL, MCP-1, p-JNK and IL-6 expression, accompanied by mitigated cerebral edema and neurobehavioral defects. RIPK3 and MCP-1 colocalized in the perinuclear region after ICH. We detected the formation of the RIPK3-MCP-1 complex in ICH brain tissue. Conclusions: There exerted an association between RIPK3 and MCP-1. The inhibition of RIPK3 alleviated MCP-1-mediated inflammation following ICH.

Intermittent hypoxia aggravates non-alcoholic fatty liver disease via RIPK3-dependent necroptosis-modulated Nrf2/NFκB signaling pathway

AimsHepatocyte necroptosis is a critical event in the progression of non-alcoholic fatty liver disease (NAFLD). Obstructive sleep apnea hypopnea syndrome (OSAHS) and chronic intermittent hypoxia (CIH) may be linked with the pathogenesis and the severity of NAFLD. However, the potential role of necroptosis in OSAHS-associated NAFLD has not been evaluated. The present study investigated whether IH could affect NAFLD progression through promoting receptor-interacting protein kinase-3 (RIPK3)-dependent necroptosis, oxidative stress, and inflammatory response, and further elucidated the underlying molecular mechanisms. Main methodsLO2 cells were treated with palmitic acid (PA) and subjected to IH, and necroptosis, oxidative stress, and inflammation were assessed. The high-fat choline-deficient (HFCD)-fed mouse model was also used to assess the effects of CIH in experimental NAFLD in vivo. Key findingsIn this study, we found that RIPK3-mediated necroptosis was activated both in the PA plus IH-treated LO2 cells and liver of HFCD/CIH mice, and which could trigger oxidative stress and inflammatory response by decreasing Nrf2 and increasing p-P65. RIPK3 downregulation significantly reduced hepatocyte necroptosis, and ameliorated oxidative stress and inflammation through modulating Nrf2/NFκB pathway in vitro and vivo. Similarly, pretreatment with TBHQ, an activator of Nrf2, effectively blocked the generation of oxidative productions and inflammatory cytokines. In addition, RIPK3 inhibitor GSK-872 or TBHQ administration obviously alleviated hepatic injury, including histology, transaminase activities, and triglyceride contents in vivo. SignificanceIH aggravates NAFLD via RIPK3-dependent necroptosis-modulated Nrf2/NFκB signaling pathway, and which should be considered as a potential therapeutic strategy for the treatment of NAFLD with OSASH.

RIP3-dependent necroptosis contributes to the pathogenesis of chronic obstructive pulmonary disease.

Necroptosis has emerged as a potential mechanism in the pathogenesis of chronic obstructive pulmonary disease (COPD). Here, we found that markers of necroptosis, including high mobility group box 1 release and phosphorylation of mixed lineage kinase domain-like protein (p-MLKL), were markedly induced in the late stage of cigarette smoking–induced (CS-induced) emphysema in mouse lung tissue as well as in lung epithelial cells and organoids with higher dosage of or more prolonged exposure to cigarette smoking extract (CSE). Apoptotic signals were also detected and maximally induced in the early stage of CS-exposed mice and CSE-treated epithelial cells. Inhibition of apoptosis by Z-VAD, a pan-caspase inhibitor, switched the cellular stress to enhanced necroptosis in lung epithelial cells and organoids treated with CSE. Depletion or inhibition of receptor-interacting protein kinase 3 (RIP3) or MLKL attenuated the CSE-induced cell death, suggesting that necroptosis contributes to CSE-induced cell death. Silencing or inhibition of RIP1 had no protective effect, indicating a RIP1-independent RIP3 activation pathway. CSE-induced necroptosis released more damage-associated molecular patterns and evoked greater engulfment but slower clearance by bone marrow–derived macrophages, leading to enhanced expression of proinflammatory cytokines Tnfα and Il6. Finally, our in vivo data verified that inhibition of necroptosis by RIP3 inhibitor GSK’872 protected mice from CS-induced emphysema and suppressed the lung inflammation. In conclusion, we provide evidence that necroptosis contributes to the pathogenesis of COPD. Targeting RIP3 and its downstream pathway may be an effective therapy for COPD.

294-OR: RIPK3-Mediated Necroptosis Is an Alternative Form of TNFa-Induced ß-Cell Death

Necroptosis is an inflammatory and immunogenic form of lytic cell death that can occur downstream of TNFα signaling. Receptor interacting protein kinase 3 (RIPK3) mediates TNFα-induced necroptosis when caspase activity is repressed, but this pathway has not been examined in insulin-producing β-cells. As TNFα contributes to β-cell death in the setting of diabetes and since we found RIPK3 is expressed in INS-1 β-cells and mouse islets, we hypothesized that β-cells are susceptible to necroptosis. To test this hypothesis, INS-1 cells were treated with TNFα (40 ng/ml) in the absence or presence of a synthetic pan-caspase inhibitor (zVAD, 50 μM) for 24 h, cell death was monitored in real-time, and caspase activity was determined. TNFα increased INS-1 cell death (1.63 ± 0.13 vs. 2.44 ± 0.13 fold, n=7, p<0.001) and caspase activity, consistent with apoptosis. When caspases were inhibited with zVAD, TNFα no longer increased caspase activity, but induced a similar degree of cell death (1.63 ± .013 vs. 2.82 ± 0.23 fold, n=7, p<0.001), suggesting an alternate mechanism under these conditions. Moreover, when TNFα-induced cell death was amplified by the SMAC/DIABLO mimetic BV6 (5 μM), we again observed similar levels of cell death with or without zVAD. To further investigate the mechanism of cell death under caspase inhibition, we used the RIPK3 inhibitor GSK ‘872 (5 μM). GSK ‘872 had no effect on TNFα+BV6-induced cell death when caspases were activated (3.35 ± 0.61 vs. 3.48 ± 1.21 fold, n=3, p=0.93), but reduced cell death when caspases were inhibited (4.69 ± 0.57 vs. 1.81 ± 0.57 fold, n=3, p=0.04). These data indicate that TNFα induces β-cell death independent of caspase activity level. However, the mechanism by which TNFα elicits β-cell death does depend on caspase activity, with apoptosis occurring when caspases are activated, and RIPK3-mediated necroptosis occurring when caspases are inhibited. Additional studies are required to understand the factors that may promote β-cell necroptosis in the setting of human diabetes. Disclosure C. J. Contreras: None. L. Lin: None. M. F. Hogan: None. A. Oberst: None. S. E. Kahn: Advisory Panel; Self; Bayer AG, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Merck & Co., Inc., Novo Nordisk, Pfizer Inc. A. T. Templin: None. Funding U.S. Department of Veterans Affairs (IK2BX004659, I01BX001060)