Abstract
Receptor-interacting protein kinase 3 (RIPK3) functions as a central regulator of necroptosis, mediating signaling transduction to activate pseudokinase mixed lineage kinase domain-like protein (MLKL) phosphorylation. Increasing evidences show that RIPK3 contributes to the pathologies of inflammatory diseases including multiple sclerosis, infection and colitis. Here, we identified a novel small molecular compound Salt-inducible Kinases (SIKs) inhibitor HG-9-91-01 inhibiting necroptosis by targeting RIPK3 kinase activity. We found that SIKs inhibitor HG-9-91-01 could block TNF- or Toll-like receptors (TLRs)-mediated necroptosis independent of SIKs. We revealed that HG-9-91-01 dramatically decreased cellular activation of RIPK3 and MLKL. Meanwhile, HG-9-91-01 inhibited the association of RIPK3 with MLKL and oligomerization of downstream MLKL. Interestingly, we found that HG-9-91-01 also trigger RIPK3-RIPK1-caspase 1-caspase 8-dependent apoptosis, which activated cleavage of GSDME leading to its dependent pyroptosis. Mechanistic studies revealed that SIKs inhibitor HG-9-91-01 directly inhibited RIPK3 kinase activity to block necroptosis and interacted with RIPK3 and recruited RIPK1 to activate caspases leading to cleave GSDME. Importantly, mice pretreated with HG-9-91-01 showed resistance to TNF-induced systemic inflammatory response syndrome. Consistently, HG-9-91-01 treatment protected mice against Staphylococcus aureus-mediated lung damage through targeting RIPK3 kinase activity. Overall, our results revealed that SIKs inhibitor HG-9-91-01 is a novel inhibitor of RIPK3 kinase and a potential therapeutic target for the treatment of necroptosis-mediated inflammatory diseases.
Highlights
Necroptosis is a regulatory form of cell death, independent of caspases activity, with cell membrane rupturing and release of damage associated molecular patterns (DAMPs) that trigger a strong innate immune response [1]
We found that Salt-inducible Kinases (SIKs) inhibitor HG-9-91-01 blocked TNF- and Toll-like receptors (TLRs)-induced necroptosis, which appeared to using the CytoTox96 lactate dehydrogenase (LDH) release kit (Promega), according to the manufacture’s instruction
SIKs inhibitor HG-9-91-01 dramatically blocks TNF- and TLRsinduced necroptosis Previous work showed that IKKi and TBK1 phosphorylated RIPK1 to inhibit TNF-induced apoptosis and RIPK1 kinase mutant mice rescued embryonic lethality of TBK1 deficiency mice [21]
Summary
Necroptosis is a regulatory form of cell death, independent of caspases activity, with cell membrane rupturing and release of damage associated molecular patterns (DAMPs) that trigger a strong innate immune response [1]. Mice with conditional deletion of caspase 8 or FADD in intestinal epithelium developed spontaneously colitis that was dependent on MLKL and RIPK3-mediated epithelial cell necroptosis [15, 16]. After 3 h, and 3 can be recruited to the necrosome complex and directly phosphorylate RIPK3 to promote TNF-induced necroptosis [22]. Cells were treated that determines whether a cell activates caspase-8 and dies by as indicated and the supernatant was collected to detect the released LDH apoptosis. There are other small molecular inhibitors that target RIPK3 kinase activity and alleviate TNF-induced systemic inflammatory response syndrome [29]. Real-time PCR RNA was obtained from cells using Trizol according to the manufacturer’s protocol. cDNA was produced by retro-transcribing RNA using PrimeScript
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