Abstract
Tumor necrosis factor (TNF) and Toll-like receptor (TLR)3/TLR4 activation trigger necroptotic cell death through downstream signaling complex containing receptor-interacting protein kinase 1 (RIPK1), RIPK3, and pseudokinase mixed lineage kinase-domain-like (MLKL). However, the regulation of necroptotic signaling pathway is far less investigated. Here we showed that c-Jun N-terminal kinases (JNK1 and JNK2) displayed kinase-dependent and -independent functions in regulating TNF- and TLRs-mediated necroptosis. We found that RIPK1 and RIPK3 promoted cell-death-independent JNK activation in macrophages, which contributed to pro-inflammatory cytokines production. Meanwhile, blocking the kinase activity of JNK dramatically reduced TNF and TLRs-induced necroptotic cell death. Consistently, inhibition of JNK activity protected mice from TNF-induced death and Staphylococcus aureus-mediated lung damage. However, depletion of JNK protein using siRNA sensitized macrophages to necroptosis that was triggered by LPS or poly I:C but still inhibited TNF-induced necroptosis. Mechanistic studies revealed that RIPK1 recruited JNK to the necrosome complex and their kinase activity was required for necrosome formation and the phosphorylation of MLKL in TNF- and TLRs-induced necroptosis. Loss of JNK protein consistently suppressed the phosphorylation of MLKL and necrosome formation in TNF-triggered necroptosis, but differentially promoted the phosphorylation of MLKL and necrosome formation in poly I:C-triggered necroptosis by promoting the oligomeration of TRIF. In conclusion, our findings define a differential role for JNK in regulating TNF- and TLRs-mediated necroptosis by their kinase or scaffolding activities.
Highlights
Necroptosis is a type of programmed cell death, independent of caspases activity, and characterized by receptor-interacting protein kinase 1 (RIPK1) and RIPK3 form a protein complex called necrosome through their RIP homotypic interaction motif (RHIM), which leads to the phosphorylation and oligomerization of RIPK37–9
RIPK3siRNA impaired the phosphorylation of JNK and P38 in peritoneal macrophages stimulated with LPS, poly I:C, or Tumor necrosis factor (TNF) plus zVAD (Fig. 1g–i)
As previous work has shown that RIPK1- and RIPK3-induced inflammation is independent on cell death from the results of MLKLdeficient macrophages[36], we found that knockdown mixed lineage kinase-domain-like (MLKL) could not change the activation of JNK and P38 in peritoneal macrophages treated with LPS plus zVAD (Fig. 1j)
Summary
Necroptosis is a type of programmed cell death, independent of caspases activity, and characterized by RIPK1 and RIPK3 form a protein complex called necrosome through their RIP homotypic interaction motif (RHIM), which leads to the phosphorylation and oligomerization of RIPK37–9. Several apoptosis-related proteins, including caspase 8, FADD, and cFLIP, inhibit the activation of necroptosis. Deficiency of these genes causes embryonic lethality in mice which is dependent on RIPK3 and MLKL-mediated necroptosis[19,20,21]. Other reports showed that several kinases, including IKKα, IKKβ, and MK2 directly phosphorylated RIPK1 and restricted the autophosphorylation of RIPK1, which inhibited the activation of TNF-induced apoptosis and necroptosis[22,23,24,25]. Necrosome recruits molecular chaperone HSP90 and CDC37 to facilitate necroptotic signaling transduction[30]
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