Figure 1. Necroptosis signaling pathway
The dynamic balance between cell death, proliferation, and differentiation is an important condition for maintaining the development and tissue homeostasis of multicellular organisms. Traditionally, apoptosis is the only form of programmed cell death, and necroptosis is the passive death of cells under extreme conditions, independent of the regulation of intracellular and extracellular signals. Apoptosis and necroptosis have different morphological features: the former exhibits cell shrinkage, chromatin condensation, formation of apoptotic bodies and phagocytosis by adjacent cells; the latter exhibits increased cell volume, organelle shrinkage, plasma membrane disintegration and other characteristics (Figure 2). More importantly, after necroptosis occurs, the cellular contents are released and cause an immune response. Then, damage-related pattern molecules (DAMPs) such as HMGB1 and mitochondrial DNA can be detected in the blood, which are markers of necroptosis. However, in recent years, more and more studies have shown that necroptosis is also a cell death pathway regulated by intracellular molecules, including programmed necroptosis, coercion, iron death, and mitochondrial permeability transition (MPT). Among them, programmed necroptosis regulated by RIP3 (receptor interacting protein kinase 3) and MLKL (mixed lineage kinase domain-like protein) is the most well-researched necrotic pathway. Programmed necroptosis may involve a series of defense processes against intracellular infections, and related studies have revealed that programmed necroptosis plays an important role in the pathology of many human diseases, such as myocardial infarction, stroke, arteriosclerosis, and ischemia-reperfusion injury.
Figure 2. Different morphological features of apoptosis and necroptosis
After necroptosis occurs, the cell contents are released and cause an immune response, and then damage-related pattern molecules (DAMPs) such as HMGB1 (nuclear high obility group box-1 proteins) and mitochondrial DNA are detected in the blood, which are signs of necroptosis. Necroptosis regulated by RIP3 (receptor interacting protein inase 3) and MLKL (mixed lineage kinase domain-like rotein) is the most well-researched necrotic pathway. The necrotic pathway is regulated by a necrotic complex, and death receptors such as tumor necroptosis factor receptor 1 (TNFR1), cell surface Toll-like receptor (TLR), and DAI (DNA-dependent activator of IFN regulatory factor) bind to their corresponding ligands, promote the assembly of necrosomes and induce the occurrence of necroptosis. RIP1 (receptor interacting rotein kinase 1) is the first identified signal molecule in the necrotic complex. It is involved in the regulation of multiple signaling pathways, including the activation of the NF-κB (nuclear factor-κB) pathway, the MAP kinase cascade, and the caspase-8-dependent apoptotic pathway. Subsequently, the discovery of the kinase RIP3 and its substrate MLKL gradually improved the key molecular composition of this pathway.
Necroptosis signaling pathway
The signaling pathways for necroptosis are mainly composed of activation and action of necroptosis signals. Necroptosis is initiated by the binding of TNF family cytokines such as TNFα, Fas/CD95 and TRAIL (TNF-related apoptosis-inducing ligand) to membrane receptors to activate intracellular RIP family kinases. In addition, LPS (lipopolysaccharide), viral DNA, and interferon can activate a programmed necroptosis signal pathway. After TNF-α binds to the TNF receptor (such as TNFR1) on the cell membrane surface, TNFR1 interacts with the death domain of the C-terminal death domain and the TNFR1-associated death domain protein, thereby recruiting RIP1. TRAF2 (TNFR-associated factor 2)/TRAF5, cIAP1 (cellular inhibitor of apoptosis protein 1)/cIAP2 and LUBAC complex (the linear ubiquitin chain assembly complex) form a TNFR1 signal complex I (complex). The complex recruits the TAK1 (TAF1-binding protein) complex and the IKK complex composed of IKK1, IKK2 and NEMO (NF-κB essential modulator) to activate the NF- κB signaling pathway and the MAP kinase cascade forms a pro-inflammatory signal and prevents cell death. The function of RIP1 in this signaling pathway is independent of its kinase activity, in which case RIP1 is a scaffolding protein of the downstream signaling pathway. Upon completion of assembly of the death receptor complex, RIP1 is rapidly ubiquitinated, which is necessary for the recruitment of IKK complexes and activation of NF-κB. TRAF2/3/5/6 and cIAPs are both E3 ubiquitin ligases, which can be modified by K63 polyubiquitin chain of RIP1. The polyubiquitination of RIP1 by cIAPs is not limited to K63 modification. When the activity of cIAP, TAK1, and NEMO is inhibited or down-regulated, TNF stimulation promotes the formation of complex IIb (complex IIb) consisting of RIP1, RIP3, FADD and pro-caspase-8, which is dependent on RIP1 kinase activity. At this time, RIP3 and RIP1 are combined with each other through their respective RHIM (homotypic interaction motif) domains. Subsequently, human RIP3 (hRIP3) is auto phosphorylated at S227, and mouse RIP3 (mRIP3) is at Ser232. Then activated RIP3 recruits MLKL to activate the necrotic pathway. Studies have shown that caspase-8 can inhibit necroptosis by cutting RIP1, RIP and CYLD, but how this process inhibits cell death is not clear. Recently, in vivo experiments with rip1, rip3, caspase-8 or FADD knockout mice confirmed the function of RIP1 to promote cell death and antagonize cell death. Knockout of rip1 or rip3 reversed the lethality of mouse embryos caused by caspase-8 or FADD knockout, thereby confirming the negative regulation of caspase-8 or FADD on necroptosis. Postnatal death can be observed in rip1 knockout mice, probably due to the knockdown of rip1, which causes the cells to lose their protective effects, a phenomenon that can be reversed by the double gene knockout of caspase-8 and RIP3. The expression level of FLIP has an important influence on the regulation of programmed necroptosis and apoptosis. High level expression of FLIP leads to the formation of the heteromeric complex caspase-8-FLIP, which has catalytic activity but does not promote complete processing of caspase-8, thereby blocking apoptosis dependent on complex IIa. The precise mechanism by which Caspase-8 promotes survival remains unclear, but the presence of FLIP and caspase-8 with catalytic activity rather than proteolytic processing activity is required. Therefore, FADD-caspase-8-FLIP-mediated regulation of complex IIb is the second important barrier to block necroptosis. Other death receptor-mediated necroptosis: In humans, there are six death receptors in the TNF superfamily: TNFR1, FAS, DR3, TRAILR1, TRAILR2, and DR6. The TNFR1-mediated signaling pathway first forms a pro-survival signaling complex, which then forms a death-inducing complex in sensitive cells, activating the death pathway. In contrast, the binding of FASL to FAS, TRAIL and TRAILR1 or TRAILR2 binds to the DD domain of the intracellular domain and the linker protein FADD, causing assembly of the death-inducing signaling complex (DISC), thereby recruiting and activating caspase-8, and causes apoptosis. In the absence of specific conditions such as cIAPs, these ligands also induce necroptosis when caspase-8 is inhibited. In addition, when cells are stressed or damaged and infected, complexes such as TLRs, NLRs, RGRs, and PKR are activated, and some receptors can also cause necroptosis. Among them, when caspase activity is inhibited, lipopolysaccharide or poly(I׃C) activates TLR4 or TLR3, respectively, and activated TLR3 forms an endosomal platform to recruit cytoplasmic linker molecule TRIF [Toll/IL-1 receptor (TIR)) domain containing adaptor protein inducing interferon (IFN)-β], and TLR4 can also transmit signals through TRIF. TRIF contains an RHIM domain that binds to RIP1 and RIP3 and induces necroptosis, but the process does not depend on RIP1. In certain types of cells, regulation of necroptosis may involve an autocrine loop. In macrophages, LPS-TLR4, TNFTNFR1, and poly(I׃C)-TLR3-regulated cells require a type I IFN-α receptor signal, indicating the presence of a type I interferon autocrine loop. The upstream signal interacts with RIP3 via other RHIM-containing proteins, and the activated RIP3 conducts necroptosis signals by phosphorylating its substrate MLKL. As a specific substrate for RIP3, MLKL is the executor of programmed cell death. Its N-terminus is a functional domain, human MLKL (hMLKL) contains 5 α-helices, and mouse MLKL (mMLKL) contains 4 α-helices. The common feature is that four spirals form a spiral bundle, the C-terminus is a kinase-like domain, and the two are linked by two alpha-helical junction domains. Unactivated MLKL is present in the cytosol in monomeric form. RIP3 is activated by phosphorylation and binds to the MLKL kinase-like domain via its kinase domain and phosphorylates the Thr357/Ser358 site of the hMLKL kinase-like domain or the Ser345/347/352 and Thr349 sites of mMLKL, thereby promoting the necroptosis program execution. Oligomerization occurs after phosphorylation of MLKL monomer, and its N-terminal helical bundle can bind to phosphatidylinositol phospholipids (PIPs) and mitochondria-specific cardiolipin (CL), which can be translocated from cytoplasm to PIP-rich or CL on the plasma membrane. The chemical inhibitor NSA binds to the 4 helixes bundle of hMLKL, disrupts the N-terminal function of MLKL, and blocks the transfer of MLKL to the plasma membrane. Similarly, inhibition of PIPs synthesis also reduces necroptosis induced by MLKL. Different PIPs can target MLKL to different cellular components. PI (4)P and PI (4,5) P2 are widely present on the plasma membrane surface, resulting in loss of plasma membrane integrity after activation of the necrotic pathway. CL is mainly distributed in the mitochondrial inner membrane and can bind to MLKL when necroptosis occurs. Similarly, other PIPs distributed in different organelles may recruit oligomeric MLKL to the corresponding membrane site. In liposome permeability experiments, MLKL (2-154) and MLKL full-length can cause quenching of sulforhodamine B fluorescence, while MLKL is lost after MLKL (2-154) or MLKL full length is incubated with NSA. The ability of the liposome contents to leak indicates that MLKL does indeed form pores in the lipid membrane. The pore-forming model also requires more research, including the acquisition of high-resolution structures of the MLKL 4-helix bundle on the plasma membrane, and whether the mechanism of MLKL permeability of the plasma membrane is like that of Bax.
In theory, it is possible to interfere with necrotic pathways at different stages, such as receptors, RIP1, RIP3, MLKL, and assembly of necroptosis complexes. Nec-1 is the earliest identified necroptosis inhibitor that specifically inhibits the kinase activity of RIP1. Nec-1 has some side effects unrelated to cell death, which is not conducive to its clinical application; and, in some animal models of RIP3 deletion, Nec-1 accelerates death. Recently, researchers have developed second-generation RIP1 kinase inhibitors with higher affinity and specificity, such as necrostatin-1s, and no accelerated disease has been observed in necrostatin-1s-treated TNF-shock mice. The pleiotropic nature of RIP1 suggests that finding inhibitors of downstream molecules is a more attractive therapeutic strategy. Among them, NSA is a direct inhibitor of human MLKL and can be used as a potential drug to block necroptosis, but its specificity, pharmacokinetics and final clinical application remain to be studied. Programmed necroptosis-mediated cell death may be directly caused by changes in plasma membrane permeability by the necrotic complex, so a new drug target may be created for the interaction of the necrotic complex with the membrane or blocking the plasma membrane channel. In addition to the development of small molecules and plasma membrane channel inhibitors that target signaling pathways, specific inhibition of necrotic receptors is also an effective means. In addition to TNFR1, other receptors and signaling molecules that induce programmed necroptosis are also being explored. Unlike our intuitive impression, programmed necroptosis is not always harmful, and it is indispensable in the normal development of an individual. Some studies have shown that necroptosis pathways are often defective in tumorigenesis and tumor progression. Under the combined induction of TNF-α and z-VAD, chronic lymphocytic leukemia (CLL) cells could not produce programmed necroptosis, and the expression levels of RIP3 and CYLD in CLL cells were significantly down-regulated. Single nucleotide polymorphisms (SNPs) of the rip3 gene were detected in 458 patients with nonHodgkin lymphoma, which was associated with an increased risk of non-Hodgkin's lymphoma. It is suggested that the rip3 gene mutation is likely to be associated with the occurrence of the disease. Studies have shown that the induction of RIP1/RIP3-dependent programmed necroptosis by Shikonin reduces osteosarcoma (osteosarcoma) abdomen metastasis. Diffusion of tumor cells uses various strategies to increase ATP levels and limit cellular ROS production, whereas RIP3 regulates TNF-induced ROS production by activating multiple metabolic enzymes in programmed necroptosis. In this case, metastasis of tumor cells requires simultaneous elimination of anoikis and programmed necroptosis. However, there are few studies on the development of programmed necroptosis in tumor development. The study on how cancer cells lose the integrity of programmed necroptosis pathway will help to understand the role of necroptosis in regulating tumorigenesis and tumor metastasis.
Necroptosis is associated with several disease pathologies and organic damage. Wild-type experimental animals have more serious conditions in diseases such as atherosclerosis caused by drugs than RIP3 knockout animals. It suggests that necroptosis plays an important role in the occurrence and development of atherosclerosis.