Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models

Takahashi, Nozomi; Duprez, Linde; Grootjans, Sasker; Cauwels, Anje; Nerinckx, Wim; DuHadaway, James B.; Goossens, Vera; Roelandt, Ria; Hauwermeiren, Filip Van; Libert, Claude; Declercq, Wim; Callewaert, Nico; Prendergast, George C.; Degterev, Alexei; Yuan, Junying; Vandenabeele, Peter

doi:10.1038/cddis.2012.176

Cited by 406 publications

(406 citation statements)

References 49 publications

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“…We assessed RIP3 activity by Western blot 10 hours after incubation with sCD74/rMIF and observed a 2‐fold increase of RIP3 phosphorylation compared with untreated control (control versus sCD74/rMIF: 100% versus 222.2±45.98%; P =0.0376, d =1.88), whereas neither rMIF nor sCD74 treatment alone affected phospho‐RIP3 levels (Figure 2C and 2D). To confirm that necroptosis contributes to cell death induction, we blocked activity of RIP1, the upstream kinase of RIP3, with the pharmacological inhibitor, 7‐Cl‐O‐Nec1 (Nec1s) 54. Surprisingly, Nec1s‐pretreated myofibroblasts demonstrated a small, but significant, decrease in cell viability following MIF treatment compared with control cells (Nec1s/control versus Nec1s/MIF: 99.72±3.04% versus 79.62±4.63%; P =0.025, d =1.81).…”

Section: Resultsmentioning

confidence: 99%

Soluble CD74 Reroutes MIF/CXCR4/AKT‐Mediated Survival of Cardiac Myofibroblasts to Necroptosis

Soppert

Kraemer

Beckers

et al. 2018

JAHA

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BackgroundAlthough macrophage migration inhibitory factor (MIF) has been demonstrated to mediate cardioprotection in ischemia/reperfusion injury and antagonize fibrotic effects through its receptor, CD74, the function of the soluble CD74 receptor ectodomain (sCD74) and its interaction with circulating MIF have not been explored in cardiac disease.Methods and ResultsCardiac fibroblasts were isolated from hearts of neonatal mice and differentiated into myofibroblasts. Co‐treatment with recombinant MIF and sCD74 induced cell death (P<0.001), which was mediated by receptor‐interacting serine/threonine‐protein kinase (RIP)1/RIP3‐dependent necroptosis (P=0.0376). This effect was specific for cardiac fibroblasts and did not affect cardiomyocytes. Gene expression analyses using microarray and RT‐qPCR technology revealed a 4‐fold upregulation of several interferon‐induced genes upon co‐treatment of myofibroblasts with sCD74 and MIF (Ifi44: P=0.011; Irg1: P=0.022; Clec4e: P=0.011). Furthermore, Western blot analysis confirmed the role of sCD74 as a modulator of MIF signaling by diminishing MIF‐mediated protein kinase B (AKT) activation (P=0.0197) and triggering p38 activation (P=0.0641). We obtained evidence that sCD74 inhibits MIF‐mediated survival pathway through the C‐X‐C chemokine receptor 4/AKT axis, enabling the induction of CD74‐dependent necroptotic processes in cardiac myofibroblasts. Preliminary clinical data revealed a lowered sCD74/MIF ratio in heart failure patients (17.47±10.09 versus 1.413±0.6244).ConclusionsThese findings suggest that treatment of cardiac myofibroblasts with sCD74 and MIF induces necroptosis, offering new insights into the mechanism of myofibroblast depletion during scar maturation. Preliminary clinical data provided first evidence about a clinical relevance of the sCD74/MIF axis in heart failure, suggesting that these proteins may be a promising target to modulate cardiac remodeling and disease progression in heart failure.

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Section: Resultsmentioning

confidence: 99%

Soluble CD74 Reroutes MIF/CXCR4/AKT‐Mediated Survival of Cardiac Myofibroblasts to Necroptosis

Soppert

Kraemer

Beckers

et al. 2018

JAHA

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“…As RT-PCR revealed that both rip1 and rip3 were upregulated in pde6c w59 mutant cone photoreceptors, we took advantage of small-molecule drugs that target specifically RIP1 34 and RIP3 kinases 35 as potential pharmacological approaches to inhibit cone cell death. Using dose-response analysis in wild-type zebrafish, 10 the highest concentration of necrostatin-1s (RIP1 inhibitor) and necrosulfonamide (RIP3 inhibitor) that could be utilized without causing toxicity to embryos was 100 and 20 mM, respectively.…”

Section: Resultsmentioning

confidence: 99%

Rip3 knockdown rescues photoreceptor cell death in blind pde6c zebrafish

et al. 2014

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Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6c w59 mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6c w59 embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed.Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6c w59 mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment.

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“…However, existing data that imply protection by interference with necroptosis in various in vivo models used the RIPK1 kinase inhibitor necrostatin (Nec)-1 to inhibit necrotic signaling (10,11,26,27). However, Nec-1 has recently been discussed to directly influence the immune system, besides its effects to block release of CDAMPs (28,29). Therefore, it remains an open question whether necroptosis in IRI is of relevance in the absence of a functional immune system.…”

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confidence: 99%

Two independent pathways of regulated necrosis mediate ischemia–reperfusion injury

Linkermann

Bräsen

Darding

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

503

456

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Regulated necrosis (RN) may result from cyclophilin (Cyp)D-mediated mitochondrial permeability transition (MPT) and receptorinteracting protein kinase (RIPK)1-mediated necroptosis, but it is currently unclear whether there is one common pathway in which CypD and RIPK1 act in or whether separate RN pathways exist. Here, we demonstrate that necroptosis in ischemia-reperfusion injury (IRI) in mice occurs as primary organ damage, independent of the immune system, and that mice deficient for RIPK3, the essential downstream partner of RIPK1 in necroptosis, are protected from IRI. Protection of RIPK3-knockout mice was significantly stronger than of CypD-deficient mice. Mechanistically, in vivo analysis of cisplatin-induced acute kidney injury and hyperacute TNF-shock models in mice suggested the distinctness of CypD-mediated MPT from RIPK1/RIPK3-mediated necroptosis. We, therefore, generated CypD-RIPK3 double-deficient mice that are viable and fertile without an overt phenotype and that survived prolonged IRI, which was lethal to each single knockout. Combined application of the RIPK1 inhibitor necrostatin-1 and the MPT inhibitor sanglifehrin A confirmed the results with mutant mice. The data demonstrate the pathophysiological coexistence and corelevance of two separate pathways of RN in IRI and suggest that combination therapy targeting distinct RN pathways can be beneficial in the treatment of ischemic injury.RIP3 | RIP1 | programmed necrosis | apoptosis

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Necrostatin-1 analogues: critical issues on the specificity, activity and in vivo use in experimental disease models

Cited by 406 publications

References 49 publications

Soluble CD74 Reroutes MIF/CXCR4/AKT‐Mediated Survival of Cardiac Myofibroblasts to Necroptosis

Soluble CD74 Reroutes MIF/CXCR4/AKT‐Mediated Survival of Cardiac Myofibroblasts to Necroptosis

Rip3 knockdown rescues photoreceptor cell death in blind pde6c zebrafish

Two independent pathways of regulated necrosis mediate ischemia–reperfusion injury

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