Cathepsins Limit Macrophage Necroptosis through Cleavage of Rip1 Kinase

Abstract

It has recently been shown that programmed necrosis, necroptosis, may play a key role in the development of inflammation. Deciphering the regulation of this pathway within immune cells may therefore have implications in pathology associated with inflammatory diseases. We show that treatment of macrophages with the pan caspase inhibitor (zVAD-FMK) results in both increased phosphorylation and decreased cleavage of receptor interacting protein kinase-1 (Rip1), leading to necroptosis that is dependent on autocrine TNF signaling. Stimulation of cells with TLR agonists such as LPS in the presence of zVAD-FMK also induced Rip1-phosphorylation via a TNFR-independent mechanism. Further examination of Rip1 expression under these stimulatory conditions revealed a regulatory cleavage of Rip1 in macrophages that is not apparently attributable to caspase-8. Instead, we provide novel evidence that cysteine family cathepsins, which are highly abundant in myeloid cells, can also cleave Rip1 kinase. Using small interfering RNA knockdown, specific cathepsin inhibitors, and cell-free cleavage assays, we demonstrate that cysteine cathepsins B and S can directly cleave Rip1. Finally, we demonstrate that only through combined inhibition of cathepsins and caspase-8 could a potent induction of macrophage necroptosis be achieved. These data reveal a novel mechanism of regulation of necroptosis by cathepsins within macrophage cells.

Type-I interferon signaling through ISGF3 complex is required for sustained Rip3 activation and necroptosis in macrophages

“…S6). Our recent work with recombinant Rip1 also confirms the specificity of the Rip1 antibody used here (52).…”

Multiple signals at the extended 8p23 locus are associated with susceptibility to systemic lupus erythematosus

“…[39][40][41] Cathepsin B was shown to regulate various immune functions; for example, persistence of memory T cells, antigen-presenting function of dendritic cells, monocyte and macrophage necrosis/necroptosis and TLR signalling. [42][43][44][45][46] The publicly available data on human monocytes and lymphoblastoid cell lines (see online supplementary table S3) have suggested cell type-specific effects/functions of the 8p23 SNPs as also supported by a recent BLK study. 47 Guthridge et al 47 performed a comprehensive analysis of the FAM167A-BLK subregion, which has revealed two functional variants that regulate alternative promoter activities in cell type-specific and developmental stage-specific manners.…”

Novel Insights into Crystal-Induced Kidney Injury

“…Conversely, indigestible lysosomal crystals can destabilize lysosomal membranes, release lysosomal enzymes into cytosol causing cell stress to engage autophagic tubular cell death. Some of the lysosomal enzymes – e.g., cathepsin B – are reported to activate necroptosis by cleaving the necroptosis inhibitor protein RIPK1 in immune cells [26]. In fact, crystals of calcium oxalate, calcium phosphate, calcium pyrophosphate, cysteine, cholesterol, and MSU induced tubular cell necroptosis in vitro by engaging RIPK3 and pseudokinase MLKL [5, 6] (Fig.…”