title: 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.
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada.
authors.forename: Scott
authors.initials: S
authors.lastname: McComb
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada.
authors.forename: Bojan
authors.initials: B
authors.lastname: Shutinoski
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada.
authors.forename: Susan
authors.initials: S
authors.lastname: Thurston
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada.
authors.forename: Erin
authors.initials: E
authors.lastname: Cessford
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada.
authors.forename: Kriti
authors.initials: K
authors.lastname: Kumar
authors.affiliation: Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, K1H 8M5, Canada Subash.sad@uottawa.ca.
authors.forename: Subash
authors.initials: S
authors.lastname: Sad
mesh_terms: D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D053181:Caspase 8 / Q000235:genetics / Q000276:immunology; D061945:Caspase Inhibitors / Q000494:pharmacology; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D002403:Cathepsins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology*; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000276:immunology*; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D059748:Proteolysis; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000235:genetics / Q000276:immunology*; D051193:Toll-Like Receptors / Q000819:agonists / Q000235:genetics / Q000276:immunology
chemical_list: D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D008070:Lipopolysaccharides; D051193:Toll-Like Receptors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C506251:Ripk1 protein, mouse; D002403:Cathepsins; C505526:Casp8 protein, mouse; D053181:Caspase 8; D002401:Cathepsin B; C533915:Ctsb protein, mouse; C028217:cathepsin S