title: Calpain Activity Is Essential for ATP-Driven Unconventional Vesicle-Mediated Protein Secretion and Inflammasome Activation in Human Macrophages.
abstract: Extracellular ATP is an endogenous danger signal that is known to activate inflammatory responses in innate immune cells, including macrophages. Activated macrophages start to secrete proteins to induce an immune response, as well as to recruit other immune cells to the site of infection and tissue damage. In this study, we characterized the secretome (i.e., the global pattern of secreted proteins) of ATP-stimulated human macrophages. We show that ATP stimulation activates robust vesicle-mediated unconventional protein secretion, including exosome release and membrane shedding, from human macrophages. Pathway analysis of the identified secreted proteins showed that calpain-related pathways were overrepresented in the secretome of ATP-stimulated cells. In accordance with this, calpains, which are calcium-dependent nonlysosomal cysteine proteases, were activated upon ATP stimulation through a P2X purinoceptor 7 receptor-dependent pathway. Functional studies demonstrated that calpain activity is essential for the P2X purinoceptor 7 receptor-mediated activation of unconventional protein secretion. Unconventional protein secretion was followed by cell necrosis and NLRP3 inflammasome-mediated secretion of the mature form of the proinflammatory cytokine IL-1β. Furthermore, ATP-driven NLRP3 inflammasome activation was also dependent on calpain activity. Interestingly, pro-IL-1β and inflammasome components ASC and caspase-1 were released by ATP-activated macrophages through a vesicle-mediated secretion pathway. In conclusion, to our knowledge, we provide the first global characterization of proteins secreted by ATP-activated human macrophages and show a pivotal role for calpains in the activation of the inflammatory response during ATP exposure.
authors.affiliation: Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland.|Finnish Institute of Occupational Health, 00250 Helsinki, Finland.
authors.forename: Elina
authors.initials: E
authors.lastname: Välimäki
authors.affiliation: Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland.
authors.forename: Wojciech
authors.initials: W
authors.lastname: Cypryk
authors.affiliation: Department of Geosciences and Geography, University of Helsinki, 00014 Helsinki, Finland.
authors.forename: Juhani
authors.initials: J
authors.lastname: Virkanen
authors.affiliation: Division of Rheumatology, Helsinki University Hospital, Helsinki University, 00015 Helsinki, Finland; and.
authors.forename: Katariina
authors.initials: K
authors.lastname: Nurmi
authors.affiliation: Department of Physiology, Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki, 00014 Helsinki, Finland.
authors.forename: Pauli M
authors.identifier: 0000-0002-3129-1678
authors.initials: PM
authors.lastname: Turunen
authors.affiliation: Division of Rheumatology, Helsinki University Hospital, Helsinki University, 00015 Helsinki, Finland; and.
authors.forename: Kari K
authors.initials: KK
authors.lastname: Eklund
authors.affiliation: Department of Physiology, Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki, 00014 Helsinki, Finland.
authors.forename: Karl E
authors.identifier: 0000-0003-4625-3081
authors.initials: KE
authors.lastname: Åkerman
authors.affiliation: Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland.
authors.forename: Tuula A
authors.initials: TA
authors.lastname: Nyman
authors.affiliation: Finnish Institute of Occupational Health, 00250 Helsinki, Finland; sampsa.matikainen@helsinki.fi.|Division of Rheumatology, Helsinki University Hospital, Helsinki University, 00015 Helsinki, Finland; and.
authors.forename: Sampsa
authors.initials: S
authors.lastname: Matikainen
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D006801:Humans; D058847:Inflammasomes / Q000276:immunology / Q000378:metabolism*; D008264:Macrophages / Q000276:immunology / Q000378:metabolism*
chemical_list: D058847:Inflammasomes; D000255:Adenosine Triphosphate; D002154:Calpain