title: Mechanisms of caspase-1 activation by P2X7 receptor-mediated K+ release.
abstract: The mechanisms underlying caspase-1 activation and IL-1beta processing during inflammatory activation of monocytes and macrophages are not well defined. Here, we describe an in vitro proteolytic processing assay that allows for comparison of caspase-1 regulatory components in a cell-free system separately from the confounding issue of IL-1beta secretion. Analysis of in vitro IL-1beta and caspase-1 processing in lysates from unstimulated Bac1 murine macrophages indicated a slow rate of basal caspase-1 activation and proteolytic maturation of IL-1beta. In contrast, brief (5 min) treatment of intact macrophages with extracellular ATP (as an activator of the P2X(7) receptor) or nigericin before cell lysis markedly accelerated the in vitro processing of caspase-1 and IL-1beta. This acceleration of in vitro processing was strictly dependent on loss of intracellular K(+) from the intact cells. The induction of in vitro caspase-1 activation by lysis per se or by K(+) loss before lysis was sensitive to pretreatment of intact macrophages with the tyrphostin AG-126 or bromoenol lactone, an inhibitor of Ca(2+)-independent phospholipase A(2). Caspase-1 activation and IL-1beta processing in lysates from unstimulated macrophages were also accelerated by addition of recombinant ASC, a previously identified adapter protein that directly associates with caspase-1. These data indicate that increased K(+) efflux via P2X(7) nucleotide receptor stimulation activates AG-126- and bromoenol lactone-sensitive signaling pathways in murine macrophages that result in stably maintained signals for caspase-1 regulation in cell-free assays.
authors.affiliation: Dept. of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
authors.forename: J Michelle
authors.initials: JM
authors.lastname: Kahlenberg
authors.forename: George R
authors.initials: GR
authors.lastname: Dubyak
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D051017:Apoptosis Regulatory Proteins; D053468:CARD Signaling Adaptor Proteins; D019556:COS Cells; D020170:Caspase 1 / Q000378:metabolism*; D002460:Cell Line; D002474:Cell-Free System; D002522:Chlorocebus aethiops; D003598:Cytoskeletal Proteins / Q000494:pharmacology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007375:Interleukin-1 / Q000378:metabolism; D007476:Ionophores / Q000494:pharmacology; D008264:Macrophages / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D009281:Naphthalenes / Q000494:pharmacology; D009550:Nigericin / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D011753:Pyrones / Q000494:pharmacology; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D011994:Recombinant Proteins / Q000494:pharmacology; D020032:Tyrphostins / Q000494:pharmacology
chemical_list: D051017:Apoptosis Regulatory Proteins; D053468:CARD Signaling Adaptor Proteins; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D007375:Interleukin-1; D007476:Ionophores; D009281:Naphthalenes; C546469:P2rx7 protein, mouse; D010726:Phosphodiesterase Inhibitors; C498534:Pycard protein, mouse; D011753:Pyrones; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D011994:Recombinant Proteins; D020032:Tyrphostins; C069028:AG 127; C068314:6-(bromomethylene)tetrahydro-3-(1-naphthaleneyl)-2H-pyran-2-one; D000255:Adenosine Triphosphate; D020170:Caspase 1; D009550:Nigericin; D011188:Potassium