ABSTRACT
Although TRAIL (tumor necrosis factor (TNF)-related apoptosis inducing ligand) is a well-known apoptosis inducer, we have previously demonstrated that acidic extracellular pH (pHe) switches TRAIL-induced apoptosis to regulated necrosis (or necroptosis) in human HT29 colon and HepG2 liver cancer cells. Here, we investigated the role of RIPK1 (receptor interacting protein kinase 1), RIPK3 and PARP-1 (poly (ADP-ribose) polymerase-1) in TRAIL-induced necroptosis in vitro and in concanavalin A (Con A)-induced murine hepatitis. Pretreatment of HT29 or HepG2 with pharmacological inhibitors of RIPK1 or PARP-1 (Nec-1 or PJ-34, respectively), or transient transfection with siRNAs against RIPK1 or RIPK3, inhibited both TRAIL-induced necroptosis and PARP-1-dependent intracellular ATP depletion demonstrating that RIPK1 and RIPK3 were involved upstream of PARP-1 activation and ATP depletion. In the mouse model of Con A-induced hepatitis, where death of mouse hepatocytes is dependent on TRAIL and NKT (Natural Killer T) cells, PARP-1 activity was positively correlated with liver injury and hepatitis was prevented both by Nec-1 or PJ-34. These data provide new insights into TRAIL-induced necroptosis with PARP-1 being active effector downstream of RIPK1/RIPK3 initiators and suggest that pharmacological inhibitors of RIPKs and PARP-1 could be new treatment options for immune-mediated hepatitis.
Subject(s)
Apoptosis/drug effects , Poly(ADP-ribose) Polymerases/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Adenosine Triphosphate/metabolism , Animals , Cell Line , Disease Models, Animal , HT29 Cells , Hep G2 Cells , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Hydrogen-Ion Concentration , Imidazoles/pharmacology , Indoles/pharmacology , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Mice , Phenanthrenes/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors , RNA Interference , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/antagonists & inhibitors , Receptor-Interacting Protein Serine-Threonine Kinases/geneticsABSTRACT
The molecular motor kinesin is an ATPase that mediates plus end-directed transport of organelles along microtubules. Although the biochemical properties of kinesin are extensively studied, conclusive data on regulation of kinesin-mediated transport are largely lacking. Previously, we showed that the proinflammatory cytokine tumor necrosis factor induces perinuclear clustering of mitochondria. Here, we show that tumor necrosis factor impairs kinesin motor activity and hyperphosphorylates kinesin light chain through activation of two putative kinesin light chain kinases. Inactivation of kinesin, hyperphosphorylation of kinesin light chain, and perinuclear clustering of mitochondria exhibit the same p38 mitogen-activated kinase dependence, indicating their functional relationship. These data provide evidence for direct regulation of kinesin-mediated organelle transport by extracellular stimuli via cytokine receptor signaling pathways.
Subject(s)
Kinesins/metabolism , Microtubule-Associated Proteins/metabolism , Mitochondria/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Animals , Cell Line , Enzyme Activation , Enzyme Inhibitors/pharmacology , Imidazoles/pharmacology , Microtubules/metabolism , Mitogen-Activated Protein Kinases/metabolism , Molecular Motor Proteins/metabolism , Phosphorylation , Pyridines/pharmacology , Receptors, Tumor Necrosis Factor/metabolism , Signal Transduction , p38 Mitogen-Activated Protein KinasesABSTRACT
It is well established that apoptosis is accompanied by activation of procaspases and by mitochondrial changes, such as decrease in mitochondrial transmembrane potential (DeltaPsim) and release of cytochrome c. We analyzed the causal relationship between activated caspases and these mitochondrial phenomena. Purified recombinant caspase-1, -11, -3, -6, -7, and -8 were incubated with mitochondria in the presence or absence of additional cellular components, after which DeltaPsim was determined. At lower caspase concentrations, only caspase-8 was able to activate a cytosolic factor, termed caspase-activated factor (CAF), which resulted in decrease in DeltaPsim and release of cytochrome c. Both CAF-mediated activities could not be blocked by protease inhibitors, including oligopeptide caspase inhibitors. CAF-induced cytochrome c release, but not decrease of DeltaPsim, was blocked in mitochondria from cells overexpressing Bcl-2. CAF is apparently involved in decrease of DeltaPsim and release of cytochrome c, whereas Bcl-2 only prevents the latter. Hence, CAF may form the link between death domain receptor-dependent activation of procaspase-8 and the mitochondrial events studied.
Subject(s)
Caspases/metabolism , Cytochrome c Group/metabolism , Mitochondria/physiology , Proteins/metabolism , T-Lymphocytes/physiology , T-Lymphocytes/ultrastructure , Animals , Caspase 8 , Caspase 9 , Cell Line , Intracellular Membranes/physiology , Membrane Potentials , Mice , Mitochondria/ultrastructureABSTRACT
Recent data show that a strong relation exists in certain cells between mitochondria and caspase activation in apoptosis. We further investigated this relation and tested whether treatment with the permeability transition (PT)-inducing agent atractyloside of Percoll-purified mitochondria released a caspase-processing activity. Following detection of procaspase-11 processing, we further purified this caspase-processing protease and identified it as cathepsin B. The purified cathepsin B, however, was found to be derived from lysosomes which were present as minor contaminants in the mitochondrial preparation. Besides procaspase-11, caspase-1 is also readily processed by cathepsin B. Procaspase-2, -6, -7, -14 are weak substrates and procaspase-3 is a very poor substrate, while procaspase-12 is no substrate at all for cathepsin B. In addition, cathepsin B induces nuclear apoptosis in digitonin-permeabilized cells as well as in isolated nuclei. All newly described activities of cathepsin B, namely processing of caspase zymogens and induction of nuclear apoptosis, are inhibited by the synthetic peptide caspase inhibitors z-VAD.fmk, z-DEVD.fmk and to a lesser extent by Ac-YVAD.cmk.
