Abstract
Actin cytoskeleton has been shown to play a regulating role in several signaling pathways, and disruption of actin filament has been reported to increase sTNF-α-induced cell death. However, whether actin is involved in tmTNF-α-mediated cytotoxicity remains unclear. Here, we demonstrated that pretreatment of HL-60 with CytD or LatA to depolymerize actin significantly suppressed tmTNF-α-mediated apoptosis. Interestingly, tmTNF-α increased the actin immunoprecipitated by anti-TNFR2 but not anti-TNFR1 antibody, and disruption of the actin filament totally blocked this effect. In addition, TNFR1 knockdown by siRNA did not affect tmTNF-α-mediated cytotoxicity and the inhibitory effect of CytD, suggesting that the involvement of actin in the tmTNF-α-induced apoptosis is linked to the TNFR2 pathway. Our results revealed further that tmTNF-α signaled the inhibition of IκB degradation and NF-κB activity by recruiting RIP1 to and uncoupling TRAF2 from the TNFR2 complex. Nevertheless, CytD totally reversed the tmTNF-α signaling and activated NF-κB by recruiting TRAF2 to and dissociating RIP1 from the TNFR2 complex. Furthermore, tmTNF-α led to activation of caspase-8 by dissociation of cFLIP from TNFR2 and inhibition of the cFLIP expression. Activated caspase-8 cleft RIP1 to suppress NF-κB activity and also mediated tmTNF-α-induced apoptosis. However, CytD blocked the tmTNF-α-induced uncoupling of cFLIP from TNFR2 and prevented caspase-8 activation and the resulting cleavage of RIP1, converting the signaling for tmTNF-α-mediated apoptosis into one for activating NF-κB to survive. These results suggest that the actin cytoskeleton functions in transmitting signals via TNFR2 to mediate tmTNF-α-induced apoptosis.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Actins / genetics
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Actins / metabolism*
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Animals
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Apoptosis / drug effects*
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Blotting, Western
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Bridged Bicyclo Compounds, Heterocyclic / pharmacology
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CASP8 and FADD-Like Apoptosis Regulating Protein / genetics
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CASP8 and FADD-Like Apoptosis Regulating Protein / metabolism
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Caspase 8 / genetics
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Caspase 8 / metabolism
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Cell Membrane / metabolism*
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Cytochalasin D / pharmacology
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Enzyme-Linked Immunosorbent Assay
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Flow Cytometry
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HL-60 Cells
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Humans
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I-kappa B Proteins / genetics
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I-kappa B Proteins / metabolism
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Immunoprecipitation
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Interleukin-8 / genetics
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Interleukin-8 / metabolism
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Mice
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NF-KappaB Inhibitor alpha
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NF-kappa B / genetics
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NF-kappa B / metabolism
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NIH 3T3 Cells
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Nuclear Pore Complex Proteins / genetics
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Nuclear Pore Complex Proteins / metabolism
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Nucleic Acid Synthesis Inhibitors / pharmacology
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RNA, Messenger / genetics
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RNA, Small Interfering / genetics
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / metabolism
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Receptors, Tumor Necrosis Factor, Type I / antagonists & inhibitors
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Receptors, Tumor Necrosis Factor, Type I / genetics
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Receptors, Tumor Necrosis Factor, Type I / metabolism*
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Receptors, Tumor Necrosis Factor, Type II / antagonists & inhibitors
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Receptors, Tumor Necrosis Factor, Type II / genetics
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Receptors, Tumor Necrosis Factor, Type II / metabolism*
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Reverse Transcriptase Polymerase Chain Reaction
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Signal Transduction / drug effects
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Thiazolidines / pharmacology
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Tumor Necrosis Factor-alpha / pharmacology*
Substances
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AGFG1 protein, human
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Actins
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Bridged Bicyclo Compounds, Heterocyclic
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CASP8 and FADD-Like Apoptosis Regulating Protein
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I-kappa B Proteins
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Interleukin-8
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NF-kappa B
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NFKBIA protein, human
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Nfkbia protein, mouse
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Nuclear Pore Complex Proteins
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Nucleic Acid Synthesis Inhibitors
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RNA, Messenger
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RNA, Small Interfering
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RNA-Binding Proteins
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Receptors, Tumor Necrosis Factor, Type I
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Receptors, Tumor Necrosis Factor, Type II
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Thiazolidines
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Tumor Necrosis Factor-alpha
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NF-KappaB Inhibitor alpha
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Cytochalasin D
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Caspase 8
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latrunculin A