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David J MacEwan

Publications and source records attributed to David J MacEwan.

8 recordsLinked to original sources
PubMed2003

Distinct regulation of cytosolic phospholipase A2 phosphorylation, translocation, proteolysis and activation by tumour necrosis factor-receptor subtypes.

The hormonally regulated Ca(2+)-dependent enzyme, cytosolic phospholipase A(2) (cPLA(2)) is activated by a range of inflammatory stimuli. Tumour necrosis factor-alpha (TNF) is one of the first known stimuli for cPLA(2) but it is not known whether both TNF receptor subtypes are involved in activating the lipase. In the present study, we show for the first time that both type I 55 kDa TNFR (TNFR1) and type II 75 kDa TNFR (TNFR2) stimulate cPLA(2) enzyme, but with distinct signalling mechanisms. TNFR1 activates mitogen-activated protein kinase (MAPK) and p38MAPK. TNFR1 then phosphorylates and activates cPLA(2) in a MAPK-dependent fashion. Furthermore, TNFR1 causes the translocation and caspase-dependent proteolysis of cPLA(2) as part of its activation profile. TNFR2, on the other hand, does not cause the phosphorylation of cPLA(2) as it does not activate MAPK or p38MAPK, but instead activates cPLA(2) by causing its translocation to plasma membrane and perinuclear subcellular regions. TNFR2 activation causes a delayed, slight increase in [Ca(2+)](i) of <50 nM that may contribute towards the translocation and activation of cPLA(2). Therefore both TNF receptor subtypes play a role in cPLA(2) activation, but by means of separate signal-transduction pathways.

Antigens, CD↗
PubMed2003

The influence of alkyl pyridinium sponge toxins on membrane properties, cytotoxicity, transfection and protein expression in mammalian cells.

The ability of two alkyl pyridinium sponge toxin preparations (poly-APS and halitoxin) to form transient pores/lesions in cell membranes and allow transfection of plasmid cDNA have been investigated using HEK 293 cells. Poly-APS and halitoxin preparations caused a collapse in membrane potential, reductions in input resistance and increased Ca2+ permeability. At least partial recovery was observed after poly-APS application but recovery was more rarely seen with halitoxin. The transfection with plasmid cDNAs for an enhanced green fluorescent protein (EGFP) and human tumour necrosis factor receptor 2 (TNFR2) was assessed for both toxin preparations and compared with lipofectamine. Stable transfection was achieved with poly-APS although it was less efficient than lipofectamine. These results show that viable cells transfected with alien cDNA can be obtained using novel transient pore-forming alkyl pyridinium sponge toxins and a simple pre-incubation protocol. This provides the first proof of principle that pore-forming alkyl pyridinium compounds can be used to deliver cDNA to the intracellular environment without permanently compromising the plasma membrane.

Animals↗
PubMed2003

Modulation by caspases of tumor necrosis factor-stimulated c-Jun N-terminal kinase activation but not nuclear factor-kappaB signaling.

Tumour necrosis factor-alpha (TNF) is capable of activating many downstream signaling molecules via its two receptors TNFR1 and TNFR2. TNF can stimulate the proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) as well as the stress induced kinase c-Jun N-terminal kinase (JNK) through mechanisms that are not fully delineated. NF-kappaB becomes activated mainly through TNFR1 while JNK can be stimulated by either TNF receptor subtype. TNF can also induce apoptosis within cells due to its ability to recruit procaspase-8 to TNFR1, which in turn induces the caspase proteolytic cascade. We provide evidence here in human cells, that TNF-induced JNK activation is under the influence of caspases while NF-kappaB activity is not. By using pharmacological inhibitors of caspases, we have shown that JNK activity is reduced following caspase inhibition, especially when caspase-3 is targeted. NF-kappaB activity, as assessed by IkappaBalpha or IkappaBbeta degradation, electrophoretic mobility shift assay and NF-kappaB gene reporter assays, is shown to be unaffected by caspase inhibition. Therefore, downstream TNF receptor signaling events are differentially influenced by caspases.

Antigens, CD↗
PubMed2003

Tumor necrosis factor-alpha-induced activation of RhoA in airway smooth muscle cells: role in the Ca2+ sensitization of myosin light chain20 phosphorylation.

Tumor necrosis factor-alpha (TNF), an inflammatory cytokine, has a potentially important role in the pathogenesis of bronchial asthma and may contribute to airway hyper-responsiveness. Recent evidence has revealed that TNF can increase the Ca(2+) sensitivity of agonist-stimulated myosin light chain(20) (MLC(20)) phosphorylation and contractility in guinea pig airway smooth muscle (ASM). In the present study, the potential intracellular pathways responsible for this TNF-induced Ca(2+) sensitization were investigated. In permeabilized cultured guinea pig ASM cells, recombinant human TNF stimulated an increase in Ca(2+)-activated MLC(20) phosphorylation under Ca(2+) "clamp" conditions. This increased MLC(20) phosphorylation was inhibited by preincubation with the Rho-kinase inhibitor Y27632. TNF also increased the proportion of GTP-bound RhoA, as measured using rhotekin Rho-binding domain, in a time course compatible with a role in the TNF-induced Ca(2+) sensitization. In cultured human ASM cells, recombinant human TNF also activated RhoA with a similar time course. In addition, TNF stimulated phosphorylation of the regulatory subunit of the myosin phosphatase, which was inhibited by Y27632. Although human ASM cells expressed both receptor subtypes, TNF-R1 and TNF-R2, the activation of RhoA was predominantly via stimulation of the TNF-R1, although RhoA did not immunoprecipitate with the TNF-R1. In conclusion, the TNF-induced increase in the Ca(2+) sensitivity of MLC(20) phosphorylation is through stimulation of the TNF-R1 receptor and via a RhoA/Rho-kinase pathway leading to inhibition of the myosin light chain phosphatase. This intracellular mechanism may contribute to TNF-induced airway hyper-responsiveness.

Animals↗
PubMed2002

Tumour necrosis factor-induced activation of c-Jun N-terminal kinase is sensitive to caspase-dependent modulation while activation of mitogen-activated protein kinase (MAPK) or p38 MAPK is not.

The activation of the extracellular signal-regulated kinases (ERKs) by tumour necrosis factor-alpha (TNF) receptors (TNFRs) is an integral part of the cytokine's pleiotropic cellular responses. Here we report differences in the caspase sensitivity and TNFR subtype activation of members of the ERK family. Inhibition in HeLa cells of caspase function by pharmacological inhibitors or the expression of CrmA (cytokine response modifier A), a viral modifier protein, blocks TNF-induced apoptosis or caspase-dependent protein kinase Cdelta and poly(ADP-ribose) polymerase protein degradation. TNFR1- or TNFR2-stimulated c-Jun N-terminal kinase (JNK) activity was attenuated in cells in which caspase activity was inhibited either by pharmacological blockers or CrmA expression. Both TNFR1- and TNFR2-stimulated JNK activity was caspase-sensitive; however, only TNFR1 was capable of stimulating p42/44 mitogen-activated protein kinase (MAPK) and p38 MAPK activities. TNFR1-stimulated p42/44 MAPK and p38 MAPK activities were insensitive to pharmacological caspase inhibition or CrmA. These findings were supported when measuring TNF-induced cytosolic phospholipase A(2) activation, which is a downstream target for MAPK and p38 MAPK. Profiling caspase enzymes activated by TNF in HeLa cells showed sequential caspase-8, -3, -7, -6 and -9 activation, with their inhibition characteristics suggesting a role for caspase-3 and/or caspase-6 in modulating JNK activity. Taken together these results show delineated ERK-activation pathways employed by TNFR subtypes.

Anisomycin↗
PubMed2002

Differential activation of nuclear factor-kappaB by tumour necrosis factor receptor subtypes. TNFR1 predominates whereas TNFR2 activates transcription poorly.

Tumour necrosis factor-alpha (TNF-alpha) signals though two receptors, TNFR1 and TNFR2. TNFR1 has a role in cytotoxicity, whereas TNFR2 regulates death responses or proliferation. TNF activates pro-inflammatory transcription factor nuclear factor-kappaB (NF-kappaB) by uncertain signalling mechanisms. Here we report the contribution of each TNFR towards the NF-kappaB activation processes. In human cells expressing endogenous or exogenous TNFR2, in addition to TNFR1, we found both TNFRs capable of activating NF-kappaB, as measured by IkappaBalpha (inhibitor of NF-kappaB) degradation, electrophoretic mobility shift assay and NF-kappaB gene reporter assays. TNFR2 activation did not degrade IkappaBbeta. However, TNF-effects on NF-kappaB activation occurred predominantly through TNFR1, with TNFR2 activating the transcription factor poorly.

Antigens, CD↗
PubMed2002

TNF receptor subtype signalling: differences and cellular consequences.

Tumour necrosis factor-alpha (TNF alpha) is a multifunctional cytokine belonging to a family of ligands with an associated family of receptor proteins. The pleiotropic actions of TNF range from proliferative responses such as cell growth and differentiation, to inflammatory effects and the mediation of immune responses, to destructive cellular outcomes such as apoptotic and necrotic cell death mechanisms. Activated TNF receptors mediate the association of distinct adaptor proteins that regulate a variety of signalling processes including kinase or phosphatase activation, lipase stimulation, and protease induction. Moreover, the cytokine regulates the activities of transcription factors, heterotrimeric or monomeric G-proteins and calcium ion homeostasis in order to orchestrate its cellular functions. This review addresses the structural basis of TNF signalling, the pathways employed with their cellular consequences, and focuses on the specific role played by each of the two TNF receptor isotypes, TNFR1 and TNFR2.

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