Steven J. Tucker

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

Tumour necrosis factor‐α (TNF‐α) 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‐κB (NF‐κB) by uncertain signalling mechanisms. Here we report the contribution of each TNFR towards the NF‐κB activation processes. In human cells expressing endogenous or exogenous TNFR2, in addition to TNFR1, we found both TNFRs capable of activating NF‐κB, as measured by IκBα (inhibitor of NF‐κB) degradation, electrophoretic mobility shift assay and NF‐κB gene reporter assays. TNFR2 activation did not degrade IκBβ. However, TNF‐effects on NF‐κB activation occurred predominantly through TNFR1, with TNFR2 activating the transcription factor poorly.

cAMP-dependent axon guidance is distinctly regulated by Epac and protein kinase A.

cAMP is a key mediator of a number of molecules that induce growth cone chemotaxis, including netrin-1 and myelin-associated glycoprotein (MAG). Endogenous neuronal cAMP levels decline during development, and concomitantly axonal growth cones switch their response to cAMP-dependent guidance cues from attraction to repulsion. The mechanisms by which cAMP regulates these polarized growth cone responses are unknown. We report that embryonic growth cone attraction to gradients of cAMP, netrin-1, or MAG is mediated by Epac. Conversely, the repulsion conferred by MAG or netrin-1 on adult growth cones is mediated by protein kinase A (PKA). Furthermore, fluorescence resonance energy transfer reveals that netrin-1 distinctly activates Epac in embryonic growth cones but PKA in postnatal neurons. Our results suggest that cAMP mediates growth cone attraction or repulsion by distinctly activating Epac or PKA, respectively. Moreover, we propose that the developmental switch in growth cone response to gradients of cAMP-dependent guidance cues from attraction to repulsion is the result of a switch from Epac- to PKA-mediated signaling pathways.

Pregnenolone-16α-carbonitrile inhibits rodent liver fibrogenesis via PXR (pregnane X receptor)-dependent and PXR-independent mechanisms

The effect of liver growth stimulation [using the rodent PXR (pregnane X receptor) activator PCN (pregnenolone-16alpha-carbonitrile)] in rats chronically treated with carbon tetrachloride to cause repeated hepatocyte necrosis and liver fibrogenesis was examined. PCN did not inhibit the hepatotoxicity of carbon tetrachloride. However, transdifferentiation of hepatic stellate cells and the extent of fibrosis caused by carbon tetrachloride treatment was significantly inhibited by PCN in vivo. In vitro, PCN directly inhibited hepatic stellate cell transdifferentiation to a profibrogenic phenotype, although the cells did not express the PXR (in contrast with hepatocytes), suggesting that PCN acts independently of the PXR. Mice with a functionally disrupted PXR gene (PXR-/-) did not respond to the antifibrogenic effects of PCN, in contrast with wild-type (PXR+/+) mice, demonstrating an antifibrogenic role for the PXR in vivo. However, PCN inhibited the transdifferentiation of PXR-/--derived mouse hepatic stellate cells in vitro, confirming that there is also a PXR-independent antifibrogenic effect of PCN through a direct interaction with hepatic stellate cells. These data suggest that the PXR is antifibrogenic in rodents in vivo and that a PXR-independent target for PXR activators exists in hepatic stellate cells that also functions to inhibit fibrosis.

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.

Elevated NF-κB responses and FLIP levels in leukemic but not normal lymphocytes: reduction by salicylate allows TNF-induced apoptosis

As its name suggests, tumor necrosis factor (TNF) is known to induce cytotoxicity in a wide variety of tumor cells and cell lines. However, its use as a chemotherapeutic drug has been limited by its deleterious side effects of systemic shock and widespread inflammatory responses. Some nonsteroidal antiinflammatory drugs, such as sodium salicylate, have been shown to have a chemopreventive role in certain forms of cancer. Here, we reveal that sodium salicylate selectively enhances the apoptotic effects of TNF in human erythroleukemia cells but does not affect primary human lymphocytes or monocytes. Sodium salicylate did not affect the intracellular distribution of TNF receptors (TNFRs) but stimulated cell surface TNFR2 shedding. Erythroleukemia cells were shown to possess markedly greater basal NF-κB responses and elevated Fas-associated protein with death domain-like IL-1converting enzyme (FLIP) levels. Sodium salicylate achieved its effects by reducing the elevated NF-κB responsiveness and FLIP levels and restoring the apoptotic response of TNF rather than the proliferative/proinflammatory effects of the cytokine in these cancer cells. Inhibition of NF-κB or FLIP levels in human erythroleukemia cells by pharmacological or molecular-biological means also resulted in switching the character of these cells from a TNF-responsive proliferative phenotype into an apoptotic one. These findings expose that the enhanced proliferative nature of human leukemia cells is caused by elevated NF-κB and FLIP responses and basal levels, reversible by sodium salicylate to allow greater apoptotic responsiveness of cytotoxic stimuli such as TNF. Such findings provide insight into the molecular mechanisms by which human leukemia cells can switch from a proliferative into an apoptotic phenotype.

Chemical synthesis and biological activities of 3-alkyl pyridinium polymeric analogues of marine toxins

Two new large poly-1,3-dodecylpyridinium salts, APS12 and APS12-2 of 12.5- and 14.7-kDa size, respectively, were synthesised and tested for their pore-forming and transfection capabilities in HEK 293 and undifferentiated mouse ES cells using patch-clamp recording, Ca2+ imaging and flow cytometry. Polymerisation reactions were enhanced by microwaves, and the product sizes were controlled by altering the irradiation time. This method can also be applied to obtain polymers with variable linking chains as shown by the preparation of poly-(1,3-octylpyridinium) salt of 11.9-kDa size. Molecular weights of the final products were determined using ESIMS analysis, which also indicated the products to be amongst the largest macro-cycles ever recorded, up to a 900-membered ring. Anti-bacterial, haemolytic and anti-acetylcholinesterase activities were also reported for the two dodecyl pyridinium polymers. These biological activities are characteristic to the structurally related marine toxin, poly-APS.

Expression of CYP2S1 in human hepatic stellate cells.

Activated stellate cells are myofibroblast‐like cells associated with the generation of fibrotic scaring in chronically damaged liver. Gene chip analysis was performed on cultured fibrotic stellate cells. Of the 51 human CYP genes known, 13 CYP and 5 CYP reduction‐related genes were detected with 4 CYPs (CYP1A1, CYP2E1, CY2S1 and CYP4F3) consistently present in stellate cells isolated from three individuals. Quantitative RT‐PCR indicated that CYP2S1 was a major expressed CYP mRNA transcript. The presence of a CYP2A‐related protein and testosterone metabolism in stellate cell cultures suggest that stellate cells express specific functional isoforms of CYP of which a major form is CYP2S1.

Unmodified calcium concentrations in tumour necrosis factor receptor subtype-mediated apoptotic cell death

Tumour necrosis factor-α (TNF) receptors mediate a variety of effects dependent on cell type. A role for Ca2+ in TNF-induced death remains uncertain. Here we investigated restricting intracellular/extracellular Ca2+ in HeLa epithelial carcinoma cells expressing low and high levels of p75TNFR receptor subtype and KYM-1 rhabdomyosarcoma cells, models of rapid TNF-induced apoptosis. Ca2+-chelators EGTA and BAPTA-AM as well as microsomal Ca2+-ATPase inhibitor thapsigargin, did not alter TNF-induced death. TNF was also unable to alter resting [Ca2+]i levels which remained < 200 nM even during times when these cells were undergoing apoptotic cell death. These findings indicate no role for modulated Ca2+ concentrations in TNF-induced apoptotic cell death.

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

The cAMP-producing agonist beraprost inhibits human vascular smooth muscle cell migration via exchange protein directly activated by cAMP

Aims During restenosis, vascular smooth muscle cells (VSMCs) migrate from the vascular media to the developing neointima. Preventing VSMC migration is therefore a therapeutic target for restenosis. Drugs, such as prostacyclin analogues, that increase the intracellular concentration of cyclic adenosine monophosphate (cAMP) can inhibit VSMC migration, but the mechanisms via which this occurs are unknown. Two main downstream mediators of cAMP are protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). This study has examined the effects of the prostacyclin analogue beraprost on VSMC migration and investigated the intracellular pathways involved. Methods and results In a chemotaxis chamber, human saphenous vein VSMC migrated towards a platelet-derived growth-factor-BB (PDGF) chemogradient. Incubation with therapeutically relevant concentrations of cAMP-producing agonist beraprost significantly decreased PDGF-induced migration. Direct activation of either PKA or Epac inhibited migration whereas inhibition of PKA did not prevent the anti-migratory effect of beraprost. Direct activation of Epac also prevented hyperplasia in ex vivo serum-treated human veins. Using fluorescence resonance energy transfer, we demonstrated that beraprost activated Epac but not PKA. The mechanisms of this Epac-mediated effect involved activation of Rap1 with subsequent inhibition of RhoA. Cytoskeletal rearrangement at the leading edge of the cell was consequently inhibited. Interestingly, Epac1 was localized to the leading edge of migrating VSMC. Conclusions These results indicate that therapeutically relevant concentrations of beraprost can inhibit VSMC migration via a previously unknown mechanism involving the cAMP mediator Epac. This may provide a novel target that could blunt neointimal formation.