A high throughput screen for neutral, magnesium-dependent sphingomyelinase (SMase) was performed. One inhibitor discovered in the screen, GW4869, functioned as a noncompetitive inhibitor of the enzyme in vitro with an IC 50 of 1 M. It did not inhibit acid SMase at up to at least 150 M. The compound was then evaluated for its ability to inhibit tumor necrosis factor (TNF)-induced activation of neutral SMase (N-SMase) in MCF7 cells. GW4869 (10 M) partially inhibited TNF-induced sphingomyelin (SM) hydrolysis, and 20 M of the compound was protected completely from the loss of SM. The addition of 10 -20 M GW4869 completely inhibited the initial accumulation of ceramide, whereas this effect was partially lost at later time points (24 h). These data therefore support the inhibitory action of GW4869 on N-SMase not only in vitro but also in a cellular model. The addition of GW4869 at both 10 and 20 M did not modify cellular glutathione levels in response to TNF, suggesting that the action of GW4869 occurred downstream of the drop in glutathione, which was shown previously to occur upstream of the activation of N-SMase. Further, whereas TNF treatment also caused a 75% increase of de novo synthesized ceramide after 20 h of incubation, GW4869, at either 10 or 20 M, had no effect on this pathway of ceramide generation. In addition, GW4869 did not significantly impair TNF-induced NF-B translocation to nuclei. Therefore, GW4869 does not interfere with other key TNF-mediated signaling effects. GW4869 was able, in a dose-dependent manner, to significantly protect from cell death as measured by nuclear condensation, caspase activation, PARP degradation, and trypan blue uptake. These protective effects were accompanied by significant inhibition of cytochrome c release from mitochondria and caspase 9 activation, therefore localizing N-SMase activation upstream of mitochondrial dysfunction. In conclusion, our results indicate that NSMase activation is a necessary step for the full development of the cytotoxic program induced by TNF.

No abstract

In an effort to define the actual function of the promiscuous putatively silent chemokine receptor D6, transfectants were generated in different cell types. Engagement of D6 by inflammatory CC chemokines elicited no calcium response nor chemotaxis, but resulted in efficient agonist internalization and degradation. Also in lymphatic endothelium, where this receptor is expressed in vivo, D6 did not elicit cellular responses other than ligand internalization and degradation. In particular, no evidence was obtained for D6-mediated transcytosis of chemokines in the apical-to-basal or basal-to-apical directions. These results indicate that D6 acts as an inflammatory chemokine scavenging nonactivatory decoy receptors and suggest that in lymphatic vessels D6 may function as a gatekeeper for inflammatory CC chemokines, by clearing them and preventing excessive diffusion via afferent lymphatics to lymph nodes.

Seven transmembrane receptors mediate diverse physiological responses including hormone action, olfaction, neurotransmission, and chemotaxis. Human D6 is a non-signaling seven-transmembrane receptor expressed on lymphatic endothelium interacting with most inflammatory CC-chemokines resulting in their rapid internalization. Here, we demonstrate that this scavenging activity is mediated by continuous internalization and constant surface expression of the receptor, a process involving the clathrin-coated pit-dependent pathway. D6 constitutively associates with the cytoplasmic adaptor ␤-arrestin, and this interaction is essential for D6 internalization. An acidic region, but not the putative phosphorylation sites in the cytoplasmic tail of D6, is critical for receptor interaction with ␤-arrestin and subsequent internalization. Neither the native D6 nor mutants uncoupled from ␤-arrestin activate any Gprotein-mediated signaling pathways. Therefore, D6 may be considered a decoy receptor structurally adapted to perform chemokine scavenging.Leukocyte recruitment into inflamed tissues is mediated by chemokines acting on a distinct subfamily of G-protein-coupled receptors (GPCR). 1 This includes 18 receptors with the ability to activate G ␣i -protein-dependent signaling events and cell migration (1-4). Two other chemokine binding molecules, with homology to chemokine receptors but defective in signaling function, called Duffy antigen and D6, have been identified (5-7) and recently classified as "silent" chemokine receptors (8). Evidence in in vitro models, gene-targeted mice, and individuals with erythrocyte-restricted deficiency suggests that the Duffy antigen silent receptor may facilitate chemokine transport across endothelial cells as well as acting as a chemokine buffering and scavenging system (5, 9 -12). D6 is highly expressed in endothelial cells lining afferent lymphatic vessels (13) and supports rapid internalization and degradation of inflammatory CC-chemokines, acting as a chemokine scavenger (7). D6 was suggested to act as a gatekeeper on afferent lymphatic endothelium, preventing excessive transfer to lymph nodes of inflammatory chemokines and disruptive leukocyte recruitment (7). The molecular mechanisms underlying this scavenging function are unknown.Upon agonist binding, most GPCRs activate a signaling cascade mediated by G-protein activation leading to receptor phosphorylation by a G-protein-coupled receptor kinase, which results in uncoupling of the receptor from G-proteins (14). This desensitization process is further facilitated by the association of the phosphorylated receptors with the cytoplasmic adaptor, ␤-arrestin (15). The receptor-␤-arrestin complex associates with clathrin and accessory proteins involved in the formation of clathrin-coated pits, ultimately leading to receptor internalization (16,17). While mediating the inhibition of G-protein functions, both G-protein-coupled receptor kinases and ␤-arrestins also activate G-protein-independent signaling events by direct association with a numb...

TSA cells engineered to release IL-12 are rejected by most mice; the ensuing immune memory for TSA parental cells, however, was less efficient than that elicited by proliferating TSA cells engineered to release other cytokines (e.g., IL-4, IL-10, and possibly interferon gamma). The immune reaction elicited by TSA-IL12 cells was the most efficient in curing mice with established TSA tumors; notably though, the same or a better cure rate was obtained with rIL-12 given intraperitoneally.

No abstract

Resveratrol (3,4',5-trans-trihydroxystilbene), a phytoalexin present in grapes and red wine, is emerging as a natural compound with potential anticancer properties. Here we show that resveratrol can induce growth inhibition and apoptosis in MDA-MB-231, a highly invasive and metastatic breast cancer cell line, in concomitance with a dramatic endogenous increase of growth inhibitory/proapoptotic ceramide. We found that accumulation of ceramide derives from both de novo ceramide synthesis and sphingomyelin hydrolysis. More specifically we demonstrated that ceramide accumulation induced by resveratrol can be traced to the activation of serine palmitoyltransferase (SPT), the key enzyme of de novo ceramide biosynthetic pathway, and neutral sphingomyelinase (nSMase), a main enzyme involved in the sphingomyelin/ceramide pathway. However, by using specific inhibitors of SPT, myriocin and L-cycloserine, and nSMase, gluthatione and manumycin, we found that only the SPT inhibitors could counteract the biological effects induced by resveratrol. Thus, resveratrol seems to exert its growth inhibitory/apoptotic effect on the metastatic breast cancer cell line MDA-MB-231 by activating the de novo ceramide synthesis pathway.

SMS [SM (sphingomyelin) synthase] is a class of enzymes that produces SM by transferring a phosphocholine moiety on to ceramide. PC (phosphatidylcholine) is believed to be the phosphocholine donor of the reaction with consequent production of DAG (diacylglycerol), an important bioactive lipid. In the present study, by modulating SMS1 and SMS2 expression, the role of these enzymes on the elusive regulation of DAG was investigated. Because we found that modulation of SMS1 or SMS2 did not affect total levels of endogenous DAG in resting cells, whereas they produce DAG in vitro, the possibility that SMSs could modulate subcellular pools of DAG, once acute activation of the enzymes is triggered, was investigated. Stimulation of SM synthesis was induced by either treatment with short-chain ceramide analogues or by increasing endogenous ceramide at the plasma membrane, and a fluorescently labelled conventional C1 domain [from PKC (protein kinase C)] enhanced in its DAG binding activity was used to probe subcellular pools of DAG in the cell. With this approach, we found, using confocal microscopy and subcellular fractionation, that modulation of SMS1 and, to a lesser extent, SMS2 affected the formation of DAG at the Golgi apparatus. Similarly, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein PKD (protein kinase D) to the Golgi. These results provide direct evidence that both enzymes are capable of regulating the formation of DAG in cells, that this pool of DAG is biologically active, and for the first time directly implicate SMS1 and SMS2 as regulators of DAG-binding proteins in the Golgi apparatus.