Paola Signorelli

Cutting Edge: Scavenging of Inflammatory CC Chemokines by the Promiscuous Putatively Silent Chemokine Receptor D6

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.

Resveratrol induces growth inhibition and apoptosis in metastatic breast cancer cells via de novo ceramide signaling

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.

Dihydroceramide intracellular increase in response to resveratrol treatment mediates autophagy in gastric cancer cells

Resveratrol has both apoptosis and autophagy-promoting activities in different cancer cells. Dihydroceramide is the immediate precursor of the apoptotic mediator ceramide in the de novo sphingolipid synthesis pathway. Here we demonstrate that resveratrol induces autophagy in HGC-27 cells, with no sign of cell death. Autophagy occurs after an increase in dihydroceramides by inhibition of dihydroceramide desaturase. The effects of resveratrol are mimicked by a dihydroceramide desaturase inhibitor. These results demonstrate that resveratrol-induced autophagy occurs with a rise in intracellular dihydroceramide levels as the result of inhibition of dihydroceramide desaturases activity and that dihydroceramide accumulation is responsible for autophagy promotion.

The chemokine system: tuning and shaping by regulation of receptor expression and coupling in polarized responses.

Chemokines are a superfamily of small proteins with a crucial role in setup of immune and inflammatory reactions (1–4). At present, about 50 chemokines have been identified in humans. These molecules share a secondary structure with a flexible N-terminal segment followed by three antiparallel, b-sheets and a C-terminal a-helix, and according to the relative position of cysteine residues have been classified in four families (CXC, CC, C and CX3C). CC chemokines are the most numerous and diverse family, including at least 25 ligands and 11 signaling receptors in humans (3–5) (Table 1). CC chemokines have been discovered following different pathways, ranging from biological and biochemical identification to direct cDNA cloning to, more recently, in silico cloning by gene-bank mining. For instance, in the case of CCL2/ MCP-1, it had already been noted in the early 1970s that supernatants of activated blood mononuclear cells contained attractants active on monocytes and neutrophils. Subsequently, a chemotactic factor active on monocytes was identified in culture supernatants of mouse (6) and human (7) tumor lines and called tumor-derived chemotactic factor (TDCF) (7,8). TDCF was at the time rather unique in that it was active on monocytes but not on neutrophils (7) and had a low molecular weight (12 kDa) (7). Moreover, correlative evidence suggested its involvement in regulation of macrophage infiltration in murine and human tumors (7, 9). A molecule with similar cellular specificity and physicochemical properties was independently identified in the culture supernatant of smooth muscle cells (10). The JE gene has been identified as an immediate-early PDGF-inducible gene in fibroblasts (1l, 12). Thus, in the mid-1980s, this gene (JE) was in search for function and a monocyte-specific attractant was waiting for molecular definition. In 1989, CCL2/ MCP-1 was successfully purified from supernatants of a human glioma (13), a human monocytic leukemia (14), and a human sarcoma (15–17): sequencing and molecular cloning revealed its relationship with the long-known JE gene (18–20). Chemokines eponymous function is represented by induction of leukocyte migration, but these molecules also affect angiogenesis, collagen production and the proliferation of hematopoietic precursors. These M. Locati, K. Otero, T. Schioppa, P. Signorelli, P. Perrier, S. Baviera, S. Sozzani, A. Mantovani Centro di Eccellenza IDET, Institute of General Pathology, University of Milan, Milan; Istituto di Ricerche Farmacologiche Mario Negri, Milan; Department of Biomedical Sciences and Biotechnology, Section of General Pathology and Immunology, University of Brescia, I-25123 Brescia, Italy

Sphingomyelin synthases regulate production of diacylglycerol at the Golgi.

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.

Dihydroceramide desaturase and dihydrosphingolipids: debutant players in the sphingolipid arena.

Sphingolipids are a wide family of lipids that share common sphingoid backbones, including (2S,3R)-2-amino-4-octadecane-1,3-diol (dihydrosphingosine) and (2S,3R,4E)-2-amino-4-octadecene-1,3-diol (sphingosine). The metabolism and biological functions of sphingolipids derived from sphingosine have been the subject of many reviews. In contrast, dihydrosphingolipids have received poor attention, mainly due to their supposed lack of biological activity. However, the reported biological effects of active site directed dihydroceramide desaturase inhibitors and the involvement of dihydrosphingolipids in the response of cells to known therapeutic agents support that dihydrosphingolipids are not inert but are in fact biologically active and underscore the importance of elucidating further the metabolic pathways and cell signaling networks involved in the biological activities of dihydrosphingolipids. Dihydroceramide desaturase is the enzyme involved in the conversion of dihydroceramide into ceramide and it is crucial in the regulation of the balance between sphingolipids and dihydrosphingolipids. Furthermore, given the enzyme requirement for O₂ and the NAD(P)H cofactor, the cellular redox balance and dihydroceramide desaturase activity may reciprocally influence each other. In this review both dihydroceramide desaturase and the biological functions of dihydrosphingolipids are addressed and perspectives on this field are discussed.

Autophagy in term normal human placentas

Autophagy is an inducible catabolic process that responds to environment and is essential for cell survival during stress, starvation and hypoxia. Its function in the human placenta it is not yet understood. We collected 14 placentas: 7 at vaginal delivery and 7 at elective caesarean section after uneventful term pregnancies. The presence of autophagy was assessed in different placental areas by immunoblotting, immunohistochemistry and electron microscopy. We found that autophagy is significantly higher in placentas obtained from cesarean section than in those from vaginal delivery. Moreover there is a significant inverse relationship between autophagy and umbilical arterial glucose concentration.

Dihydroceramide delays cell cycle G1/S transition via activation of ER stress and induction of autophagy.

Dihydroceramides, the precursors of ceramides in the de novo sphingolipid synthesis, have been recently implicated in active signalling. We previously demonstrated that dihydroceramide accumulation, in response to treatment with the dihydroceramide desaturase inhibitor XM462, induced autophagy with no sign of cell death in the gastric carcinoma HCG27 cell line. Here we show that XM462 treatment induces a transient early increase in dihydroceramides that are successively metabolized into other sphingolipids. Dihydroceramides accumulation is associated with cyclin D1 expression modulation, delayed G1/S transition of cell cycle and increased autophagy. Moreover, XM462 treatment induces ER stress via the activation of the translation inhibitor eIF2α and the pro-survival transcriptional factor Xbp1. Exogenous addition of a short chain dihydroceramide analog reproduces the effects of endogenous accumulation of dihydroceramides, causing cell cycle delay of the G1/S transition, autophagy enhancement, eIF2α activation and Xbp1 splicing. Blocking autophagy with 3-methyladenine abrogates the effect of XM462 on cell cycle and reduces cell survival to XM462 treatment. Furthermore, the XM462-induced survival response is able to reduce etoposide toxicity in HCG27 and HCT116 cancer cells. Our data suggest a role of dihydroceramide in regulating cell proliferation and survival.

Ceramide inhibition of NF-κB activation involves reverse translocation of classical protein kinase C (PKC) isoenzymes: requirement for kinase activity and carboxyl-terminal phosphorylation of PKC for the ceramide response

Protein kinase C (PKC) is known to activate NF‐κB whereas the lipid mediator ceramide was recently shown to inhibit activation of this transcription factor (1, 2). In this study, the mechanisms by which ceramide interferes with this pathway were examined in Jurkat leukemia and MCF‐7 breast cancer cells. Both exogenous and endogenous ceramide inhibited selectively PKC‐mediated activation of NF‐κBby reverting PKC translocation to the membrane. Next, confocal and immunofluorescence studies were performed to evaluate the direct effects of ceramide on PKC. These studies showed that ceramide inhibited translocation of a green fluorescent protein (GFP)‐PKCβ2 fusion protein in response to PMA. A mutant PKC in which autophosphorylation sites were mutated to alanine (PKC‐DA) was resistant to ceramide. A kinase‐inactive mutant (PKC‐KR) was also resistant to ceramide action, and the results were supported using kinase inhibitors of the enzyme. Finally, overexpression of PKC‐DA prevented, at least partly, the ability of ceramide to inhibit activation of NF‐κB. Taken together, these studies show that ceramide has acute effects on translocation of PKC by inducing reverse translocation, and this reversal requires both the kinase activity of PKC and phosphorylation of the autophos‐phorylation sites.—Signorelli, P., Luberto, C., Hannun, Y. A. Ceramide inhibition of NF‐kB activation involves reverse translocation of classical protein kinase C (PKC) isoenzymes: requirement for kinase activity and carboxyl‐terminal phosphorylation of PKC for the ceramide response. FASEB J. 15, 2401–2414 (2001)

Role of Sphingolipids in the Pathobiology of Lung Inflammation

Sphingolipid bioactivities in the respiratory airways and the roles of the proteins that handle them have been extensively investigated. Gas or inhaled particles or microorganisms come into contact with mucus components, epithelial cells, blood barrier, and immune surveillance within the airways. Lung structure and functionality rely on a complex interplay of polar and hydrophobic structures forming the surfactant layer and governing external-internal exchanges, such as glycerol-phospholipids sphingolipids and proteins. Sphingolipids act as important signaling mediators involved in the control of cell survival and stress response, as well as secreted molecules endowed with inflammation-regulatory activities. Most successful respiratory infection and injuries evolve in the alveolar compartment, the critical lung functional unit involved in gas exchange. Sphingolipid altered metabolism in this compartment is closely related to inflammatory reaction and ceramide increase, in particular, favors the switch to pathological hyperinflammation. This short review explores a few mechanisms underlying sphingolipid involvement in the healthy lung (surfactant production and endothelial barrier maintenance) and in a selection of lung pathologies in which the impact of sphingolipid synthesis and metabolism is most apparent, such as acute lung injury, or chronic pathologies such as cystic fibrosis and chronic obstructive pulmonary disease.