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Biomedical subjects

A Santoni

Publications and source records attributed to A Santoni.

At least 19 recordsLinked to original sources
PubMed2002

Signaling pathways activated by chemokine receptor CXCR2 and AMPA-type glutamate receptors and involvement in granule cells survival.

We show that treatment of cerebellar granules with interleukin-8 (IL-8), growth-related gene product beta (GRObeta) or AMPA induced activation of PI3-K/Akt and of ERK pathways, the latter being independent of PI3-K and dependent on PTX-sensitive G proteins. We also show that AMPA-mediated neuron survival was abolished both by ERK kinase inhibitor PD98059 and AMPA-Rs blocker CNQX, and that chemokine-mediated survival was blocked by the PI3-K inhibitors LY294002 and wortmannin. We conclude that the neurotrophic effects of AMPA need the contemporary activation of ERKs and stimulation of AMPA-Rs, and that PI3-K/Akt activation is a determinant pathway for the IL-8/GRObeta anti-apoptotic activity.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed2001

Ubiquitination and degradation of Syk and ZAP-70 protein tyrosine kinases in human NK cells upon CD16 engagement.

Syk and ZAP-70 nonreceptor protein tyrosine kinases (PTKs) are essential elements in several cascades coupling immune receptors to intracellular responses. The critical role of these kinases in promoting the propagation of intracellular signaling requires a tight regulation of their activity, thus the existence of a negative feedback loop regulating their expression can be hypothesized. Herein, we have investigated whether ubiquitin-dependent proteolysis could be a mechanism responsible for controlling the fate of Syk and ZAP-70 after their immunoreceptor-induced activation. We found that both Syk and ZAP-70 become ubiquitinated in response to aggregation of the low affinity Fc receptor for IgG (CD16) on human natural killer cells. We confirmed the identity of the major in vivo ubiquitinated kinase species by performing an in vitro ubiquitination assay. In addition, we found that after CD16 stimulation, ubiquitinated forms of Syk and ZAP-70 associate with the receptor complex. After CD16 engagement, we also observed a decrease in the stability of Syk and ZAP-70 PTKs that is counteracted by pretreatment with either proteasome or lysosomal inhibitors. Moreover, in the presence of the proteasome inhibitor, epoxomicin, we observed an accumulation of ubiquitinated forms of both kinases. Our findings provide evidence of ligand-induced ubiquitination of nonreceptor PTKs belonging to the Syk family and propose the ubiquitin-dependent proteasome-mediated degradation pathway as a mechanism for attenuating the propagation of intracellular signaling initiated by immune receptor engagement.

Cells, Cultured↗
PubMed2001

The adaptor protein shc is involved in the negative regulation of NK cell-mediated cytotoxicity.

The activation of protein tyrosine kinase(s) (PTK) is a critical event required for the development of NK cell-mediated cytotoxicity. Here we demonstrate that the adaptor protein shc undergoes tyrosine phosphorylation during the generation of antibody-dependent cellular cytotoxicity (ADCC) and natural killing. In addition, we report that, upon direct or antibody-dependent target cell interaction, shc coprecipitates with the Src homology 2 (SH2)-containing inositol phosphatase, SHIP. To gain information on the functional role of shc in NK cytotoxicity, we overexpressed wild-type or dominant negative shc constructs in the human NKL cell line. Our findings show a consistent shc-mediated down-regulation of ADCC and natural killing. Such functional effect correlates with a perturbation of the phosphoinositide (PI) metabolism by means of a shc-mediated negative regulation of inositol 1,4,5 triphosphate (IP3) generation and intracellular calcium flux upon CD16 ligation. Furthermore, our data show that dominant-negative shc-mediated perturbation of shc/SHIP interaction leads to inhibition of ligand-dependent SHIP recruitment to CD16 zeta chain. We suggest that shc plays a role of negative adaptor by modulating SHIP recruitment to activation receptors involved in the generation of NK cytotoxic function.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Natural killer cells and nitric oxide.

Natural killer (NK) cells and nitric oxide (NO) are both important components of the natural or innate immune response. NK cells are large granular lymphocytes capable of destroying cells infected by virus or bacteria and susceptible tumor cells without prior sensitization and restriction by MHC antigens. They are abundant in blood, spleen, liver and lungs and are distinct from both T and B lymphocytes in their circulation patterns, profile of surface antigens, receptor repertoire and the way in which they discriminate between self and non-self. Uniquely, NK cells express receptors that can recognize and discriminate between normal and altered MHC class I determinants. NK cell cytotoxic activity is strongly induced by cytokines such as IL-2 and IL-12, and this activation is associated with synthesis of NO. Inhibitors of NO synthesis impair NK cell-mediated target cell killing, demonstrating a role for NO in NK cell function. Furthermore, NO itself can regulate NK cell activation. In this article, evidence that NO is a mediator of NK cell-mediated target cell killing, and that NO is a regulator of NK cell activation will be reviewed. Results of NO synthase gene deletion studies will be discussed, and rodent and human NK cells will be compared.

Animals↗
PubMed2000

I-309 binds to and activates endothelial cell functions and acts as an angiogenic molecule in vivo.

Several chemokines have been shown to act as angiogenic molecules or to modulate the activity of growth factors such as fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor (VEGF). The detection of the CC chemokine receptor (CCR) 8 message in human umbilical vein endothelial cells (HUVECs) by reverse transcription- polymerase chain reaction (RT-PCR) and RNase protection assay (RPA), prompted us to investigate the potential role exerted by the CC chemokine I-309, a known ligand of such receptor, in both in vitro and in vivo angiogenesis assays. We show here that I-309 binds to endothelial cells, stimulates chemotaxis and invasion of these cells, and enhances HUVEC differentiation into capillary-like structures in an in vitro Matrigel assay. Furthermore, I-309 is an inducer of angiogenesis in vivo in both the rabbit cornea and the chick chorioallantoic membrane assay (CAM).

Allantois↗
PubMed2000

The chemokine growth-related gene product beta protects rat cerebellar granule cells from apoptotic cell death through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors.

Cultured cerebellar granule neurons are widely used as a cellular model to study mechanisms of neuronal cell death because they undergo programmed cell death when switched from a culture medium containing 25 mM to one containing 5 mM K(+). We have found that the growth-related gene product beta (GRObeta) partially prevents the K(+)-depletion-induced cell death, and that the neuroprotective action of GRObeta on granule cells is mediated through the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type of ionotropic glutamate receptors. GRObeta-induced survival was suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione, which is a specific antagonist of AMPA/kainate receptors; it was not affected by the inhibitor of N-methyl-D-aspartate receptors, 2-amino-5-phosphonopentanoic acid, and was comparable to the survival of granule cells induced by AMPA (10 microM) treatment. Moreover, GRObeta-induced neuroprotection was abolished when granule cells were treated with antisense oligonucleotides specific for the AMPA receptor subunits, which significantly reduced receptor expression, as verified by Western blot analysis with subunit-specific antibodies and by granule cell electrophysiological sensitivity to AMPA. Our data demonstrate that GRObeta is neurotrophic for cerebellar granule cells, and that this activity depends on AMPA receptors.

2-Amino-5-phosphonovalerate↗
PubMed2000

bcl-2 over-expression enhances NF-kappaB activity and induces mmp-9 transcription in human MCF7(ADR) breast-cancer cells.

bcl-2 expression is often associated with poor prognosis in several types of tumors; however, the role of this molecule in breast cancer is still controversial. We found earlier that over-expression of bcl-2 in a human breast-cancer cell line (MCF7(ADR)) enhances its tumorigenicity and metastatic potential by inducing metastasis-associated properties such as increased secretion of the matrix metalloproteinase-9 (mmp-9). In the present study, we investigated the effect of bcl-2 over-expression on the activity of the transcription factor NF-kappaB, an important regulator of genes involved in tumor progression and invasion. Transient transfection experiments indicate that over-expression of bcl-2 in the MCF7(ADR) cell line, enhances NF-kappaB-dependent transcriptional activity. Mobility-shift analysis revealed an increase of NF-kappaB DNA-binding in bcl-2-over-expressing clones that correlated with lower levels of the NF-kappaB cytoplasmic inhibitor IkappaBalpha. Moreover, point mutations of 2 highly conserved residues within the BH1 and BH2 domains that abrogate the interaction of bcl-2 with bax, or deletion of the N-terminal BH4 domain, completely eliminate the ability of this molecule to up-regulate NF-kappaB-dependent transactivation. Since mmp-9 is a NF-kappaB-regulated gene, we also investigated whether bcl-2 over-expression up-regulated mmp-9 transcription. We found that induction of mmp-9 mRNA correlates with the activation of an mmp-9-promoter-reporter-gene construct in transient transfection assay, and a mutation of the (-600)mmp-9-NF-kappaB binding element abolishes this effect. The overall data indicate that bcl-2-mediated regulation of NF-kappaB-transcription-factor activity may represent an important mechanism for the promotion of malignant behavior in MCF-7(ADR) cells.

Binding Sites↗
PubMed2000

Cutting edge: functional role for proline-rich tyrosine kinase 2 in NK cell-mediated natural cytotoxicity.

Protein tyrosine kinase activation is one of the first biochemical events in the signaling pathway leading to activation of NK cell cytolytic machinery. Here we investigated whether proline-rich tyrosine kinase 2 (Pyk2), the nonreceptor protein tyrosine kinase belonging to the focal adhesion kinase family, could play a role in NK cell-mediated cytotoxicity. Our results demonstrate that binding of NK cells to sensitive target cells or ligation of beta2 integrins results in a rapid induction of Pyk2 phosphorylation and activation. By contrast, no detectable Pyk2 tyrosine phosphorylation is found upon CD16 stimulation mediated by either mAb or interaction with Ab-coated P815 cells. A functional role for Pyk2 in natural but not Ab-mediated cytotoxicity was demonstrated by the use of recombinant vaccinia viruses encoding the kinase dead mutant of Pyk2. Finally, we provide evidence that Pyk2 is involved in the beta2 integrin-triggered extracellular signal-regulated kinase activation, supporting the hypothesis that Pyk2 plays a role in the natural cytotoxicity by controlling extracellular signal-regulated kinase activation.

Antibody-Dependent Cell Cytotoxicity↗
PubMed2000

Dichotomic effects of IFN-gamma on the development of systemic lupus erythematosus-like syndrome in MRL-lpr / lpr mice.

Systemic lupus erythematosus (SLE)-prone female MRL-lpr / lpr (MRL-lpr) mice were treated with mouse or rat IFN-gamma under different experimental conditions, both prophylactically in 6- to 8 week-old animals and therapeutically in 12- to 18-week-old SLE-affected mice. It was found that IFN-gamma heterogeneously modulated the course of the disease in MRL-lpr mice. When administered prophylactically, IFN-gamma favorably modulated the histological, serological and clinical signs of the disease. Relative to untreated or PBS-treated control animals, the MRL-lpr mice which received IFN gamma were virtually free of inflammatory infiltration of the kidneys and the lungs, had lower levels of azotemia with reduction of both circulating IgG1, IgG2a and IgG3 and anti-double strand (ds) and single strand (ss) DNA antibodies, milder skin vasculitis, significantly reduced enlargement of their lymph nodes and lower weight of the spleens. IFN-gamma also lowered the rate of mortality of MRL-lpr mice. In contrast to these findings, therapeutically administered IFN-gamma worsened the course of the disease in MRL-lpr mice, which exhibited increased proteinuria, higher levels of IgG2a and IgG3 and anti-ds and -ss DNA antibodies, more aggressive nephritis and died at an earlier age than PBS-treated control mice. The dichotomic effect of IFN-gamma on disease manifestation in MRL-lpr mice offers new insights into the complex role of this cytokine in the regulation of systemic autoimmunity such as SLE.

Animals↗
PubMed2000

CD69-triggered ERK activation and functions are negatively regulated by CD94 / NKG2-A inhibitory receptor.

CD69 represents a functional triggering molecule on activated NK and T cells, capable of inducing cytotoxic activity and costimulating cytokine production. It belongs to the C-lectin type superfamily, and its gene maps in the NK gene complex, close to other genes coding for NK receptors. CD94 / NKG2-A complex is the inhibitory receptor for the non classical MHC class I molecule HLA-E on human NK cells. To investigate CD69-initiated signal transduction pathways, and to evaluate CD94 / NKG2-A interference on CD69 triggering ability, we have generated transfectants expressing both receptors in the RBL cell line. Here we report that CD69 engagement leads to the activation of extracellular signal-regulated kinase (ERK) enzymes belonging to the MAPK family, and that this event is required for CD69-mediated cell degranulation. Moreover, we show that the co-engagement of CD94 / NKG2-A inhibitory receptor effectively suppresses both CD69-triggered cell degranulation in RBL transfectants, through the inhibition of ERK activation, and CD69-induced cytotoxicity in human NK cells. Thus, here we provide new information on the molecular mechanisms initiated by CD69 activation receptor, and show that CD69-initiated signaling pathways and functional activity are negatively regulated by CD94 / NKG2-A inhibitory complex.

Animals↗
PubMed2000

RAC1/P38 MAPK signaling pathway controls beta1 integrin-induced interleukin-8 production in human natural killer cells.

The MAP kinase (MAPK) p38 plays a key role in regulating inflammatory responses. Here, we demonstrate that beta1 integrin ligation on human NK cells results in the activation of the p38 MAPK signaling pathway, which is required for integrin-triggered IL-8 production. In addition, we identified some of the upstream events accompanying the beta1 integrin-mediated p38 MAPK activation, namely, the activation of the Rac guanine nucleotide exchange factor (GEF) p95 Vav, the small G protein Rac1, and the cytoplasmic kinases Pak1 and MKK3. Finally, we provide direct evidence that p95 Vav and Rac control the activation of p38 MAPK triggered by beta1 integrins.

Cells, Cultured↗
PubMed2000

SDF-1alpha-mediated modulation of synaptic transmission in rat cerebellum.

The functional expression of the seven-transmembrane domain G protein-coupled chemokine receptor CXCR-4/fusin in rat nerve cell was demonstrated by staining with a polyclonal anti-CXCR-4 Ab, and by evaluating the calcium responses to the physiological agonist stromal-derived cell factor-1alpha (SDF-1alpha) in both cerebellar granule cells in culture and Purkinje neurons (PNs) in cerebellar slices. Cerebellar glial, granule and Purkinje cells showed a pronounced staining for CXCR-4. Furthermore, cultured granule cells exhibited Ca2+ transients elicited by the application of SDF-1alpha, both in cell bodies and in neuronal processes. Whole-cell patch-clamped PNs in cerebellar slices responded to SDF-1alpha application by a slow inward current followed by an increase of both intracellular Ca2+ level and spontaneous synaptic activity. In particular, the SDF-1alpha-induced slow inward current was considerably reduced by ionotropic glutamate receptor blockers, but developed fully in a medium in which synaptic transmission was inhibited, indicating that this current might be, at least in part, mediated by extrasynaptic glutamate, possibly released from the surrounding glial and/or nerve cells. Taken together, these findings indicate a functional involvement of CXCR-4 in the modulation of synaptic transmission, adding another member to the repertoire of the chemokine receptors exerting a neuromodulatory role in the cerebellum.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1999

The growth-related gene product beta induces sphingomyelin hydrolysis and activation of c-Jun N-terminal kinase in rat cerebellar granule neurones.

The growth-related gene product beta (GRObeta) is a small chemoattractant cytokine that belongs to the CXC chemokine family, and GRObeta receptors are expressed in the brain, including the cerebellum. We demonstrate that rat cerebellar granule neurones express the GRObeta receptor CXCR2. We also show that, in addition to the known stimulation of a phosphoinositide-specific phospholipase C, GRObeta activates both neutral (N-) and acidic (A-) sphingomyelinases (SMase) and the stress-activated c-Jun N-terminal kinase 1 (JNK1). Although both exogenous ceramide and bacterial SMase stimulate JNK1, GRObeta-induced JNK1 activation is an event probably independent of ceramide generated by A-SMase, since it is maintained in the presence of compounds that block A-SMase activity. This is the first report on the activation of the SMase pathway by chemokines.

Animals↗
PubMed1999

Mu3 opiate receptor expression in lung and lung carcinoma: ligand binding and coupling to nitric oxide release.

The mu3 opiate receptor subtype is expressed in human surgical specimens of both normal lung and non-small-cell lung carcinoma. Nitric oxide (NO) release is mediated through the mu3 receptor, and in lung carcinoma, morphine-stimulated NO release is significantly higher and prolonged than in normal lung. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis we show that specific mu opioid receptor transcripts are present in lung carcinoma and other cells with the mu3 profile. Our findings identify a unique role for the mu3 opiate receptor in opiate-mediated NO release and suggest that endogenous opiates, through their release of NO, may play a role in cancer progression.

Dihydromorphine↗
PubMed1999

CD94/NKG2-A inhibitory complex blocks CD16-triggered Syk and extracellular regulated kinase activation, leading to cytotoxic function of human NK cells.

The CD94/NKG2-A complex is the inhibitory receptor for the nonclassical MHC class I molecule HLA-E on human NK cells. Here we studied the molecular mechanisms underlying the inhibitory activity of CD94/NKG2-A on NK cell functions by analyzing its interference on CD16-initiated signaling pathways involved in the control of cytolytic activity. Both tyrosine phosphorylation and activation of Syk kinase together with tyrosine phosphorylation of CD16 receptor zeta subunit are markedly inhibited by the coengagement of CD94/NKG2-A complex. As a downstream consequence, CD94/NKG2-A cross-linking impairs the CD16-induced activation of extracellular regulated kinases (ERKs), a pathway involved in NK cytotoxic function. The block of ERK activation is exerted at an early, PTK-dependent stage in the events leading to p21ras activation, as the CD16-induced tyrosine phosphorylation of Shc adaptor protein and the formation of Shc/Grb-2 complex are abrogated by CD94/NKG2-A simultaneous engagement. Our observations indicate that CD94/NKG2-A inhibits the CD16-triggered activation of two signaling pathways involved in the cytotoxic activity of NK cells. They thus provide molecular evidence to explain the inhibitory function of CD94/NKG2-A receptor on NK effector functions.

Animals↗
PubMed1999

Interleukin-2-activated rat natural killer cells express inducible nitric oxide synthase that contributes to cytotoxic function and interferon-gamma production.

Natural killer (NK) cells are large granular lymphocytes capable of destroying cells infected by virus or bacteria and susceptible tumor cells without prior sensitization and restriction by major histocompatability complex (MHC) antigens. Their cytotoxic activity could be strongly enhanced by interleukin-2 (IL-2). Previous findings, even if obtained with indirect experimental approaches, have suggested a possible involvement of the inducible nitric oxide (iNOS) pathway in the NK-mediated target cell killing. The aim of the present study was first to directly examine the induction of iNOS in IL-2-activated rat NK cells isolated from peripheral blood (PB-NK) or spleen (S-NK), and second to investigate the involvement of the iNOS-derived NO in the cytotoxic function of these cells. Our findings clearly indicate the induction of iNOS expression in IL-2-activated PB-NK and S-NK cells, as evaluated either at mRNA and protein levels. Accordingly, significantly high levels of iNOS activity were shown, as detected by the L-arginine to L-citrulline conversion in appropriate assay conditions. The consequent NO generation appears to partially account for NK cell-mediated DNA fragmentation and lysis of sensitive tumor target cells. In fact, functional inhibition of iNOS through specific inhibitors, as well as the almost complete abrogation of its expression through a specific iNOS mRNA oligodeoxynucleotide antisense, significantly reduced the lytic activity of IL-2-activated NK cells. Moreover, IL-2-induced interferon-gamma production appears also to be dependent, at least in part, on iNOS induction.

Animals↗
PubMed1999

Dexamethasone-induced thymocyte apoptosis: apoptotic signal involves the sequential activation of phosphoinositide-specific phospholipase C, acidic sphingomyelinase, and caspases.

Glucocorticoid hormones (GCH) have been implicated as regulators of T-lymphocyte growth and differentiation. In particular, it has been reported that GCH can induce thymocyte apoptosis. However, the molecular mechanisms responsible for this GCH-induced death have not been clarified. In this work, the biochemical events associated with apoptosis induced by Dexamethasone (Dex), a synthetic GCH, in normal mouse thymocytes, have been analyzed. Results indicate that Dex-induced thymocyte apoptosis is attributable to an early ceramide generation caused by the activation of an acidic sphingomyelinase (aSMase). Caspase activity plays a crucial role in Dex-induced apoptosis and is downstream the aSMase activation in that inhibition of the early ceramide generation inhibits caspase activation and thymocyte death. Moreover, Dex treatment rapidly induces diacylglycerol (DAG) generation, through a protein kinase C (PKC) and G-protein-dependent phosphatidylinositol-specific phospholipase C (PI-PLC), an event which precedes and is required for aSMase activation. Indeed, PI-PLC inhibition by U73122 totally prevents Dex-induced aSMase activity, ceramide generation, and consequently, caspase activation and apoptosis. All these effects require Dex interaction with GCH receptor (GR), are countered by the GR antagonist RU486, and precede the GCH/GR-activated transcription and protein synthesis. These observations indicate that GCH activates thymocyte death through a complex signaling pathway that requires the sequential activation of different biochemical events.

Animals↗