Rosa Molfetta

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.

CIN85 regulates the ligand-dependent endocytosis of the IgE receptor: A new molecular mechanism to dampen mast cell function

Ligation of the high-affinity receptor for IgE (FcεRI), constitutively expressed on mast cells and basophils, promotes cell activation and immediate release of allergic mediators. Furthermore, FcεRI up-regulation on APC from atopic donors is involved in the pathophysiology of allergic diseases. In consideration of the clinical relevance of the IgE receptor, the down-modulation of FcεRI expression in mast cells may represent a potential target for handling atopic diseases. In an effort to identify new molecular mechanisms involved in attenuating FcεRI expression and signaling, we focused our attention on CIN85, a scaffold molecule that regulates, in concert with the ubiquitin ligase Cbl, the clathrin-mediated endocytosis of several receptor tyrosine kinases. In the present study, we show that endogenous CIN85 is recruited in Cbl-containing complexes after engagement of the FcεRI on a mast cell line and drives ligand-induced receptor internalization. By confocal microscopic analysis, we provide evidence that CIN85 directs a more rapid receptor sorting in early endosomes and delivery to a lysosomal compartment. Furthermore, biochemical studies indicate that CIN85 plays a role in reducing the expression of receptor complex. Finally, we demonstrate that CIN85-overexpressing mast cells are dramatically impaired in their ability to degranulate following Ag stimulation, suggesting that the accelerated internalization of activated receptors by perturbing the propagation of FcεRI signaling may contribute to dampen the functional response. This role of CIN85 could be extended to include other multimeric immune receptors, such as the T and B cell receptors, providing a more general molecular mechanism for attenuating immune responses.

Lipid raft-dependent FcεRI ubiquitination regulates receptor endocytosis through the action of ubiquitin binding adaptors

The best characterized role for ubiquitination of membrane receptors is to negatively regulate signaling by targeting receptors for lysosomal degradation. The high affinity receptor for IgE (FcεRI) expressed on mast cells and basophils is rapidly ubiquitinated upon antigen stimulation. However, the nature and the role of this covalent modification are still largelly unknown. Here, we show that FcεRI subunits are preferentially ubiquitinated at multiple sites upon stimulation, and provide evidence for a role of ubiquitin as an internalization signal: under conditions of impaired receptor ubiquitination a decrease of receptor entry is observed by FACS analysis and fluorescence microscopy. We also used biochemical approaches combined with fluorescence microscopy, to demonstrate that receptor endocytosis requires the integrity of specific membrane domains, namely lipid rafts. Additionally, by RNA interference we demonstrate the involvement of ubiquitin-binding endocytic adaptors in FcεRI internalization and sorting. Notably, the triple depletion of Eps15, Eps15R and Epsin1 negatively affects the early steps of Ag-induced receptor endocytosis, whereas Hrs depletion retains ubiquitinated receptors into early endosomes and partially prevents their sorting into lysosomes for degradation. Our results are compatible with a scenario in which the accumulation of engaged receptor subunits into lipid rafts is required for receptor ubiquitination, a prerequisite for efficient receptor internalization, sorting and delivery to a lysosomal compartment.

NGF-dependent and tissue-specific transcription of vgf is regulated by a CREB–p300 and bHLH factor interaction

Neurotrophins support neuronal survival, development, and plasticity through processes requiring gene expression. We studied how vgf target gene transcription is mediated by a critical promoter region containing E‐box, CCAAT and cAMP response element (CRE) sites. The p300 acetylase was present in two distinct protein complexes bound to this region. One complex, containing HEB (ubiquitous basic helix–loop–helix (bHLH)), bound the promoter in non‐neuronal cells and was involved in repressing vgf expression. Neurotrophin‐dependent transcription was mediated by the second complex, specific for neuronal cells, which included CRE binding protein and MASH1 (neuro‐specific bHLH), bound the CCAAT motif, and was target of neurotrophin signalling. The interaction, mediated by p300, of different transcription factors may add specificity to the neurotrophin response.

Nitric oxide donors increase PVR/CD155 DNAM-1 ligand expression in multiple myeloma cells: role of DNA damage response activation

BackgroundDNAX accessory molecule-1 (DNAM-1) is an activating receptor constitutively expressed by macrophages/dendritic cells and by T lymphocytes and Natural Killer (NK) cells, having an important role in anticancer responses; in this regard, combination therapies able to enhance the expression of DNAM-1 ligands on tumor cells are of therapeutic interest. In this study, we investigated the effect of different nitric oxide (NO) donors on the expression of the DNAM-1 ligand Poliovirus Receptor/CD155 (PVR/CD155) in multiple myeloma (MM) cells.MethodsSix MM cell lines, SKO-007(J3), U266, OPM-2, RPMI-8226, ARK and LP1 were used to investigate the activity of different nitric oxide donors [DETA-NO and the NO-releasing prodrugs NCX4040 (NO-aspirin) and JS-K] on the expression of PVR/CD155, using Flow Cytometry and Real-Time PCR. Western-blot and specific inhibitors were employed to investigate the role of soluble guanylyl cyclase/cGMP and activation of the DNA damage response (DDR).ResultsOur results indicate that increased levels of nitric oxide can upregulate PVR/CD155 cell surface and mRNA expression in MM cells; in addition, exposure to nitric oxide donors renders myeloma cells more efficient to activate NK cell degranulation and enhances their ability to trigger NK cell-mediated cytotoxicity. We found that activation of the soluble guanylyl cyclase and increased cGMP concentrations by nitric oxide is not involved in the up-regulation of ligand expression. On the contrary, treatment of MM cells with nitric oxide donors correlated with the activation of a DNA damage response pathway and inhibition of the ATM /ATR/Chk1/2 kinase activities by specific inhibitors significantly abrogates up-regulation.ConclusionsThe present study provides evidence that regulation of the PVR/CD155 DNAM-1 ligand expression by nitric oxide may represent an additional immune-mediated mechanism and supports the anti-myeloma activity of nitric oxide donors.

c-Cbl regulates MICA- but not ULBP2-induced NKG2D down-modulation in human NK cells.

The NKG2D activating receptor on human NK cells mediates “altered self” recognition, as its ligands (NKG2DLs) are upregulated on target cells in a variety of stress conditions. Evidence collected in the past years shows that, even though expression of NKG2DLs acts as a danger signal that renders tumor cells susceptible to cytotoxicity, chronic exposure to soluble or membrane‐bound NKG2DLs can lead to down‐modulation of receptor expression and impairment of NKG2D‐mediated cell functions. Here, we evaluated whether different cell‐bound NKG2DLs, namely MICA and ULBP2, are equivalently able to induce NKG2D down‐modulation on human NK cells. We found that although both ligands reduce NKG2D surface expression, MICA promotes a stronger receptor down‐modulation than ULBP2, leading to a severe impairment of NKG2D‐dependent NK‐cell cytotoxicity. We also provide evidence that the ubiquitin pathway and c‐Cbl direct MICA‐induced but not ULBP2‐induced NKG2D internalization and degradation, thus identifying a molecular mechanism to explain the differential effects of MICA and ULBP2 on NKG2D expression. A better understanding of the molecular mechanisms employed by the different NKG2DLs to control NKG2D surface expression could be useful for the development of anti‐tumor strategies to restore a normal level of NKG2D receptors on human NK cells.

The Adaptor Molecule CIN85 Regulates Syk Tyrosine Kinase Level by Activating the Ubiquitin-Proteasome Degradation Pathway

Triggering of mast cells and basophils by IgE and Ag initiates a cascade of biochemical events that lead to cell degranulation and the release of allergic mediators. Receptor aggregation also induces a series of biochemical events capable of limiting FcεRI-triggered signals and functional responses. Relevant to this, we have recently demonstrated that Cbl-interacting 85-kDa protein (CIN85), a multiadaptor protein mainly involved in the process of endocytosis and vesicle trafficking, regulates the Ag-dependent endocytosis of the IgE receptor, with consequent impairment of FcεRI-mediated cell degranulation. The purpose of this study was to further investigate whether CIN85 could alter the FcεRI-mediated signaling by affecting the activity and/or expression of molecules directly implicated in signal propagation. We found that CIN85 overexpression inhibits the FcεRI-induced tyrosine phosphorylation of phospholipase Cγ, thus altering calcium mobilization. This functional defect is associated with a substantial decrease of Syk protein levels, which are restored by the use of selective proteasome inhibitors, and it is mainly due to the action of the ubiquitin ligase c-Cbl. Furthermore, coimmunoprecipitation experiments demonstrate that CIN85 overexpression limits the ability of Cbl to bind suppressor of TCR signaling 1 (Sts1), a negative regulator of Cbl functions, while CIN85 knockdown favors the formation of Cbl/Sts1 complexes. Altogether, our findings support a new role for CIN85 in regulating Syk protein levels in RBL-2H3 cells through the activation of the ubiquitin-proteasome pathway and provide a mechanism for this regulation involving c-Cbl ligase activity.

Ubiquitin-dependent endocytosis of NKG2D-DAP10 receptor complexes activates signaling and functions in human NK cells

Signaling by an internalized natural killer cell receptor is required for the secretion of cytolytic granules and interferon-γ. Turning on natural killer cells Ligands present on the surface of tumor cells stimulate and activate natural killer (NK) cells of the innate immune system, which then trigger tumor cell death. Quatrini et al. found that the ligand-dependent internalization of a complex containing the stimulatory receptor NKG2D and the adaptor protein DAP10 depended on DAP10 ubiquitylation. Internalized receptor complexes in endosomes stimulated signaling by the kinase ERK, which was required for the secretion of cytolytic granules. Mutation of DAP10 to prevent its ubiquitylation resulted in decreased receptor internalization and defective cytotoxic responses, indicating that internalized receptors stimulate NK cell functions. Cytotoxic lymphocytes share the presence of the activating receptor NK receptor group 2, member D (NKG2D) and the signaling-competent adaptor DNAX-activating protein 10 (DAP10), which together play an important role in antitumor immune surveillance. Ligand stimulation induces the internalization of NKG2D-DAP10 complexes and their delivery to lysosomes for degradation. In experiments with human NK cells and cell lines, we found that the ligand-induced endocytosis of NKG2D-DAP10 depended on the ubiquitylation of DAP10, which was also required for degradation of the internalized complexes. Moreover, through combined biochemical and microscopic analyses, we showed that ubiquitin-dependent receptor endocytosis was required for the activation of extracellular signal–regulated kinase (ERK) and NK cell functions, such as the secretion of cytotoxic granules and the inflammatory cytokine interferon-γ. These results suggest that NKG2D-DAP10 endocytosis represents a means to decrease cell surface receptor abundance, as well as to control signaling outcome in cytotoxic lymphocytes.

Genotoxic Stress Induces Senescence-Associated ADAM10-Dependent Release of NKG2D MIC Ligands in Multiple Myeloma Cells

Genotoxic stress can promote antitumor NK cell responses by upregulating the surface expression of activating ligands on cancer cells. Moreover, a number of studies suggested a role for soluble NK group 2D ligands in the impairment of NK cell tumor recognition and killing. We investigated whether genotoxic stress could promote the release of NK group 2D ligands (MHC class I–related chain [MIC]A and MICB), as well as the molecular mechanisms underlying this event in human multiple myeloma (MM) cells. Our results show that genotoxic agents used in the therapy of MM (i.e., doxorubicin and melphalan) selectively affect the shedding of MIC molecules that are sensitive to proteolytic cleavage, whereas the release of the short MICA*008 allele, which is frequent in the white population, is not perturbed. In addition, we found that a disintegrin and metalloproteinase 10 expression is upregulated upon chemotherapeutic treatment both in patient-derived CD138+/CD38+ plasma cells and in several MM cell lines, and we demonstrate a crucial role for this sheddase in the proteolytic cleavage of MIC by means of silencing and pharmacological inhibition. Interestingly, the drug-induced upregulation of a disintegrin and metalloproteinase 10 on MM cells is associated with a senescent phenotype and requires generation of reactive oxygen species. Moreover, the combined use of chemotherapeutic drugs and metalloproteinase inhibitors enhances NK cell–mediated recognition of MM cells, preserving MIC molecules on the cell surface and suggesting that targeting of metalloproteinases in conjunction with chemotherapy could be exploited for NK cell–based immunotherapeutic approaches, thus contributing to avoid the escape of malignant cells from stress-elicited immune responses.

Anti-CD20 Therapy Acts via FcγRIIIA to Diminish Responsiveness of Human Natural Killer Cells

Natural killer (NK) immune cells mediate antibody-dependent cellular cytotoxicity (ADCC) by aggregating FcγRIIIA/CD16, contributing significantly to the therapeutic effect of CD20 monoclonal antibodies (mAb). In this study, we show that CD16 ligation on primary human NK cells by the anti-CD20 mAb rituximab or ofatumumab stably impairs the spontaneous cytotoxic response attributable to cross-tolerance of several unrelated NK-activating receptors (including NKG2D, DNAM-1, NKp46, and 2B4). Similar effects were obtained from NK cells isolated from patients with chronic lymphocytic leukemia in an autologous setting. NK cells rendered hyporesponsive in this manner were deficient in the ability of these cross-tolerized receptors to phosphorylate effector signaling molecules critical for NK cytotoxicity, including SLP-76, PLCγ2, and Vav1. These effects were associated with long-lasting recruitment of the tyrosine phosphatase SHP-1 to the CD16 receptor complex. Notably, pharmacologic inhibition of SHP-1 with sodium stibogluconate counteracted CD20 mAb-induced NK hyporesponsiveness, unveiling an unrecognized role for CD16 as a bifunctional receptor capable of engendering long-lasting NK cell inhibitory signals. Our work defines a novel mechanism of immune exhaustion induced by CD20 mAb in human NK cells, with potentially negative implications in CD20 mAb-treated patients where NK cells are partly responsible for clinical efficacy.