Mario Piccoli

RAC1/P38 MAPK Signaling Pathway Controls β1 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.

Negative Regulation of CD95 Ligand Gene Expression by Vitamin D3 in T Lymphocytes

Fas (APO-1/CD95) and its ligand (FasL/CD95L) are cell surface proteins whose interaction activates apoptosis of Fas-expressing targets. In T lymphocytes, the Fas/FasL system regulates activation-induced cell death, a fundamental mechanism for negative selection of immature T cells in the thymus and for maintenance of peripheral tolerance. Aberrant expression of Fas and FasL has also been implicated in diseases in which the lymphocyte homeostasis is compromised, and several studies have described the pathogenic functions of Fas and FasL in vivo, particularly in the induction/regulation of organ-specific autoimmune diseases. The 1,25(OH)2D3 is a secosteroid hormone that activates the nuclear receptor vitamin D3 receptor (VDR), whose immunosuppressive activities have been well studied in different models of autoimmune disease and in experimental organ transplantation. We and others have recently described the molecular mechanisms responsible for the negative regulation of the IFN-γ and IL-12 genes by 1,25(OH)2D3 in activated T lymphocytes and macrophages/dendritic cells. In this study, we describe the effect of 1,25(OH)2D3 on the activation of the fasL gene in T lymphocytes. We show that 1,25(OH)2D3 inhibits activation-induced cell death, fasL mRNA expression, and that 1,25(OH)2D3-activated VDR represses fasL promoter activity by a mechanism dependent on the presence of a functional VDR DNA-binding domain and ligand-dependent transcriptional activation domain (AF-2). Moreover, we identified a minimal region of the promoter containing the transcription start site and a noncanonical c-Myc-binding element, which mediates this repression. These results place FasL as a novel target for the immunoregulatory activities of 1,25(OH)2D3, and confirm the interest for a possible pharmacological use of this molecule and its derivatives.

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.

Proline-rich tyrosine kinase 2 and Rac activation by chemokine and integrin receptors controls NK cell transendothelial migration

Protein tyrosine kinase activation is an important requisite for leukocyte migration. Herein we demonstrate that NK cell binding to endothelium activates proline-rich tyrosine kinase 2 (Pyk-2) and the small GTP binding protein Rac that are coupled to integrin and chemokine receptors. Chemokine-mediated, but not integrin-mediated, Pyk-2 and Rac activation was sensitive to pretreatment of NK cells with pertussis toxin, a pharmacological inhibitor of Gi protein-coupled receptors. Both Pyk-2 and Rac are functionally involved in chemokine-induced NK cell migration through endothelium or ICAM-1 or VCAM-1 adhesive proteins, as shown by the use of recombinant vaccinia viruses encoding dominant negative mutants of Pyk-2 and Rac. Moreover, we found that Pyk-2 is associated with the Rac guanine nucleotide exchange factor Vav, which undergoes tyrosine phosphorylation upon integrin triggering. Finally, we provide direct evidence for the involvement of Pyk-2 in the control of both chemokine- and integrin-mediated Rac activation. Collectively, our results indicate that Pyk-2 acts as a receptor-proximal link between integrin and chemokine receptor signaling, and the Pyk-2/Rac pathway plays a pivotal role in the control of NK cell transendothelial migration.

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 β2 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 β2 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.

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.

Src-Dependent Syk Activation Controls CD69-Mediated Signaling and Function on Human NK Cells

CD69 C-type lectin receptor represents a functional triggering molecule on activated NK cells, capable of directing their natural killing function. The receptor-proximal signaling pathways activated by CD69 cross-linking and involved in CD69-mediated cytotoxic activity are still poorly understood. Here we show that CD69 engagement leads to the rapid and selective activation of the tyrosine kinase Syk, but not of the closely related member of the same family, ZAP70, in IL-2-activated human NK cells. Our results indicate the requirement for Src family kinases in the CD69-triggered activation of Syk and suggest a role for Lck in this event. We also demonstrate that Syk and Src family tyrosine kinases control the CD69-triggered tyrosine phosphorylation and activation of phospholipase Cγ2 and the Rho family-specific exchange factor Vav1 and are responsible for CD69-triggered cytotoxicity of activated NK cells. The same CD69-activated signaling pathways are also observed in an RBL transfectant clone, constitutively expressing the receptor. These data demonstrate for the first time that the CD69 receptor functionally couples to the activation of Src family tyrosine kinases, which, by inducing Syk activation, initiate downstream signaling pathways and regulate CD69-triggered functions on human NK cells.

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.

Evidence for αvβ3 and αvβ5 Integrin-like Vitronectin (VN) Receptors in Candida albicans and Their Involvement in Yeast Cell Adhesion to VN

The expression of integrin vitronectin (VN) receptors on Candida albicans yeasts and their involvement in the adhesion to VN were investigated. By immunofluorescence and cytofluorimetric analysis, several antibodies directed against human alphav, beta3, beta5, alphavbeta3, or alphavbeta5 integrin positively stained C. albicans yeasts. Biochemical analysis on yeast lysates with anti-human alphav, beta3, or beta5 antibody revealed molecular species of 130, 110, 100, and 84 kDa. The 130-kDa band was identified as alphav, whereas the doublet of 110/100 kDa and the 84-kDa band likely correspond to the beta3 and beta5 subunits, respectively. Some 48%-54% of Candida yeasts specifically adhered to VN, and this binding was strongly inhibited by anti-human alphav, beta3, alphavbeta3, and alphavbeta5 antibodies and by RGD- but not RGE-containing peptides. In addition, VN inhibited C. albicans adherence to a human endothelial cell line. Thus, C. albicans in the yeast phase expresses VN receptors antigenically related to the vertebrate alphavbeta3 and alphavbeta5 integrins, which mediate its adhesion to VN.

Hyperthermia Enhances CD95-Ligand Gene Expression in T Lymphocytes

Hyperthermia represents an interesting therapeutic strategy for the treatment of tumors. Moreover, it is able to regulate several aspects of the immune response. Fas (APO-1/CD95) and its ligand (FasL) are cell surface proteins whose interaction activates apoptosis of Fas-expressing targets. In T cells, the Fas-Fas-L system regulates activation-induced cell death, is implicated in diseases in which lymphocyte homeostasis is compromised, and plays an important role during cytotoxic and regulatory actions mediated by these cells. In this study we describe the effect of hyperthermia on activation of the fas-L gene in T lymphocytes. We show that hyperthermic treatment enhances Fas-L-mediated cytotoxicity, fas-L mRNA expression, and fas-L promoter activity in activated T cell lines. Our data indicate that hyperthermia enhances the transcriptional activity of AP-1 and NF-κB in activated T cells, and this correlates with an increased expression/nuclear translocation of these transcription factors. Moreover, we found that heat shock factor-1 is a transactivator of fas-L promoter in activated T cells, and the overexpression of a dominant negative form of heat shock factor-1 may attenuate the effect of hyperthermia on fas-L promoter activity. Furthermore, overexpression of dominant negative mutants of protein kinase Cε (PKCε) and PKCθ partially inhibited the promoter activation and, more importantly, could significantly reduce the enhancement mediated by hyperthermia, indicating that modulation of PKC activity may play an important role in this regulation. These results add novel information on the immunomodulatory action of heat, in particular in the context of its possible use as an adjuvant therapeutic strategy to consider for the treatment of cancer.