Alessandra Eva

THE PLECKSTRIN HOMOLOGY DOMAIN MEDIATES TRANSFORMATION BY ONCOGENIC DBL THROUGH SPECIFIC INTRACELLULAR TARGETING

The pleckstrin homology (PH) domain is an ~100 amino acid structural motif found in many cellular signaling molecules, including the Dbl oncoprotein and related, putative guanine nucleotide exchange factors (GEFs). Here we have examined the role of the Dbl PH (dPH) domain in the activities of oncogenic Dbl. We report that the dPH domain is not involved in the interaction of Dbl with small GTP-binding proteins and is incapable of transforming NIH 3T3 fibroblasts. On the other hand, co-expression of the dPH domain with oncogenic Dbl inhibits Dbl-induced transformation. A deletion mutant of Dbl that lacks a significant portion of the PH domain retains full GEF activity, but is completely inactive in transformation assays. Replacement of the PH domain by the membrane-targeting sequence of Ras is not sufficient for the recovery of transforming activity. However, subcellular fractionations of Dbl and Dbl mutants revealed that the PH domain is necessary and sufficient for the association of Dbl with the Triton X-100-insoluble cytoskeletal components. Thus, our results suggest that the dPH domain mediates cellular transformation by targeting the Dbl protein to specific cytoskeletal locations to activate Rho-type small GTP-binding proteins.

Hypoxia modulates the gene expression profile of immunoregulatory receptors in human mature dendritic cells: identification of TREM-1 as a novel hypoxic marker in vitro and in vivo

Dendritic cells (DCs) are a heterogeneous group of professional antigen-presenting cells functioning as sentinels of the immune system and playing a key role in the initiation and amplification of innate and adaptive immune responses. DC development and functions are acquired during a complex differentiation and maturation process influenced by several factors present in the local milieu. A common feature at pathologic sites is represented by hypoxia, a condition of low pO(2), which creates a unique microenvironment affecting cell phenotype and behavior. Little is known about the impact of hypoxia on the generation of mature DCs (mDCs). In this study, we identified by gene expression profiling a significant cluster of genes coding for immune-related cell surface receptors strongly up-regulated by hypoxia in monocyte-derived mDCs and characterized one of such receptors, TREM-1, as a new hypoxia-inducible gene in mDCs. TREM-1 associated with DAP12 in hypoxic mDCs, and its engagement elicited DAP12-linked signaling, resulting in ERK-1, Akt, and IκBα phosphorylation and proinflammatory cytokine and chemokine secretion. Finally, we provided the first evidence that TREM-1 is expressed on mDCs infiltrating the inflamed hypoxic joints of children affected by juvenile idiopathic arthritis, representing a new in vivo marker of hypoxic mDCs endowed with proinflammatory properties.

Transcriptome of Hypoxic Immature Dendritic Cells: Modulation of Chemokine/Receptor Expression

Hypoxia is a condition of low oxygen tension occurring in inflammatory tissues. Dendritic cells (DC) are professional antigen-presenting cells whose differentiation, migration, and activities are intrinsically linked to the microenvironment. DCs will home and migrate through pathologic tissues before reaching their final destination in the lymph node. We studied the differentiation of human monocytes into immature DCs (iDCs) in a hypoxic microenvironment. We generated iDC in vitro under normoxic (iDCs) or hypoxic (Hi-DCs) conditions and examined the hypoxia-responsive element in the promoter, gene expression, and biochemical KEGG pathways. Hi-DCs had an interesting phenotype represented by up-regulation of genes associated with cell movement/migration. In addition, the Hi-DC cytokine/receptor pathway showed a dichotomy between down-regulated chemokines and up-regulated chemokine receptor mRNA expression. We showed that CCR3, CX3CR1, and CCR2 are hypoxia-inducible genes and that CCL18, CCL23, CCL26, CCL24, and CCL14 are inhibited by hypoxia. A strong chemotactic response to CCR2 and CXCR4 agonists distinguished Hi-DCs from iDCs at a functional level. The hypoxic microenvironment promotes the differentiation of Hi-DCs, which differs from iDCs for gene expression profile and function. The most prominent characteristic of Hi-DCs is the expression of a mobility/migratory rather than inflammatory phenotype. We speculate that Hi-DCs have the tendency to leave the hypoxic tissue and follow the chemokine gradient toward normoxic areas where they can mature and contribute to the inflammatory process. (Mol Cancer Res 2008;6(2):175–85)

Human dendritic cells differentiated in hypoxia down-modulate antigen uptake and change their chemokine expression profile

Dendritic cells (DCs) are the most potent antigen‐presenting cells and fine‐tune the immune response. We have investigated hypoxias effects on the differentiation and maturation of DCs from human monocytes in vitro, and have shown that it affects DC functions. Hypoxic immature DCs (H‐iDCs) significantly fail to capture antigens through down‐modulation of the RhoA/Ezrin‐Radixin‐Moesin pathway and the expression of CD206. Moreover, H‐iDCs released higher levels of CXCL1, VEGF, CCL20, CXCL8, and CXCL10 but decreased levels of CCL2 and CCL18, which predict a different ability to recruit neutrophils rather than monocytes and create a proinflammatory and proangiogenic environment. By contrast, hypoxia has no effect on DC maturation. Hypoxic mature DCs display a mature phenotype and activate both allogeneic and specific T cells like normoxic mDCs. This study provides the first demonstration that hypoxia inhibits antigen uptake by DCs and profoundly changes the DC chemokine expression profile and may have a critical role in DC differentiation, adaptation, and activation in inflamed tissues.

The integrin cytoplasmic domain-associated protein ICAP-1 binds and regulates Rho family GTPases during cell spreading

Using two-hybrid screening, we isolated the integrin cytoplasmic domain-associated protein (ICAP-1), an interactor for the COOH terminal region of the β1A integrin cytoplasmic domain. To investigate the role of ICAP-1 in integrin-mediated adhesive function, we expressed the full-length molecule in NIH3T3 cells. ICAP-1 expression strongly prevents NIH3T3 cell spreading on extracellular matrix. This inhibition is transient and can be counteracted by coexpression of a constitutively activated mutant of Cdc42, suggesting that ICAP-1 acts upstream of this GTPase. In addition, we found that ICAP-1 binds both to Cdc42 and Rac1 in vitro, and its expression markedly inhibits activation of these GTPases during integrin-mediated cell adhesion to fibronectin as detected by PAK binding assay. In the attempt to define the molecular mechanism of this inhibition, we show that ICAP-1 reduces both the intrinsic and the exchange factor–induced dissociation of GDP from Cdc42; moreover, purified ICAP-1 displaces this GTPase from cellular membranes. Together, these data show for the first time that ICAP-1 regulates Rho family GTPases during integrin-mediated cell matrix adhesion, acting as guanine dissociation inhibitor.

Recruitment of Dbl by ezrin and dystroglycan drives membrane proximal Cdc42 activation and filopodia formation

Dystroglycan is an essential laminin binding cell adhesion molecule which is also an adaptor for several SH2 domain-containing signalling molecules and as a scaffold for the ERK-MAP kinase cascade. Loss of dystroglycan function is implicated in muscular dystrophies and the aetiology of epithelial cancers. We have previously demonstrated a role for dystroglycan and ezrin in the formation of filopodia structures. Here we demonstrate the existence of a dystroglycan:ezrin:Dbl complex that is targeted to the membrane by dystroglycan where it drives local Cdc42 activation and the formation of filopodial. Deletion of an ezrin binding site in dystroglycan prevented the association with ezrin and Dbl and the formation of filopodia. Furthermore, expression of the dystroglycan cytoplasmic domain alone had a dominant-negative effect on filopodia formation and Cdc42 activation by sequestering ezrin and Dbl away from the membrane. Depletion of dystroglycan inhibited Cdc42-induced filopodia formation. For the first time we also demonstrate co-localisation of Cdc42 and dystroglycan at the tips of dynamic filopodia.

p130Cas is an essential transducer element in ErbB2 transformation

The ErbB2 oncogene is often overexpressed in breast tumors and associated with poor clinical outcome. p130Cas represents a nodal scaffold protein regulating cell survival, migration, and proliferation in normal and pathological cells. The functional role of p130Cas in ErbB2‐dependent breast tumorigenesis was assessed by its silencing in breast cancer cells derived from mouse mammary tumors overexpressing ErbB2 (N202‐1A cells), and by its reexpression in ErbB2‐transformed p130Cas‐null mouse embryonic fibroblasts. We demonstrate that p130Cas is necessary for ErbB2‐dependent foci formation, anchorage‐independent growth, and in vivo growth of orthotopic N202‐1A tumors. Moreover, intranipple injection of p130Cas‐stabilized siRNAs in the mammary gland of Balbc‐NeuT mice decreases the growth of spontaneous tumors. In ErbB2‐transformed cells, p130Cas is a crucial component of a functional molecular complex consisting of ErbB2, c‐Src, and Fak. In human mammary cells, MCF10A.B2, the concomitant activation of ErbB2, and p130Cas overexpression sustain and strengthen signaling, leading to Rac1 activation and MMP9 secretion, thus providing invasive properties. Consistently, p130Cas drives N202‐1A cell in vivo lung metastases colonization. These results demonstrate that p130Cas is an essential transducer in ErbB2 transformation and highlight its potential use as a novel therapeutic target in ErbB2 positive human breast cancers.—Cabodi, S., Tinnirello, A., Bisaro, B., Tornillo, G., Camacho‐Leal, M. P., Forni, G., Cojoca, R., Iezzi, M., Amici, A., Montani, M., Eva, A., Di Stefano, P., Muthuswamy, S. K., Tarone, G., Turco, E., Defilippi, P. p130Cas is an essential transducer element in ErbB2 transformation. FASEB J. 24, 3796–3808 (2010). www.fasebj.org

Chronic hypoxia reprograms human immature dendritic cells by inducing a proinflammatory phenotype and TREM‐1 expression

DCs are powerful antigen‐presenting cells central in the orchestration of innate and acquired immunity. DC development, migration, and activities are intrinsically linked to the microenvironment. DCs migrate through pathologic tissues before reaching their final destination in the lymph nodes. Hypoxia, a condition of low partial oxygen pressure, is a common feature of many pathologic situations, capable of modifying DC phenotype and functional behavior. We studied human monocyte‐derived immature DCs generated under chronic hypoxic conditions (H‐iDCs). We demonstrate by gene expression profiling the upregulation of a cluster of genes coding for antigen‐presentation, immunoregulatory, and pattern recognition receptors, suggesting a stimulatory role for hypoxia on iDC immunoregulatory functions. In particular, we show that H‐iDCs express triggering receptor expressed on myeloid cells(TREM‐1), a member of the Ig superfamily of immunoreceptors and an amplifier of inflammation. This effect is reversible because H‐iDC reoxygenation results in TREM‐1 down‐modulation. TREM‐1 engagement promotes upregulation of T‐cell costimulatory molecules and homing chemokine receptors, typical of mature DCs, and increases the production of proinflammatory, Th1/Th17‐priming cytokines/chemokines, resulting in increased T‐cell responses. These results suggest that TREM‐1 induction by the hypoxic microenvironment represents a mechanism of regulation of Th1‐cell trafficking and activation by iDCs differentiated at pathologic sites.

The hypoxic environment reprograms the cytokine/chemokine expression profile of human mature dendritic cells

Myeloid dendritic cells (DCs) are professional antigen-presenting cells critical for the orchestration of immunity and maintenance of self-tolerance. DC development and functions are tightly regulated by a complex network of inhibitory and activating signals present in the tissue microenvironment, and dysregulated DC responses may result in amplification of inflammation, loss of tolerance, or establishment of immune escape mechanisms. Generation of mature (m)DCs from monocytic precursors recruited at pathological sites occurs under condition of low partial oxygen pressure (pO(2)). However, the way in which the hypoxic microenvironment modulates the functions of these cells is still not clear. We demonstrate that chronic hypoxia (4 days, 1% O(2)) promotes the onset of a highly proinflammatory gene expression profile in mDCs generated from primary human monocytes, characterized by the modulation of a significant cluster of genes coding for proinflammatory chemokines/cytokines and/or their receptors. Within the chemokine system, strong upregulation of genes encoding proteins chemotactic for neutrophils, such as CXCL2, CXCL3, CXCL5, CXCL6, and CXCL8, and for activated/memory T lymphocytes, monocytes, and immature (i) DCs, e.g. CCL20, CCL3 and CCL5, was observed, concomitant with decreased expression of genes coding for naive/resting T cells chemoattractants, CCL18 and CCL23. Other hypoxia-inducible genes coded for cytokines with a primary role in inflammation and angiogenesis, including osteopontin, vascular endothelial growth factor, and IL-1β. mRNA modulation was paralleled by protein secretion. These results suggest that conditions of reduced O(2) availability reprograms mDCs toward a proinflammatory direction by tuning the cytokine/chemokine repertoire, thus affecting their ability to regulate leukocyte trafficking and activation at pathological sites, with potential implications for the pathogenesis of chronic inflammatory diseases.

Defective Dendrite Elongation but Normal Fertility in Mice Lacking the Rho-Like GTPase Activator Dbl

ABSTRACT Dbl is the prototype of a large family of GDP-GTP exchange factors for small GTPases of the Rho family. In vitro, Dbl is known to activate Rho and Cdc42 and to induce a transformed phenotype. Dbl is specifically expressed in brain and gonads, but its in vivo functions are largely unknown. To assess its role in neurogenesis and gametogenesis, targeted deletion of the murine Dbl gene was accomplished in embryonic stem cells. Dbl-null mice are viable and did not show either decreased reproductive performances or obvious neurological defects. Histological analysis of mutant testis showed normal morphology and unaltered proliferation and survival of spermatogonia. Dbl-null brains indicated a correct disposition of the major neural structures. Analysis of cortical stratification indicated that Dbl is not crucial for neuronal migration. However, in distinct populations of Dbl-null cortical pyramidal neurons, the length of dendrites was significantly reduced, suggesting a role for Dbl in dendrite elongation.