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Mireia Vallespinós,
David Fernández,
Lorena Rodríguez,
Josué Alvaro-Blanco,
Esther Baena,
Maitane Ortiz,
Daniela Dukovska,
Dolores Martínez,
Ana Rojas, Miguel R Campanero,
Ignacio Moreno de Alborán
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ABSTRACT: c-Myc, a member of the Myc family of transcription factors, is involved in numerous biological functions including the regulation of cell proliferation, differentiation, and apoptosis in various cell types. Of all of its functions, the role of c-Myc in cell differentiation is one of the least understood. We addressed the role of c-Myc in B lymphocyte differentiation. We found that c-Myc is essential from early stages of B lymphocyte differentiation in vivo and regulates this process by providing B cell identity via direct transcriptional regulation of the ebf-1 gene. Our data show that c-Myc influences early B lymphocyte differentiation by promoting activation of B cell identity genes, thus linking this transcription factor to the EBF-1/Pax-5 pathway.
The Journal of Immunology 06/2011; 186(12):6726-36. · 5.79 Impact Factor
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Roberto Martín Jean-Mairet,
Celia López-Menéndez,
Lucía Sánchez-Ruiloba,
Sandra Sacristán,
María Rodríguez-Martínez,
Lorena Riol-Blanco,
Paloma Sánchez-Mateos,
Francisco Sánchez-Madrid,
José Luis Rodríguez-Fernández, Miguel R Campanero,
Teresa Iglesias
European Journal of Immunology 04/2011; 41(4). · 5.10 Impact Factor
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Roberto Martín Jean-Mairet,
Celia López-Menéndez,
Lucía Sánchez-Ruiloba,
Sandra Sacristán,
María Rodríguez-Martínez,
Lorena Riol-Blanco,
Paloma Sánchez-Mateos,
Francisco Sánchez-Madrid,
José Luis Rodríguez-Fernández, Miguel R. Campanero,
Teresa Iglesias
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ABSTRACT: Kinase D interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is a protein that is mainly expressed in brain and neural cells where its function is only starting to be characterized. Here, we show that Kidins220/ARMS is also expressed in T lymphocytes where it is highly concentrated at the uropod of polarized T cells. In this cellular model, Kidins220/ARMS colocalizes with typical uropod T-cell molecules and coimmunoprecipitates with ICAM-3. Furthermore, Kidins220/ARMS associates with raft domains at the uropod and coimmunoprecipitates with caveolin-1, a molecule we show here to be also expressed in T cells. Importantly, induction of morphological polarization in primary T lymphocytes and Jurkat cells enhances Kidins220/ARMS colocalization with ICAM-3. Conversely, disruption of cell polarity provokes Kidins220/ARMS redistribution from the uropod to other cellular regions and drastically impairs its association with ICAM-3 in a protein kinase C-dependent manner. Finally, Kidins220/ARMS knockdown in human polarized T-cell lines promotes both basal and stromal cell-derived factor-1α-induced directed migration, identifying a novel function for this molecule. Altogether, our findings show that Kidins220/ARMS is a novel component of the uropod involved in the regulation of T-cell motility, an essential process for the immune response.
European Journal of Immunology 03/2011; 41(4):1035 - 1046. · 5.10 Impact Factor
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Roberto Martín Jean-Mairet,
Celia López-Menéndez,
Lucía Sánchez-Ruiloba,
Sandra Sacristán,
María Rodríguez-Martínez,
Lorena Riol-Blanco,
Paloma Sánchez-Mateos,
Francisco Sánchez-Madrid,
José Luis Rodríguez-Fernández, Miguel R Campanero,
Teresa Iglesias
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ABSTRACT: Kinase D interacting substrate of 220 kDa (Kidins220), also known as ankyrin repeat-rich membrane spanning (ARMS), is a protein that is mainly expressed in brain and neural cells where its function is only starting to be characterized. Here, we show that Kidins220/ARMS is also expressed in T lymphocytes where it is highly concentrated at the uropod of polarized T cells. In this cellular model, Kidins220/ARMS colocalizes with typical uropod T-cell molecules and coimmunoprecipitates with ICAM-3. Furthermore, Kidins220/ARMS associates with raft domains at the uropod and coimmunoprecipitates with caveolin-1, a molecule we show here to be also expressed in T cells. Importantly, induction of morphological polarization in primary T lymphocytes and Jurkat cells enhances Kidins220/ARMS colocalization with ICAM-3. Conversely, disruption of cell polarity provokes Kidins220/ARMS redistribution from the uropod to other cellular regions and drastically impairs its association with ICAM-3 in a protein kinase C-dependent manner. Finally, Kidins220/ARMS knockdown in human polarized T-cell lines promotes both basal and stromal cell-derived factor-1α-induced directed migration, identifying a novel function for this molecule. Altogether, our findings show that Kidins220/ARMS is a novel component of the uropod involved in the regulation of T-cell motility, an essential process for the immune response.
European Journal of Immunology 02/2011; 41(4):1035-46. · 5.10 Impact Factor
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ABSTRACT: Current treatments of sporadic Burkitt's lymphoma (sBL) are associated with severe toxicities. A better understanding of sBL formation would facilitate development of less toxic therapies. The etiology of sBL remains, however, largely unknown, C-MYC up-regulation being the only lesion known to occur in all sBL cases. Several studies examining the role of C-MYC in the pathogenesis of BL have concluded that C-MYC translocation is not the only critical event and that additional unidentified factors are expected to be involved in the formation of this tumor. We herein report that a gene distinct from C-MYC, E2F1, is involved in the formation of all or most sBL tumors. We found that E2F1 is highly expressed in Burkitt's lymphoma cell lines and sBL lymphoma specimens. Our data indicate that its elevated expression is not merely the consequence of the presence of more cycling cells in this tumor relative to other cell lines or to other neoplasias. In fact, we show that reduction of its expression in sBL cells inhibits tumor formation and decreases their proliferation rate. We also provide data suggesting that E2F1 collaborates with C-MYC in sBL formation. E2F1 expression down-regulation did not affect, however, the proliferation of human primary diploid fibroblasts. Because E2F1 is not needed for cell proliferation of normal cells, our results reveal E2F1 as a promising therapeutic target for sBL.
Cancer Research 06/2009; 69(9):4052-8. · 7.86 Impact Factor
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ABSTRACT: Although C-MYC is overexpressed in a number of tumors, the mechanisms governing its expression in normal or tumor cells are not completely understood. Recruitment of the Retinoblastoma protein family members to gene promoters by E2F factors has a dominant negative effect on their activity during the G(0) and G(1) phases of the cell cycle. Despite the presence of an E2F-binding site on the C-MYC promoter, it escapes the repressive effect of E2F-Retinoblastoma complexes through unknown mechanisms during exit from quiescence. We hypothesized that occupancy of E2F elements by factors distinct from E2F might account for this escape. To test this hypothesis, we investigated whether the E2F element in the C-MYC promoter is regulated differently than E2F elements in promoters that are activated at the G(1)-S transition. Employing gel shift analysis, the E2F element from the C-MYC promoter was found to form a unique non-E2F complex, referred to as E2F C-MYC Specific (EMYCS), which is not observed with E2F elements from several other promoters. The DNA contact residues for EMYCS are distinct but overlapping with residues required for binding of E2F proteins. Finally, the approximate estimated molecular weight of the DNA-binding component of EMCYS is 105 kDa. Functional studies indicate that EMYCS has transcriptional transactivation capacity and suggest that it is required to activate the C-MYC promoter during exit from quiescence.
Carcinogenesis 02/2009; 30(3):440-8. · 5.70 Impact Factor
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Miguel R. Campanero,
Alicia G. Arroyo,
Rafael Pulido,
Angeles Ursa,
Milagros S. de Matías,
Paloma Sánchez-Mateos,
Paul D. Kassner,
Bosco M. C. Chan,
Martin E. Hemler,
Angel L. Corbí,
Manuel O. de Landázuri,
Francisco Sánchez-Madrid
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ABSTRACT: Whereas all of the integrins in the VLA protein subfamily are involved in cell-extracellular matrix interactions, only VLA-4 (through the α4 subunit) has been implicated in the triggering of intercellular adhesion. Here we describe that the VLA protein β1 subunit (CD29) is also involved in the induction of homotypic cell aggregation. We have obtained three novel anti-β1 monoclonal antibodies (mAb) with the ability to induce cell aggregation on different leukocyte cell types. These mAb recognize an antigenic site on the common β1 chain of VLA proteins which is topographically and/or functionally distinct from other epitopes previously defined by several prototype anti-β1 mAb. Induction of cell aggregation by anti-β1 mAb is epitope specific, isotype and Fc independent, and displays kinetics similar to α4-mediated aggregation. This cell aggregation requires an intact cellular metabolism, the presence of divalent cations in the extracellular medium, and the integrity of the cytoskeleton. We also have found that the Na+/H+ antiporter may be essential for this process. For Ramos cells, which bear only the VLA α4/β1 heterodimer, intercellular adhesion induced through the VLA-β1 chain could be selectively inhibited by other anti-β1 mAb as well as by anti-α4 mAb. Interestingly, anti-β1 mAb which induced strong aggregation of VLA-α2- or VLA-α4-transfected K562 cells, had minimal effect on the α2− α4− α5+ K562 cell line. Furthermore, the β1-mediated induction of cell aggregation on α2-K562- and α4-K562-transfected cells was blocked by preincubation with either anti-α2 or anti-α4 mAb, respectively, as well as by other anti-β1 mAb. Interestingly, parental K562 cells were able to interact with both α2- and α4-transfected K562 cells, thus suggesting that counter-receptors for both integrins (VLA-2 and VLA-4) might exist on these cells. Together these results provide strong evidence supporting the involvement of α2/β1 and α4/β1 heterodimers in intercellular interactions and underline the pivotal role of the common β1 chain of VLA proteins in the integrin-mediated induction of cell aggregation.
European Journal of Immunology 11/2005; 22(12):3111 - 3119. · 5.10 Impact Factor
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ABSTRACT: Very-late antigen (VLA)-4 (CD49d/CD29) constitutes the only member of the β1 integrin family that plays a role in the interaction of lymphoid cells with both extracellular matrix and endothelial cells through two identified ligands: fibronectin (FN) and VCAM-1, respectively. The expression and functional activity of VLA-4 has been studied in different maturation and activation stages of B cells from several cellular compartments. Resident B lymphocytes of different lymphoid organs were almost negative for VLA-4 as detected by both immunoperoxidase staining and flow cytometry analysis. However, a high expression of both chains of this heterodimer was observed when tonsillar B cells were activated in vitro with different stimuli, such as phorbol esters or Staphylococcus aureus Cowan I (SAC). Both nonactivated and in vitro activated B cells from peripheral blood constitutively expressed high levels of this surface antigen. The induced expression of VLA-4 after activation of tonsillar B lymphocytes was accompanied by the acquisition of the capacity to bind to a 38-kDa proteolytic fragment, containing the connecting segment I domain, of FN. Interestingly, nonactivated peripheral blood B cells were unable to attach to this FN fragment, in spite of their constitutive expression of VLA-4, and only acquired this functional capacity after cell activation with phorbol esters and SAC. This FN-binding acquisition was not affected by preincubation with inhibitors of protein and RNA synthesis. These results underline that the FN-binding activity of VLA-4 is dependent on processes affecting cellular activation as described for other members of the integrin family. By contrast, VLA-4-mediated homotypic aggregation of peripheral blood B cells could be triggered by anti-4 monoclonal antibodies independently of the cell activation state.
European Journal of Immunology 11/2005; 21(10):2437 - 2445. · 5.10 Impact Factor
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ABSTRACT: The myelodysplastic/myeloproliferative diseases (MDS/MPDs) are a heterogeneous group of myeloid neoplasms that share characteristics with chronic myeloproliferative diseases and myelodysplastic syndromes. The broad spectrum of clinical manifestations makes MDS/MPDs extremely difficult to diagnose and treat, with a median survival time of 1-5 years. No single gene defect has been firmly associated with MDS/MPDs, and no animal models have been developed for these diseases. The association of deletions on chromosome 20q with myeloid malignancies suggests the presence of unidentified tumor suppressor genes in this region. Here we show that the recently identified death inducer-obliterator (Dido) gene gives rise to at least 3 polypeptides (Dido1, Dido2, and Dido3) through alternative splicing, and we map the human gene to the long arm of chromosome 20. We found that targeting of murine Dido caused a transplantable disease whose symptoms and signs suggested MDS/MPDs. Furthermore, 100% of human MDS/MPD patients analyzed showed Dido expression abnormalities, which we also found in other myeloid but not lymphoid neoplasms or in healthy donors. Our findings suggest that Dido might be one of the tumor suppressor genes at chromosome 20q and that the Dido-targeted mouse may be a suitable model for studying MDS/MPD diseases and testing new approaches to their diagnosis and treatment.
Journal of Clinical Investigation 10/2005; 115(9):2351-62. · 15.39 Impact Factor
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ABSTRACT: Cyclin G2 is an unconventional cyclin highly expressed in postmitotic cells. Unlike classical cyclins that promote cell cycle progression, cyclin G2 blocks cell cycle entry. Here we studied the mechanisms that regulate cyclin G2 mRNA expression during the cell cycle. Analysis of synchronized NIH 3T3 cell cultures showed elevated cyclin G2 mRNA expression levels at G(0), with a considerable reduction as cells enter cell cycle. Downregulation of cyclin G2 mRNA levels requires activation of phosphoinositide 3-kinase, suggesting that this enzyme controls cyclin G2 mRNA expression. Because the phosphoinositide 3-kinase pathway inhibits the FoxO family of forkhead transcription factors, we examined the involvement of these factors in the regulation of cyclin G2 expression. We show that active forms of the forkhead transcription factor FoxO3a (FKHRL1) increase cyclin G2 mRNA levels. Cyclin G2 has forkhead consensus motifs in its promoter, which are transactivated by constitutive active FoxO3a forms. Finally, interference with forkhead-mediated transcription by overexpression of an inactive form decreases cyclin G2 mRNA expression levels. These results show that FoxO genes regulate cyclin G2 expression, illustrating a new role for phosphoinositide 3-kinase and FoxO transcription factors in the control of cell cycle entry.
Molecular and Cellular Biology 04/2004; 24(5):2181-9. · 5.53 Impact Factor
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ABSTRACT: The regulation of the cell surface expression of ICAM-3 (CD50) was investigated in human neutrophils. Immunofluorescence flow cytometry analysis revealed a remarkable and very rapid down-regulation of the ICAM-3 cell surface expression upon neutrophil activation with stimulating agents such as phorbol myristate acetate (PMA) or calcium ionophore. A similar low expression of ICAM-3 was observed on neutrophils from patients undergoing hemodialysis with cell-activating cellulosic membranes. Internalization assays with 125I-labeled anti-ICAM-3 monoclonal antibody (mAb) suggested that ICAM-3-down-regulation was due to antigen release from the cell surface towards the outer milieu, rather than to antigen internalization. Immunoprecipitation studies confirmed this down-regulatory effect, and revealed the presence of ICAM-3 in cell-free supernatants from activated neutrophils. Furthermore, the presence of a soluble form of ICAM-3 with a range of concentrations of 0–296 ng/ml in the plasma from healthy human volunteers was detected by using a two-site mAb radioimmunoassay. A proteolytic mechanism likely accounts for this process since protease inhibitors virtually abrogated the PMA-induced down-regulation of ICAM-3. Functional studies showed that anti-ICAM-3 mAb were able to trigger homotypic neutrophil aggregation both before and after ICAM-3 down-regulation, indicating that the fraction of ICAM-3 molecules remaining on the neutrophil surface upon activation are still capable of sustaining cell adhesion. In contrast, the loss of L-selectin (CD62L) on activated neutrophils was almost complete, thus leading to an impairment of L-selectin-mediated neutrophil-endothelial cell adhesion. These results indicate that ICAM-3 is released to the medium upon neutrophil stimulation and that both ICAM-3 and L-selectin have a role in the neutrophil adhesive phenomena.
European Journal of Immunology 10/1994; 24(11):2586 - 2594. · 5.10 Impact Factor
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ABSTRACT: The involvement of the CD2 (T11) molecule, an alternative activation pathway for T lymphocytes, in the regulation of tumor necrosis factor (TNF)-α/TNF receptor system in human T lymphocytes has been investigated. It has been found that both TNF-α synthesis and secretion were induced after incubation of purified T lymphocytes with the appropriate mitogenic combination of antibodies specific for two different epitopes on the CD2 molecule. Moreover, TNF-α secretion was also observed by activation of T lymphocytes either through CD3 or CD69 molecular pathways, or with other stimulating agents such as Ca2+ ionophore in combination with phorbol esters. The expression of TNF receptors has been studied in both nonactivated and CD2-activated T lymphocytes. Unstimulated T cells weakly expressed a functional 75-kDa receptor form, whereas they lacked detectable levels of the 55-kDa receptor form. Triggering of T cell activation through the CD2 molecule also markedly increased the expression of the p75-kDa TNF receptor form, but did not exert any inductive effect on the expression of the p55-kDa TNF receptor. In addition, we have found that TNF-α enhanced the proliferative response triggered by the mixture of anti-CD2 monoclonal antibodies. Taken together, these results support a role for the CD2 activation pathway in the functional regulation of TNF-α/TNF receptor system in T lymphocytes, and reinforce the view of CD2 as an alternative pathway for regulation of the cytokine network that modulates the function of T lymphocytes.
European Journal of Immunology 11/1992; 22(12):3155 - 3160. · 5.10 Impact Factor
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ABSTRACT: The structure-function relationship of the human integrin VLA-4 (α4/β1; CD49d/CD29), has been studied in the human B-cell line Ramos by immunochemical and functional analysis. Ramos cells expressed the 150-kDa non-proteolyzed form of the α4 chain, which could be digested upon mild trypsin treatment to generate the 80- and 65-kDa proteolyzed forms, as well as α4 polypeptides of 55 and 50 kDa. In addition, treatment of Ramos cells with high doses of pronase predominantly yielded the 55- and 50-kDa α4 peptides. The trypsin-generated 80- and 65-kDa α4 polypeptides, but not the 55- and 50-kDa fragments, were able to associate with the β1 chain. Distinct anti-VLA-4 mAb against four different α4 epitopes, referred to as epitopes A, B1, B2, and C, recognized the 150-kDa α4 chain both associated or non-associated with the β1 chain. The α4 proteolytic forms of 80, 65 and 50 kDa were precipitated by the anti-α4 mAb directed against the four different α4 epitopes. On the other hand, the 55-kDa α4 peptide was present in precipitates from anti-α4 mAb specific for epitopes A, B1 and C, but absent in precipitates from the anti-α4 mAb specific for epitope B2. The different adhesive capacities of the VLA-4 integrin, namely the interaction with a 38-kDa fibronectin fragment containing the CS-1 region of plasma fibronectin (Fn-38), the binding to the vascular cell adhesion molecule-1 (VCAM-1), or the ability to mediate the anti-α4-induced cell aggregation, were not altered on VLA-4 from cells upon mild trypsin treatment, when compared to non-treated cells. However, the 55- and 50-kDa α4 forms generated by high-dose pronase cell treatment, failed to mediate cell interaction with Fn-38 or VCAM-1 ligands, and cell aggregation could not be triggered through VLA-4 under these conditions.
FEBS Letters.
