In endothelial cells, we are analyzing the signaling pathways and the role of transcription factors involved in the activation induced by Vascular Endothelial Growth Factor (VEGF), a very potent mitogen and angiogenic factor for endothelial cells. Recently, we have found that VEGF induces the transcriptional activation of NFAT in human endothelial cells, which is required for Cyclooxygenase-2 –gene expression. Given the role of this gene in angiogenesis and cancer, we are analyzing the effect of NFAT inhibitors in corneal angiogenesis in mice and on the ability of endothelial cells to form capillary-like structures. Initially, we have found that the inhibition of NFAT by Cyclosporin A results in the selective inhibition of VEGF-mediated angiogenesis. These results have also encouraged us to investigate whether NFAT inhibitors display therapeutic properties in diseases that occurs with neovascularization mediated by VEGF such as the diabetic and the prematurity retinopathies.

Our research interest is focussed on the study of the regulation of gene expression in the activation of lymphocyte and endothelial cells. We are analyzing the mechanisms that integrate extracellular signal transduction with the activation of transcription factors which regulate specific gene expression programs.
A major goal of our research is the characterization of the molecular mechanisms that control the subcellular localization of the NFAT family of transcription factors. This family is composed of at least four members that are found in the cytoplasm of resting cells and translocate to the nucleus in response to calcium signals triggered upon cell activation. Initially, this process involves the calcineurin-mediated dephosphorylation of NFAT, and later on , once calcium signals are curtailed NFAT is exported from the nucleus to the cytoplasm through the action of nuclear kinases not completely identified . We have demonstrate that the activation of p38 and JNK results in the nuclear exclusion and transactivation of NFAT, respectively. We are mapping the NFAT regions that interact with MAPKs to perform site directed mutagenesis of the NFAT sites phosphorylated by MAPKs to further identify the mutants that result in changes in the shuttling of NFAT members in vivo. These analyses may help to define new mechanisms by which eukaryotic transcription factors are deactivated and contribute to the identification of new therapeutic targets for immnosuppression. As an additional aim, by using mass spectrometry and stable transfectants overexpressing the different family members we are analyzing the cellular complexes that integrate NFAT-interacting proteins.

Selected publications
Martínez-Martínez S, Redondo JM. Inhibitors of the Calcineurin/NFAT Pathway. Curr. Med. Chem. 11, 997-1007 (2004)
Ruiz-Ruiz C, Ruiz de Almodóvar C, Rodríguez A, Ortiz-Ferrón G, Redondo JM, López-Rivas A. The up-regulation of human caspase-8 by interferon-gamma in breast tumor cells requires the induction and action of the transcription factor Interferon Regulatory Factor-1. J. Biol. Chem. 279, 19712-19720 (2004).
Ruiz de Almodóvar C, López-Rivas A, Redondo JM, Rodríguez A. Transcriptional regulation of the TRAIL-R3 gene. Vitamins and Hormones 67, 51-63 (2004)
Ruiz de Almodóvar C, Ruiz-Ruiz C, Rodríguez A, Ortiz-Ferrón, Redondo JM, López-Rivas A. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) decoy receptor TRAIL-R3 is up-regulated by p53 in breast tumor cells through a mechanism involving an intronic p53-binding site. J. Biol. Chem. 279, 4093–4101 (2004).
Ruiz-Ruiz C, Robledo G, Cano E, Redondo JM, López-Rivas A. Characterization of p53-mediated up-regulation of CD95 gene expression upon genotoxic treatment in human breast tumor cells. J. Biol. Chem. 278, 31667–31675 (2003).
Iñiguez MA , Rodríguez A, Volpert OV, Fresno M, Redondo JM. Cyclooxygenase-2: a therapeutic target in angiogenesis. Trends Mol. Med. 9, 73-78 (2003)
Ruiz de Almodóvar C, López-Rivas A, Redondo JM, Rodríguez A. Transcription initiation sites and promoter structure of the human TRAIL-R3 gene. FEBS letters 531, 304-308 (2002)
Lorenzo E, Ruiz-Ruiz C, Quesada AJ, Hernández G, López-Rivas A, Redondo JM. Doxorubicin induces apoptosis and CD95 gene expression in human primary endothelial cells through a p53-dependent mechanism. J. Biol. Chem. 17,10883-10892 (2002)
Hernández G, Volpert OV, Iñiguez MA, Lorenzo E, Martínez-Martínez S, Grau R, Fresno M, Redondo JM. Selective inhibition of VEGF-mediated angiogenesis by cyclosporin A: Roles of NFAT and cyclooxygenase-2. J. Exp. Med. 193, 607-620 (2001)
Iñiguez MA, Martínez-Martínez S, Punzón C, Redondo JM, Fresno M. An essential role of the nuclear factor of activated T cells in the regulation of the expression of the cyclooxygenase-2 gene in human T lymphocytes. J. Biol. Chem. 275, 23627-23635 (2000)
Gomez del Arco P, Martinez-Martinez S, Maldonado JL, Ortega-Perez I, Redondo JM. A role for the p38 MAP kinase pathway in the nuclear shuttling of NFATp. J.Biol.Chem. 275, 13872-13878 (2000)
Lauzurica P, Martínez-Martínez S, Marazuela M, Gómez del Arco P, Martínez AC, Sánchez-Madrid F, Redondo JM. Pyrrolidine dithiocarbamate protects mice from lethal shock induced by LPS or TNF-a. Eur. J. Immunol. 29,1890-1900 (1999)
Armesilla AL, Lorenzo E, Gómez del Arco P, Martínez-Martínez S, Alfranca A, Redondo JM. VEGF activates nuclear factor of activated T cells (NFAT) in human endothelial cells; a role for tissue factor gene expression. Mol. Cell. Biol. 19, 2032-2043 (1999)

Collaborations
Jesús Vázquez & Manuel Fresno, Centro de Biología Molecular, CSIC (Madrid)
Francisco Sánchez-Madrid, Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid
Manuel López-Cabrera, Unidad de Biología Molecular, Hospital de la Princesa, Universidad Autónoma de Madrid
Abelardo López-Rivas, Instituto de Parasitología y Biomedicina, CSIC (Granada)
Pura Muñoz, Institut de Recerca Oncológica (Barcelona)
Benilde Jimenez, Instituto de Investigaciones Biomedicas (UAM-CSIC) (Madrid)
Olga Volpert, Northwestern University (Chicago, IL, USA