Sara Martínez-Martínez

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

We have previously reported that transcriptional induction of cyclooxygenase-2 (COX-2) isoenzyme occurs early after T cell receptor triggering, suggesting functional implications of cyclooxygenase activity in this process. Here, we identify the cis-acting elements responsible for the transcriptional activation of this gene in human T lymphocytes. COX-2 promoter activity was induced upon T cell activation both in primary resting T lymphocytes and in Jurkat cells. This induction was abrogated by inhibition of calcineurin phosphatase with the immunosuppressive drug cyclosporin A, whereas expression of an active calcineurin catalytic subunit enhanced COX-2 transcriptional activation. Moreover, cotransfection of nuclear factor of activated T cells (NFAT) wild type protein transactivated COX-2 promoter activity. Conversely, dominant negative mutants of NFATc or c-Jun proteins inhibited COX-2 induction. Electrophoretic mobility shift assays and site-directed mutagenesis allowed the identification of two regions of DNA located in the positions −117 and −58 relative to the transcriptional start site that serves as NFAT recognition sequences. These results emphasize the central role that the Ca2+/calcineurin pathway plays in COX-2 transcriptional regulation in T lymphocytes pointing to NFAT/activator protein-1 transcription factors as essential for COX-2 promoter regulation in these cells.

Vascular endothelial growth factor activates nuclear factor of activated T cells in human endothelial cells: a role for tissue factor gene expression.

ABSTRACT Vascular endothelial growth factor (VEGF) is a potent angiogenic inducer that stimulates the expression of tissue factor (TF), the major cellular initiator of blood coagulation. Here we show that signaling triggered by VEGF induced DNA-binding and transcriptional activities of nuclear factor of activated T cells (NFAT) and AP-1 in human umbilical vein endothelial cells (HUVECs). VEGF also induced TF mRNA expression and gene promoter activation by a cyclosporin A (CsA)-sensitive mechanism. As in lymphoid cells, NFAT was dephosphorylated and translocated to the nucleus upon activation of HUVECs, and these processes were blocked by CsA. NFAT was involved in the VEGF-mediated TF promoter activation as evidenced by cotransfection experiments with a dominant negative version of NFAT and site-directed mutagenesis of a newly identified NFAT site within the TF promoter that overlaps with a previously identified κB-like site. Strikingly, this site bound exclusively NFAT not only from nuclear extracts of HUVECs activated by VEGF, a stimulus that failed to induce NF-κB-binding activity, but also from extracts of cells activated with phorbol esters and calcium ionophore, a combination of stimuli that triggered the simultaneous activation of NFAT and NF-κB. These results implicate NFAT in the regulation of endothelial genes by physiological means and shed light on the mechanisms that switch on the gene expression program induced by VEGF and those regulating TF gene expression.

Inhibitors of the Calcineurin / NFAT Pathway

The well known calcium-sensitive phosphatase calcineurin is implicated in many eukaryotic activation and developmental programmes, including lymphocyte activation, heart-valve morphogenesis, angiogenesis, and neural and muscle development. The importance of this phosphatase is graphically illustrated by the observation that the immunosuppressive actions of the microbial drugs Cyclosporin A (CsA) and FK506 arise from their inhibition of calcineurin. As substrates of calcineurin, transcription factors of the NFAT family play an essential role in lymphocyte activation, and it follows that their function is also inhibited by CsA and FK506. Although the use of these drugs has been crucial for the success of organ transplantation, their therapeutic use is associated with severe side effects. There is, therefore a need to develop better, less toxic immunosuppressive agents. In recent years, a number of endogenous calcineurin inhibitor proteins have been identified that bind calcineurin and block its phosphatase activity. In some cases the calcineurin interaction domains of these proteins, or their corresponding docking sites on calcineurin, have been described. However, their mode of action and regulatory mechanisms are not completely known. In a more recent development, specific amino acidic sequences implicated in the interaction between calcineurin and NFAT have been identified. It is of special interest that specific disruption of this pathway has been obtained through the expression of peptides based on some of these sequences. A more profound analysis of these issues could open up new perspectives in immunosuppressive therapy; promising compounds with features of endogenous calcineurin inhibitors (and thus likely to have fewer toxic effects than CsA and FK506), or selective blockers of calcineurin-NFAT interactions that would not alter the functioning of other calcineurin substrates.

A Conserved Docking Surface on Calcineurin Mediates Interaction with Substrates and Immunosuppressants

The phosphatase calcineurin, a target of the immunosuppressants cyclosporin A and FK506, dephosphorylates NFAT transcription factors to promote immune activation and development of the vascular and nervous systems. NFAT interacts with calcineurin through distinct binding motifs: the PxIxIT and LxVP sites. Although many calcineurin substrates contain PxIxIT motifs, the generality of LxVP-mediated interactions is unclear. We define critical residues in the LxVP motif, and we demonstrate its binding to a hydrophobic pocket at the interface of the two calcineurin subunits. Mutations in this region disrupt binding of mammalian calcineurin to NFATC1 and the interaction of yeast calcineurin with substrates including Rcn1, which contains an LxVP motif. These mutations also interfere with calcineurin-immunosuppressant binding, and an LxVP-based peptide competes with immunosuppressant-immunophilin complexes for binding to calcineurin. These studies suggest that LxVP-type sites are a common feature of calcineurin substrates, and that immunosuppressant-immunophilin complexes inhibit calcineurin by interfering with this mode of substrate recognition.

A robust method for quantitative high-throughput analysis of proteomes by 18O labeling

MS-based quantitative proteomics plays an increasingly important role in biological and medical research and the development of these techniques remains one of the most important challenges in mass spectrometry. Numerous stable isotope labeling approaches have been proposed. However, and particularly in the case of 18O-labeling, a standard protocol of general applicability is still lacking, and statistical issues associated to these methods remain to be investigated. In this work we present an improved high-throughput quantitative proteomics method based on whole proteome concentration by SDS-PAGE, optimized in-gel digestion, peptide 18O-labeling, and separation by off-gel isoelectric focusing followed by liquid chromatography-LIT-MS. We demonstrate that the off-gel technique is fully compatible with 18O peptide labeling in any pH range. A recently developed statistical model indicated that partial digestions and methionine oxidation do not alter protein quantification and that variances at the scan, peptide, and protein levels are stable and reproducible in a variety of proteomes of different origin. We have also analyzed the dynamic range of quantification and demonstrated the practical utility of the method by detecting expression changes in a model of activation of Jurkat T-cells. Our protocol provides a general approach to perform quantitative proteomics by 18O-labeling in high-throughput studies, with the added value that it has a validated statistical model for the null hypothesis. To the best of our knowledge, this is the first report where a general protocol for stable isotope labeling is tested in practice using a collection of samples and analyzed at this degree of statistical detail.

Blockade of NFAT activation by the second calcineurin-binding site

Activation of NFAT transcription factors requires their dephosphorylation by the phosphatase calcineurin (CN). NFATs contain two CN binding motifs: PxIxIT and CnBP-B/CNBR2 (which we call LxVP). Here we carry out a detailed comparative analysis of the CN binding activity displayed by the PxIxIT and LxVP sites from different NFATs. Dose-response CN binding experiments with GST fusion proteins of NFATc1 and NFATc2 showed that NFATc1 binds CN in vitro more efficiently than does NFATc2. This difference in binding appears to be caused by the different CN binding potencies of the corresponding LxVP sites; thus while the LxVPc2 peptide fused to GST did not bind CN, GST-LxVPc1 bound it more efficiently than did GST-PxIxITc1 or GST-PxIxITc2. Furthermore, an NFATc2 chimera protein containing the LxVP motif from NFATc1 interacted with CN much more potently than did wild-type NFATc2. Free peptides spanning the LxVP motifs from NFATc1, c3 or c4 displaced CN from GST-NFATc1 and GST-NFATc2 more efficiently than any PxIxIT peptide. PxIxITc2 and LxVPc1 peptides were each able to cross-compete GST-LxVPc1-CN and GST-PxIxITc2-CN binding. In contrast with PxIxITc2, the LxVP peptide not only blocked CN-NFAT binding but also inhibited CN phosphatase activity in vitro. Furthermore, exogenous LxVPc1 blocked NFATc2 phosphorylation and nuclear translocation in vivo. These results suggest a model in which the different CN binding characteristics of the PxIxIT and LxVP sites enable different NFAT members to influence each others activities in cells where they are co-expressed.

Blockade of T-cell activation by dithiocarbamates involves novel mechanisms of inhibition of nuclear factor of activated T cells.

Dithiocarbamates (DTCs) have recently been reported as powerful inhibitors of NF-kappaB activation in a number of cell types. Given the role of this transcription factor in the regulation of gene expression in the inflammatory response, NF-kappaB inhibitors have been suggested as potential therapeutic drugs for inflammatory diseases. We show here that DTCs inhibited both interleukin 2 (IL-2) synthesis and membrane expression of antigens which are induced during T-cell activation. This inhibition, which occurred with a parallel activation of c-Jun transactivating functions and expression, was reflected by transfection experiments at the IL-2 promoter level, and involved not only the inhibition of NF-kappaB-driven reporter activation but also that of nuclear factor of activated T cells (NFAT). Accordingly, electrophoretic mobility shift assays (EMSAs) indicated that pyrrolidine DTC (PDTC) prevented NF-kappaB, and NFAT DNA-binding activity in T cells stimulated with either phorbol myristate acetate plus ionophore or antibodies against the CD3-T-cell receptor complex and simultaneously activated the binding of AP-1. Furthermore, PDTC differentially targeted both NFATp and NFATc family members, inhibiting the transactivation functions of NFATp and mRNA induction of NFATc. Strikingly, Western blotting and immunocytochemical experiments indicated that PDTC promoted a transient and rapid shuttling of NFATp and NFATc, leading to their accelerated export from the nucleus of activated T cells. We propose that the activation of an NFAT kinase by PDTC could be responsible for the rapid shuttling of the NFAT, therefore transiently converting the sustained transactivation of this transcription factor that occurs during lymphocyte activation, and show that c-Jun NH2-terminal kinase (JNK) can act by directly phosphorylating NFATp. In addition, the combined inhibitory effects on NFAT and NF-KB support a potential use of DTCs as immunosuppressants.

Cooperative Synergy between NFAT and MyoD Regulates Myogenin Expression and Myogenesis

Calcineurin/NFAT signaling is involved in multiple aspects of skeletal muscle development and disease. The myogenic basic helix-loop-helix transcription factors, MyoD, myogenin, Myf5, and MRF4 specify the myogenic lineage. Here we show that calcineurin/NFAT (nuclear factor of activated T cells) signaling is required for primary myogenesis by transcriptional cooperation with the basic helix-loop-helix transcription factor MyoD. Calcineurin/NFAT signaling is involved in myogenin expression in differentiating myoblasts, where the myogenic regulatory factor MyoD synergistically cooperates with NFATc2/c3 at the myogenin promoter. Using gel shift and chromatin immunoprecipitation assays, we identified two conserved NFAT binding sites in the myogenin promoter that were occupied by NFATc3 upon skeletal muscle differentiation. The transcriptional integration between NFATc3 and MyoD is crucial for primary myogenesis in vivo, as myogenin expression is weak in myod:nfatc3 double null embryos, whereas myogenin expression is unaffected in embryos with null mutations for either factor alone. Thus, the combined findings provide a novel transcriptional paradigm for the first steps of myogenesis, where a calcineurin/NFATc3 pathway regulates myogenin induction in cooperation with MyoD during myogenesis.

ANTIOXIDANTS AND AP-1 ACTIVATION : A BRIEF OVERVIEW

Activity of the transcription factor AP-1 is controlled by different MAPK cascades that regulate the different AP-1 components at the transcriptional and posttranscriptional level. Recently, AP-1 has been shown to behave as a redox-sensitive transcription factor that can be induced under both pro-oxidative and antioxidative conditions. In this overview we summarize the signaling pathways that converge on the activation of AP-1 and the components of these pathways that have been shown to be targets of antioxidants. The activation of AP-1 by antioxidants may account for the expression of a number of genes that mediate important functions under physiological conditions.

The RCAN carboxyl end mediates calcineurin docking-dependent inhibition via a site that dictates binding to substrates and regulators

Specificity of signaling kinases and phosphatases toward their targets is usually mediated by docking interactions with substrates and regulatory proteins. Here, we characterize the motifs involved in the physical and functional interaction of the phosphatase calcineurin with a group of modulators, the RCAN protein family. Mutation of key residues within the hydrophobic docking-cleft of the calcineurin catalytic domain impairs binding to all human RCAN proteins and to the calcineurin interacting proteins Cabin1 and AKAP79. A valine-rich region within the RCAN carboxyl region is essential for binding to the docking site in calcineurin. Although a peptide containing this sequence compromises NFAT signaling in living cells, it does not inhibit calcineurin catalytic activity directly. Instead, calcineurin catalytic activity is inhibited by a motif at the extreme C-terminal region of RCAN, which acts in cis with the docking motif. Our results therefore indicate that the inhibitory action of RCAN on calcineurin-NFAT signaling results not only from the inhibition of phosphatase activity but also from competition between NFAT and RCAN for binding to the same docking site in calcineurin. Thus, competition by substrates and modulators for a common docking site appears to be an essential mechanism in the regulation of Ca2+-calcineurin signaling.