Enric Espel

Post-transcriptional regulation of TNF-α during in vitro differentiation of human monocytes/macrophages in primary culture

Tumor necrosis factor α (TNF‐α), a proinflammatory cytokine, is produced abundantly by monocytes and macrophages. We have compared LPS‐stimulated TNF‐α production and regulation in freshly isolated human monocytes and macrophages differentiated in vitro. A significant increase in LPS‐induced TNF‐α protein secretion was observed in macrophages over freshly isolated monocytes without comparable differences in TNF‐α mRNA induction. Polysome gradient analysis showed polysome‐mRNA distribution did not change, whereas TNF‐α mRNA stability increased in macrophages. Tristetraprolin mRNA expression was constitutive and decreased with differentiation‐linked kinetics. Blockable LPS‐inducible MAP kinase activity (p38, ERK) affected TNF‐α biosynthesis differentially at the transcriptional and post‐transcriptional level throughout the culture period. We suggest that the increase in TNF‐α secretion in macrophages relates to changes in post‐transcriptional processing, which is regulated indirectly by the expression of RNA‐binding proteins. Changes in gene expression throughout monocytic differentiation equip the cell to act as a more potent producer of this proinflammatory cytokine.

NARROW A/T-RICH ZONES PRESENT AT THE DISTAL 5'-FLANKING SEQUENCES OF THE ZEIN GENES ZC1 AND ZC2 BIND A UNIQUE 30 KDA HMG-LIKE PROTEIN

Nuclear extracts from maize endosperm were used to investigate protein-DNA interactions in the 5′-upstream region of the Zc1 and Zc2 genes. These genes encode for zeins of apparent molecular mass (MWapp) 16 and 28 kDa, respectively, which accumulate in the endosperm during seed maturation. Binding assays revealed specific binding of a nuclear protein to three A/T-rich elements, 0.9–1.0 kbp upstream from the initiation codon. One of these elements (41 bp, 88% A/T), present in Zc1, contained a 13 nucleotide duplication. The other two (28 bp, 86% A/T; 42 bp alternating A-T) are consecutive elements in Zc2. Competition experiments strongly suggest that the three elements bind to the same protein. Protein-DNA interaction was detected in endosperm nuclear extracts of 8 to 21 days after pollination (DAP), as well as in 25 DAP embryos and in different tissues from plantlets. The protein factor has an MWapp of ca. 30 kDa. This factor has properties suggesting it is an HMG-like protein. These results are consistent with a growing accumulation of data for a number of genes indicating that A/T-rich elements, located at distal and proximal zones of the 5′-flanking sequences, interact with HMG-like proteins.

The PDZ-binding domain of syndecan-2 inhibits LFA-1 high-affinity conformation.

Syndecans are cell membrane proteoglycans that can modulate the activity and dynamics of some growth factor receptors and integrins. Here, we show the down-regulation of integrin lymphocyte function-associated antigen-1 (LFA-1) and inhibition of adhesion of Jurkat T cells transfected with syndecan-2. The PDZ-binding domain in the cytoplasmic region of syndecan-2 was necessary to block the LFA-1 high-affinity conformation, and to reduce cellular adhesion. A second cytoplasmic motif comprising tyrosines 179 and 191, and serines 187 and 188 contributed also to reduce LFA-1 function and cellular adhesion. Inhibition of the LFA-1 high-affinity conformation by syndecan-2 was independent of the expression of the talin head domain and RhoA, Rac1 and Cdc42 GTPases. These results demonstrate the importance of PDZ-binding domain of syndecan-2 for controlling LFA-1 affinity and cell adhesion.

Integrating signals from T-cell receptor and serum by T cells enhance translation of tumour necrosis factor-α

Tumour necrosis factor‐α (TNF‐α) is a proinflammatory cytokine produced by several cell types, including T cells upon antigen stimulation. Its production is crucial for the development of an early defence against many pathogens, but its beneficial effects are dependent on the strength and duration of its expression. In this paper we present evidence indicating that serum increases translational efficiency of TNF‐α in human peripheral blood mononuclear cells stimulated with superantigen. The increase in translation of TNF‐α due to serum could be inhibited by the phosphatidylinositol (PI) 3‐K inhibitors, wortmannin and LY294002, suggesting that PI 3‐K is involved in the translational control of TNF‐α by serum. Similarly to primary T cells, stimulation of Jurkat T cells with superantigen led to TNF‐α secretion and this was up‐regulated by serum. Transfection of Jurkat cells with a constitutively active form of PI 3‐Kα increased the production of TNF‐α in cells stimulated with superantigen. Additionally, we used the specific inhibitors targeting ERK kinase and p38 mitogen‐activated protein kinase (MAPK), potentially downstream of PI 3‐kinase, PD98059 and SB203580. Differently from with PI 3‐K inhibitors, the accumulation of TNF‐α mRNA was inhibited by PD98059 or SB203580. These results suggest that, in T cells, activation of PI 3‐K is an important step in controlling TNF‐α protein synthesis in response to growth factors.

Dual role of ERK5 in the regulation of T cell receptor expression at the T cell surface

Regulation of the levels of the TCR/CD3 complex at the cell surface is critical to proper T cell development and mature T cell activation. We provide evidence that the MAPK ERK5 regulates the surface expression of the TCR/CD3 complex by controlling the degradation of the CD3ζ chain and the recovery of the complex after anti‐CD3ɛ stimulation. ERK5 knockdown led to TCR/CD3 up‐regulation at the cell surface and increased amounts of the CD3ζ chain. Inhibition of the MEK5‐dependent phosphorylation status of the kinase domain of ERK5 in human T CD4+ cells reduced CD3ζ ubiquitination and degradation, limiting TCR/CD3 down‐regulation in anti‐CD3‐stimulated cells. Moreover, TCR/CD3 recovery at the cell surface, after anti‐CD3ɛ treatment, is impaired by ERK5 knockdown or pharmacological inhibition of autophosphorylation in the ERK5 C‐terminal region. ERK5 loss in thymocytes augmented cellular CD3ζ and increased cell surface levels of TCR/CD3 on CD4+CD8+ thymocytes. This correlated with enhanced generation of CD4+CD8−CD25+ thymocytes. Our findings define ERK5 as a novel kinase that modulates the levels of TCR/CD3 at the cell surface by promoting CD3ζ degradation and TCR/CD3 recovery after TCR stimulation.

Erk5 contributes to maintaining the balance of cellular nucleotide levels and erythropoiesis

An adequate supply of nucleotides is essential for accurate DNA replication, and inappropriate deoxyribonucleotide triphosphate (dNTP) concentrations can lead to replication stress, a common source of DNA damage, genomic instability and tumourigenesis. Here, we provide evidence that Erk5 is necessary for correct nucleotide supply during erythroid development. Mice with Erk5 knockout in the haematopoietic lineage showed impaired erythroid development in bone marrow, accompanied by altered dNTP levels and increased DNA mutagenesis in erythroid progenitors as detected by exome sequencing. Moreover, Erk5-depleted leukemic Jurkat cells presented a marked sensitivity to thymidine-induced S phase stalling, as evidenced by increased H2AX phosphorylation and apoptosis. The increase in thymidine sensitivity correlated with a higher dTTP/dCTP ratio. These results indicate that Erk5 is necessary to maintain the balance of nucleotide levels, thus preventing dNTP misincorporation and DNA damage in proliferative erythroid progenitors and leukemic Jurkat T cells.

Syndecan-2 can promote clearance of T-cell receptor/CD3 from the cell surface

T cells express the heparan sulphate proteoglycans syndecan‐2 and syndecan‐4. Syndecan‐4 plays a T‐cell inhibitory role; however, the function of syndecan‐2 is unknown. In an attempt to examine this function, syndecan‐2 was expressed constitutively in Jurkat T cells. Interestingly, the expression of syndecan‐2 decreased the surface levels of T‐cell receptor (TCR)/CD3 complex, concomitant with intracellular retention of CD3ε and partial degradation of the TCR‐ζ chain. Immunofluorescence microscopy revealed that intracellular CD3ε co‐located with Rab‐4 endosomes. However, the intracellular pool of CD3ε did not recycle to the cell surface. The lower TCR/CD3 surface levels caused by syndecan‐2 led to reduced TCR/CD3 responsiveness. We show that the cytosolic PDZ‐binding domain of syndecan‐2 is not necessary to elicit TCR/CD3 down‐regulation. These results identify a previously unrecognized means of controlling surface TCR/CD3 expression by syndecan‐2.

Neutralization of Measles Virus Infectivity and Antibody-Dependent Cell-Mediated Cytotoxicity Activity against an Epstein-Barr Virus-Infected Cell Line by Intravenous Administration of Immunoglobulin G

ABSTRACT Patients with antibody deficiency disorders are highly susceptible to microbial infections. Intravenous (i.v.) immunoglobulin concentrates were originally developed as replacement therapy for such patients. The present study assesses the measles virus neutralizing antibody titers and the antibody-dependent cell-mediated cytotoxicity (ADCC) capacities against Epstein-Barr virus (EBV)-infected cells of immunoglobulin G (IgG) preparations produced for i.v. use (i.v. IgG). The level of neutralizing antibodies against measles virus was determined by a syncytium neutralization test with Vero cells as targets. The measles virus neutralizing antibody titers of the i.v. IgG preparations were >3 × 102 and were an average of 1.0 log higher than the titers in pooled plasma from healthy subjects. The two IgG preparations tested showed similar ADCC activities against EBV-infected Raji cells, being active at concentrations of 3 mg/ml or higher. i.v. IgG bound to Raji cells but not to the EBV-negative Ramos cells, as evaluated by flow cytometry. Our in vitro findings may provide further support for the use of i.v. IgG for the prevention and treatment of infections caused by specific viral pathogens.

Role of LFA-1 and ICAM-1 in Cancer

The lymphocyte function-associated antigen-1 (LFA-1) (also known as CD11a/CD18 and αLβ2), is just one of many integrins in the human body, but its significance is derived from its exclusive presence in leukocytes. In this review, we summarize the studies relating LFA-1 and its major ligand ICAM-1 (or CD54) with cancer, through the function of lymphocytes and myeloid cells on tumor cells. We consider how LFA-1 mediates the interaction of leukocytes with tumors and the role of ICAM-1 in tumor dynamics, which can be independent of its interaction with LFA-1. We also offer a more detailed examination of the role of LFA-1 within B-cell chronic lymphocytic leukemia. Finally, we discuss the role that exosomes harboring LFA-1 play in tumor growth and metastasis.

Absence of ERK5/MAPK7 delays tumorigenesis in Atm -/- mice

Ataxia-telangiectasia mutated (ATM) is a cell cycle checkpoint kinase that upon activation by DNA damage leads to cell cycle arrest and DNA repair or apoptosis. The absence of Atm or the occurrence of loss-of-function mutations in Atm predisposes to tumorigenesis. MAPK7 has been implicated in numerous types of cancer with pro-survival and pro-growth roles in tumor cells, but its functional relation with tumor suppressors is not clear. In this study, we show that absence of MAPK7 delays death due to spontaneous tumor development in Atm−/− mice. Compared with Atm−/− thymocytes, Mapk7−/−Atm−/− thymocytes exhibited an improved response to DNA damage (increased phosphorylation of H2AX) and a restored apoptotic response after treatment of mice with ionizing radiation. These findings define an antagonistic function of ATM and MAPK7 in the thymocyte response to DNA damage, and suggest that the lack of MAPK7 inhibits thymic lymphoma growth in Atm−/− mice by partially restoring the DNA damage response in thymocytes.