The nuclear DNA-binding protein DEK is an autoantigen that has been implicated in the regulation of transcription, chromatin architecture, and mRNA processing. We demonstrate here that DEK is actively secreted by macrophages and is also found in synovial fluid samples from patients with juvenile arthritis. Secretion of DEK is modulated by casein kinase 2, stimulated by interleukin-8, and inhibited by dexamethasone and cyclosporine A, consistent with a role as a proinflammatory molecule. DEK is secreted in both a free form and in exosomes, vesicular structures in which transcription-modulating factors such as DEK have not previously been found. Furthermore, DEK functions as a chemotactic factor, attracting neutrophils, CD8؉ T lymphocytes, and natural killer cells. Therefore, the DEK autoantigen, previously described as a strictly nuclear protein, is secreted and can act as an extracellular chemoattractant, suggesting a direct role for DEK in inflammation.DEK is a mammalian oncoprotein and putative autoantigen whose primary biological function has not been previously elucidated, despite literature linking it to the regulation of transcription, chromatin architecture, and mRNA processing (1,2,11,15,16,24,35,51,65). The DEK protein is widely conserved among species and is transcribed at high levels, especially in hematopoietically derived cells (14,(62)(63)(64). DEK was first characterized as part of the protein product of the DEK-CAN fusion oncogene, resulting from a t(6;9) translocation found in a subset of patients with acute myelogenous leukemia (64). DEK is overexpressed in several different malignancies, including melanoma, hepatocellular carcinoma, glioblastoma, retinoblastoma, bladder cancer, T-cell large granular lymphocyte leukemia, and acute myelogenous leukemia independent of the t(6;9) translocation (7,10,19,20,29,30,32,39,64). Although DEK has previously been described as a strictly nuclear protein, DEK autoreactivity has been identified as a major component of the autoantibody profile in patients with juvenile idiopathic arthritis (JIA) and is also seen in patients with other autoimmune diseases (8,21,38,49,54,55). In the studies presented in this paper, we show that the nuclear protein DEK can be actively secreted by inflammatory cells, is found in synovial fluid samples from JIA patients, and can function as a chemoattractant for inflammatory cells, suggesting a potential role for DEK in immunity and/or autoimmunity. MATERIALS AND METHODSCell preparation. Monocyte-derived macrophages (MDM) were prepared as previously described (31). Briefly, heparinized venous blood samples were collected from healthy volunteers following a protocol approved by the institutional review board, and peripheral blood mononuclear cells were separated by FicollHypaque (Amersham Pharmacia Biotech AB, Uppsala, Sweden) density gradient centrifugation. MDM were purified by adherence to plastic for 2 h at 37°C at a concentration of 5 ϫ 10 5 /ml. Adherent cells were consistently Ͼ90% monocytes (31). Adherence-purified human mon...

Human immunodeficiency virus type 2 (HIV-2) gene expression is regulated by upstream promoter elements, including the peri-Ets (pets) site, which mediate enhancer stimulation following treatment with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). We previously showed that the oncoprotein DEK binds to the pets site in a site-specific manner. In this report, we show that binding to the HIV-2 pets site is modulated by treatment of U937 monocytic cells with TPA, an activator of protein kinase C. TPA treatment resulted in a reduction in the levels of DEK and the formation of a faster migrating pets complex in gel shift assays. We show further that the actions of TPA on pets binding can be duplicated by phosphatase treatment of nuclear proteins and is blocked with okadaic acid, a protein phospatase-2A (PP2A) inhibitor. Finally, we demonstrate that ectopic expression of the catalytic domain of PP2A can activate the HIV-2 enhancer/promoter alone or in synergy with TPA, an effect mediated in part through the pets site. These results suggest that, through an interaction with the protein kinase C pathway, PP2A is strongly involved in regulating HIV-2 enhancer-mediated transcription. This is a consequence of its effects on DEK expression and binding to the pets site, as well as its effects on other promoter elements. These findings have implications not only for HIV-2 transcription but also for multiple cellular processes involving DEK or PP2A.

The viral replication rate in patients infected with human immunodeficiency virus type 1 (HIV-1) is controlled in part by regulation of the transcription of viral genes. The rate of transcription is determined by a complex interplay between cellular and viral proteins and the promoter elements found in the long terminal repeats. Protein phosphatase 2A (PP2A) is a phosphoprotein that plays important roles in the regulation of signal transduction and cell growth. In this report, we demonstrate that overexpression of the catalytic subunit of protein phosphatase 2A (PP2Ac) increases the basal activity of the HIV-1 promoter and, especially, enhances the promoter's response to the protein kinase C (PKC) activator 12-O-tetradecanoyl phorbol-13-acetate (PMA). Additionally, ectopic PP2Ac enhances activation of HIV-1 provirus by PMA. Okadaic acid, a potent inhibitor of PP2A, markedly reduces both HIV-1 enhancer and proviral activation. Fostriecin, a PP2A inhibitor which has been used as an antineoplastic agent in clinical trials, is also able to inhibit PMA-stimulated HIV-1 proviral activation. These observations demonstrate a role for the important cellular phosphatase PP2A in HIV-1 transcription and replication and also suggest that PKC can potentiate the activity of PP2A. PP2A is a potential target for therapeutic intervention in patients infected with HIV-1.

T lymphocytes up-regulate the synthesis of ligands for E- and P-selectin during proliferative responses in vivo and in vitro. Previous studies from our laboratories indicated that the α(1,3)-fucosyltransferase FucT-VII regulates the synthesis of E-selectin ligands and sialylated Lewisx-related epitopes (sLex-related epitopes) in human T lymphoblasts. The current report shows that production of both P- and E-selectin ligands is FucT-VII dependent, but peak synthesis of each occurs at different levels of fucosyltransferase activity in intact cells. In brief, FucT-VII mRNA levels were higher in cultured T lymphoblasts expressing sLex-related epitopes and both selectin ligands than in cells expressing P-selectin ligands alone. However, synthesis of the epitopes and both selectin ligands required the FucT-VII enzyme in transfected Molt-4 cells. In contrast, neither constitutive nor transfection-enhanced levels of the FucT-IV enzyme generated active P-selectin ligands in these lines. In addition, targeted deletion of the FucT-VII gene in mice markedly inhibited the synthesis of both P- and E-selectin ligands during blast transformation in vitro. Finally, the optimal synthesis of active P-selectin ligands occurred at lower level of FucT-VII activity than required for synthesis of equally active E-selectin ligands in both cultured T lymphoblasts and FucT-VII transfectants. Consequently, the FucT-VII enzyme is essential for the synthesis of both P- and E-selectin ligands by T lymphoblasts, and its activity determines whether P-selectin ligands are expressed alone or in conjunction with E-selectin ligands and sLex-related epitopes on human T cells.