Vimentin is a widely expressed intermediate filament protein thought to be involved mainly in structural processes, such as wound healing. We now demonstrate that activated human macrophages secrete vimentin into the extracellular space. The maturation of blood-derived monocytes into macrophages involves several signalling pathways. We show that secretion of vimentin, which is phosphorylated at serine and threonine residues, is enhanced by the phosphatase inhibitor okadaic acid and blocked by the specific protein kinase C inhibitor GO6983. These findings are consistent with previous observations that phosphorylation of vimentin affects its intracellular localization and that vimentin is a substrate for protein kinase C (PKC). We also show that the anti-inflammatory cytokine interleukin-10 (IL-10), which inhibits PKC activity, blocks secretion of vimentin. In contrast, the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-alpha) can trigger secretion of vimentin. Finally, we found that extracellular vimentin is involved in bacterial killing and the generation of oxidative metabolites, two important functions of activated macrophages. These data establish that vimentin is secreted by macrophages in response to pro-inflammatory signalling pathways and is probably involved in immune function.

The natural product withaferin A (WFA) exhibits antitumor and antiangiogenesis activity in vivo, which results from this drug's potent growth inhibitory activities. Here, we show that WFA binds to the intermediate filament (IF) protein, vimentin, by covalently modifying its cysteine residue, which is present in the highly conserved alpha-helical coiled coil 2B domain. WFA induces vimentin filaments to aggregate in vitro, an activity manifested in vivo as punctate cytoplasmic aggregates that colocalize vimentin and F-actin. WFA's potent dominant-negative effect on F-actin requires vimentin expression and induces apoptosis. Finally, we show that WFA-induced inhibition of capillary growth in a mouse model of corneal neovascularization is compromised in vimentin-deficient mice. These findings identify WFA as a chemical genetic probe of IF functions, and illuminate a potential molecular target for withanolide-based therapeutics for treating angioproliferative and malignant diseases.

DEK is a nuclear phosphoprotein implicated in oncogenesis and autoimmunity and a major component of metazoan chromatin. The intracellular cues that control the binding of DEK to DNA and its pleiotropic functions in DNA-and RNA-dependent processes have remained mainly elusive so far. Our recent finding that the phosphorylation status of DEK is altered during death receptor-mediated apoptosis suggested a potential involvement of DEK in stress signaling. In this study, we show that in cells committed to die, a portion of the cellular DEK pool is extensively posttranslationally modified by phosphorylation and poly(ADP-ribosyl)ation. Through interference with DEK expression, we further show that DEK promotes the repair of DNA lesions and protects cells from genotoxic agents that typically trigger poly(ADP-ribose) polymerase activation. The posttranslational modification of DEK during apoptosis is accompanied by the removal of the protein from chromatin and its release into the extracellular space. Released modified DEK is recognized by autoantibodies present in the synovial fluids of patients affected by juvenile rheumatoid arthritis/juvenile idiopathic arthritis. These findings point to a crucial role of poly(ADP-ribosyl)ation in shaping DEK's autoantigenic properties and in its function as a promoter of cell survival.Human DEK is an abundant and highly conserved nuclear protein that has long been implicated in carcinogenesis and autoimmune disorders (for a review, see references 50 and 60). Originally isolated from a specific subtype of acute myeloid leukemia (55), the gene encoding DEK is expressed under the control of transcription factors E2F and YY1 (10, 49). High levels of DEK support cell immortalization and inhibit both senescence and apoptosis, as shown in cells infected with highrisk human papillomavirus E7 (1,62,63). DEK is also upregulated in a variety of aggressive human tumors, including retinoblastoma, colon and bladder cancer, and melanoma (e.g., see references 10, 19, 28, 30, and 37).In the nucleus, DEK is involved in a variety of DNA-and RNA-dependent processes, such as DNA replication (2), splice site recognition (51), and gene transcription. Here it can function as either an activator (9) or a repressor (16,20,45). The diversity of these effects is in line with DEK's described function as a possible regulator of chromatin architecture, which may affect genome activity at various levels in a highly contextdependent manner. In fact, DEK has been shown in vitro to be a modifier of DNA higher-order structure, acting in concert with topoisomerase I to introduce constrained positive supercoils in closed circular DNA plasmids and simian virus 40 (SV40) minichromosomes (24,25,58,59). Accordingly, DEK was shown to bind to DNA in a structure-specific rather than sequence-specific manner and to reduce the accessibility of chromatin to components of the replication machinery (2, 58). Beyond its effects on DNA topology, DEK can modulate the activity of other chromatin-associated proteins, such as P/CAF and p3...

Type 3 secretion systems (T3SSs) of bacterial pathogens translocate bacterial effector proteins that mediate disease into the eukaryotic cytosol. Effectors traverse the plasma membrane through a translocon pore formed by T3SS proteins. In a genome-wide selection, we identified the intermediate filament vimentin as required for infection by the T3SS-dependent pathogen Shigella flexneri. We found that vimentin is required for efficient T3SS translocation of effectors by S. flexneri and other pathogens that use T3SS, Salmonella Typhimurium and Yersinia pseudotuberculosis. Vimentin and the intestinal epithelial intermediate filament keratin 18 interact with the C-terminus of the Shigella translocon pore protein IpaC. Vimentin and its interaction with IpaC are dispensable for pore formation, but are required for stable docking of S. flexneri to cells; moreover, stable docking triggers effector secretion. These findings establish that stable docking of the bacterium specifically requires intermediate filaments, is a process distinct from pore formation, and is a prerequisite for effector secretion.

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...

During mammalian vascular development, endothelial cells form a complex array of vessels that differ markedly in structure and function, but the molecular basis for this vascular complexity is poorly understood. Recent insights into endothelial diversity have come from the identification of molecular markers expressed on distinct endothelial cell populations. One such marker, the PAL-E antibody, has been used for almost 20 years to distinguish blood and lymphatic vessels, but the identity of the protein recognized by PAL-E has been unknown. In the present study we have used protein purification and tandem mass spectrometry analysis of tryptic peptides to identify the PAL-E antigen as a secreted form of vimentin. Vimentin has been well characterized as an intracellular intermediate filament protein expressed broadly in mesenchymal cells. In contrast, PAL-E-reactive vimentin is secreted extracellularly, its synthesis is restricted to a distinct population of blood endothelial cells and activated macrophages, and PAL-E-reactive vimentin is found in circulating human blood. PAL-E-reactive vimentin does not arise from an endothelial cell-specific mRNA transcript but is the product of cell-specific posttranslational modification. The PAL-E antibody therefore defines secretion of vimentin as a molecular distinction among endothelial cells and exposes a novel, extracellular role for vimentin in the blood vasculature.

Listeria monocytogenes is a facultative intracellular bacterial pathogen that is frequently associated with food-borne infection. Of particular concern is the ability of L. monocytogenes to breach the blood-brain barrier, leading to life-threatening meningitis and encephalitis. The mechanisms used by bacterial pathogens to infect the brain are not fully understood. Here we show that L. monocytogenes is able to utilize vimentin for invasion of host cells. Vimentin is a type III intermediate filament protein within the cytosol but is also expressed on the host cell surface. We found that L. monocytogenes interaction with surface-localized vimentin promoted bacterial uptake. Furthermore, in the absence of vimentin, L. monocytogenes colonization of the brain was severely compromised in mice. The L. monocytogenes virulence factor InlF was found to bind vimentin and was necessary for optimal bacterial colonization of the brain. These studies reveal a novel receptor-ligand interaction that enhances infection of the brain by L. monocytogenes and highlights the importance of surface vimentin in host-pathogen interactions.

Objective. DEK is a nuclear phosphoprotein and autoantigen in a subset of children with juvenile idiopathic arthritis (JIA). Autoantibodies to DEK are also found in a broad spectrum of disorders associated with abnormal immune activation. We previously demonstrated that DEK is secreted by macrophages, is released by apoptotic T cells, and attracts leukocytes.Since DEK has been identified in the synovial fluid (SF) of patients with JIA, this study was undertaken to investigate how DEK protein and/or autoantibodies may contribute to the pathogenesis of JIA.Methods. DEK autoantibodies, immune complexes (ICs), and synovial macrophages were purified from the SF of patients with JIA. DEK autoantibodies and ICs were purified by affinity-column chromatography and analyzed by 2-dimensional gel electrophoresis, immunoblotting, and enzyme-linked immunosorbent assay. DEK in supernatants and exosomes was purified by serial centrifugation and immunoprecipitation with magnetic beads, and posttranslational modifications of DEK were identified by nano-liquid chromatography tandem mass spectrometry (nano-LC-MS/MS).Results. DEK autoantibodies and protein were found in the SF of patients with JIA. Secretion of DEK by synovial macrophages was observed both in a free form and via exosomes. DEK autoantibodies (IgG2) may activate the complement cascade, primarily recognize the C-terminal portion of DEK protein, and exhibit higher affinity for acetylated DEK. Consistent with these observations, DEK underwent acetylation on an unprecedented number of lysine residues, as demonstrated by nano-LC-MS/MS.Conclusion. These results indicate that DEK can contribute directly to joint inflammation in JIA by generating ICs through high-affinity interaction between DEK and DEK autoantibodies, a process enhanced by acetylation of DEK in the inflamed joint.Juvenile idiopathic arthritis (JIA), a polymorphic chronic inflammatory disease of unknown etiology, is