Nirit Mor-Vaknin

Vimentin is secreted by activated macrophages

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 GÖ6983. 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 α (TNF-α) 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.

DEK Is a Poly(ADP-Ribose) Acceptor in Apoptosis and Mediates Resistance to Genotoxic Stress

ABSTRACT 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 DEKs autoantigenic properties and in its function as a promoter of cell survival.

The Endothelial Cell-Specific Antibody PAL-E Identifies a Secreted Form of Vimentin in the Blood Vasculature

ABSTRACT 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.

The DEK Nuclear Autoantigen Is a Secreted Chemotactic Factor

ABSTRACT 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.

Effect of dietary fat on early morphological intestinal adaptation in a rat with short bowel syndrome

Among factors promoting mucosal hyperplasia after bowel resection, long-chain fatty acids may have a special role. The purpose of the present study was to evaluate the effects of high-fat diet (HFD) on early intestinal adaptation in rats with short bowel syndrome (SBS). Male Sprague-Dawley rats underwent either a bowel transection with re-anastomosis (Sham rats) or 75% small bowel resection (SBS rats). Animals were randomly assigned to one of three groups: Sham rats fed normal chow (Sham-NC); SBS rats fed NC (SBS-NC); and SBS rats fed HFD (SBS-HFD). Rats were killed on days 3 or 14. Body weight and parameters of intestinal adaptation (overall bowel and mucosal weight, mucosal DNA and protein, villus height, and crypt depth) were determined at time of killing. By day 3, SBS-HFD rats demonstrated higher duodenal and jejunal bowel and mucosal weights and ileal villus height and jejunal crypt depth vs SBS-NC rats. By day 14 SBS-HFD rats continued to demonstrate increased duodenal and jejunal bowel weight and duodenal mucosal weight vs SBS-NC animals. We conclude that early exposure to HFD both augmented and accelerated structural bowel adaptation in a rat model of SBS.

DEK in the synovium of patients with juvenile idiopathic arthritis: characterization of DEK antibodies and posttranslational modification of the DEK autoantigen.

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.

Leukocytic promotion of prostate cellular proliferation

Histological evidence of pervasive inflammatory infiltrate has been noted in both benign prostatic hyperplasia/hypertrophy (BPH) and prostate cancer (PCa). Cytokines known to attract particular leukocyte subsets are secreted from prostatic stroma consequent to aging and also from malignant prostate epithelium. Therefore, we hypothesized that leukocytes associated with either acute or chronic inflammation attracted to the prostate consequent to aging or tumorigenesis may promote the abnormal cellular proliferation associated with BPH and PCa.

Murine Colitis is Mediated by Vimentin

Vimentin, an abundant intermediate filament protein, presumably has an important role in stabilizing intracellular architecture, but its function is otherwise poorly understood. In a vimentin knockout (Vim KO) mouse model, we note that Vim KO mice challenged with intraperitoneal Escherichia coli control bacterial infection better than do wild-type (WT) mice. In vitro, Vim KO phagocytes show significantly increased capacity to mediate bacterial killing by abundant production of reactive oxygen species (ROS) and nitric oxides, likely due to interactions with the p47phox active subunit of NADPH oxidase. In acute colitis induced by dextran sodium sulfate (DSS), Vim KO mice develop significantly less gut inflammation than do WT mice. Further, Vim KO mice have markedly decreased bacterial extravasation in the setting of DSS-induced acute colitis, consistent with decreased intestinal disease. Our results suggest that vimentin impedes bacterial killing and production of ROS, thereby contributing to the pathogenesis of acute colitis.

Intercellular trafficking of the nuclear oncoprotein DEK

DEK is a biochemically distinct, conserved nonhistone protein that is vital to global heterochromatin integrity. In addition, DEK can be secreted and function as a chemotactic, proinflammatory factor. Here we show that exogenous DEK can penetrate cells, translocate to the nucleus, and there carry out its endogenous nuclear functions. Strikingly, adjacent cells can take up DEK secreted from synovial macrophages. DEK internalization is a heparan sulfate-dependent process, and cellular uptake of DEK into DEK knockdown cells corrects global heterochromatin depletion and DNA repair deficits, the phenotypic aberrations characteristic of these cells. These findings thus unify the extracellular and intracellular activities of DEK, and suggest that this paracrine loop involving DEK plays a role in chromatin biology.

Effect of dietary fat on fat absorption and concomitant plasma and tissue fat composition in a rat model of short bowel syndrome

The aim of this study was to investigate the effect of dietary fat on the time course of changes in fat absorption and tissue and plasma lipid composition in a rat model of short bowel syndrome (SBS). Male Sprague-Dawley rats underwent either a bowel transection with re-anastomosis (Sham rats) or 75% small bowel resection (SBS rats). Animals were randomly assigned to one of three groups: Sham rats fed normal chow (Sham-NC), SBS rats fed normal chow (SBS-NC), or SBS rats fed a high-fat diet (SBS-HFD). Rats were sacrificed on day 3 or 14. Body weight, food intake, food clearance (dry fecal mass), and fat clearance (total fecal fat) were measured twice a week. Fat and energy intakes were calculated according to the amount of ingested food. Food and fat absorbability were calculated as intake minus clearance and were expressed as percent of intake. Serum cholesterol, triglyceride, and albumin were measured. Total lipid composition of the liver, epididymal adipose tissue, and the small intestine was determined. Statistical analysis was performed by a Student’s test, with p values <0.05 considered significant. Both food and fat absorbability diminished after bowel resection in rats fed NC. This was accompanied by a decrease in body weight gain, plasma triglyceride and protein levels, and total lipid content of the liver at day 3 and of a decrease in adipose tissue at day 14 following operation. SBS-HFD rats experienced a significant increase ( p <0.05) in food absorbability after 7 days and fat absorbability after 3 days compared with Sham-NC and SBS-NC rats ( p <0.05), as well as increases in serum cholesterol, triglycerides, and glucose compared with SBS-NC rats. On day 14, plasma lipid levels in SBS-HFD rats were not different from SBS-NC or control rats; however, albumin levels were higher. A high-fat diet increased total fat content of the liver early after operation. In conclusion, in a rat model of SBS, an early high-fat diet increased the absorptive capacity of the intestinal remnant as seen by increased food and fat absorbability. These findings suggest a benefit of a high-fat diet on intestinal adaptation in general and on lipid absorption in particular.