Bernd R. Binder

Protective role of phospholipid oxidation products in endotoxin-induced tissue damage

Lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria, interacts with LPS-binding protein and CD14, which present LPS to toll-like receptor 4 (refs 1, 2), which activates inflammatory gene expression through nuclear factor κB (NFκB) and mitogen-activated protein-kinase signalling. Antibacterial defence involves activation of neutrophils that generate reactive oxygen species capable of killing bacteria; therefore host lipid peroxidation occurs, initiated by enzymes such as NADPH oxidase and myeloperoxidase. Oxidized phospholipids are pro-inflammatory agonists promoting chronic inflammation in atherosclerosis; however, recent data suggest that they can inhibit expression of inflammatory adhesion molecules. Here we show that oxidized phospholipids inhibit LPS-induced but not tumour-necrosis factor-α-induced or interleukin-1β-induced NFκB-mediated upregulation of inflammatory genes, by blocking the interaction of LPS with LPS-binding protein and CD14. Moreover, in LPS-injected mice, oxidized phospholipids inhibited inflammation and protected mice from lethal endotoxin shock. Thus, in severe Gram-negative bacterial infection, endogenously formed oxidized phospholipids may function as a negative feedback to blunt innate immune responses. Furthermore, identified chemical structures capable of inhibiting the effects of endotoxins such as LPS could be used for the development of new drugs for treatment of sepsis.

Signaling Molecules of the NF-κB Pathway Shuttle Constitutively between Cytoplasm and Nucleus

We aimed to investigate the dynamics of the NF-κB signaling pathway in living cells using GFP variants of p65-NF-κB, IκBα, tumor necrosis factor-receptor associated factor 2 (TRAF2), the NF-κB inducing kinase (NIK) and IκB kinases (IKK1 and IKK2). Detailed kinetic analysis of constitutive nucleocytoplasmic shuttling processes revealed that IκBα enters the nucleus faster than p65. Examination of signaling molecules upstream of NF-κB and IκBα revealed a predominant cytoplasmic localization at steady state. However, after addition of leptomycin B, NIK rapidly accumulated in the nucleus, whereas we could not detect any significant effect on TRAF2 or IKK2. Using various truncation mutants of NIK, we identified a functional nuclear export signal within the COOH-terminal region 795–805, which counteracts the inherent NLS at amino acids 143–149. Prolonged incubation in the presence of LMB also leads to nuclear accumulation of IKK1, which was dependent on a lysine residue at position 44, which is also essential for kinase activity. Investigation of endogenous protein levels by immunofluorescence staining and Western blots verified the results obtained with GFP chimeras. We conclude that NF-κB·IκB complexes and the upstream signaling kinases NIK and IKK1 shuttle between cytoplasm and nucleus of nonactivated cells and that this process leads to a basal transcriptional activity of NF-κB.

Activation of NF-κB by XIAP, the X chromosome-linked inhibitor of apoptosis, in endothelial cells involves TAK1

Exposure of endothelial and many other cell types to tumor necrosis factor α generates both apoptotic and anti-apoptotic signals. The anti-apoptotic pathway leads to activation of the transcription factor NF-κB that regulates the expression of genes such as A20 or members of the IAP gene family that protect cells from tumor necrosis factor α-mediated apoptosis. In turn, some anti-apoptotic genes have been shown to modulate NF-κB activity. Here we demonstrate that XIAP, a NF-κB-dependent member of the IAP gene family, is a strong stimulator of NF-κB. Expression of XIAP leads to increased nuclear translocation of the p65 subunit of NF-κB via a novel signaling pathway that involves the mitogen-activated protein kinase kinase kinase TAK1. We show that TAK1 physically interacts with NIK and with IKK2, and both XIAP or active TAK1 can stimulate IKK2 kinase activity. Thus, XIAP may be part of a system of regulatory loops that balance a cells response to environmental stimuli.

uPAR – uPA – PAI-1 interactions and signaling: A vascular biologist’s view

The urokinase-type plasminogen activator (uPA), its inhibitor PAI-1 and its cellular receptor (uPAR), play a pivotal role in pericellular proteolysis. In addition, through their interactions with extracellular matrix proteins as well as with transmembrane receptors and other links to the intracellular signaling machinery, they modulate cell migration, cell-matrix interactions and signaling pathways. A large body of experimental evidence from in-vitro and in-vivo data as well as from the clinics indicates an important role of the uPA-uPAR-PAI-1 systems in cancer. In addition to their role in tumor cell biology, the uPA-uPAR-PAI-1 systems are also important for vascular biology by modulating angiogenesis and by altering migration of smooth muscle cells and fibrin deposition in atherosclerosis and restenosis. This review will focus on the general mechanism of uPAR/uPA/PAI-1 interactions and signaling and the possible relevance of this system in vascular biology.

Oxidized phospholipids induce expression of human heme oxygenase-1 involving activation of cAMP-responsive element-binding protein.

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation, protects against oxidative stress, and shows potent anti-inflammatory effects. Oxidized phospholipids, which are generated during inflammation and apoptosis, modulate the inflammatory response by inducing the expression of several genes including HO-1. Here we investigated the signaling pathways and transcriptional events involved in the induction of HO-1 gene expression by oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) in human umbilical vein endothelial cells. OxPAPC up-regulated HO-1 mRNA and protein in a time- and concentration-dependent manner, whereas pro-inflammatory agents like TNF-α and lipopolysaccharide did not significantly induce HO-1 expression in human umbilical vein endothelial cells. Signaling pathways involved in the OxPAPC-mediated HO-1 induction included protein kinases A and C, as well as the mitogen-activated protein kinases p38 and ERK. The cAMP-responsive element-binding protein (CREB) was phosphorylated via these pathways in response to OxPAPC treatment and expression of a dominant-negative mutant of CREB inhibited OxPAPC-induced activity of a human heme oxygenase-1 promoter-driven luciferase reporter construct. We identified a cAMP-responsive element and a Maf recognition element to be involved in the transcriptional activation of the HO-1 promoter by OxPAPC. In gel shift assays we observed binding of CREB to the cAMP-responsive element after OxPAPC treatment. Induction of HO-1 expression by lipid oxidation products via CREB may represent a feedback mechanism to limit inflammation and associated tissue damage.

Specificity, diversity, and convergence in VEGF and TNF-α signaling events leading to tissue factor up-regulation via EGR-1 in endothelial cells

Tissue factor (TF) has been shown to be up‐regulated in endothelial cells by the inflammatory cytokine tumor necrosis factor a (TNF‐α) as well as by the main angiogenic factor VEGF. Since both stimuli induce the transcription factor EGR‐1, which is critically involved in TF gene regulation, we used EGR‐1‐dependent TF induction as a model to identify potential cross‐talks between the various signal transduction cascades initiated by VEGF and TNF‐α. The data show that at the MAP kinase level, VEGF mainly activates ERK1/2 and p38 MAP kinases in human endothelial cells. TNF‐α is able to activate all three MAP kinase cascades as well as the classical inflammatory IκB/NFκB pathway. Furthermore, the MEK/ERK module of MAP kinases appears to act as the convergence point of VEGF‐ and TNF‐α‐initiated signaling cascades, which lead to the activation of EGR‐1 and subsequent TF expression, whereas the upstream signals are distinct. We found that induction of TF by VEGF via EGR‐1 is strongly PKC dependent. The TNF‐α‐initiated MEK/ERK cascade connected to EGR‐1 and TF expression is clearly less sensitive to PKC inhibition. TNF‐α‐mediated activation of MEK/ERK and EGR‐1 can be blocked by adenoviral expression of a dominant negative mutant of IKK2, whereas the VEGF signaling pathway is unaffected. Thus, our data demonstrate a new link between the classical inflammatory IKK/IκB and the MEK/ERK cascades triggered by TNF‐α. The additional finding that EGF induces ERK and EGR‐1 in a PKC independent manner and that this signal is not sufficient to up‐regulate TF emphasizes the importance of a VEGF‐specific signaling pattern for the induction of TF.— Mechtcheriakova, D., Schabbauer, G., Lucerna, M., Clauss, M., de Martin, R., Binder, B. R., Hofer, E. Specificity, diversity, and convergence in VEGF and TNF‐α signaling events leading to tissue factor up‐regulation via EGR‐1 in endothelial cells. FASEB J. 15, 230–242 (2001)

Expression of Heme Oxygenase-1 in Human Vascular Cells Is Regulated by Peroxisome Proliferator-Activated Receptors

Objective—Activation of peroxisome proliferator-activated receptors (PPARs) by lipid-lowering fibrates and insulin-sensitizing thiazolidinediones inhibits vascular inflammation, atherosclerosis, and restenosis. Here we investigate if the vasculoprotective and anti-inflammatory enzyme heme oxygenase-1 (HO-1) is regulated by PPAR ligands in vascular cells. Methods and Results—We show that treatment of human vascular endothelial and smooth muscle cells with PPAR ligands leads to expression of HO-1. Analysis of the human HO-1 promoter in transient transfection experiments together with mutational analysis and gel shift assays revealed a direct transcriptional regulation of HO-1 by PPAR&agr; and PPAR&ggr; via 2 PPAR responsive elements. We demonstrate that a clinically relevant polymorphism within the HO-1 promoter critically influences its transcriptional activation by both PPAR isoforms. Moreover, inhibition of HO-1 enzymatic activity reversed PPAR ligand-mediated inhibition of cell proliferation and expression of cyclooxygenase-2 in vascular smooth muscle cells. Conclusion—We demonstrate that HO-1 expression is transcriptionally regulated by PPAR&agr; and PPAR&ggr;, indicating a mechanism of anti-inflammatory and antiproliferative action of PPAR ligands via upregulation of HO-1. Identification of HO-1 as a target gene for PPARs provides new strategies for therapy of cardiovascular diseases and a rationale for the use of PPAR ligands in the treatment of other chronic inflammatory diseases.

Urokinase Receptor Is Associated with the Components of the JAK1/STAT1 Signaling Pathway and Leads to Activation of This Pathway upon Receptor Clustering in the Human Kidney Epithelial Tumor Cell Line TCL-598

The urokinase-type plasminogen activator (uPA) binds to cells via a specific receptor attached to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Despite the lack of a transmembrane domain, the urokinase receptor (uPAR) is capable of transducing extracellular signals affecting growth, migration, and adhesion. Several Tyr kinases of the src family as well as β1, β2, and β3 integrins were found to be associated with the uPAR. We found that in the human kidney epithelial line TCL-598, also components of the JAK1/STAT1 signal transduction pathway including gp130, are associated with uPAR as revealed by coimmunoprecipitation and are co-localized in caveolae. Upon clustering of uPA·uPAR complex by a monoclonal antibody, JAK1 associates with uPAR, which in turn leads to STAT1 phosphorylation, dimerization, specific binding to DNA, and gene activation. To prove the dependence of STAT1 activation on the uPAR, TCL-598 cells were treated with sense and antisense uPAR oligonucleotides. In antisense-treated cells in which uPAR expression was reduced to less then one third, activation of STAT1 by the clustering antibody was abolished while STAT1 activation by interferon-γ was unaffected. Therefore, in this cell line, uPA·uPAR also utilizes the JAK1/STAT1 pathway for signaling, and gp130 might be the transmembrane adapter for this signal transduction pathway.

M6P/IGFII‐receptor complexes urokinase receptor and plasminogen for activation of transforming growth factor‐β1

Transforming growth factor‐β1 (TGF‐β1) is a critical cytokine for cell proliferation and differentiation. It is secreted by many cells in a latent pro‐form (LTGF‐β1) from which biologically active TGF‐β1 is released by an in vivo mechanism that is not known. Here we show that the mannose‐6‐phosphate/insulin‐like growth factor II‐receptor (M6P/IGFII‐R), which binds LTGF‐β1, complexes with urokinase (plasminogen activator)‐receptor (uPA‐R) on the surface of human monocytes and directly binds plasminogen (Plg). Plasmin generated from Plg in the complex mediates release of TGF‐β1 when M6P/IGFII‐R is associated with uPA‐R. Thus, this interaction of M6P/IGFII‐R and uPA‐R suggests a potential mechanism for the generation of TGF‐β1 by cells.

Antiinflammatory activity of astragaloside IV is mediated by inhibition of NF-κB activation and adhesion molecule expression

The regulated expression of adhesion molecules on the surface of endothelial cells is a key process in the pathogenesis of inflammation. The saponin astragaloside IV (AS-IV), a 3-O-beta-D-xylopyranosyl-6-O-beta-D-glucopyranosylcycloastragenol purified from the Chinese medical herb Astragalus membranaceus (Fisch) Bge. has been shown to have anti-inflammatory effects in vivo. In this study we have investigated the effect of AS-IV on cytokine-and LPS-stimulated expression of adhesion molecules in and leukocyte adhesion to endothelial cells. We have demonstrated that AS-IV significantly reduced the adhesion promoting activity of LPS-stimulated HUVECs for polymorph-nuclear leukocytes (PMNs) and the monocytic cell line THP-1. Furthermore, by using specific cell ELISAs we could show that AS-IV decreased the LPS-induced expression of E-selectin and VCAM-1 on the surface of HUVECs in a dose and time dependent manner, whereas the expression of ICAM-1 was not affected by AS-IV. AS-IV also inhibits TNFalpha-induced VCAM-1 expression. The saponin octyl-D-glucopyranoside had no effect on the LPS-induced expression of E-selectin and VCAM-1 excluding an unspecific detergent-like effect of AS-IV. Moreover, AS-IV significantly inhibited LPS- and TNFalpha-induced specific mRNA levels for E-selectin and VCAM-1. Finally, we could show that AS-IV completely abolished LPS- and TNFalpha-induced nuclear translocation of NF-kappaB and NF-kappaB DNA binding activity in endothelial cells. We conclude that the ability of AS-IV to inhibit the NF-kappaB pathway might be one under-lying mechanism contributing to its anti-inflammatory potential in vivo.