Diana Mechtcheriakova

Phosphorylation of the Immunomodulatory Drug FTY720 by Sphingosine Kinases

The immunomodulatory drug FTY720 is phosphorylated in vivo, and the resulting FTY720 phosphate as a ligand for sphingosine-1-phosphate receptors is responsible for the unique biological effects of the compound. So far, phosphorylation of FTY720 by murine sphingosine kinase (SPHK) 1a had been documented. We found that, while FTY720 is also phosphorylated by human SPHK1, the human type 2 isoform phosphorylates the drug 30-fold more efficiently, because of a lower Km of FTY720 for SPHK2. Similarly, murine SPHK2 was more efficient than SPHK1a. Among splice variants of the human SPHKs, an N-terminally extended SPHK2 isoform was even more active than SPHK2 itself. Further SPHK superfamily members, namely ceramide kinase and a “SPHK-like” protein, failed to phosphorylate sphingosine and FTY720. Thus, only SPHK1 and 2 appear to be capable of phosphorylating FTY720. Using selective assay conditions, SPHK1 and 2 activities in murine tissues were measured. While activity of SPHK2 toward sphingosine was generally lower than of SPHK1, FTY720 phosphorylation was higher under conditions favoring SPHK2. In human endothelial cells, while activity of SPHK1 toward sphingosine was 2-fold higher than of SPHK2, FTY720 phosphorylation was 7-fold faster under SPHK2 assay conditions. Finally, FTY720 was poorly phosphorylated in human blood as compared with rodent blood, in line with the low activity of SPHK1 and in particular of SPHK2 in human blood. To conclude, both SPHK1 and 2 are capable of phosphorylating FTY720, but SPHK2 is quantitatively more important than SPHK1.

Dimethylfumarate Inhibits TNF-Induced Nuclear Entry of NF-κB/p65 in Human Endothelial Cells

Fumaric acid esters, mainly dimethylfumarate (DMF), have been successfully used to treat psoriasis. Based on previous observations that DMF inhibited expression of several TNF-induced genes in endothelial cells, we wished to explore the molecular basis of DMF function in greater detail. In first experiments we analyzed DMF effects on tissue factor expression in human endothelial cells in culture, because tissue factor is expressed by two independent sets of transcription factors, by NF-κB via TNF and by early gene response-1 transcription factor via vascular endothelial growth factor (VEGF). We show that DMF inhibits TNF-induced tissue factor mRNA and protein expression as well as TNF-induced DNA binding of NF-κB proteins, but not VEGF-induced tissue factor protein, mRNA expression, or VEGF-induced early gene response-1 transcription factor/DNA binding. To determine where DMF interferes with the TNF/NF-κB signaling cascade, we next analyzed DMF effects on IκB and on the subcellular distribution of NF-κB. DMF does not inhibit TNF-induced IκBα phosphorylation and IκB degradation; thus, NF-κB is properly released from IκB complexes even in the presence of DMF. Importantly, DMF inhibits the TNF-induced nuclear entry of NF-κB proteins, and this effect appears selective for NF-κB after the release from IκB, because the constitutive shuttling of inactive NF-κB/IκB complexes into and out from the nucleus is not blocked by DMF. Moreover, DMF does not block NF-κB/DNA binding. In conclusion, DMF appears to selectively prevent the nuclear entry of activated NF-κB, and this may be the basis of its beneficial effect in psoriasis.

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)

FTY720 Rescue Therapy in the Dark Agouti Rat Model of Experimental Autoimmune Encephalomyelitis: Expression of Central Nervous System Genes and Reversal of Blood‐Brain‐Barrier Damage

FTY720 (fingolimod) is an oral sphingosine‐1 phosphate (S1P) receptor modulator in phase III development for the treatment of multiple sclerosis. To further investigate its mode of action, we analyzed gene expression in the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE). FTY720 downregulated inflammatory genes in addition to vascular adhesion molecules. It decreased the matrix metalloproteinase gene MMP‐9 and increased its counterregulator—tissue inhibitor of metalloproteinase, TIMP‐1—resulting in a proteolytic balance that favors preservation of blood‐brain‐barrier (BBB) integrity. Furthermore, FTY720 reduced S1P lyase that increases the S1P concentration in the brain, in line with a marked reversal of neurological deficits and raising the possibility for enhanced triggering of S1P receptors on resident brain cells. This is accompanied by an increase in S1P1 and S1P5 in contrast with the attenuation of S1P3 and S1P4. Late‐stage rescue therapy with FTY720, even up to 1 month after EAE onset, reversed BBB leakiness and reduced demyelination, along with normalization of neurologic function. Our results indicate rapid blockade of ongoing disease processes by FTY720, and structural restoration of the CNS parenchyma, which is likely caused by the inhibition of autoimmune T cell infiltration and direct modulation of microvascular and/or glial cells.

Oxidized Phospholipids Stimulate Angiogenesis Via Autocrine Mechanisms, Implicating a Novel Role for Lipid Oxidation in the Evolution of Atherosclerotic Lesions

Angiogenesis is a common feature observed in advanced atherosclerotic lesions. We hypothesized that oxidized phospholipids (OxPLs), which accumulate in atherosclerotic vessels can stimulate angiogenesis. We found that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) stimulated the formation of sprouts from endothelial cell spheroids and promoted growth of capillaries into Matrigel plugs in mice. OxPLs stimulated expression of vascular endothelial growth factor (VEGF) in vivo and in several normal and tumor cell types in vitro. In addition, OxPAPC upregulated cyclooxygenase (COX)-2 and interleukin (IL)-8. COX-2 inhibitors, as well as blocking antibodies to IL-8 suppressed activation of sprouting by OxPAPC. We conclude that OxPAPC stimulates angiogenesis via autocrine mechanisms involving VEGF, IL-8, and COX-2–generated prostanoids. Our data suggest that accumulation of OxPLs may contribute to increased growth of blood capillaries in advanced lesions, thus leading to progression and destabilization of atherosclerotic plaques.

Early activation of sphingosine kinase in mast cells and recruitment to FcεRI are mediated by its interaction with Lyn kinase

ABSTRACT Sphingosine kinase has been recognized as an essential signaling molecule that mediates the intracellular conversion of sphingosine to sphingosine-1-phosphate. In mast cells, induction of sphingosine kinase and generation of sphingosine-1-phosphate have been linked to the initial rise in Ca2+, released from internal stores, and to degranulation. These events either precede or are concomitant with the activation of phospholipase C-γ and the generation of inositol trisphosphate. Here we show that sphingosine kinase type 1 (SPHK1) interacts directly with the tyrosine kinase Lyn and that this interaction leads to the recruitment of this lipid kinase to the high-affinity receptor for immunoglobulin E (FcεRI). The interaction of SPHK1 with Lyn caused enhanced lipid and tyrosine kinase activity. After FcεRI triggering, enhanced sphingosine kinase activity was associated with FcεRI in sphingolipid-enriched rafts of mast cells. Bone marrow-derived mast cells from Lyn−/ − mice, compared to syngeneic wild-type cells, were defective in the initial induction of SPHK1 activity, and the defect was overcome by retroviral Lyn expression. These findings position the activation of SPHK1 as an FcεRI proximal event.

Characterisation of an engineered trastuzumab IgE antibody and effector cell mechanisms targeting HER2/neu-positive tumour cells

Trastuzumab (Herceptin®), a humanized IgG1 antibody raised against the human epidermal growth factor receptor 2 (HER2/neu), is the main antibody in clinical use against breast cancer. Pre-clinical evidence and clinical studies indicate that trastuzumab employs several anti-tumour mechanisms that most likely contribute to enhanced survival of patients with HER2/neu-positive breast carcinomas. New strategies are aimed at improving antibody-based therapeutics like trastuzumab, e.g. by enhancing antibody-mediated effector function mechanisms. Based on our previous findings that a chimaeric ovarian tumour antigen-specific IgE antibody showed greater efficacy in tumour cell killing, compared to the corresponding IgG1 antibody, we have produced an IgE homologue of trastuzumab. Trastuzumab IgE was engineered with the same light- and heavy-chain variable-regions as trastuzumab, but with an epsilon in place of the gamma-1 heavy-chain constant region. We describe the physical characterisation and ligand binding properties of the trastuzumab IgE and elucidate its potential anti-tumour activities in functional assays. Both trastuzumab and trastuzumab IgE can activate monocytic cells to kill tumour cells, but they operate by different mechanisms: trastuzumab functions in antibody-dependent cell-mediated phagocytosis (ADCP), whereas trastuzumab IgE functions in antibody-dependent cell-mediated cytotoxicity (ADCC). Trastuzumab IgE, incubated with mast cells and HER2/neu-expressing tumour cells, triggers mast cell degranulation, recruiting against cancer cells a potent immune response, characteristic of allergic reactions. Finally, in viability assays both antibodies mediate comparable levels of tumour cell growth arrest. These functional characteristics of trastuzumab IgE, some distinct from those of trastuzumab, indicate its potential to complement or improve upon the existing clinical benefits of trastuzumab.

Neutropenia with impaired immune response to Streptococcus pneumoniae in ceramide kinase-deficient mice.

In mammals, ceramide kinase (CerK)-mediated phosphorylation of ceramide is the only known pathway to ceramide-1-phosphate (C1P), a recently identified signaling sphingolipid metabolite. To help delineate the roles of CerK and C1P, we knocked out the gene of CerK in BALB/c mice by homologous recombination. All in vitro as well as cell-based assays indicated that CerK activity is completely abolished in Cerk−/− mice. Labeling with radioactive orthophosphate showed a profound reduction in the levels of de novo C1P formed in Cerk−/− macrophages. Consistently, mass spectrometry analysis revealed a major contribution of CerK to the formation of C16-C1P. However, the significant residual C1P levels in Cerk−/− animals indicate that alternative routes to C1P exist. Furthermore, serum levels of proapoptotic ceramide in these animals were significantly increased while levels of dihydroceramide as the biosynthetic precursor were reduced. Previous literature pointed to a role of CerK or C1P in innate immune cell function. Using a variety of mechanistic and disease models, as well as primary cells, we found that macrophage- and mast cell-dependent readouts are barely affected in the absence of CerK. However, the number of neutrophils was strikingly reduced in blood and spleen of Cerk−/− animals. When tested in a model of fulminant pneumonia, Cerk−/− animals developed a more severe disease, lending support to a defect in neutrophil homeostasis following CerK ablation. These results identify ceramide kinase as a key regulator of C1P, dihydroceramide and ceramide levels, with important implications for neutrophil homeostasis and innate immunity regulation.

The isoprostane 8-iso-PGF2α stimulates endothelial cells to bind monocytes: differences from thromboxane-mediated endothelial activation

Increased isoprostane levels were found in patients with vascular disease and in atherosclerotic lesions. Isoprostanes interact with vascular cells via thromboxane receptors (TPs) and block TP‐ reduced atherosclerosis in apoE‐deficient mice. This effect was, however, independent of thromboxane formation, suggesting involvement of isoprostanes. We investigated the stimulation of human endothelial cells (ECs) by the isoprostane 8‐iso‐PGF2α with respect to induced monocyte adhesion, a key initiating event in atherogenesis. The isoprostane 8‐iso‐PGF2α stimulated ECs to specifically bind monocytes. The TP agonist U46619 induced monocyte and neutrophil binding and expression of E‐selectin and vascular cell adhesion molecule‐1 (VCAM‐ 1). The monocyte adhesion induced by 8‐iso‐PGF2α was independent of VCAM‐1 and could be blocked by a PKA (H89) and a MEK‐1 inhibitor (PD98059), whereas U46619‐induced monocyte adhesion was mediated by VCAM‐1 and could be blocked by a PKC inhibitor (bisindolylmaleimide I). The effects of 8‐iso‐PGF2α and U46619 could be blocked by a TP antagonist and did not involve NF‐MB translocation but required activation of p38MAPK. Our data demonstrate that 8‐iso‐PGF2α induces monocyte adhesion in ECs by activating TPs or a TP‐ related receptor but via signaling mechanisms that are critically different from those activated by U46619. Therefore, generation of 8‐iso‐PGF2α may induce atherosclerotic lesion formation by activation of ECs to bind monocytes.

Sustained Expression of Early Growth Response Protein-1 Blocks Angiogenesis and Tumor Growth

Transient induction of the transcription factor early growth response protein-1 (EGR-1) plays a pivotal role in the transcriptional response of endothelial cells to the angiogenic growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which are produced by most tumors and are involved in the angiogenic switch. We report here that sustained expression of EGR-1 by recombinant adenoviruses in endothelial cells, however, leads to the specific induction of potent feedback inhibitory mechanisms, including strong up-regulation of transcriptional repressors, negative cell cycle check point effectors, proteins with established antiangiogenic activity, and several proapoptotic genes. Sustained EGR-1 expression consistently leads to an antiangiogenic state characterized by an altered responsiveness to VEGF and bFGF and a striking inhibition of sprouting and tubule formation in vitro. Furthermore, EGR-1-expressing viruses potently inhibit cell invasion and vessel formation in the murine Matrigel model and repress tumor growth in a murine fibrosarcoma model. We propose that gene therapy involving sustained EGR-1 expression may constitute a novel therapeutic principle in the treatment of cancer due to the simultaneous induction of multiple pathways of antiangiogenesis, growth arrest, and apoptosis induction in proliferating cells leading to preferential inhibition of angiogenesis and tumor growth.