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 K m 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.FTY720 is an immunomodulatory drug, which is highly efficacious in models of transplantation and of autoimmune diseases (1). It was recently found to be effective in kidney transplantation in humans (2). FTY720 elicits a lymphopenia resulting from the reversible redistribution of lymphocytes from the circulation to secondary lymphoid organs, without leading to general immunosuppression (3, 4). Conversion of FTY720 to its monophosphate appears to be essential for the effects of the drug on lymphocyte homing, since FTY720 phosphate acts as an agonist at four of the five G-protein-coupled receptors for sphingosine-1-phosphate (S1P) 1 (5, 6); it is assumed that at least one S1P receptor is critical to the lymphopenic response induced by FTY720 treatment (2). More recently, FTY720 was found to stimulate multidrug transporterdependent T-cell chemotaxis to lymph nodes (7); in this instance, FTY720 phosphate as the active metabolite is hypothesized to be responsible for stimulation of efflux activity of the lipid transporter Abcb1. FTY720 has been reported to be phosphorylated ex vivo by rodent lymphoid tissues (5) and whole blood of several species (6), and is rapidly phosphorylated in vivo (5, 6). After oral application of FTY720 to rats, the blood levels of the monophosphate exceeded those of the parent compound 2-4 fold (5). FTY720 was shown to be a substrate for recombinant murine sphingosine kinase 1a (muSPHK1a) (5). Studies with chiral analogs of FTY720 (namely the R-an...

FTY720, a potent immunomodulatory drug in phase 2/3 clinical trials, induces rapid and reversible sequestration of lymphocytes into secondary lymphoid organs, thereby preventing their migration to sites of inflammation. As prerequisite for its function, phosphorylation of FTY720 to yield a potent agonist of the sphingosine-1-phosphate receptor S1P 1 is required in vivo, catalyzed by an as-yet-unknown kinase. Here, we report on the generation of sphingosine kinase 2 (SPHK2) knockout mice and demonstrate that this enzyme is essential for FTY720 phosphate formation in vivo. Consequently, administration of FTY720 does not induce lymphopenia in SPHK2-deficient mice. After direct dosage of FTY720 phosphate, lymphopenia is only transient in this strain, indicating that SPHK2 is constantly required to maintain FTY720 phosphate levels in vivo. IntroductionNaive T cells regularly circulate between the bloodstream and lymphatic tissue in search for foreign antigen, as well as for tumor and autoantigen. Their activation in secondary lymphoid organs followed by regulated egress back into the circulation to reach sites of inflammation is a prerequisite for any adaptive immune response in the T-cell compartment. Recently, one of the G protein-coupled receptors for sphingosine-1-phosphate (S1P), namely the S1P 1 receptor, was shown to be crucial for the tempo-spatial trafficking of T cells into and out of the secondary peripheral lymphoid organs. 1 The importance of S1P 1 in lymphocyte trafficking became clear through studies with FTY720, an analog of sphingosine. FTY720, after phosphorylation in vivo to FTY720 phosphate (FTY720-P), induces a reversible sequestration of lymphocytes into lymph nodes and Peyer patches. 2,3 FTY720-P thereby acts as a functional antagonist of the S1P 1 receptor, thus inducing aberrant internalization and consequently rendering T cells unresponsive to the obligatory egress signal provided by S1P. 1,[4][5][6] FTY720 has emerged as a potent immunomodulatory agent with usefulness in the control of organ transplant rejection and for treatment of autoimmune diseases. In animals, FTY720 is efficacious in prolonging graft survival, as well as in models of multiple sclerosis, acute lung injury, autoimmune diabetes, atherosclerosis, and renal ischemia-reperfusion injury. 7 Promising results have been obtained from human trials on FTY720 for indications in renal transplantation and multiple sclerosis. 7,8 Since FTY720 prodrug activation is essential for its action on T (and B) cells, understanding how the drug gets phosphorylated in vivo is of high interest, in particular for the design of novel analogs with altered pharmacologic properties.FTY720 is known to be phosphorylated in vitro by the 2 mammalian sphingosine kinases SPHK1 and 2, with SPHK2 being considerably more efficient. [9][10][11] As shown by a recent study in SPHK1-deficient mice, 12 this enzyme appears to be dispensable for the action of FTY720 in vivo, as Sphk1-null mice are still rendered lymphopenic by the drug.In this study, we describe th...

Osteoporosis is a major cause of fractures and associated morbidity in the aged population. The pathogenesis of osteoporosis is multifactorial; whereas traditional pathophysiological concepts emphasize endocrine mechanisms, it has been recognized that also components of the immune system have a significant impact on bone. Since 2000, when the term ‘osteoimmunology' was coined, novel insights into the role of inflammatory cytokines by influencing the fine-tuned balance between bone resorption and bone formation have helped to explain the occurrence of osteoporosis in conjunction with chronic inflammatory reactions. Moreover, the phenomenon of a low-grade, chronic, systemic inflammatory state associated with aging has been defined as ‘inflamm-aging' by Claudio Franceschi and has been linked to age-related diseases such as osteoporosis. Given the tight anatomical and physiological coexistence of B cells and the bone-forming units in the bone marrow, a role of B cells in osteoimmunological interactions has long been suspected. Recent findings of B cells as active regulators of the RANK/RANKL/OPG axis, of altered RANKL/OPG production by B cells in HIV-associated bone loss or of a modulated expression of genes linked to B-cell biology in response to estrogen deficiency support this assumption. Furthermore, oxidative stress and the generation of advanced glycation end products have emerged as links between inflammation and bone destruction.

Tissue factor (TF) has been shown to be up-regulated in endothelial cells by the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) 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-alpha. The data show that at the MAP kinase level, VEGF mainly activates ERK1/2 and p38 MAP kinases in human endothelial cells. TNF-alpha is able to activate all three MAP kinase cascades as well as the classical inflammatory IkappaB/NFkappaB pathway. Furthermore, the MEK/ERK module of MAP kinases appears to act as the convergence point of VEGF- and TNF-alpha-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-alpha-initiated MEK/ERK cascade connected to EGR-1 and TF expression is clearly less sensitive to PKC inhibition. TNF-alpha-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/IkappaB and the MEK/ERK cascades triggered by TNF-alpha. 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.

IntroductionTissue factor (TF) is a cell surface receptor initiating blood coagulation, 1 thereby promoting thrombotic events in atherosclerosis, sepsis, and cancer. 2,3 Enhanced endothelial TF expression has been demonstrated in atherosclerotic plaques, 4,5 a process that may account for thrombotic events associated with early and advanced atherosclerosis. TF expression in endothelial cells (ECs) can be induced by a variety of agonists, including inflammatory cytokines, angiogenic growth factors, infectious agents, and minimally modified low-density lipoprotein (MM-LDL). 1,6 MM-LDL regulates TF expression at the level of transcription 5 ; however, the signaling pathways and transcription factors involved in this process are not known. Some of the effects of MM-LDL can be mimicked by oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC). 7 Three biologically active components of oxidized PAPC (Ox-PAPC) have been structurally identified as 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine (PGPC), 8 and 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphorylcholine (PEIPC). 9 Which of these components of MM-LDL is responsible for induction of TF is not known.In contrast to interleukin-1 (IL-1) or tumor necrosis factor ␣ (TNF-␣), MM-LDL neither up-regulates E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1), nor stimulates neutrophil binding to human ECs. 7 This suggests that classical inflammatory agonists and MM-LDL activate different signaling mechanisms. The major pathway induced by inflammatory cytokines activates transcription factors of the nuclear factor-B (NF-B) family. 10 Whether NF-B is activated by MM-LDL is a subject of controversy. 6,11 In fact, it was shown that MM-LDL and some of its components were capable of down-regulating NF-B-mediated transcription induced by inflammatory cytokines. 12 Thus, the role of the NF-B pathway in inflammatory activation of ECs by oxidized lipids requires further investigation.Apart from NF-B, 13 transcription of the TF gene can be promoted by early growth response factor 1 (EGR-1) and nuclear factor of activated T cells (NFAT). 14,15 Whereas inflammatory cytokines induce NF-B as well as EGR-1, vascular endothelial In the present study, we investigated signaling pathways and transcription factors mediating induction of TF expression in human ECs by biologically active oxidized phospholipids. We show that expression of TF is elevated by OxPAPC, and that this induction was mainly mediated by EGR-1-and NFAT-dependent transcription, but was independent of NF-B activation. Upstream mechanisms activated by OxPAPC were elevation of cytosolic Ca ϩϩ , activation of protein kinase C (PKC), and the mitogenactivated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK MAP kinase cascade. Materials and methods MaterialsCyclosporin A was purchased from Novartis (Vienna, Austria); TNF-␣ from Genzyme (Cambridge...

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.

Abstract-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. Key Words: oxidized phospholipids Ⅲ atherosclerosis Ⅲ angiogenesis Ⅲ plaque destabilization T he adventitial layer of human coronary arteries contains a network of microvasculature, termed vasa vasorum, that delivers oxygen and nutrients to the outer layers of the arterial wall. In contrast to the adventitia, the intima and the inner media of normal large arteries usually do not contain capillaries. However, during atherosclerotic plaque formation, microvessels appear in the thickened intima and media of more that 40% of lesions. 1,2 These neovessels originate mainly from the adventitial vasa vasorum and support growth of atherosclerotic intima beyond the critical thickness limited by diffusion from the major arterial lumen. 3 It has been widely hypothesized that neovascularization is a causative factor for atherosclerotic plaque growth and destabilization. Although these issues are difficult to address in a direct experiment (discussed in Khurana et al 4 ), there is much indirect evidence suggesting that neovascularization can influence the evolution of atheroma via several mechanisms. The density of vasa vasorum strongly correlates with the number of infiltrating mononuclear cells, suggesting that neovessels are an important route for the entry of leukocytes into advanced lesions. 2,5 Furthermore, intraplaque microvascular hemorrhages provide blood cell-derived lipids that deposit in the lipid core. 6,7 Microvessels are functionally important in atherogenesis as illustrated by the ability of angiogenesis inhibitors angiostatin and TNP-470 to reduce angiogenesis and inhibit the development of lesions in apoE knockout mice. 5,8 In contrast, angiogenic stimuli such as vascular endothelial growth factor (VEGF) promote lesion development. 9 Such data strongly suggest that formation of vasa vasorum stimulates the progression of atherosclerotic lesions.In addition to a role in plaque growth, the formation of neovessels potentially can decrease the stability of atheroma because of digestion of plaque tissue by metalloproteases secreted by growing capillaries. Indeed, unstable and ruptured atherosclerotic plaques are characterized by an increased density of plaque microvessels. 10,11 Hypoxia is among the strongest known angiogenic stimuli also thought to play a role in the development of intimal neovascularization. 12 However, additional microenvironmental factors independent of lesion thickness may also be important for plaque angiogenesis. In apoE-deficient mice,

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