Yannick Schreiber

Long chain ceramides and very long chain ceramides have opposite effects on human breast and colon cancer cell growth.

Ceramides are known to be key players in intracellular signaling and are involved in apoptosis, cell senescence, proliferation, cell growth and differentiation. They are synthesized by ceramide synthases (CerS). So far, six different mammalian CerS (CerS1-6) have been described. Recently, we demonstrated that human breast cancer tissue displays increased activity of CerS2, 4, and 6, together with enhanced generation of their products, ceramides C(16:0), C(24:0), and C(24:1). Moreover, these increases were significantly associated with tumor dignity. To clarify the impact of this observation, we manipulated cellular ceramide levels by overexpressing ceramide synthases 2, 4 or 6 in MCF-7 (breast cancer) and HCT-116 (colon cancer) cells, respectively. Overexpression of ceramide synthases 4 and 6 elevated generation of short chain ceramides C(16:0), C(18:0) and C(20:0), while overexpression of ceramide synthase 2 had no effect on ceramide production in vivo, presumably due to limited substrate availability, because external addition of very long chain acyl-CoAs resulted in a significant upregulation of very long chain ceramides. We also demonstrated that upregulation of CerS4 and 6 led to the inhibition of cell proliferation and induction of apoptosis, whereas upregulation of CerS2 increased cell proliferation. On the basis of our data, we propose that a disequilibrium between ceramides of various chain length is crucial for cancer progression, while normal cells require an equilibrium between very long and long chain ceramides for normal physiology.

Ceramide Synthase 6 Plays a Critical Role in the Development of Experimental Autoimmune Encephalomyelitis

Ceramides are mediators of apoptosis and inflammatory processes. In an animal model of multiple sclerosis (MS), the experimental autoimmune encephalomyelitis (EAE) model, we observed a significant elevation of C16:0-Cer in the lumbar spinal cord of EAE mice. This was caused by a transiently increased expression of ceramide synthase (CerS) 6 in monocytes/macrophages and astroglia. Notably, this corresponds to the clinical finding that C16:0-Cer levels were increased 1.9-fold in cerebrospinal fluid of MS patients. NO and TNF-α secreted by IFN-γ–activated macrophages play an essential role in the development of MS. In murine peritoneal and mouse-derived RAW 264.7 macrophages, IFN-γ–mediated expression of inducible NO synthase (iNOS)/TNF-α and NO/TNF-α release depends on upregulation of CerS6/C16:0-Cer. Downregulation of CerS6 by RNA interference or endogenous upregulation of C16:0-Cer mediated by palmitic acid in RAW 264.7 macrophages led to a significant reduction or increase in NO/TNF-α release, respectively. EAE/IFN-γ knockout mice showed a significant delay in disease onset accompanied by a significantly less pronounced increase in CerS6/C16:0-Cer, iNOS, and TNF-α compared with EAE/wild-type mice. Treatment of EAE mice with l-cycloserine prevented the increase in C16:0-Cer and iNOS/TNF-α expression and caused a remission of the disease. In conclusion, CerS6 plays a critical role in the onset of MS, most likely by regulating NO and TNF-α synthesis. CerS6 may represent a new target for the inhibition of inflammatory processes promoting MS development.

The equilibrium between long and very long chain ceramides is important for the fate of the cell and can be influenced by co-expression of CerS.

Ceramides are synthesized by six different ceramide synthases (CerS1-6), which differ in their specificity to produce ceramides of distinct chain length. We investigated the impact of CerS-co-transfection on ceramide production and apoptosis and proliferation in HCT-116 cells. Over-expression of CerS4 and CerS6 enhanced the level of C(16:0)-Cer twofold, that of C(18:0)- and C(20:0)-Cer up to sevenfold, in comparison to vector control transfected cells, whereas over-expression of CerS2 had no effect on the level of very long chain ceramide C(24:0)- and C(24:1)-Cer. Instead over-expression of CerS2 together with CerS4 or CerS6 increased the activity of CerS2 against very-long-chain ceramides about twofold. In contrast, co-expression of CerS4 with CerS6 inhibited slightly the production of C20:0-ceramide in comparison to cells over-expressing CerS4 alone, whereas the activity of CerS6 seemed not to be affected by other CerS. Interestingly, down-regulation of ELOVL1 had a comprehensive effect on the synthesis of very long chain ceramides which possibly point to a requirement for ELOVL1 expression for full CerS2-activity. Co-expression of CerS2 with CerS4/CerS6 reversed the inhibitory effect of long chain ceramides on cell proliferation and the induction of apoptosis. Even though we observed a twofold increase in total ceramide levels after co-expression of CerS2 with CerS4/CerS6, we detected no effect on cell proliferation. These data indicate that an increase in ceramide production per se is not critical for cell survival, but the equilibrium between long and very long chain ceramides and possibly protein/protein interactions determine the fate of the cell.

Synthesis of lipid mediators during UVB-induced inflammatory hyperalgesia in rats and mice.

Peripheral sensitization during inflammatory pain is mediated by a variety of endogenous proalgesic mediators including a number of oxidized lipids, some of which serve endogenous modulators of sensory TRP-channels. These lipids are eicosanoids of the arachidonic acid and linoleic acid pathway, as well as lysophophatidic acids (LPAs). However, their regulation pattern during inflammatory pain and their contribution to peripheral sensitization is still unclear. Here, we used the UVB-model for inflammatory pain to investigate alterations of lipid concentrations at the site of inflammation, the dorsal root ganglia (DRGs) as well as the spinal dorsal horn and quantified 21 lipid species from five different lipid families at the peak of inflammation 48 hours post irradiation. We found that known proinflammatory lipids as well as lipids with unknown roles in inflammatory pain to be strongly increased in the skin, whereas surprisingly little changes of lipid levels were seen in DRGs or the dorsal horn. Importantly, although there are profound differences between the number of cytochrome (CYP) genes between mice and rats, CYP-derived lipids were regulated similarly in both species. Since TRPV1 agonists such as LPA 18∶1, 9- and 13-HODE, 5- and 12-HETE were elevated in the skin, they may contribute to thermal hyperalgesia and mechanical allodynia during UVB-induced inflammatory pain. These results may explain why some studies show relatively weak analgesic effects of cyclooxygenase inhibitors in UVB-induced skin inflammation, as they do not inhibit synthesis of other proalgesic lipids such as LPA 18∶1, 9-and 13-HODE and HETEs.

Prostacyclin mediates neuropathic pain through interleukin 1β-expressing resident macrophages.

Summary Prostacyclin synthesized at the sites of nerve injuries regulates the accumulation of resident macrophages. These cells express IL1&bgr;, which supports the development of neuropathic pain. ABSTRACT Prostacyclin is an important mediator of peripheral pain sensation. Here, we investigated its potential participation in mediating neuropathic pain and found that prostacyclin receptor (IP) knockout mice exhibited markedly decreased pain behavior. Application of an IP antagonist to the injury site or selective IP deficiency in myeloid cells mimicked the antinociceptive effect observed in IP knockout mice. At the site of nerve injury, IP was expressed in interleukin (IL) 1&bgr;‐containing resident macrophages, which were less common in IP knockout mice. Local administration of the IP agonist cicaprost inhibited macrophage migration in vitro and promoted accumulation of IP‐ and IL1&bgr;‐expressing cells as well as an increase of IL1&bgr; concentrations at the application site in vivo. Fittingly, the IL1‐receptor antagonist anakinra (IL‐1ra) decreased neuropathic pain behavior in wild‐type mice but not in IP knockout mice. Finally, continuous, but not single administration, of the cyclooxygenase inhibitor meloxicam early after nerve injury decreased pain behavior and the number of resident macrophages. Thus, early synthesis of prostacyclin at the site of injury causes accumulation of IL1&bgr;‐expressing macrophages as a key step in neuropathic pain after traumatic injury.

Analysis of sphingolipid and prostaglandin synthesis during zymosan-induced inflammation

Sphingosine-1-phosphate (S1P) is generated through phosphorylation of sphingosine by two sphingosine kinases (SPHK-1 and -2). As extra- and intracellular messenger S1P fulfils multiple roles in inflammation such as mediating proinflammatory inputs or acting as chemoattractant. In addition, S1P induces cyclooxygenase-2 (COX-2) expression and the synthesis of proinflammatory prostanoids in several cell types. Here, we analysed in vivo the regulation of S1P level as well as potential interactions between S1P and COX-dependent prostaglandin synthesis during zymosan-induced inflammation. S1P and prostanoid levels were determined in the blood and at the site of inflammation under basal conditions and during zymosan-induced inflammation using wild type and SPHK-1 and -2 knockout mice. We found that alterations in S1P levels did not correlate with changes in plasma- or tissue-concentrations of the prostanoids as well as COX-2 expression. In the inflamed tissue S1P and prostanoid concentrations were reciprocally regulated. Prostaglandin levels increased over 6h, while S1P and sphingosine level decreased during the same time, which makes an induction of prostanoid synthesis by S1P in zymosan-induced inflammation unlikely. Additionally, despite altered S1P levels wild type and SPHK knockout mice showed similar behavioural nociceptive responses and oedema sizes suggesting minor functions of S1P in this inflammatory model.

PGE2/EP4 signaling in peripheral immune cells promotes development of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated inflammatory autoimmune disease model of multiple sclerosis (MS). The inflammatory process is initiated by activation and proliferation of T cells and monocytes and by their subsequent migration into the central nervous system (CNS), where they induce demyelination and neurodegeneration. Prostaglandin E2 (PGE2) - synthesized by cyclooxygenase 2 (COX-2) - has both pro- and anti-inflammatory potential, which is translated via four different EP receptors. We hypothesized that PGE2 synthesized in the preclinical phase by peripheral immune cells exerts pro-inflammatory properties in the EAE model. To investigate this, we used a bone marrow transplantation model, which enables PGE2 synthesis or EP receptor expression to be blocked specifically in peripheral murine immune cells. Our results reveal that deletion of COX-2 or its EP4 receptor in bone marrow-derived cells leads to a significant delay in the onset of EAE. This effect is due to an impaired preclinical inflammatory process indicated by a reduced level of the T cell activating interleukin-6 (IL-6), reduced numbers of T cells and of the T cell secreted interleukin-17 (IL-17) in the blood of mice lacking COX-2 or EP4 in peripheral immune cells. Moreover, mice lacking COX-2 or EP4 in bone marrow-derived cells show a reduced expression of matrix metalloproteinase 9 (MMP9), which results in decreased infiltration of monocytes and T cells into the CNS. In conclusion, our data demonstrate that PGE2 synthesized by monocytes in the early preclinical phase promotes the development of EAE in an EP4 receptor dependent manner.

Regulation of ceramide synthase 6 in a spontaneous experimental autoimmune encephalomyelitis model is sex dependent

Ceramides (Cer) are mediators of inflammatory processes. In a chronic experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS), we observed a significant elevation of C16-Cer and its synthesizing enzyme, ceramide synthase(CerS)6, in the lumbar spinal cord. In the present study, we have confirmed that C16-Cer and CerS6 are also upregulated in the lumbar spinal cord in a spontaneous relapse-remitting EAE model, using SJL mice overexpressing a transgenic T cell receptor (TCR1640). CerS6 was found to be expressed in macrophages, T cells and B cells in EAE lesions. In macrophages, we demonstrated that interferon gamma (IFN-γ)-induced CerS6 upregulation was amplified by 17ß-estradiol, an action that was further accompanied by increased upregulation of tumor necrosis factor alpha (TNF-α). Accordingly, CerS6 and TNF-α expression was upregulated predominantly in the spinal cord in female TCR1640 mice, which usually develop the relapse-remitting form of EAE, while male TCR1640 mice showed an attenuated regulation of CerS6 and TNF-α and exhibit mostly chronic disease progression. Furthermore, expression of TNFR2, one of two receptors of TNF-α, which is linked to neuroprotection and remyelination, was also upregulated to a greater extent during EAE in female TCR1640 mice in comparison to male TCR1640 mice. Taken together, our results confirm the upregulation of CerS6 and C16-Cer in an adjuvant-independent, physiological EAE model and further suggest an anti-inflammatory role of CerS6 in the regulation of the disease course in female TCR1640 mice via TNF-α/TNFR2.

Targeting CYP2J to reduce paclitaxel-induced peripheral neuropathic pain.

Significance Chemotherapy-induced peripheral neuropathic pain (CIPNP) is a severe side effect that affects up to 80% of patients during cancer treatment. Pharmacological treatment options are poor, as CIPNP differs mechanistically from other chronic pain states. Here, we describe a mechanism by which pronociceptive oxidized lipid mediators are generated by the cytochrome P450-epoxygenase CYP2J2 in sensory neurons during CIPNP. Blocking the synthesis of these oxidized lipids with telmisartan, which we identified as a potent CYP2J2 inhibitor in a large drug repurposing screen, caused a robust reduction of paclitaxel-induced pain in mice in vivo. We therefore consider targeting CYP2J2 with telmisartan as a treatment option for chemotherapy-induced peripheral neuropathic pain in patients. Chemotherapy-induced peripheral neuropathic pain (CIPNP) is a severe dose- and therapy-limiting side effect of widely used cytostatics that is particularly difficult to treat. Here, we report increased expression of the cytochrome-P450-epoxygenase CYP2J6 and increased concentrations of its linoleic acid metabolite 9,10-EpOME (9,10-epoxy-12Z-octadecenoic acid) in dorsal root ganglia (DRGs) of paclitaxel-treated mice as a model of CIPNP. The lipid sensitizes TRPV1 ion channels in primary sensory neurons and causes increased frequency of spontaneous excitatory postsynaptic currents in spinal cord nociceptive neurons, increased CGRP release from sciatic nerves and DRGs, and a reduction in mechanical and thermal pain hypersensitivity. In a drug repurposing screen targeting CYP2J2, the human ortholog of murine CYP2J6, we identified telmisartan, a widely used angiotensin II receptor antagonist, as a potent inhibitor. In a translational approach, administration of telmisartan reduces EpOME concentrations in DRGs and in plasma and reverses mechanical hypersensitivity in paclitaxel-treated mice. We therefore suggest inhibition of CYP2J isoforms with telmisartan as a treatment option for paclitaxel-induced neuropathic pain.

Antinociceptive effects of FTY720 during trauma-induced neuropathic pain are mediated by spinal S1P receptors.

Abstract FTY720 (fingolimod) is, after its phosphorylation by sphingosine kinase (SPHK) 2, a potent, non-selective sphingosine-1-phosphate (S1P) receptor agonist. FTY720 has been shown to reduce the nociceptive behavior in the paclitaxel model for chemotherapy-induced neuropathic pain through downregulation of S1P receptor 1 (S1P1) in microglia of the spinal cord. Here, we investigated the mechanisms underlying the antinociceptive effects of FTY720 in a model for trauma-induced neuropathic pain. We found that intrathecal administration of phosphorylated FTY720 (FTY720-P) decreased trauma-induced pain behavior in mice, while intraplantar administered FTY720-P had no effect. FTY720-P, but not FTY720, reduced the nociceptive behavior in SPHK2-deficient mice, suggesting the involvement of S1P receptors. Fittingly, intrathecal administration of antagonists for S1P1 or S1P3, W146 and Cay10444 respectively, abolished the antinociceptive effects of systemically administered FTY720, demonstrating that activation of both receptors in the spinal cord is necessary to induce antinociceptive effects by FTY720. Accordingly, intrathecal administration of S1P1 receptor agonists was not sufficient to evoke an antinociceptive effect. Taken together, the data show that, in contrast to its effects on chemotherapy-induced neuropathy, FTY720 reduces trauma-induced neuropathic pain by simultaneous activation of spinal S1P1 and S1P3 receptor subtypes.