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Ceramide synthases (CerS) are important enzymes of the sphingolipid pathway, responsible for the production of ceramides with distinct chain lengths. In human breast cancer tissue, we detected a significant increase in CerS4 and CerS6 mRNA in estrogen receptor positive (ER+) cancer tissue. To clarify the molecular mechanism of this upregulation, we cloned CerS2, -4, -5 and CerS6 promoter and 3'-UTR fragments into luciferase reporter gene plasmids and determined luciferase activity in MCF-7 (ERα/β) and MDA-MB-231 (ERβ) cells after 17β-estradiol treatment. Only the activities of CerS4 and CerS5 promoter Luc constructs, as well as CerS2- and CerS5-3'-UTR Luc constructs increased after estradiol treatment in MCF-7 cells, and this could be inhibited by the anti-estrogen fulvestrant. Co-transfection with the G protein-coupled estrogen receptor 1 (GPER1) also enhanced CerS2, CerS4 and CerS6 promoter activity whereas CerS5 promoter activity was inhibited in both cell lines. Promoter deletion and mutation constructs from CerS4 and CerS5 promoters revealed that estradiol and GPER1 mediate their effects on both promoters by activating AP-1, most likely through dimerization of c-Jun and c-Fos. At least we could show, that cell proliferation induced by estradiol could be blocked by co-treatment with Fumonisin B1, indicating that upregulation of CerS in breast cancer cells by estrogen is important for cell proliferation and possibly tumor development. In conclusion, our data highlight transcriptional and posttranscriptional mechanisms regulating CerS expression in human cells which provide the basis for further studies investigating the regulation of CerS expression and ceramide synthesis after diverse stimuli in physiological and pathophysiological processess., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)