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

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Abstract

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 C16:0-Cer twofold, that of C18:0- and C20:0-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 C24:0- and C24:1-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.

Introduction

Ceramides (Cer) and their complex derivatives are important components of mammalian membranes and are key players in intracellular signaling, involved in apoptosis, cell senescence, proliferation, cell growth and differentiation (Pettus et al., 2002). They are synthesized at the endoplasmic reticulum by the action of ceramide synthases (CerS). Mammalian ceramide synthases are a family of six transmembrane proteins (Levy and Futerman, 2010). They display specificity towards the fatty acyl CoA moiety used for N-acylation of sphingosine or sphinganine, respectively, leading either to ceramides or dihydroceramides with distinct chain length (Levy and Futerman, 2010). For example, CerS2 produces mainly C22–C24-Cer, whereas CerS5 and CerS6 synthesize C16:0-Cer (Laviad et al., 2008, Mizutani et al., 2005, Mizutani et al., 2006, Riebeling et al., 2003). Ceramide levels increase after treatment of cells with diverse apoptosis-inducing agents including ionizing radiation, UV light, TNF-α and chemotherapeutic agents and seem to be involved in several cell death pathways (Deng et al., 2008, Hannun and Luberto, 2000, Maziere et al., 2001, Mesicek et al., 2010, Mullen et al., 2011, Vit and Rosselli, 2003). Unfortunately, all these studies investigated the role of ceramides in combination with a cell stress inducing agent, which has the disadvantage that also other signaling pathways are induced. To circumvent this disadvantage, we recently investigated the exclusive effect of ceramides on cell proliferation, survival and apoptosis by over-expressing distinct CerS enzymes in human breast and colon cancer cells without any co-stimulation (Hartmann et al., 2012). Our previous data demonstrated that in human colon and breast cancer cells, overproduction of long chain ceramides induces apoptosis and inhibits cell cycle progression, leading to inhibition of cell proliferation and cell death. On the other hand, overproduction of very long chain ceramides had a slight proliferative effect in these cells. Unfortunately, overexpressed CerS2 was only active when very long chain acyl-CoAs were externally added. Very recently, it has been shown that the production of very long chain ceramides is dependent on dimerization of CerS2 with other CerS (Laviad et al., 2012). Another group demonstrated that the activity of CerS2 is also associated with factors of the elongase complex, such as ELOVL1, 3-ketoacyl-CoA reductase or trans-2,3-enoyl-CoA reductase (Ohno et al., 2010). Very long chain fatty acids are produced from certain long chain fatty acids, provided through diet or generated by fatty acid synthase. Elongation is mediated by overall seven fatty acid elongase subtypes (ELOVL 1–7) in humans. Each of them has distinct substrate specificities and are tissue specific expressed (Ohno et al., 2010). Here, we investigated the impact of ELOVL1, which is mainly responsible for the elongation of C20- to C22–C26-fatty acid, on the activity of CerS2 and the outcome of CerS co-expression on proliferation and apoptosis in human colon cancer cells.

Section snippets

Cells and reagents

The human colon cancer cell line HCT-116 was ordered from “Deutsche Sammlung für Mikroorganismen und Zellkulturen” (DSMZ, Braunschweig, Germany). HCT-116 cells were incubated in McCoy's 5A (Invitrogen, Darmstadt, Germany) supplemented with 100 units/ml penicillin G, 100 μg/ml streptomycin and 10% FCS (fetal calf serum). Cells were cultured at 37 °C in an atmosphere containing 5% CO2. Fugene® HD transfection reagent was purchased from Promega (Mannheim, Germany). The sphingolipids (standards) were

Co-transfection of CerS and impact on ceramide levels

Co-transfection experiments with pTARGET™-CerS2, pTARGET™-CerS4 or pTARGET™-CerS6 revealed that all CerS were highly overexpressed in human HCT-116 colon cancer cells (Fig. 1A), which resulted in an up to two fold increase of total ceramide levels in these cells (Fig. 1B). Overexpressed CerSs are co-localized with an ER marker in HCT-116 cell, indicating that also the transfected CerSs are expressed in the same compartment as the endogenous one (Supplement 1). The analysis of the chain length

Discussion

According to the data published by Laviad et al. (2012), we were able to show that CerS2 activity especially is enhanced by co-transfection of either CerS4 or CerS6 (Fig. 2). Additionally to these data we also investigated the effect of co-transfection on the activity of CerS4 and CerS6. Because CerS1 and CerS3 are not expressed in HCT-116 cells we didnot include these enzymes in our investigations. The in vivo CerS-activity assay demonstrated that in CerS2 transfected cells, the addition of C

Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) Forschergruppe FOG 784/TP5 (GR2011/2-1) and DFG projects GR2011/3-1, GR2011/3-2, DFG GE 695/3-1 and the LOEWE Lipid Signaling Forschungszentrum Frankfurt (LiFF). We thank Mike Parnham for critical reading of the manuscript.

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