The International Journal of Biochemistry & Cell Biology
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
Graphical abstract
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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