Ceramide Compartments and Protein Interaction: Structure Meets Function

Abstract

Ceramide is a key sphingolipid, regulating a variety of critical cellular processes. Although exosomes and cilia are derivatives of the membrane, little is known about the role of lipids in their formation. Here we examined the novel role of ceramide in two ceramide-enriched, subcellular compartments: 1) secreted, extracellular vesicles (EVs) termed exosomes, and 2) cell membrane protrusions termed cilia. Firstly, we attempted to address the role of ceramide in exosome secretion and breast cancer. Breast cancer cells acquire multidrug resistance (MDR) mediated by ABC transporters such as breast cancer resistance protein (BCRP). We show that incubation of human breast cancer MDA-MB-231 cells with the farnesoid X receptor antagonist guggulsterone (gug) and retinoid X receptor agonist bexarotene (bex) elevated ceramide, which is known to induce exosome secretion. Ceramide elevation by combined treatment with gug and bex induced BCRP secretion in exosomes and reduced cellular BCRP in cancer and cancer stem-like cells. Consistent with reduced BCRP, ABC transporter assays showed that gug+bex treatment increased doxorubicin retention and that the combination of gug+bex with doxorubicin enhanced cell death. Our results suggest a novel mechanism by which ceramide induces BCRP secretion and reduces MDR, which may be useful as adjuvant drug treatment for sensitizing breast cancer cells and cancer stem cells to chemotherapy. Secondly, to investigate the role of ceramide in ciliogenesis, in particular motile cilia, we used Chlamydomonas reinhardtii (Chlamydomonas) and murine ependymal cells as models. Motile cilia are specialized organelles formed by cell membrane protrusions to function in movement of body fluids. We show for the first time that Chlamydomonas expresses serine palmitoyl transferase (SPT), the first enzyme in the sphingolipid biosynthetic pathway. Ceramide depletion, by the SPT inhibitor myriocin and a neutral sphingomyelinase deficiency (fro/fro mouse), led to glycogen synthase kinase-3 (GSK3) dephosphorylation and defective flagella and cilia, respectively. A novel activation mechanism for GSK3 by the sphingolipids phytoceramide and ceramide is shown to be critical for ciliogenesis in Chlamydomonas and ependymal cells, respectively. We conclude that ceramide promotes exosome secretion to reduce MDR in MDA-MB-231 cells and regulates GSK3-mediated ciliogenesis in Chlamydomonas and murine ependymal cells.