2 '-Hydroxy C16-Ceramide Induces Apoptosis-Associated Proteomic Changes in C6 Glioma Cells

Journal of Proteome Research (Impact Factor: 4.25). 08/2013; 12(10). DOI: 10.1021/pr4003432
Source: PubMed


Ceramide is a bioactive sphingolipid involved in regulation of numerous cell signaling pathways. Evidence is accumulating that difference in ceramide structure, such as N-acyl chain length and desaturation of sphingoid base, determines the biological activities of ceramide. Using synthetic (R) 2'-hydroxy-C16-ceramide, which is the naturally occurring stereoisomer, we demonstrate this ceramide has more potent pro-apoptotic activity compared to its (S) isomer or nonhydroxylated C16-ceramide. Upon exposure to (R) 2'-hydroxy-ceramide, C6 glioma cells rapidly underwent apoptosis as indicated by caspase-3 activation, PARP cleavage, chromatin condensation, and annexin V stain. A 2D-gel proteomics analysis identified 28 proteins whose levels were altered during the initial 3 hours of exposure. Using the list of 28 proteins, we performed a software-assisted pathway analysis to identify possible signaling events that would result in the observed changes. The result indicated that Akt and MAP kinase pathways are among the possible pathways regulated by (R) 2'-hydroxy-ceramide. Experimental validation confirmed that 2'-hydroxy-ceramide significantly altered phosphorylation status of Akt and its downstream effector GSK3β, as well as P38, ERK1/2, and JNK1/2 MAP kinases. Unexpectedly, robust phosphorylation of Akt was observed within 1 hr of exposure to 2'-hydroxy-ceramide, followed by dephosphorylation. Phosphorylation status of MAPKs showed a complex pattern, in which rapid phosphorylation of ERK1/2 was followed by dephosphorylation of p38 and ERK1/2 and phosphorylation of the 46 kDa isoform of JNK1/2. These data indicate that (R) 2'-hydroxy-ceramide regulates multiple signaling pathways by affecting protein kinases and phosphatases with kinetics distinct from that of the extensively studied non-hydroxy-ceramide or its unnatural stereoisomer.

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Available from: Venkatesh Kota, Sep 30, 2014
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    ABSTRACT: Ceramide is a precursor of complex sphingolipids and also plays important roles in cell signaling. With the advances in lipid analytical technologies, the structural diversity of ceramide species have become evident, and the complexity of cellular metabolism and function associated with distinct ceramide species is beginning to be revealed. One of the common structural variations of ceramide is 2'-hydroxylation of the N-acyl chain. Fatty acid 2-hydroxylase (FA2H) is one of the enzymes that introduce the hydroxyl group during de novo synthesis of ceramide. FA2H is essential for the normal functioning of the nervous system, as evidenced by demyelinating disorder associated with FA2H mutations in humans and mice. Studies of Fa2h mutant mice indicate that lack of 2'-hydroxy galactosylceramide in the myelin membrane results in loss of long-term stability of myelin and eventual demyelination. FA2H also regulates differentiation of various cell types (epidermal keratinocytes, schwannoma cells, adipocytes). When provided exogenously, ceramide induces apoptosis in many cell types. Interestingly, the effective concentration of 2'-hydroxy ceramide that induces apoptosis is significantly lower compared to non-hydroxy ceramide, and cells die much more rapidly, suggesting that 2'-hydroxy ceramide can mediate proapoptotic signaling distinct from non-hydroxy ceramide. Collectively, current evidence clearly shows that 2'-hydroxy ceramide and 2'-hydroxy complex sphingolipids have unique functions in membrane homeostasis and cell signaling that could not be substituted by non-hydroxy counterparts.
    10/2013; 54(1). DOI:10.1016/j.jbior.2013.09.012
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