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ARF6 knockdown increases cellular glucosylceramide levels through increased glucosylceramide synthase activity in Neuro-2a cells. (A) Schematic overview of the sphingolipid synthesis pathway. (B-D) Neuro-2a cells were transfected with control (c) or ARF6 siRNA in medium with 10% FCS and analyzed after 48 h. (B) mRNA expression of glucosylceramide synthase (GCS) in RNA extracted from Neuro-2a cells (n = 6 per group). (C) Representative image of a TLC plate from a GCS activity assay showing increased levels of synthesized NBDglucosylceramide in Neuro-2a cells transfected with ARF6 siRNA. (D) Quantification of increased glucosylceramide synthase activity after ARF6 knockdown (n = 4 per group). *P,0.05, **P,0.01, ***P,0.001 vs control. doi:10.1371/journal.pone.0060118.g003
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The small GTPase ADP ribosylation factor 6 (ARF6) mediates endocytosis and has in addition been shown to regulate neuron differentiation. Here we investigated whether ARF6 promotes differentiation of Neuro-2a neuronal cells by modifying the cellular lipid composition. We showed that knockdown of ARF6 by siRNA in Neuro-2a cells increased neuronal ou...
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... and sphingomyelin are synthesized from ceramide in the Golgi apparatus ( Figure 3A) [26,27]. Because ceramide levels were unchanged by ARF6 knockdown, we speculated that the shift in the relative levels of sphingomyelin and glucosylceramide could be caused by an effect of ARF6 knockdown on glucosylceramide synthase. ...
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... ceramide levels were unchanged by ARF6 knockdown, we speculated that the shift in the relative levels of sphingomyelin and glucosylceramide could be caused by an effect of ARF6 knockdown on glucosylceramide synthase. In agreement with our hypothesis, we observed significantly increased mRNA expression of glucosylceramide synthase in Neuro-2a cells transfected with ARF6 siRNA ( Figure 3B). In addition, we tested whether glucosylceramide synthase activity was affected by ARF6 knock- down by following the synthesis of glucosylceramide from fluorescently labeled NBD-C6-ceramide. ...
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... addition, we tested whether glucosylceramide synthase activity was affected by ARF6 knock- down by following the synthesis of glucosylceramide from fluorescently labeled NBD-C6-ceramide. Importantly, we found that glucosylceramide synthase activity was increased by 80-100% after ARF6 knockdown (Figure 3C-D). ...
Citations
... For blockade of CER metabolic pathways, CFPAC-1 cells were pretreated for 4 h with 10 M D-MAPP (Biomol) (26), 2 M DMS (Enzo Life Sciences) (27), 20 M fumonisin B1 (SigmaAldrich) (28), 10 M D-PDMP (Biomol) (29), or 1 M NVP-231 (Sigma-Aldrich) (30), followed by a 20-h incubation with vehicle or 10 M C4-CER in the presence or absence of the above CER metabolic pathway inhibitors. For inhibition of PI3K kinase activity, CFPAC-1 cells were pretreated for 4 h with 0.2 M wortmannin (Calbiochem) (31), 8 M LY294002 (Calbiochem) (32), or 2 M PI-103 (Tocris Bioscience) (33), followed by a 20-h incubation with vehicle or 10 M C4-CER in the presence or absence of the above PI3K inhibitors. ...
Cystic fibrosis (CF) is due to a folding defect in the CF transmembrane conductance regulator (CFTR) protein. The most common
mutation, ΔF508, prevents CFTR from trafficking to the apical plasma membrane. Here we show that activation of the PDK1/SGK1
signaling pathway with C4-ceramide (C4-CER), a non-toxic small molecule, functionally corrects the trafficking defect in both
cultured CF cells and primary epithelial cell explants from CF patients. The mechanism of C4-CER action involves a series
of mutual autophosphorylation and phosphorylation events between PDK1 and SGK1. Detailed mechanistic studies indicate that
C4-CER initially induces autophosphorylation of SGK1 at Ser422. SGK1[Ser(P)422] and C4-CER coincidently bind PDK1 and permit PDK1 to autophosphorylate at Ser241. Then PDK1[Ser(P)241] phosphorylates SGK1[Ser(P)422] at Thr256 to generate fully activated SGK1[Ser422, Thr(P)256]. SGK1[Ser(P)422,Thr(P)256] phosphorylates and inactivates the E3 ubiquitin ligase Nedd4-2. ΔF508-CFTR is thus free to traffic to the plasma membrane.
Importantly, C4-CER-mediated activation of both PDK1 and SGK1 is independent of the PI3K/Akt/mammalian target of rapamycin
signaling pathway. Physiologically, C4-CER significantly increases maturation and stability of ΔF508-CFTR (t½ ∼10 h), enhances cAMP-activated chloride secretion, and suppresses hypersecretion of interleukin-8 (IL-8). We suggest that
candidate drugs for CF directed against the PDK1/SGK1 signaling pathway, such as C4-CER, provide a novel therapeutic strategy
for a life-limiting disorder that affects one child, on average, each day.
The plasma membrane of cells contains a diverse array of lipids that provide important structural and biological features. Glycolipids are typically a minor component of the cell membrane and consist primarily of glycosphingolipids (GSLs). GSLs in vertebrates contain a multifarious assortment of glycan headgroups, which can be important to biological functions based on lipid–lipid and lipid–protein interactions. The design of probes to study these complex targets requires advanced synthetic methodologies. In this Review, we will discuss recent advances in chemical and chemoenzymatic synthesis of GSLs in conjunction with the use of these approaches to design new probes. Examples using either chemical or enzymatic semisynthesis methods starting from isolated GSLs will also be reviewed. Focusing primarily on vertebrate glycolipids, we will highlight examples of radionuclide, fluorophore, photoresponsive, and bioorthogonal tagged GSL probes.
Sphingosine-1-phosphate is synthesized by two sphingosine kinases, cytosolic SK1 and nuclear SK2 but SK2 expression was much higher than SK1in mouse skin fibroblasts. However, in SK2-/- cells, SK1 expression was markedly increased to SK2 levels whereas in SK1-/- cells, SK2 expression was unaffected. Ceramide, glucosylceramide and sphingosine levels were all increased in SK1-/- but less so in SK2-/- cells and S1P levels were not significantly reduced in either SK1-/- or SK2-/- cells. Greatly increased Ceramide glucosyltransferase expression was observed in SK1-/- cells but less so in SK2-/- cells suggested a role in drug resistance. SK2-/- cells grew faster than control and SK1-/-. The cell division gene PCNA was significantly overexpressed in SK2-/- cells, suggesting a cross regulation between SKs and Ceramide glucosyltransferase.
The present work reports the first solid phase synthesis of biologically interesting d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (d-threo-PDMP) derivatives. This synthetic strategy includes facile preparation of versatile azido intermediate (5) in a relatively short sequence and the subsequent derivatization of 5, which led to a series of sulfonamide, urea and heterocycle substituted PDMP analogs (10 and 10′). With this method, a 5280-member compound library has been successfully built by IRORI Nanokan© system.
