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ARTICLE IN PRESSG ModelC 4315 1–6

The International Journal of Biochemistry & Cell Biology xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

The International Journal of Biochemistry& Cell Biology

jo ur nal home page: www.elsev ier .com/ locate /b ioce l

eramide and its metabolites modulate time-dependently the activityf the Na+/K+ ATPase in HepG2 cells

awsan Ibrahim Kreydiyyeh ∗, Zeina Dakroubepartment of Biology, Faculty of Arts & Sciences, American University of Beirut, Beirut, Lebanon

r t i c l e i n f o

rticle history:eceived 25 December 2013eceived in revised form 17 April 2014ccepted 30 April 2014vailable online xxx

eywords:a+/K+ ATPaseeramideeramide one phosphate

a b s t r a c t

Ceramide is involved in the regulation of many cellular processes including cell proliferation and apo-ptosis, which are accompanied respectively with a decrease and an increase in the activity of the Na+/K+

ATPase. These antagonistic effects may be time-dependent and due to different signaling pathwaysrequiring different time intervals to be activated. While we showed previously a ceramide-induced inhi-bition of the ATPase in HepG2 cells during the first hour, we study here the effect of ceramide thereafter.Ceramide stimulated the Na+/K+ ATPase between 1 and 4 h with a peak at 2 h. This stimulation was main-tained in the simultaneous presence of an inhibitor of ceramidase (CAY 10466) but disappeared whenceramide kinase was inhibited, suggesting a role of ceramide-1-phosphate (cer-1-P) in the observedeffect. Exogenous cer-1-P caused a similar stimulation of the ATPase which was not affected by an inhibi-

F-�BNKepG2 cells

tion of JNK but changed into a decrease in presence of PDTC, a specific inhibitor of NF-�B, and disappearedwhen NF-�B and JNK were inhibited simultaneously. It was concluded that cer-1-P activates both JNK andNF-�B. While JNK exerts an inhibitory effect on the ATPase, NF-�B increases its activity and abrogates thestimulatory effect of the sphingolipid on JNK leading thus to an additional increase in the ATPase activity.

© 2014 Published by Elsevier Ltd.

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. Introduction

Ceramide is involved in the regulation of many cellularrocesses including cell proliferation, differentiation, death, senes-ence (Minamino et al., 2003; Gómez-Munoz, 2006; Ruvolo, 2001;hevissen et al., 2006), autophagy (Taha et al., 2006), necrosisMorales et al., 2007), cell cycle, and inflammation. Ceramide lev-ls increase in response to stress stimuli (Gulbins et al., 2004) likeeat shock, ionizing radiation, oxidative stress, chemotherapeuticgents, and cytokines such as interleukin 1 and TNF-� (Lin et al.,006; Hannun, 1994a,b). Ceramide mediates pro-apototic signals

n most cell types but can exert also survival functions in others,ike in neurons and kidneys (Gómez-Munoz, 2006; Verdoorn et al.,010).

Ceramide is synthesized via three pathways, the De novo,atabolic and salvage pathway.

In the de novo pathway, the enzyme serine palmitoyltrans-

Please cite this article in press as: Kreydiyyeh SI, Dakroub Z. Ceramidthe Na+/K+ ATPase in HepG2 cells. Int J Biochem Cell Biol (2014), http:

erase allows the condensation of serine and palmitoyl-CoA toorm ketosphinganine which is reduced to dihydrosphingosine.he latter is then changed to dihydroceramide after acylation by

∗ Corresponding author. Tel.: +961 1 350000; fax: +961 1 744461.E-mail address: Sawkreyd@aub.edu.lb (S.I. Kreydiyyeh).

ttp://dx.doi.org/10.1016/j.biocel.2014.04.027357-2725/© 2014 Published by Elsevier Ltd.

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ceramide synthase. Dihydroceramide undergoes oxidation by dihy-droceramide reductase which introduces a trans-4,5 double bondleading to ceramide (Hannun et al., 1994).

The catabolic or the sphingomyelin pathway involves the gen-eration of ceramide by hydrolysis of the phosphodiester bond ofsphigomyelin a phospholipid present in plasma membrane.

In the salvage pathway ceramide generation involves break-down of complex sphingolipids into sphingosine which afterre-acylation produce ceramide (Kitatani et al., 2008).

Ceramide levels are modulated by the enzyme ceramidase thatmediates deacylation of ceramide into sphingosine and by theenzyme ceramide kinase generating Ceramide-1-phosphate (cer-1-P) (Gómez-Munoz, 2006; Hannun and Luberto, 2000; Woodcock,2006) which is involved in cell proliferation (Chalfant and Spiegel,2005; Lebman and Spiegel, 2008). Whether cell survival or celldeath is induced, depends on the level of cer-1-P and ceramide.

One important hallmark of apoptosis is cell shrinkage. Changesin cell volume are directly related to changes in cellular ionic con-centrations especially those of Na+, K+ and chloride. Depletion ofintracellular K+ was shown to be the factor responsible for trigger-

e and its metabolites modulate time-dependently the activity of//dx.doi.org/10.1016/j.biocel.2014.04.027

ing apoptotic processes like caspase cleavage, cytochrome c releaseand endonuclease activation (Bortner and Cidlowski, 1998; Hugheset al., 1997). These alterations in K+ homeostasis are induced bychanges in the activity of the Na+/K+ ATPase. Inhibition of the pump

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ith ouabain promoted apoptosis and necrosis (Xiao et al., 2002)hile an increased activity promoted cell survival and proliferation

Karitskaya et al., 2010).In the liver, the Na+/K+ ATPase establishes in addition, the Na+

radient needed for the transport of bile salts (Erlinger, 1982),nd drives the activity of many other transporters like the Na/Hxchanger and the Na+–K+2Cl− cotransporter involved respectivelyn the regulation of intracellular pH and cell volume. The Na+/H+

xchanger serves to extrude protons, is stimulated by intracellu-ar acidification and up-regulated following partial hepatectomyDallenbach et al., 1994) and rat biliary cirrhosis (Elsing et al., 1994).epatocytes express the NHE-1 isoform (Orlowski et al., 1992)hich is modulated by changes in cell volume. Hypertonic stress

r cell shrinkage stimulate the uptake of Na+, K+, and Cl− ions viaa+/K+ pump, Na+/K+/2Cl− symporter, ion channels, and Na+/H+

xchange coupled to Cl−/HCO3− and Na+/HCO3− exchange leading

o an increase in cell salt and water content (Friedrich et al., 2006).hanges in hepatocyte’s cell volume are associated with changes

n, bile flow and bile acid transport, cytoplasmic and endosomalH, metabolism of carbohydrates, proteins and lipids, transcrip-ion and translation, and alteration of cytoskeleton componentsGraf and Haussingerm, 1996). The Na+/K+ ATPase is modulatedy many factors including hormones, changes in substrate concen-rations, phosphorylation state and alterations in the biophysicalroperties of the membrane (Zhang et al., 2008). These factorsct by activating many mediators like MAPKs (mitogen-activatedrotein kinases), PKA, PKC, PKG, Ca2+/calmodulin (CaM), and ROSZhang et al., 2008). In the rat liver the Na+/K+ATPase is stimulatedy dopamine (Abu-Jayyab and Mahgoub, 1987), inhibited by estro-en (Davis et al., 1978) and activated by phenylephrine, an alpha

adrenergic agonist, for the first 5 min and inhibited thereafterSmart and Deth, 1988).

Because of a wide array of activities, the Na+/K+ pump plays keyoles in the regulation of cellular physiology. Any alteration in itsctivity is expected to impair liver functions leading to disease oro the promotion of cell death or proliferation.

Literature on the effect of ceramide or its metabolites on thea+/K+ ATPase is very scarce, and the results are controversial and

issue specific. Cabral et al. (2010) reported a ceramide-inducednhibition of the furosemide sensitive Na+ ATPase (i.e. NaKCl2otransporter) in kidney proximal tubule, but failed to observe anyffect on the Na+/K+ ATPase.

In another study, Colina et al. (2002) showed in human erythro-ytes, an inhibitory effect of ceramide on another P-type ATPaseamely the Ca++ ATPase. The effect of ceramide on hepatic Na+/K+

TPase is still unclear.Whether ceramide modulates the activity of the Na+/K+ ATPase

n hepatocytes is a question that we have partially addressedefore by studying the short term (0–60 min) effect of ceramiden the ATPase in HepG2 cells (Dakroub and Kreydiyyeh, 2012). Weypothesize that the ability of ceramide to induce dual oppositeffects on cell survival may be time dependent and due to differentetabolites and signaling pathways that need different time inter-

als to be activated. In this work we study the effect of ceramiden the Na+/K+ ATPase in HepG2 cells beyond 60 min and attempto delineate the signaling pathway involved.

. Materials and methods

.1. Materials

Please cite this article in press as: Kreydiyyeh SI, Dakroub Z. Ceramidthe Na+/K+ ATPase in HepG2 cells. Int J Biochem Cell Biol (2014), http:

The human liver carcinoma cell line (HepG2) was pur-hased from American Type Culture Collection (ATCC), VA,SA. Dulbecco modified Eagle’s medium (DMEM), fetal bovine

erum (FBS) and anti-I�B� antibody were purchased from

PRESSf Biochemistry & Cell Biology xxx (2014) xxx–xxx

Invitrogen (CA, USA). Ceramidase inhibitor N-[(1R,2R)-2-hydroxy-1-(hydroxymethyl)-2-(4-nitrophenyl) ethyl]-tetradecanamide(CAY10466/D-NMAPPD) was purchased from Cayman (MI, USA).The protein assay reagent was purchased from Bio-Rad (München,Germany). Rabbit Na+/K+ ATPase was purchased from Upstate (SC,USA). The TransAM1 NF-�B p65 kit was purchased from ActiveMotif, Carlsbad, CA. The ceramide kinase inhibitor NVP231 waspurchased from Calbiochem EMD Millipore (Darmstadt, Germany).C2 cer-1-P was obtained from Santa Cruz Biotec Inc. (CA, USA).The cell permeable C2 ceramide, N-acetyl-d-shingosine, the NF-�Binhibitor Pyrrolidinedithiocarbamate (PDTC), the JNK inhibitor SP600125, and all other chemicals were purchased from Sigma (MO,USA).

2.2. Methods

2.2.1. Culture and treatment of HepG2 cellsHepG2 cells were grown in DMEM supplemented with 10% FBS

and 1% streptomycin in a humidified incubator (5% CO2). Cells weretreated at 60–70% confluence, after an overnight serum starvation.A time-response study on the effect of ceramide (3 �M) on theactivity of the Na+/K+ ATPase revealed a stimulatory effect thatpeaked at 120 min. Consequently, all treatments with ceramidewere conducted for a period of 120 min.

Ceramide and the ceramidase inhibitor CAY10466/D-NMAPPDwere dissolved in DMSO and used at a respective final concentra-tion of 3, and 100 �M. Cer-1-P (applied for 15 min) was dissolved ina mixture of ethanol:dodecane (98:2, v/v) and used at a concentra-tion of 3 �M. The ceramide kinase inhibitor NVP231 was dissolvedin DMSO and added to the cells at a final concentration of 0.1 �M.The involvement of NF-�B and JNK in the response to ceramide wasstudied by pre-treating the cells with their respective inhibitors,PDTC (100 �M) and SP600125 (50 �M). All inhibitors were addedto cells 30 min before the addition of the sphingolipid.

At the end of the incubation period, HepG2 cells were collected,washed, lyzed and homogenized on ice. Proteins were quantifiedusing the BioRad Reagent.

2.2.2. NF-�B activationThe activation of NF-�B was tested using the TransAM® NF-�B

p65 kit (Active Motif, Carlsbad, CA, USA) as recommended by themanufacturer. The assay relies on the binding of activated NF-�Bin nuclear extracts to oligonucleotides containing NF-�B consen-sus binding sites immobilized on 96 well plates. The bound NF-�Bp65 is then recognized by a primary antibody which is detectedby a secondary antibody conjugated to horseradish peroxidase,providing thus a signal that can be quantified with a microplatespectrophotometer at 450 nm. Nuclear extracts were preparedusing the TransAM® nuclear extract kit. 10 �g of nuclear extractswere added to each well and the assay was run in triplicates.

2.2.3. Assay for Na+/K+ ATPase activityThe activity of the Na+/K+ ATPase was assayed by measuring the

amount of inorganic phosphate liberated in presence and absenceof Na+/K+ ATPase substrates and cofactors, as described by Esmann(1998). The cell homogenate was diluted in histidine (150 mM)pH = 7.4 to a protein concentration of 2 �g/�l. Samples were thenwithdrawn and incubated with saponin (0.2%) for 30 min at roomtemperature in presence of 0.03 M glycerophosphate and 0.03 Mpyrophosphate. The reaction was then initiated by the additionof ATP (30 mM) in presence and absence of KCl (200 mM), NaCl(1240 mM), and MgCl2 (40 mM) and continued for 30 min at 37 ◦C.

e and its metabolites modulate time-dependently the activity of//dx.doi.org/10.1016/j.biocel.2014.04.027

Since conformational changes of the enzyme and its phosphoryla-tion and dephosphorylation are dependent respectively on Na+ andK+, these ions were omitted from the incubation medium togetherwith Mg++ a known co-activator (Jorgensen et al., 2003). Choline

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hloride was added in replacement of the omitted salts to main-ain the same chloride concentration. The reaction was stoppedy the addition of trichloroacetic acid and samples were spun at000 × g for 5 min. The amount of inorganic phosphate liberated

n the supernatant was measured colorimetrically according tohe method of Taussky and Shor (1953). Enzymatic activity wasetermined by calculating the difference in inorganic phosphatePi) liberated in the presence and absence of the substrates andofactors (Na+/K+ and Mg2+) and reported as a percentage of thealue observed in the control untreated group.

.2.4. Western blots analysisEqual amounts of protein were resolved on 10% acrylamide gel

hen transferred to PVDF membranes which were incubated with primary anti-I�B, anti-c-Jun, or anti P-c-Jun, followed by incuba-ion with a secondary antibody. Signals were detected by enhancedhemiluminescence using luminol reagent.

In all electrophoresis work, equal loading was checked by deter-ination of GAPDH protein expression.

.2.5. Statistical analysisThe data were analyzed by ANOVA followed by Tukey Kramer

est using GraphPad InStat software.

. Results

.1. Time response study

HepG2 cells treated with 3 �M ceramide for different time inter-als showed an increase in Na+/K+ ATPase activity. This increaseppeared at 60 min, peaked at 120 min, and was still significantlyigher than the control value at 240 min (Fig. 1).

Please cite this article in press as: Kreydiyyeh SI, Dakroub Z. Ceramidthe Na+/K+ ATPase in HepG2 cells. Int J Biochem Cell Biol (2014), http:

.2. Involvement of ceramide-1-phoshpate

To see if the observed increase in ATPase activity is triggered byphingosine, a metabolite of ceramide, its synthesis was blocked by

ig. 2. Involvement of Cer-1P in ceramide action on the Na+/K+ ATPase. Effect of ceramin inhibitor of ceramidase, (B) NVP 231, an inhibitor of ceramide kinase. The inhibitors wommon letter are not significantly different from each other at P < 0.01. (C) Effect of Cer

= 5. Bars sharing a common letter are not significantly different from each other at P < 0

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the Na+/K+ ATPase. Values are means ± SEM. N = 4. **Significantly different from thecontrol at P < 0.01. *Significantly from the control value at P < 0.05.

pre-treating HepG2 cells with CAY 10644, an inhibitor of cerami-dase. In this case, the ceramide-induced increase in the Na+/K+

ATPase activity appeared unchanged (Fig. 2A), but was signifi-cantly reduced when ceramide kinase was inhibited with a specificinhibitor NVP 231 [29] (Fig. 2B). Treating the cells with exogenouscer-1-P resulted in 240% stimulation of the pump (Fig. 2C), an effectsimilar to that of ceramide.

3.3. Involvement of JNK

Since the above results implicate cer-1-P as the effectormolecule, its mechanism of action was investigated by studyingthe involvement of JNK. The stimulatory effect of cer-1-P on theNa+/K+ ATPase still appeared in presence of SP600125 (Fig. 3A),an inhibitor of JNK, but was abrogated completely when both JNKand NF-�B were inhibited simultaneously (Fig. 3B). In addition, the

e and its metabolites modulate time-dependently the activity of//dx.doi.org/10.1016/j.biocel.2014.04.027

level of phosphorylated c-jun in HepG2 cells was reduced by cer-1-P treatment but enhanced by the simultaneous treatment of thecells with cer-1-P and the NF-�B inhibitor PDTC (Fig. 3C).

de (3 �M, 2 h) on the activity of the Na+/K+ ATPase in presence of (A) CAY 10466,ere added 30 min before ceramide. Values are means ± SEM. N = 5. Bars sharing a

-1-P (3 �M, 15 min) on the activity of the Na+/K+ ATPase. Values are means ± SEM..01.

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Fig. 3. Involvement of JNK in Cer-1-P action on the Na+/K+ ATPase. Effect of cer-1-P (3 �M, 15 min) on the activity of the Na+/K+ ATPase in presence of (A) SP600125, ani inhiba of cero

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nhibitor of JNK. (B) SP600125 and PDTC, respective inhibitors of JNK and NF-�B. The common letter are not significantly different from each other at P < 0.01. (C) Effectf c-jun. The blot is a representative of an experiment repeated 3 times.

.4. Involvement of NF-�B

Cer-1-P caused a significant increase in the activity of the tran-cription factor (Fig. 4A). In support of this finding, a decrease in therotein expression of I�B was observed in HepG2 cells treated wither-1-P (Fig. 4B). The increase in Na+/K+ ATPase activity induced byer-1-P changed into a decrease upon pre-treatment with PDTC, annhibitor of NF-�B (Fig. 4C).

.5. Cross talk between JNK and NF-�B

To position NF-�B and JNK with respect to each other on theignaling pathway, the effect of cer-1-P on c-jun phosporylationhen NF-�B is inhibited was studied. When the transcription fac-

or was not active, cer-1-P exerted a stimulatory effect on JNKFig. 3C). Cer-1-P activation of NF-�B was maintained when JNKas inhibited (Fig. 4A).

. Discussion

Ceramide emerged recently, as an important effector andignaling molecule. It forms microdomains, which coalesce intoarger platforms where receptors and signaling molecules are reor-anized allowing better interaction between the signal transducingolecules involved in the activated pathway. Ceramide plays thus

n important role in facilitating signal transduction and transmis-ion (Castro et al., 2014). Its production is enhanced by stress stimulind as such, it may play an important role in the modulation ofellular responses to stressful situations. In a previous study weemonstrated a significant inhibitory effect of exogenous ceramiden the Na+/K+ ATPase in HepG2 cells (Dakroub and Kreydiyyeh,012). This effect was greatest at 15 min but disappeared by 1 h,

Please cite this article in press as: Kreydiyyeh SI, Dakroub Z. Ceramidthe Na+/K+ ATPase in HepG2 cells. Int J Biochem Cell Biol (2014), http:

nd was ascribed to sphingosine one phosphate. In this work weemonstrate an opposite significant stimulatory effect that startedppearing after 1 h, indicating that it is mediated by other ceramideetabolites than the ones generated at early times.

itors were added 30 min before Cer-1-P. Values are means ± SEM. N = 5. Bars sharing-1P in presence and absence of inhibitors of NF-�B and JNK, on the phosphorylation

Since the observed up regulation of the pump still appearedwhen HepG2 cells were treated with ceramide in presence of aninhibitor of ceramidase, the possibility of having sphingosine orsphingosine-1-P as mediators of ceramide action can be ruled out.In presence of NVP231, a specific inhibitor of ceramide kinase (Grafet al., 2008), the up-regulatory effect of ceramide was significantlyreduced, inferring that it is due, at least partially to ceramide onephosphate.

The results suggest that the ceramide signaling pathway prevail-ing in the early response and which is mediated via S1P, is differentfrom the one involved at later times and in which ceramideacts via cer-1-P generated by phosphorylation of ceramide byceramide kinase. The longer time needed for the cer-1-P effect to bemanifested may be due to the time required for the activation/up-regulation of ceramide kinase. In support of this hypothesis, C2ceramide was shown by Scheid et al. (1999) to induce, in MC/9cells,an increase in the phosphorylation of the transcription factorCREB, and phosphorylated CREB was found to up-regulate ceramidekinase (Euskirchen et al., 2004). Thus the delay in the appearanceof the effect of Cer1P may be ascribed to the time needed for theexpression of ceramide kinase. Future work needs to be conductedto test this hypothesis.

Our results demonstrate that NF-�B, which is activated byceramide/cer-1-P, is not the only player responsible for theobserved ceramide-induced up-regulation of the Na+/K+ ATPase.Had this been the case, then the activity of the pump would havebeen reduced back to its basal level upon inhibition of the tran-scription factor, and would not have dropped below control value.In addition, the fact that ceramide decreased the phosphorylationof c-jun but increased it when NF-�B was inhibited, implicates arole for NF-�B in modulating JNK activity, and indicates a maskedstimulatory effect of the sphingolipid on JNK. This stimulatory effect

e and its metabolites modulate time-dependently the activity of//dx.doi.org/10.1016/j.biocel.2014.04.027

appeared only when NF-�B was inhibited, because the transcrip-tion factor when active, inhibits JNK and overrides the stimulatoryeffect of the sphingolipid(s), leading thus to up-regulation of thepump.

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ig. 4. Involvement of NF-�B in cer-1-P action on the Na /K ATPase. (A) Effect of ceo each well 10 �g of nuclear extracts were added and optical density was read at 4ifferent from each other at P < 0.01. (B) Effect of cer-1-P (3 �M, 15 min) on the protn experiment repeated 3 times. (C) Effect of Cer-1P in presence of PDTC, an inhibit

The proposed signaling pathway is indicated in Fig. 5.From the current and previous work (Dakroub and Kreydiyyeh,

012), we can conclude that ceramide exerts in HepG2 cells a dualime-dependent and opposite effect on the Na+/K+ ATPase thats mediated by different ceramide metabolites. Sphingosine one

Please cite this article in press as: Kreydiyyeh SI, Dakroub Z. Ceramidthe Na+/K+ ATPase in HepG2 cells. Int J Biochem Cell Biol (2014), http:

hosphate is responsible for the down regulation observed at earlyimes and ceramide one phosphate is responsible for the up reg-lation induced at 2 hrs. While inhibition of the Na+/K+ ATPase isssociated with apoptosis (Nobel et al., 2000; Arrebola et al., 2005),

Fig. 5. Suggested signaling pathway of ceramide.

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in presence and absence of SP600125, an inhibitor of JNK, on the activity of NF-�B.. Values are means ± SEM. N = 4. Bars sharing a common letter are not significantlypression of I�B in presence and absence of SP600125. The blot is representative ofF-�B, on the activity of the Na+/K+ ATPase.

an increase in the Na+/K+ ATPase activity is associated with proli-ferative effects in liver cells (Martínez-Mas et al., 1995) and hencemediates cell survival. Ceramide one phosphate seems thus to playa pro-survival role in HepG2 cells. Such a role has been recognizedby other works. Ceramide one phosphate was shown to stimu-late cell division in rat fibroblasts (Gomez-Munoz et al., 1995) andinhibit apoptosis in macrophages by inhibiting acid sphingomyeli-nase (Gomez-Munoz et al., 2004). It was also found to stimulatein macrophages, the PI3K pathway, leading to activation of thetranscription factor NF-�B (Gomez-Munoz et al., 2005) and JNK(Gangoiti et al., 2008). Cross-talks between NF-�B and JNK have alsobeen reported. The transcription factor induced the expression ofsome genes whose products inhibit JNK. Among these can be listedthe growth arrest DNA damage-inducible gene 45� (GADD45�), thecaspase inhibitor XIAP, and the zinc finger protein A20 (Wullaertet al., 2006; Tang et al., 2001). GADD45� inhibits JNK by inhibitingits upstream activator MKK7 (De Smaele et al., 2001).

The involvement of NF-�B in the up-regulatory effect of cer-1-P on the Na+/K+ ATPase is hence in line with previous findingsreported in the literature. What remains unclear however, is theeffect of time in tipping the balance in favor of one metabolite oranother and consequently in favor of cell death or cell survival.

Conflict of interest

No conflict of interest to disclose.

Acknowledgement

This work was supported by a grant from the University

Research Board.

e and its metabolites modulate time-dependently the activity of//dx.doi.org/10.1016/j.biocel.2014.04.027

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