1521-0111/91/4/373–382$25.00 http://dx.doi.org/10.1124/mol.116.107169MOLECULAR PHARMACOLOGY Mol Pharmacol 91:373–382, April 2017U.S Government work not protected by U.S. copyright

Ceramide 1-Phosphate Increases P-Glycoprotein TransportActivity at the Blood-Brain Barrier via ProstaglandinE2 Signaling s

Emily V. Mesev, David S. Miller, and Ronald E. CannonSignal Transduction Laboratory, National Institute of Environmental Health Sciences, National Institutes of Health, ResearchTriangle Park, North Carolina

Received October 7, 2016; accepted January 19, 2017

ABSTRACTP-glycoprotein, an ATP-driven efflux pump, regulates perme-ability of the blood-brain barrier (BBB). Sphingolipids, endoge-nous to brain tissue, influence inflammatory responses and cellsurvival in vitro. Our laboratory has previously shown thatsphingolipid signaling by sphingosine 1-phosphate decreasesbasal P-glycoprotein transport activity. Here, we investigated thepotential for another sphingolipid, ceramide 1-phosphate (C1P),to modulate efflux pumps at the BBB. Using confocal micros-copy and measuring luminal accumulation of fluorescentsubstrates, we assessed the transport activity of several effluxpumps in isolated rat brain capillaries. C1P treatment inducedP-glycoprotein transport activity in brain capillaries rapidlyand reversibly. In contrast, C1P did not affect transport activityof two other major efflux transporters, multidrug resistance

protein 2 and breast cancer resistance protein. C1P inducedP-glycoprotein transport activity without changing transporterprotein expression. Inhibition of the key signaling componentsin the cyclooxygenase-2 (COX-2)/prostaglandin E2 signalingcascade (phospholipase A2, COX-2, multidrug resistanceprotein 4, and G-protein–coupled prostaglandin E2 receptors1 and 2), abolished P-glycoprotein induction by C1P. We showthat COX-2 and prostaglandin E2 are required for C1P-mediated increases in P-glycoprotein activity independentof transporter protein expression. This work describes howC1P activates a signaling cascade to dynamically regulateP-glycoprotein transport at the BBB and offers potentialclinical targets to modulate neuroprotection and drug deliveryto the CNS.

IntroductionThe blood-brain barrier (BBB), located in the endothelium of

brain capillaries, protects the central nervous system (CNS)from neurotoxins. A specialized network of microvessels, theBBB forms a chemical and structural barrier between thebrain and circulatory system. Tight junction complexes re-strict paracellular flow of solutes, and substrate-specific ATP-driven efflux transporters regulate levels of endogenousmetabolites and xenobiotics (Begley, 2004). When modified,the activity of these efflux transporters alters the permeabilityof the BBB (Miller, 2010).

Themost studied and highest expressed efflux transporter atthe BBB is P-glycoprotein. Expressed luminally in brainendothelial cells, P-glycoprotein exports a vast range of sub-strates out of brain tissue and into the circulatory system,making it an essential part of neuroprotection and a redoubt-able obstacle in drug delivery (Begley, 2004; Miller et al.,2008). Some therapeutic strategies are designed to decreaseP-glycoprotein activity to improve drug delivery into the CNS,and others attempt to increase P-glycoprotein activity torestrict the movement of toxins across the BBB and enhanceCNS protection. Specifically, in certain cases of CNS disease orinjury, such as cerebral ischemia, traumatic brain injury, orsubarachnoid hemorrhage, studies have proposed preservingthe integrity of the BBB to protect against further cellulardamage (Alfieri et al., 2011; Zhang et al., 2013).Evenwithout clinical intervention, P-glycoprotein transport

activity is dynamic. Studies show that P-glycoprotein trans-port increases or decreases in response to cellular injury, such

This work was supported by the Intramural Research Program of theNational Institutes of Health National Institute of Environmental HealthSciences.

dx.doi.org/10.1124/mol.116.107169.s This article has supplemental material available at molpharm.

aspetjournals.org.

ABBREVIATIONS: AA, arachidonic acid; AH-6809, 9-oxo-6-propan-2-yloxyxanthene-2-carboxylic acid; BBB, blood-brain barrier; BCRP, breastcancer resistance protein; BODIPY prazosin, boron[1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-[3-[5-[(3,5-dimethyl-2H-pyrrol-2-ylidene-kN)methyl]-1H-pyrrol-2-yl-kN]-1-oxopropyl]piperazinato]difluoro-(T-4)-175799-93-6; BSA, bovine serum albumin; CERK, ceramide kinase; CNS,central nervous system; C1P, ceramide 1-phosphate; COX-2, cyclooxygenase-2; EP1, prostaglandin E2 receptor 1; EP2, prostaglandin E2 receptor2; KO143, (3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino-[1ʹ,2ʹ:1,6]pyrido[3,4-b]indole-3-propanoicacid 1,1-dimethylethyl ester; MRP, multidrug resistance protein; NBD-CSA, [N-«-(4-nitrobenzofurazan-7-yl)-D-Lys8]cyclosporine A; NS-398, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; PBS, phosphate-buffered saline; NVP-231, N-(2-benzamido-1,3-benzothiazol-6-yl)adamantane-1-carboxamide; PF-04418948, 1-(4-fluorobenzoyl)-3-[(6-methoxynaphthalen-2-yl)oxymethyl]azetidine-3-carboxylic acid; PGE2, prostaglandin E2;PLA2, phospholipase A2; PSC833, valspodar; SC-51089 hydrate, 3-chloro-Nʹ-(3-pyridin-4-ylpropanoyl)-6H-benzo[b][1,4]benzoxazepine-5-carbohydrazide; hydrate; hydrochloride; S1P, sphingosine 1-phosphate.

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as inflammation or oxidative stress (Seelbach et al., 2007;Miller et al., 2008; Chodobski et al., 2011; Wang et al., 2014).Increased P-glycoprotein activity has also been observed inanimals with certain neurologic and neuroinflammatorydisorders, such as epilepsy and amyotrophic lateral sclerosis(Brandt et al., 2006; Bauer et al., 2008; Milane et al., 2010;Jablonski et al., 2012). Understanding the mechanisms thatregulate P-glycoprotein and how basal P-glycoprotein ismodulated will help the development of clinical targets forboth enhanced neuroprotection and drug delivery.Sphingolipids are signaling molecules that are endogenous to

brain tissue and involved in inflammatory responses. However,despite observations that inflammation in brain tissue can alterBBB efflux transport, research regarding the involvement ofsphingolipids at the BBB remains limited. Structurally, sphin-golipids contain a sphingoid backbone acetylated at the Nterminus with a fatty acid chain specific to one of many ceramidespecies (Maceyka and Spiegel, 2014). One of the most commonlystudied sphingolipids is ceramide, which can be converted tomany other species. The membrane-bound enzyme ceramidekinase (CERK) phosphorylates ceramide intracellularly to pro-duce the proinflammatorymolecule ceramide 1-phosphate (C1P)(Lamour and Chalfant, 2008). Although the physiologic role ofC1P is not fully understood, in vitro studies suggest that C1Pinduces proinflammatory cascades, decreases apoptosis, in-creases cell survival, increases cell migration, and is releasedin high levels from damaged cells (Granado et al., 2009; Aranaet al., 2010; Gómez-Muñoz et al., 2010; Kim et al., 2013).Our laboratory has previously documented the ability of

another sphingolipid, sphingosine 1-phosphate (S1P), to regulateP-glycoprotein transport activity at the BBB (Cannon et al.,2012). In this study,we investigatedwhetherC1P could similarlyregulate transport at the BBB, especially since its formativeenzyme, CERK, is highly active in brain tissue (Van Overloopet al., 2006). Our study explores the ability of C1P to modifyP-glycoprotein activity at the BBB. In contrast to S1P, whichdecreases P-glycoprotein activity, we found that exposure of ratbrain capillaries to C1P rapidly increases P-glycoprotein trans-port activity. The effect is reversible, transporter-specific, andoccurs with no change to transporter protein expression. Furthercharacterization revealed that the effect of C1PonP-glycoproteintransport activity is mediated via the cyclooxygenase-2 (COX-2)and prostaglandin E2 (PGE2) signaling cascade. With thesefindings, we propose a model for C1P-mediated signaling thatinduces P-glycoprotein transport activity quickly and reversiblyto render the BBB impermeable to toxins or drugs.

Materials and MethodsChemicals. C18:1 ceramide 1-phosphate (d18:1/18:1) and sphin-

gosine 1-phosphate (d18:1) were purchased from Avanti Polar Lipids(Alabaster, AL). Stock solution of C1P was prepared in 2:1 chloro-form/methanol. NBD-CSA, [N-«-(4-nitrobenzofurazan-7-yl)-D-Lys8]-cyclosporine A, was custom synthesized. PSC-833 (valspodar), aspecific inhibitor of P-glycoprotein, was provided by Novartis (Basel,Switzerland). Mouse monoclonal C219 antibody to P-glycoprotein forWestern blotting was purchased from Thermo Fisher Scientific(Waltham, MA). Alexa Fluor-488-conjugated goat anti-mouse IgGwas purchased from Invitrogen (Carlsbad, CA). NS-398 [N-(2-cyclo-hexyloxy-4-nitrophenyl)methanesulfonamide] was purchased fromSanta Cruz Biotechnology (Dallas, TX). Rabbit monoclonal PGE2

receptor EP2 antibody and ceefourin 1 were purchased from Abcam(Cambridge, MA). AH-6809 (9-oxo-6-propan-2-yloxyxanthene-2-

carboxylic acid) was purchased from Cayman Chemical (Ann Arbor,MI). BODIPY prazosin [boron[1-(4-amino-6,7-dimethoxy-2-quinazo-linyl)-4-[3-[5-[(3,5-dimethyl-2H-pyrrol-2-ylidene-kN)methyl]-1H-pyrrol-2-yl-kN]-1-oxopropyl]piperazinato]difluoro-(T-4)-175799-93-6] waspurchased from Life Technologies (Carlsbad, CA). KO143 [(3S,6S,12aS)-1,2,3,4,6,7,12,12a-octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino-[1ʹ,2ʹ:1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester],a specific inhibitor of breast cancer resistance protein (BCRP), waspurchased from Enzo Life Sciences (Ann Arbor, MI). G-protein antag-onist peptide was purchased from Tocris Bioscience (Minneapolis, MN).Ceramide from bovine spinal cord, mouse monoclonal b-actinantibody, chlorpromazine hydrochloride, Ficoll PM 400, cyclohexi-mide, SC-51089 hydrate [3-chloro-Nʹ-(3-pyridin-4-ylpropanoyl)-6H-benzo[b][1,4]benzoxazepine-5-carbohydrazide;hydrate;hydrochloride],PF-04418948 [1-(4-fluorobenzoyl)-3-[(6-methoxynaphthalen-2-yl)-oxymethyl]azetidine-3-carboxylic acid], celecoxib, NVP-231 [N-(2-benzamido-1,3-benzothiazol-6-yl)adamantane-1-carboxamide], andall other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).

Animals. All experiments were performed in compliance with theNational Institutes of Health animal care and use guidelines andapproved by the Animal Care and Use Committee of National Instituteof Environmental Health Sciences. Sprague-Dawley male retired ratswere obtained from Taconic Biosciences (Hudson, NY). COX-2-deficientmice and littermate controls on a 129B6F1 background were obtainedfrom Dr. Artiom Gruzdev at the National Institute of EnvironmentalHealth Sciences (Morham et al., 1995). All animals were housed intemperature-controlled rooms under a 12-hour light/dark cycle with adlibitum access to food and water. The animals were euthanized by CO2

inhalation followed by decapitation. For transport assays and immuno-histochemistry, brain capillaries were harvested and used immediately;for Western blot analysis, capillaries were treated and stored at 280°Cfor further analysis.

Brain Capillary Isolation. The procedure for rat brain capillaryisolation has been detailed elsewhere (Miller et al., 2000; Hartz et al.,2004). In summary, brains were stripped of their midbrain, meninges,choroid plexus, olfactory lobes, and white matter. The remaining braintissue was homogenized in cold 1x phosphate-buffered saline (PBS)supplementedwith glucose and sodiumpyruvate (2.7mMKCL, 1.5mMKH2PO4, 136.9 mM NaCl, 8.1 mM Na2HPO4×7H20, 1 mM CaCl2×2H20,1 mM MgCl2×6H20, 5 mM D-glucose, and 1 mM sodium pyruvate). Anequal volume of 30% Ficoll was added to the homogenate, and thesolution was centrifuged at 6800 rpm for 20 minutes at 4°C toseparate the capillaries from the parenchyma. The resulting capil-lary pellet was resuspended in 1% bovine serum albumin (BSA) inPBS, passed over 30 mm-mesh filters (pluriStrainer; pluriSelect,Leipzig, Germany), and washed with PBS before immediate use.

Transport Assay. Details about transport activity assays usingisolated brain capillaries may be found in previous studies (Hartz et al.,2004; Bauer et al., 2007). Experiments in this study were performed atroom temperature in chambered coverglass (Lab-Tek; Thermo FisherScientific) filled with 1xPBS. Isolated rat brain capillaries were allowedto adhere to the coverglass for at least 20 minutes in plain 1x PBS,followed by incubation for 40minutes in a fluorescent substrate specificto one BBB efflux transporter: 2mMNBD-CSA for P-glycoprotein; 2mMTexas Red for multidrug resistance protein 2 (MRP2); and 2 mMBODIPY prazosin for BCRP (Hartz et al., 2004; Wang et al., 2010).Capillaries were treated with C1P plus NBD-CSA for a further20 minutes. In select cases, capillaries were pretreated for 40 minuteswith an inhibitor or antagonist plus NBD-CSA before exposure to C1P.

For each experiment, one chamber contained 10 mM PSC833, aspecific P-glycoprotein inhibitor. Images of 10–20 capillaries wereacquired per chamber using a Zeiss 510 confocal microscope (CarlZeiss Inc., Thornwood, NY) with a 40� water-immersion objective.Luminal fluorescence (as a measure of substrate accumulation) wasanalyzed via National Institutes of Health ImageJ software (https://imagej.nih.gov/ij/). Specific P-glycoprotein activity was calculated asthe difference between total luminal fluorescence and the fluorescenceof capillaries exposed to PSC833.

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Western Blotting. Whole capillary isolates were collected andassayed for protein concentration using the Bradford method. Proteinaliquots were mixed with 4x loading buffer, added to a 4–12% Bis-Trisgel for electrophoresis, and transferred to an Immobilon-FL mem-brane (EMD Millipore, Billerica, MA). Membranes were blocked inOdyssey blocking buffer (Li-Cor Biosciences, Lincoln, NE) for 30 min-utes at room temperature and incubated overnight at 4°C withprimary antibody: for P-glycoprotein, C219 antibody (1/200 dilution,predicted band size 180 kDal, secondary goat anti-mouse); for EP2,antiprostaglandin E receptor EP2 antibody (1/1000 dilution, predictedband size 40 kDal, secondary goat anti-rabbit). As a loading control,membranes were also immunoblotted with b-actin (1/10,000 dilution;predicted band size 42 kDa; secondary goat anti-mouse).

Before imaging, membranes were washed in PBS with 0.1% Tween20 and treated with corresponding secondary antibody IRDye 680RDfor 1 hour. Imaging was performed with the Li-Cor Odyssey InfraredImaging System.

Immunohistochemistry. Isolated capillaries were allowed toadhere to chambered coverglass and were then fixed with 4%paraformaldehyde/0.2% glutaraldehyde for 15 minutes. Chamberswere washed with 1x PBS, permeabilized in 0.1% Triton X-100 (inPBS) for 30 minutes, and blocked in 1% BSA (in PBS) for30 minutes. Capillaries were incubated with prostaglandin Ereceptor EP2 antibody (rabbit; monoclonal; 1/500 dilution) over-night at 4°C. The negative control omitted the primary antibody.Isotype control capillaries were incubated with rabbit monoclonalIgG primary antibody (1/25,000 dilution).

Before imaging, capillaries were washed in 1x PBS, followed byexposure to Alexa Fluor-488-conjugated goat anti-mouse IgG for1 hour at 37°C. Imaging was performed by confocal microscopy usinga Zeiss 510 microscope with a 40� water-immersion objective.

Statistical Analysis. Data are expressed as mean 6 standarderror. Statistical analyses between groups were calculated by one-wayanalysis of variance with Tukey-Kramer post test (multiple compar-isons) or Student’s unpaired t test with GraphPad Prism 6 software(GraphPad Software, San Diego, CA). P , 0.05 was considered astatistically significant difference between means.

ResultsC1P Increases P-Glycoprotein Activity Ex Vivo. Our

laboratory has previously established a confocal microscopy-based assay to measure the activity of transporters at the BBBin isolated rat brain capillaries (Miller et al., 2000; Hartz et al.,2004). Specific transporter activity is determined by measur-ing the luminal accumulation of fluorescent substratesspecific to a particular transporter. To assess the activity ofP-glycoprotein in brain capillaries, we measured the lumi-nal accumulation of 2 mM NBD-CSA at steady state (approx-imately 40 minutes of exposure). To quantify specificP-glycoprotein activity, we subtracted the nonspecific fluo-rescence of capillaries treated with 10 mM PSC833, a potentP-glycoprotein inhibitor.Using this method, we determined the effect of C1P

exposure on BBB efflux transporter activity by exposingfreshly isolated rat brain capillaries to 250 nM C1P for20 minutes. Figure 1A shows representative confocal imagesof rat brain capillaries after 1 hour of exposure to 2 mM NBD-CSA (control), 250 nM C1P (40 minutes of blank NBD-CSAfollowed by 20 minutes C1P concurrently with NBD-CSA), or

Fig. 1. C1P induces P-glycoprotein transport activity atthe blood-brain barrier. (A) Representative confocalimages showing that accumulation of NBD-CSA in thelumen of isolated rat brain capillaries increases after20 minutes of exposure to 250 nM C1P. (B) Quantifica-tion of luminal NBD-CSA fluorescence in isolated ratbrain capillaries treated for 90 minutes with 10 mMPSC833 (specific inhibitor of P-glycoprotein) or for20 minutes with 250 nM C1P. (C) PSC833-sensitiveluminal fluorescence of NBD-CSA expressed as specificP-glycoprotein transport activity. Shown are mean 6S.E.M. for 10–20 capillaries from single preparation(pooled brains from 3–5 rats). ****P,0.0001, signifi-cantly different than control.

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10 mMPSC833 (30 minutes of PSC833 pretreatment, followedby 1 hour of PSC833 concurrently with NBD-CSA). Figure 1Bshows quantitatively that the luminal accumulation ofPSC833-treated capillaries decreased significantly by 50%–

60%. These data are consistent with prior studies that showPSC833 maximally inhibits P-glycoprotein transport of NBD-CSA; any residual fluorescence after PSC833 treatmentresults from nonspecific luminal entry (Hartz et al., 2004).Figure 1 also shows the changes in luminal fluorescence of

isolated rat brain capillaries exposed to 250 nM C1P for20 minutes. The luminal fluorescence of capillaries exposed toC1P increased significantly by approximately 50% (Fig. 1B).The PSC833-sensitive NBD-CSA luminal fluorescence incapillaries exposed to 250 nM C1P was 2-fold higher than inthe control capillaries (Fig. 1C). The PSC833-sensitive lumi-nal fluorescence of another P-glycoprotein substrate, rhoda-mine 123, was also found to increase 2-fold after C1Pexposure (Supplemental Fig. 1). These data show that specificP-glycoprotein transport activity doubles in response to short-term 250 nM C1P exposure.Ceramide Is Converted to C1P via CERK to Induce

P-Glycoprotein. We tested whether ceramide, the intracel-lular precursor to C1P, could similarly affect P-glycoproteinactivity. Exposing isolated rat brain capillaries to 250 nMceramide increased P-glycoprotein transport activity after20 minutes; however, compared with C1P, the effect wasmodest (Fig. 2A). For further comparison between ceram-ide and C1P, we analyzed the time course required for bothsphingolipids to increase P-glycoprotein transport activity.Capillaries treated with 250 nM C1P reached maximalP-glycoprotein induction in under 5 minutes (Fig. 2B),while capillaries treated with ceramide required between

15 and 40 minutes to reach peak P-glycoprotein induction(Fig. 2C).These results prompted us to analyze whether the delay in

ceramide-mediated P-glycoprotein induction resulted fromintracellular conversion of ceramide to C1P. Given that CERKconverts ceramide into C1P, we treated isolated brain capil-laries with a CERK inhibitor (50 nM NVP-231) and measuredP-glycoprotein activity. We found that CERK inhibitionblocked the ability of ceramide to increase P-glycoproteinactivity, indicating that ceramide must first be converted toC1P to induce P-glycoprotein transport activity (Fig. 2D).C1P Action Is Rapid, Reversible, and Transporter

Specific. To characterize the induction of P-glycoproteinactivity caused by C1P, we exposed capillaries to concentra-tions of C1P ranging from 50 nM to 250 nM. After 20 minutes,C1P increased P-glycoprotein activity in a concentration-dependent manner, with peak induction occurring between100 nM and 250 nM (Fig. 3A). As such, we exposed capillariesto 250 nMC1P in all subsequent experiments. To confirm thatthe changes in capillary fluorescence were biologic, weanalyzed the interaction between C1P and NBD-CSA fluores-cence in the absence of brain capillaries. An assessment of thebaseline fluorescence of NBD-CSA with and without 250 nMC1P confirmed that C1P does not chemically affect theintensity of NBD-CSA (Fig. 3B). This indicates that thechanges observed in the luminal fluorescence of capillariestreated with NBD-CSA and C1P result from alterationsin P-glycoprotein transport rather than from a chemicalinteraction.To investigate whether C1P affects any other efflux trans-

porters at the BBB, we tested the effects of C1P exposure(250 nM; 20minutes) onMRP2 and BCRP in brain capillaries.

Fig. 2. Exposure to C1P or ceramide inducesP-glycoprotein transport activity at the blood-brainbarrier. (A) Dose response of 20 minutes ceramidetreatment, showing that ceramide increases specificP-glycoprotein activity in a concentration-dependentmanner. Capillaries were exposed to 2 mM NBD-CSAfor 40minutes followed by 20 minutes exposure to eitherC1P or ceramide concurrently NBD-CSA. (B) Timecourse of C1P-mediated P-glycoprotein induction, show-ing that C1P increases P-glycoprotein activity in under5 minutes. (C) Time course of ceramide-mediatedP-glycoprotein induction, showing that ceramide re-quires approximately 15–40 minutes to take significanteffect. (D) Inhibiting ceramide kinase with NVP-231abolishes P-glycoprotein induction caused by ceramide.Shown are mean 6 S.E.M. for 10–20 capillaries fromsingle preparation (pooled brains from 3–5 rats).*P,0.05, **P,0.01, ****P,0.0001, significantly higherthan control.

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After exposure to C1P, we saw no changes in the luminalaccumulation of the fluorescent substrates for these trans-porters: Texas Red forMRP2 andBODIPYPrasozin for BCRP.Quantitatively, we saw no change in the specific activity ofthese transporters (Fig. 3C). Because C1P exposure elicited nochanges to these transporters, we focused exclusively onP-glycoprotein for the remainder of our study.In time course experiments, C1P induced P-glycoprotein

transport in under 5 minutes and sustained the induction forup to 90 minutes, provided that C1P remained in the capillarytreatment buffer (Fig. 3D). When C1P was removed from thetreatment buffer, P-glycoprotein transport activity returnedto control levels in 1 hour (Fig. 3D). These results suggest thatC1P acts on P-glycoprotein in a rapid and reversible manner.

Our laboratory has previously shown that translationalevents in brain capillaries require several hours to measur-ably affect transporter activity (Wang et al., 2014). Hence, wehypothesized that C1P increased P-glycoprotein indepen-dently of transcription or translation. Western blots showedthat neither 20 minutes nor 4 hours of exposure to C1Pincreased the total protein expression of P-glycoprotein inisolated rat brain capillaries (Fig. 4, A and B and Supplemen-tal Fig. 2).Further, we performed transport assays with capillaries

that had been pretreated with an inhibitor of either transcrip-tion (1 mM actinomycin D) or translation (50 mg/ml cyclohex-imide) before treatment with C1P. Neither inhibitor blockedC1P-mediated P-glycoprotein induction (Fig. 4C). Together,

Fig. 3. Effects of C1P exposure on efflux transporteractivity in isolated rat brain capillaries. (A) C1P concen-tration response in regards to P-glycoprotein activity.Capillaries were exposed to 2 mM NBD-CSA for 40 min-utes followed by exposure to C1P concurrently withNBD-CSA for 20 minutes. (B) C1P treatment does notaffect baseline NBD-CSA fluorescence in the absence oftransporters. (C) C1P exposure for 20 minutes does notaffect the transport activity of MRP2 or BCRP. (D) Timecourse and washout of C1P action, showing that C1Pinduction of P-glycoprotein is rapid and reversible.Capillaries were treated with NBD-CSA for 45 minutesto reach steady state, followed by exposure to 250 nMC1P plus NBD-CSA for time shown. P-glycoproteininduction can be completely reversed 1 hour after C1Pis removed from treatment solution. Shown are means6S.E.M. for 10–20 capillaries from single preparation(pooled brains from 3–5 rats). *P,0.05, ****P,0.0001,significantly higher than control.

Fig. 4. C1P does not increase overall protein levels ofP-glycoprotein in isolated rat brain capillaries. (A, B)Western blots of whole capillary lysates (brain capillar-ies pooled from 8–10 rats) show that exposure to C1P for20 minutes or 4 hours does not increase P-glycoproteinprotein expression. (C) Pretreatment of 40 minutes withan inhibitor of transcription, 1mMactinomycin D (ActD),or translation, 50 mg/ml cycloheximide (CHX), does notaffect the ability of C1P to increase P-glycoproteinactivity in 20 minutes. (D) Pretreatment of 40 minuteswith an inhibitor of vesicle trafficking, 400 nM brefeldinA (BFA), blocks C1P-mediated P-glycoprotein induction.Shown are mean 6 S.E.M. for 10–20 capillaries fromsingle preparation (pooled brains from 3–5 rats).****P,0.0001, significantly higher than control.

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these data strongly suggest that C1P increases P-glycoproteinactivity without increasing overall transporter proteinexpression.To explore an alternative mechanism of how P-glycoprotein

is up-regulated, we pretreated capillaries with 400 nM bre-feldin A, an inhibitor of vesicle formation that preventsintracellular protein trafficking (Klausner et al., 1992). Brel-dein A is certainly not specific for P-glycoprotein, but it haspreviously been shown to prevent the trafficking of intracel-lular P-glycoprotein to the plasmamembrane (Fu et al., 2004).Our study found that pretreatment with brefeldin A blockedC1P-mediated P-glycoprotein induction (Fig. 4D). It is possiblethat exposure to C1P may lead to relocation of P-glycoprotein,causing increased P-glycoprotein activity at the BBB. Alter-natively, vesicle trafficking may be involved elsewhere in theC1P-initiated signaling cascade.C1P Requires Phospholipase A2 and COX-2 Signaling.

We sought to identify the signaling cascade through whichC1P increases P-glycoprotein activity. Previous studies in celllines indicate that C1P stimulates the release of arachidonicacid (AA) by activating cytosolic phospholipase A2 (PLA2)(Pettus et al., 2003, 2004; Nakamura et al., 2006). Further-more, both AA and cyclooxygenase-2 (COX-2), the enzyme thatconverts AA into prostaglandins, have previously been asso-ciated with increased P-glycoprotein transport activity(Bauer et al., 2008; Zibell et al., 2009). As such, we investi-gated whether C1P depends on activation of an AA/COX-2-associated signaling cascade to alter P-glycoprotein activity.

We inhibited PLA2 by pretreating capillaries with chlor-promazine (200 nM; 40 minutes), which blocked the ability ofC1P to increase P-glycoprotein activity (Fig. 5A). Next, weblocked COX-2 with two selective inhibitors: celecoxib(100 nM; 40 minutes) and NS-398 (5 mM; 40 minutes), whichsimilarly blocked the action of C1P (Fig. 5, B and C).To further confirm that COX-2 was required for C1P-

mediated P-glycoprotein induction, we performed transportassays using COX-2-deficient mice. Figure 5D shows thatC1P treatment on wild-type mice brain capillaries resultedin a 2-fold fluorescence increase of P-glycoprotein activitycomparable to the increases observed in wild-type rat braincapillaries. C1P exposure in brain capillaries isolated fromCOX-2–deficient mice produced no change in the luminalaccumulation of NBD-CSA (Fig. 5D). These results indicatethat COX-2 is necessary for C1P to increase P-glycoproteinactivity.C1P Pathway Involves PGE2 Receptor. Previous re-

search in cell lines has proposed the existence of an as-yet-unidentified G-protein–coupled C1P-specific receptor(Granado et al., 2009). In our study, blocking Gi, Go, and Gs

activation with a G-protein antagonist peptide prevented C1Pfrom inducing P-glycoprotein activity (Fig. 6A). However,because no C1P-specific receptor has yet been identified inbrain capillaries, we sought to investigate alternative expla-nations for this observation. We thus explored downstreamsignaling events in the PLA2/COX-2 pathway that involveG-protein–coupled receptors.

Fig. 5. Involvement of PLA2 and COX-2signaling on C1P-mediated P-glycoproteininduction. (A) Inhibiting PLA2 with 200 nMchlorpromazine blocks P-glycoprotein induc-tion caused by C1P treatment. (B) Pretreat-ment of 40 minutes with a COX-2 inhibitor(100 nM celecoxib) blocks the increases inP-glycoprotein activity caused by C1P treat-ment. (C) Another COX-2 inhibitor (5 mMNS-398) similarly blocks the increases inP-glycoprotein activity caused by C1P treat-ment. (D) Exposing COX-2 deficientmouse brain capillaries to 250 nM C1P for20 minutes resulted in no induction ofP-glycoprotein activity. Wild-type (WT)mouse brain capillaries exposed to 250 nMC1P for 20 minutes exhibit an increase inP-glycoprotein activity comparable to thatof wild-type rat brain capillaries. Shownare mean 6 S.E.M. for 10–20 capillariesfrom single preparation (pooled brainsfrom 3–5 rats or 4–6 mice). ***P,0.001,****P,0.0001, significantly higher thancontrol.

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The enzyme COX-2 produces prostaglandin H2 from AA,which is then converted to various prostaglandins, includingprostaglandin E2 (PGE2). PGE2 is transported extracellularlyby MRP4 where it activates four G-protein–coupled receptors:EP1, EP2, EP3, and EP4 (Coleman et al., 1994; Reid et al.,2003). Research has previously associated PGE2 productionwith S1P and C1P (Pettus et al., 2005), and EP1 and EP2receptors have been associated with BBB health and mainte-nance (McCullough et al., 2004; Pekcec et al., 2009). Morespecifically, several studies have implicated the EP2 receptorin inflammation, cell migration, proliferation, apoptosis, andangiogenesis—processes associated with C1P or BBB trans-port (Sung et al., 2005; Kamiyama et al., 2006; Liang et al.,2008; Jiang and Dingledine, 2013).We tested whether C1P signaled through any PGE2 recep-

tors to increase P-glycoprotein activity. First, we targetedMRP4, the transporter that moves PGE2 from the intracellu-lar to the extracellular matrix (Reid et al., 2003). Pretreat-ment with an inhibitor ofMRP4, ceefourin (1 mM; 40minutes),blocked the ability of C1P to induce P-glycoprotein activity

(Fig. 6B). Second, we focused on the EP1 and EP2 receptors.Considerable levels of EP1 have already been noted in braincapillary membranes (Pekcec et al., 2009). To test whetherEP2 is similarly present in brain capillaries, we performedWestern blot analyses on cytosolic and membrane lysatefractions from isolated capillaries using anti-prostaglandin Ereceptor EP2 antibody (rabbit, monoclonal, 1/1000 dilution).EP2 protein was found to be expressed in both fractions (Fig.6C). Immunohistochemical analysis confirmed that the EP2receptor is expressed in rat brain capillaries and is mostabundantly localized in the luminal membrane (Fig. 6D andFig. 6E).Transport assays revealed that pretreatment with AH-6809

(835 nM; 40 minutes), a dual-antagonist for EP1 and EP2,completely inhibited the ability of C1P to alter P-glycoproteinactivity in brain capillaries (Fig. 6F). Pretreatment with anEP1-specific antagonist, SC51089 (1 mM; 40 minutes), onlypartially inhibited the action of C1P without also loweringcontrol activity of P-glycoprotein (Fig. 6G). On the other hand,pretreating with an EP2-specific antagonist, PF-04418948

Fig. 6. Involvement of PGE2 in C1P-mediated P-glycoprotein induction. (A)Inhibiting Gi, Go, and Gs activation with25 mM G-protein antagonist peptide(GPAnt-2) abolishes the increase ofP-glycoprotein activity caused by 20 min-utes of exposure to 250 nm C1P. S1P,known to act through aG-protein–coupledreceptor, is included as a positive control.(B) An inhibitor of MRP4 (1 mM ceefourin)abolishes C1P-mediated P-glycoproteintransport induction. (C) EP2 is expressedin cytosolic and membrane fractions ofisolated rat brain capillaries. (D) Repre-sentative immunohistochemical images oflocalization of PGE2 receptor EP2 in ratbrain capillaries, using prostaglandin Ereceptor EP2 antibody (rabbit, monoclo-nal, 1/500 dilution). (E) Quantification ofEP2 localization in isolated rat braincapillaries shows that EP2 is foundmost abundantly at the luminal mem-brane of rat brain capillaries. (F) Target-ing PGE2 receptors EP1 and EP2 withdual antagonist (835 nM AH-6809) abol-ishes C1P-mediated P-glycoprotein trans-port induction. (G) A specific antagonist ofEP1 (1 mM SC-51089) partially blocks theability of C1P to increase P-glycoproteinactivity. (H) Specifically targeting the EP2receptor with an antagonist (100 nMPF-044189448) completely reduces C1P-mediated P-glycoprotein transport induc-tion in a dose-dependent manner. Shownare mean 6 S.E.M. for 10–20 capillariesfrom single preparation (pooled brainsfrom 3–5 rats). *P,0.05 ***P,0.001,****P,0.0001, significantly higher thancontrol.

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(10 nM and 100 nM; 40 minutes), blocked C1P action in aconcentration-dependent manner, with the highest concen-tration completely abolishing C1P action without affectingcontrol P-glycoprotein activity (Fig. 6H).In all, these data suggest that C1P requires the action of

PLA2, COX-2, PGE2, and EP2 to increase P-glycoproteinactivity. To a lesser extent, C1P may also require the actionof EP1. Figure 7 shows a complete working model of thisproposed pathway.

DiscussionEfflux transporters at the BBB remain an obstacle to CNS

pharmacotherapy yet are an effective form of neuroprotection.Understanding the biologic mechanisms that regulate thebasal activity of these transporters is critical in developingclinical targets for improved drug delivery to the brain orincreasing CNS protection in cases of cellular injury or stress(Miller, 2010). In this study, we demonstrate that short-termexposure to 18-carbon C1P, an endogenous sphingolipid inbrain tissue, increases P-glycoprotein transport activity inisolated rat and mouse brain capillaries. Furthermore, weshow the requirement for signaling through PLA2, COX-2,and PGE2. Given that C1P acts rapidly and is highlyexpressed in brain tissue, we speculate that C1P levelsregulate a signaling pathway that rapidly increases basalactivity of P-glycoprotein, regulatingminute-by-minute trans-port at the BBB.Exposure to long-chain (18-carbon) ceramide, the precursor

for C1P, increases P-glycoprotein activity similarly to18-carbon C1P. However, unlike C1P, ceramide requires theactivity of CERK, the enzyme that converts ceramide intoC1P. Furthermore, ceramide requires considerably more timethan C1P to increase P-glycoprotein activity. Both observa-tions have led us to speculate that exogenous ceramide mustfirst undergo conversion into C1P before alteringP-glycoprotein activity and that ceramide alone has nomeasurable, short-term effect on P-glycoprotein.Prior research has shown that CERK is highly active in

brain tissue and acts optimally on ceramides with 12-acylcarbon chains or longer (Wijesinghe et al., 2005; Van Overloopet al., 2006). Unsurprisingly, the most abundant C1P speciesfound in brain tissue contain 16-carbon chains or longer(Yamashita et al., 2016). Therefore, CERK activity maymodulate basal P-glycoprotein transport activity at theBBB by converting long-chain ceramide species into C1P.Given that drug efflux pumps such as P-glycoprotein

contribute substantially to multidrug resistance in certaindiseases, these findings point to a potential role for C1P andCERK in drug resistance. Down-regulation of ceramide, animportant mediator of apoptosis, has been associated withpoor prognosis and multidrug resistance in tumors, possiblyas a result of dysfunctionalmetabolism into other sphingolipidspecies (Senchenkov et al., 2001; Koybasi et al., 2004). As such,enzymes that metabolize ceramide, such as CERK, could be

targeted for tumor therapy (Reynolds et al., 2003; Payne et al.,2014). In light of our findings, the phosphorylation of ceramideby CERK should be investigated in cases of multidrug re-sistance to determine whether such resistance results fromelevations in P-glycoprotein activity caused by C1P.Our experiments using fluorescent substrates for other

transporters suggest that C1P acts selectively on P-glycoprotein.After C1P treatment, we observed no changes in the accumu-lation of luminal fluorescence of capillaries incubated withTexas Red (substrate for MRP2) or BODIPY prazosin (sub-strate for BCRP). These results indicate that 1) C1P does notaffect the transport activity of MRP2 or BCRP and 2) thestructural integrity of endothelial tight junctions remains un-affected in capillaries exposed to C1P. In the scope of our study,C1P seems to act exclusively on the transport activity ofP-glycoprotein.In time course experiments, we determined that the effect of

C1P on P-glycoprotein occurs in approximately 5–15 minutesand reverses fully within 1 hour after C1P is removed fromcapillaries. Previously, our laboratory has shown that anothersphingolipid, S1P, decreases P-glycoprotein activity in acomparably rapid and reversible manner (Cannon et al.,2012). Such immediate action suggests that endogenousC1P, S1P, and possibly other sphingolipids in brain tissueregulate minute-by-minute activity of P-glycoprotein at theBBB.Our study also indicates that C1P acts through signaling

and does not increase the overall protein expression ofP-glycoprotein in rat brain capillaries. Western blot analysesrevealed that the total protein expression of P-glycoprotein isnot induced after treatment with C1P, and transport assaysconfirmed that C1P does not require the processes of tran-scription or translation to affect P-glycoprotein transportactivity. To our knowledge, this is the first time a moleculehas been shown to increase P-glycoprotein activity with nochange to overall transporter protein expression. This may bean important component of basal P-glycoprotein activity reg-ulation. In cases when the BBBmust be rendered immediatelyimpermeable to environmental toxins or harmful xenobiotics,C1P could produce a rapid increase of efflux from the BBB,resulting in greater neuroprotection.To begin elucidating a possible mechanism that is indepen-

dent of protein expression, we found that inhibiting proteintrafficking blocks the ability of C1P to increase P-glycoproteinactivity. P-glycoprotein has been shown to exist not only at theplasma membrane but also within intracellular compart-ments, including the endoplasmic reticulum, Golgi, andcytoplasmic vesicles (Bendayan et al., 2006; Fu and Arias,2012). Some studies suggest that accumulation of intracellu-lar P-glycoprotein caused by inhibition of protein traffickingcan interfere with P-glycoprotein function (Fu et al., 2004;McCaffrey et al., 2012). Our results suggest that C1P exposuremay promote trafficking of intracellular P-glycoprotein to thecapillary membrane, which could increase transport activityof P-glycoprotein at the BBB without increasing overalltransporter protein expression. However, because other com-ponents in the C1P signaling cascade may also be subject totrafficking, thismechanismmust be explored in future studieswith less general inhibitors of protein traffic.Using specific signaling inhibitors, immunohistochemistry

analysis, and COX-2–deficient mice, we have characterizedthe involvement of the PLA2/COX-2/PGE2 inflammatory

Fig. 7. Proposed signaling cascade for the induction of P-glycoproteinactivity by ceramide and C1P.

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signaling pathway in C1P-mediated P-glycoprotein induction.Previous cell models show that C1P targets and activatescytosolic PLA2 (Pettus et al., 2003; Nakamura et al., 2006),which subsequently stimulates a high release of AA (Pettuset al., 2004). Downstream in this pathway, COX-2 converts AAinto prostaglandins. Studies have implicated COX-2 inP-glycoprotein up-regulation (Bauer et al., 2008; Zibell et al.,2009). Together, these findings led us to speculate that a connec-tion exists between C1P and COX-2-mediated P-glycoproteininduction. Indeed, our results showed that COX-2 activity isnecessary for C1P to increase the activity of P-glycoprotein inisolated rat and mouse brain capillaries.We further identified that EP2, a G-protein–coupled re-

ceptor for PGE2, is involved in the induction of P-glycoproteincaused by C1P. In cell lines, C1P has been shown to increasePGE2 production, even at concentrations as low as 300 nM(Pettus et al., 2005). Additional studies have proposed theinvolvement of G-protein coupled receptors in C1P signaling(Granado et al., 2009). While our study did not identify anyC1P-specific receptors in brain capillaries, we found that C1Paction on P-glycoprotein did require the general activity of aG-protein coupled receptor. More specifically, EP2 recep-tor antagonists blocked the ability of C1P to increaseP-glycoprotein activity. Our experiments also indicate possi-ble involvement of EP1, althoughEP1 inhibitors only partiallyattenuated P-glycoprotein induction.Previous studies associate EP receptors with the BBB

(McCullough et al., 2004; Pekcec et al., 2009; Jiang andDingledine, 2013). EP2, in particular, shares biologic charac-teristics with C1P; both EP2 and C1P have been implicatedwith decreased apoptosis, increased angiogenesis, and thepromotion of inflammatory responses through COX-2 (Sunget al., 2005; Kamiyama et al., 2006; Liang et al., 2008; Kimet al., 2013; Rivera et al., 2015). Our results show that EP2exists almost exclusively on the luminal membrane of ratbrain capillaries, suggesting that in our model PGE2 activatesits receptor in the capillary lumen. Signaling componentsdownstream of PGE2 should be identified to determine the fullpathway throughwhich C1P increases P-glycoprotein activity.Drug resistance in disorders such as brain cancer, epilepsy,

and depression show associations with higher activity of effluxtransporters at the BBB, including P-glycoprotein (Löscherand Potschka, 2005; Brandt et al., 2006). It is not uncommonfor such diseases to also exhibit increased levels of theenzymes involved in C1P production, such as CERK andsphingomyelinase D. Higher levels of CERK have beenassociated with tumor recurrence (Payne et al., 2014), andpatients suffering from depression have presented with raisedlevels of sphingomyelinase D (Kornhuber et al., 2005). Giventhe results of our study, future work should explore theassociations between C1P-mediated P-glycoprotein inductionand drug resistance associated with precursors of C1P. Drugresistance in certain diseases might be linked with C1P-mediated up-regulation of transporters that restrict drugaccess to the CNS.On the other hand, P-glycoprotein is critical for brain

homeostasis and limits the passage of harmful metabolitesand xenobiotics into the CNS. Studies have shown that in-flammation can change the activity of P-glycoprotein (Baueret al., 2007;Miller et al., 2008; Chodobski et al., 2011), and somespeculate that up-regulation of P-glycoprotein in response toinflammation may actually provide neuroprotection (Seelbach

et al., 2007; Alfieri et al., 2011). Over recent years, targetingsphingolipids has become an attractive clinical possibility forthe treatment of various health conditions; for example, studiespropose targeting C1P and CERK for tissue regeneration andthe treatment of inflammatory diseases, cancer, and otherconditions (Zeidan and Hannun, 2007; Granado et al., 2009;Arana et al., 2010; Gómez-Muñoz et al., 2010; Kim et al., 2013;Maceyka and Spiegel, 2014; Pastukhov et al., 2014; Baudißet al., 2016). Given our results, the neuroprotective functions ofC1P, CERK, and other sphingolipids should be considered inthese cases. While inhibition of C1P or CERK might be aneffective therapeutic strategy certain cases, it might also havethe potential to lower CNS protection against further cellularharm.From our results, we propose that endogenous production of

C1P via CERK in brain tissue increases the basal activity ofP-glycoprotein and contributes to general neuroprotection inhealthy brains. In cases of cellular injury or stress, it ispossible that increases in C1P would protect against addi-tional cellular damage. Conversely, in disease states whereinthe levels of C1P and CERK increase, the activation of thePLA2/COX-2/PGE2 pathway may contribute to drug re-sistance. As such, the ability of C1P and CERK to restrictBBB transport could either be targeted for enhanced drugdelivery or exploited for neuroprotection. Also important isthe potential for enzymatic interconversion of the sphingo-lipids (C1P and S1P) and their ability to rapidly alterP-glycoprotein activity in opposite directions. Controllingthe balance of C1P and S1P cellular levels would provide amechanism for dynamic regulation of P-glycoprotein trans-port activity at the BBB. In all, our data support that C1Pis an important regulator of P-glycoprotein activity andhas the potential to be a versatile molecule for clinicalmanipulation.

Acknowledgments

The authors thank the members of the Miller laboratory fortechnical support: Dr. Gary Chan, Rebecca Evans, David Banks, andJoyce Blaisdell. The authors also thank Dr. Artiom Gruzdev and Dr.Matthew Edin for their involvement and advice, and the staff at theNIEHS animal facility for providing care to the animals used in thisstudy.

Authorship Contributions

Participated in research design: Mesev, Miller, Cannon.Conducted experiments: Mesev.Performed data analysis: Mesev, Cannon.Wrote or contributed to the writing of the manuscript: Mesev,

Cannon.

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Address correspondence to: Dr. Ronald E. Cannon, 111 TW AlexanderDrive, Research Triangle Park, NC 27709. E-mail: cannon1@niehs.nih.gov

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