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NR4A orphan nuclear receptors influence retinoic acid and docosahexaenoic acid signaling via up-regulation of fatty acid binding protein 5

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NR4A orphan nuclear receptors influence retinoic acid and docosahexaenoic acid signaling via up-regulation of fatty acid binding protein 5
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  NR4A orphan nuclear receptors influence retinoic acid and docosahexaenoicacid signaling via up-regulation of fatty acid binding protein 5 Nikolaos Volakakis a , Eliza Joodmardi a , Thomas Perlmann a,b, * a Ludwig Institute for Cancer Research Ltd., Box 240, S-17177 Stockholm, Sweden b The Department of Cell and Molecular Biology, Karolinska Institute, S-17177 Stockholm, Sweden a r t i c l e i n f o  Article history: Received 20 October 2009Available online 25 October 2009 Keywords: Nuclear receptorNurr1 FABP5 Gene expression a b s t r a c t The orphan nuclear receptor (NR) Nurr1 is expressed in the developing and adult nervous system and isalso induced as an immediate early gene in a variety of cell types.  In silico  analysis of human promotersidentified  fatty acid binding protein 5 (FABP5),aproteinshowntoenhanceretinoicacid-mediatedPPAR  b / d signaling, asapotential Nurr1target gene. Nurr1haspreviouslybeenimplicatedinretinoidsignaling viaits heterodimerization partner RXR. Since NRs are commonly involved in cross-regulatory control wedecided to further investigate the regulatory relationship between Nurr1 and FABP5. FABP5 expressionwas up-regulated by Nurr1 and other NR4A NRs in HEK293 cells, and Nurr1 was shown to activateand bind to the  FABP5  promoter, supporting that  FABP5  is a direct downstream target of NR4A NRs.We also show that the RXR ligand docosahexaenoic acid (DHA) can induce nuclear translocation of FABP5. Moreover, via up-regulation of   FABP5  Nurr1 can enhance retinoic acid-induced signaling of PPAR  b / d  and DHA-induced activation of RXR. We also found that other members of the NR4A orphanNRs can up-regulate FABP5. Thus, our findings suggest that NR4A orphan NRs can influence signalingevents of other NRs via control of FABP5 expression levels.   2009 Elsevier Inc. All rights reserved. Introduction NRs comprise a family of transcription factors that have criticalfunctionsduringdevelopmentandinadultphysiology.NRsincludesteroid hormone receptors and receptors for other lipophilic li-gands such as vitamin D3 and retinoids [1]. Several members of the NR family lack identified ligands and are referred to as orphanreceptors [2]. Nurr1 is an orphan receptor, which together withNGFI-B and Nor1, constitutes the NR4A orphan NRs [3]. Nurr1 isexpressed within the embryonic central nervous system where itplays a key role for the development of dopamine neurons [4]and continues to be expressed in the adult brain where it may becritical for dopamine neuron survival and other functions [5,6].Nurr1, NGFI-B and Nor1 are unique within the NR family in beingencodedbyimmediateearlygenesthatarerapidlyinducedbyvar-ious stimuli such as growth factors, ischemia and seizures [7].The structural features of the Nurr1 and DHR38 (NR4A-homo-logue in  Drosophila ) ligand binding domain show that the NR4Afamily members lack a hydrophobic pocket for ligand bindingand thus function as ligand-independent NRs [8,9]. NR4A proteinsbindtoDNAeitherasmonomersorhomodimersandpromotecon-stitutive activation of transcription [10,11]. In addition, Nurr1 andNGFI-B, but not Nor1, can form heterodimers with the retinoid Xreceptor(RXR),whichhavetheabilitytopromotestrongtranscrip-tional activation after binding to RXR ligands like 9- cis -retinoicacid [12] or fatty acids such as docosahexaenoic acid [13]. Inthis studyweidentify  fatty acid binding protein 5  ( FABP5 ) as aNurr1-regulated gene. Fatty acid binding proteins are cytosolicproteins that bind long-chain fatty acids [14].  FABP5  is expressedin a variety of tissues [15] and has been implicated in regulationof water permeability barrier of the skin, neurite outgrowth [16]andfatty acidtransport duringneuronal regeneration[17,18]. Ret-inoicacidbindingtoFABP5leadstoitstranslocationtothenucleusand enhancement of retinoic acid-induced activation of PPAR  b / d ,which in turn can promote cellular survival [19]. DHA, a ligandfor RXR, has also been shown to bind to FABP5 [20]. Since Nurr1is indirectly linked to retinoid and DHA-signaling via its ability toform heterodimers with RXR we were interested to further inves-tigate if Nurr1-mediated regulation of   FABP5  could influencecross-regulation between Nurr1 and other NR-mediated signalingpathways. OurexperimentsindicatethatNurr1canregulate FABP5 expression via direct binding to the  FABP5  promoter and we alsoprovideevidenceindicatingthatNurr1canmodulateRXRsignalingviathisregulation.Thus, FABP5 couldbearegulatedtargetofNurr1and the two other NR4A NRs in an immediate early context andthereby influence signaling by ligands binding to RXR. 0006-291X/$ - see front matter    2009 Elsevier Inc. All rights reserved.doi:10.1016/j.bbrc.2009.10.116 *  Corresponding author. Address: Ludwig Institute for Cancer Research Ltd., Box240, S-17177 Stockholm, Sweden. Fax: +46 8 332812. E-mail address:  thomas.perlmann@licr.ki.se (T. Perlmann).Biochemical and Biophysical Research Communications 390 (2009) 1186–1191 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc  Materials and methods Chemicals.  Retinoic acid and DHA ( cis -4,7,10,13,16,19-docosa-hexanoic acid, C22:6) were purchased from Sigma–Aldrich.LG268 was kindly provided by Mark Leibowitz at LigandPharmaceuticals. siRNA.  A control siRNA and a pool of three siRNAs designed toknock down human  FABP5  expression were purchased from SantaCruz Biotechnology. Cells were transfected in 24-well plates with20pM siRNA per well using Lipofectamine 2000. Plasmids. pCMX-Nurr1,-Nor1,-NGFI-B,-Nurr1 R334A and-Nurr1 dim have been described by [21]. The luciferase reporter MH100-tk-luccontainsfourcopiesoftheGAL4bindingsiteupstreamoftheherpessimplexvirusthymidinekinasepromoter[12].pCMX-RXR  a ,-LacZ,-GAL4-Nurr1 and -GAL4-Nurr1 dim are described by [22]. PPREx3-tk-luc has been described by [23], while PSG5-PPAR  b / d  was providedby Dr. Desvergne (University of Lausanne, Switzerland). The  FABP5 promoterfragmentwasamplifiedfromgenomicDNAextractedfromHEK293cellsusingQuickExtractDNAextractionsolution(EpicentreBiotechnologies).Thefollowingprimerswereused:5 0 -AAAAGCTAGCGTGGTCTGATTTCATAAGGT-3 0 and5 0 -AAAAAGATCTGCACCCGGCGCCGGCGGCTG-3 0 . The PCR-amplified promoter fragment was ligatedintothepGL2basicplasmid(Promega).The FABP5 promoterconstructwiththemutatedNBREsitewasgeneratedwithaPfuultrapolymer-ase(Stratagene)reactionwiththewild-typepromoterconstructasatemplate and the following primers: F: 5 0 -GCGAGGAGCAGAAGGAAAGGGAGGCACCGTAG-3 0 and R: 5 0 -CTACGGTGCCTCCCTTTCCTTCTGCTCCTCGC-3 0 . The human  FABP5  cDNAwas amplified fromthe IR-AVp968H094 cDNA clone (RZPD, Accession No. BC002008) usingthefollowingprimers:5 0 -AAAAAAGCTTATGGCCAGTCTTAAGGATCT-3 0 and 5 0 -AAAAGCTAGCTCATTGCACCTTCTCATAGA-3 0 . The PCR-amplifiedfragmentwasclonedintothepCMXvector. Cell culture and transfections.  Human embryonic kidney (HEK)293 cells and chorion carcinoma JEG-3 cells were maintained inDMEM and MEM (Invitrogen), respectively. Media were supple-mented with 10% heat-inactivated fetal calf serum (Invitrogen) at37  C under 5% CO 2  humidifiedatmosphere. Cells were transfectedwith the appropriate plasmids using Lipofectamine Plus reagent(Invitrogen) according to the manufacturer’s instructions. Cellswere lysed in 0.65% NP40, 10mM Tris HCl pH 8.0, 1mM EDTApH 8.0 and cell extracts were assayed for luciferase and  b -galacto-sidase activity using Lucy luminometer (Anthos Labtec Instru-ments). The ratio of luciferase activity to  b -galactosidase activitywas calculated to normalize the luciferase value. For all transienttransfections, values represent the mean of nine samples. Real-time PCR.  Total RNA was prepared using the RNAeasy minikit (Qiagen) and reverse-transcribed using SuperScriptII (Invitro-gen). Real-time PCR analysis was performed on the Rotor-Gene3000A(CorbettResearch)usingSYBRPCRMastermix(AppliedBio-systems). The following primers were used:  FABP5  F: 5 0 -GGAAGATGGCGCCTGGTGGA-3 0 ,  FABP5  R: 5 0 -CCGAGTACAGGTGACATTGT-3 0 ,GAPDH F: 5 0 -CATGGCCTTCCGTGTTCCTA-3 0 and GAPDH R: 5 0 -GCGGCACGTCAGATCCA-3 0 . All values were normalized against thehousekeepinggeneGAPDHandthenpresentedasrelativeincreaseof  FABP5  expression. Dataarepresentedasthemean+SEMofqua-druplicate determinations of a representative experiment. Similarresultswereobtainedinatleastthreeindependentexperiments. Western blot analysis. HEK293cellswerelysedin10mMHepes–KOH pH 7.9, 0.4M NaCl, 1mM EDTA, 5% glycerol). For theexperiment described in Fig. 4C, extracts were performed usingthe protocol described by [24]. Equal amounts of extracts wereelectrophoresed on 10% SDS–polyacrylamide gels and blotted ontoPVDF membranes (BioRad). After transfer, the membrane waswashed with PBS pH 7.6 and saturated with 5% dry milk in PBS0.1%Tween20(PBS-T)for1hatroomtemperature.Themembranewasthenincubatedwithanti-human FABP5 (Biovendor),anti-GAP-DHandanti-histone1(SantaCruzBiotechnology)rabbitpolyclonalantibodies in PBS-T 0.5% milk for 18h at 4  C. The membrane wasincubated with horseradish peroxidase-coupled anti-rabbit IgGantibody (Pierce) for 60min at room temperature. Labeling wasperformed as described in the ECL Plus detection kit (Amersham). Chromatin immunoprecipitation.  Chromatin immunoprecipita-tion was performed using the ChIP assay kit (Upstate). The pro-tein–DNA complexes were cross-linked using 37% formaldehyde(Sigma–Aldrich), sheared by sonication and immunoprecipitatedovernightat4  Cwith2 l grabbitanti-Nurr1antibody(E-20,SantaCruzbiotechnology)orrabbitIgGantibody(R&DSystems).Theanti-bodieswereelutedfromtheDNA–proteincomplexesandthecross-links were reversed by overnight incubation at 65  C with 0.2MNaCl.Sampleswerepurified(MinElutePCRpurificationkit,Qiagen) Fig. 1.  Nurr1 regulates endogenous FABP5 expression in HEK293 cells. (A) Total RNA was extracted at different time points post transfection with cmx-Nurr1 and theexpressionlevels of FABP5mRNAweredeterminedwithreal-time PCR. (B)HEK293cells weretransientlytransfectedwiththeindicatedplasmids. Cells wereharvested 24hafter transfection and real-time PCR analysis of FABP5 expression was performed. (C) HEK293 cells were transfected with the indicated plasmids. Whole cell extracts wereprepared after 24h. Resolved on SDS–PAGE and probed with anti-FABP5 antibody. N. Volakakis et al./Biochemical and Biophysical Research Communications 390 (2009) 1186–1191  1187  and1 l lChIPDNAwasusedforPCRwithprimersamplifyingaregioncontaining the NBRE in the  FABP5  promoter (F: 5 0 -GATGGCGAGAGCAGGTTCTC-3 0 andR:5 0 -CGCCGGACTCGGACTGCAGG-3 0 )andacon-trolregionlocated4260bpdownstreamofthetranscriptionalstartsite of the  FABP5  gene (F: 5 0 -GGGGCAGTCTTGAGGGTACT-3 0 and R:5 0 -TCAAATAGTATAGGGCAAGC-3 0 ). Results Transcriptional regulation of the FABP5 gene in HEK293 cells To identify potential Nurr1-regulated genes we searched thehuman promoters included in a mammalian promoter database(MpromDb) [25] for Nurr1-binding sites situated within 1kb 5 0 the transcriptional start sites. One of the hits scored positive forNurr1-binding was  FABP5 . By transfection in HEK293 cells  FABP5 could be verified as a Nurr1-regulated gene since  FABP5  mRNAexpression, was up-regulated in cells transfected with a Nurr1expression vector (Fig. 1A).Next,weanalyzediftherelatedNRsNGFI-BandNor1werealsoable to induce  FABP5  expression. Nurr1, NGFI-B and Nor1 expres-sion vectors were transiently transfected into HEK293 cells.Twenty-four hours post transfectioncellswere harvestedandana-lyzed for  FABP5  mRNA expression. NGFI-B and Nor1 were alsocapable of inducing  FABP5  mRNA expression but somehow lessefficiently as compared to Nurr1 (Fig. 1B). Furthermore, HEK293cells were transiently transfected with different Nurr1 derivativesfollowed by analysis of   FABP5  mRNA levels. Nurr1 dim , a mutantlacking the ability to form heterodimers with RXR  [22], functionsequally efficient as wild-type Nurr1 in inducing  FABP5  mRNAexpression. As expected, a DNA-binding deficient mutant of Nurr1(Nurr1 R334A ;substitutesarginine334foranalanine)wasincapableof inducing  FABP5  expression. These results indicate that Nurr1monomers or homodimers, but not heterodimers with RXR are re-quired for the induction of   FABP5 . Fig. 2.  Nurr1activatestheFABP5promoterthroughaNBREsite. (A)SchematicpictureofthefragmentofthehumanFABP5promoter constructthat wasusedandindicationof the mutation that was introduced in the putative Nurr1-binding site (NBRE). (B, C) HEK293 cells were transiently transfected with the wild-type promoter constructtogether with the indicated plasmids and harvested 24h post transfection. (D) HEK293 cells were transiently transfected with the indicated promoter constructs togetherwith mock or Nurr1. Cells were harvested 24h post transfection. (E) Schematic overview of the location of primers used in the ChIP assay. (F) After 24h of mock or Nurr1transfection, HEK293 cells were harvested and immunoprecipitated with Nurr1 or IgG antibody. PCR was performed with primers directed towards the NBRE site in theFABP5 promoter or a control region of the FABP5 gene.1188  N. Volakakis et al./Biochemical and Biophysical Research Communications 390 (2009) 1186–1191  To establish that FABP5 protein was also induced by Nurr1 wechecked the steady-state protein levels of FABP5 in HEK293 cellstransfected with Nurr1, Nor1, NGFI-B, Nurr1 dim or Nurr1 R334A . Asseen in Fig. 1C, Nurr1, Nor1 and Nurr1 dim significantly up-regu-lated the FABP5 protein level, while NGFI-B over-expressionresultedinalessrobustup-regulation.Asexpected,theDNA-bind-ing mutant Nurr1 R334A did not affect FABP5 expression. The human FABP5 promoter is regulated by Nurr1 A NBRE site in the  FABP5  promoter (GAAGGTCA) is situated atposition   583 to   576 in relation to the putative  FABP5  transcrip-tional start site. A fragment containing 1133bp of the humangenomic sequence upstream of the  FABP5  transcriptional start site( FABP5 -1133, Fig. 2A) was isolated and cloned into a luciferase re-porter plasmid. In transient transfections in HEK293 cells, Nurr1activated the  FABP5 -1133 construct about 2-fold (Fig. 2B). Consis-tent with measurements of   FABP5  mRNA expression, Nurr1 mono-mers or homodimers, but not RXR heterodimers are required forincreased  FABP5 -1133 reporter gene activity (Fig. 2C). The DNA-binding mutant of Nurr1 (Nurr1 R334A ) was also unable to increasereporter gene activity.To investigate whether the putative NBRE is important forNurr1-dependent activation of the  FABP5  promoter, adenine sub-stitutions that disrupt this sequencewere introduced in the repor-ter construct. These mutations reduced the ability of Nurr1 toactivate the promoter (Fig. 2D) suggesting that Nurr1 recruitmenttotheintactNBREsiteisimportantforactivationofthe FABP5  pro-moter. To verify that Nurr1 is recruited to this site at the endoge-nous promoter, we performed a chromatin immunoprecipitationassay in HEK293 cells. After 24h in the presence or absence of transiently transfected Nurr1, proteins were cross-linked to chro-matin and immunoprecipitated with an antibody against Nurr1.PCR primers amplifying a region containing the NBRE were used(Fig.2E).AsshowninFig.2F,Nurr1wasrecruitedtotheNBRE-con- taining region but not to a control region located 4260bp down-stream of the  FABP5  transcriptional start site. Nurr1 enhances RA-induced PPAR b  /  d  signaling and DHA-induced RXRsignaling by inducing FABP5 expression FABP5 can bind retinoic acid, translocate to the nucleus inresponse to it and enhance retinoic acid-induced activation of PPAR  b / d  [19]. To test if Nurr1-mediated up-regulation of FABP5enhances RA-induced PPAR  b / d  signaling we transfected humanPPAR  b / d  together with a reporter containing PPAR binding sites(PPREx3-tk-luc).Asexpectedfrompreviousresults,co-transfectionofFABP5enhancedRA-inductionofthePPARreporter.Nurr1couldalso enhance the reporter but this effect was blunted when Nurr1was co-transfected with siRNA against  FABP5 , suggesting thatNurr1, via the induction of FABP5 expression, can enhance RA-in-ducedactivationof PPAR  b / d  (Fig. 3A). SinceFABP5hasbeenshowntobindthepolyunsaturatedfattyacidDHA[20]andsinceDHAisaligandforthenuclearreceptorRXR [13]wewishedtoinvestigateif FABP5 influences DHA-induced RXR signaling. Since RXR can formheterodimers with Nurr1 we transiently transfected RXR  a  andGAL4-Nurr1 and a GAL4-reporter in human JEG-3 cells [12].GAL4-Nurr1-RXR heterodimers can activate the reporter in re-sponse to RXR ligands and thus function as sensors for RXR activa-tion. Co-expression of FABP5 leads to increased DHA activation of the GAL4-Nurr1-RXR heterodimers indicating that FABP5, similarto its influence on RA-induced activation of PPAR  b / d , can promoteRXR signaling. In contrast, FABP5 was not able to enhance activa-tionbyasyntheticligand(LG268)thathasdifferentstructurefromfatty acids that can bind to FABP5. Enhanced activation of the re-porter was also observed when the RXR-dimerization mutant of Nurr1, but not when the DNA-binding mutant of Nurr1, was co-transfected (Fig. 4B). Nurr1 dim can induce the expression of FABP5(Fig 1B) but cannot compete with GAL4-Nurr1 for interactionwithRXR. In cells over-expressing siRNA against  FABP5 , the enhance-ment of the reporter activation by Nurr1 dim was blunted, suggest-ing that the induction of FABP5 expressionby Nurr1 is responsiblefor the enhancement of DHA-induced activation of RXR. Finally,treatmentofJEG-3cellswith200 l MDHAfor1hresultedintrans-location of over-expressed FABP5 from the cytoplasm to the nu-cleus (Fig. 4C) suggesting that FABP5 delivers DHA to the nucleuswhere it can exert its transcriptional effects. Discussion Our results show that  FABP5  mRNA and protein expression areup-regulated in Nurr1-over-expressing HEK293 cells. Nurr1 alsoregulates the  FABP5  promoter (including 1133bp of the  FABP5  up-stream sequence) in reporter assays, but the activation of the pro-moter by Nurr1 is relatively weak since the  FABP5  promoteralready has a basal activity in HEK293 cells, emphasized by detec-tion of basal  FABP5  mRNA and protein levels (Fig. 1C). Fig. 3.  Nurr1 enhances RA-induced PPAR  b / d  signaling. (A) JEG-3 cells were transiently transfected with the luciferase reporter PPREx3-tk-luc, pSG5-PPAR  b / d  and emptyvector (mock) or cmx-FABP5 or cmx-Nurr1. Cells transfected with Nurr1 were pre-transfected for 24h with control siRNA or siRNA against FABP5. 9- cis -Retinoic acid (RA)was added 5h post transfection and the cells were harvested 20h post RA addition. (B) Efficiency of down-regulation of FABP5 expression by FABP5 siRNA. N. Volakakis et al./Biochemical and Biophysical Research Communications 390 (2009) 1186–1191  1189  We have shown that Nurr1 up-regulates  FABP5  expression in vitro  and that it can bind to the endogenous  FABP5  promoterin HEK293 cells. However, it is unlikely that Nurr1 or other NR4Amembers are critical for the basal  FABP5  expression  in vivo  sincetissueswithhighlevelsof  FABP5  and NR4A  mRNAsdonotcorrelate([15]; data not shown). Although both  FABP5  and  Nurr1  areexpressed in the mouse brain our analyses showed that they arenot co-localized under basal conditions and expression of   FABP5 mRNA is unaffected in Nurr1 knock-out brain (data not shown).Thus, we favor that  FABP5  is downstream Nurr1 in stress responsepathwaysinitiatedbystressstimulisuchasbraininjury,seizureorinflammation. Potential links between Nurr1 and  FABP5  favor thishypothesis. First, both  Nurr1  and  FABP5  are induced in the hippo-campus by kainic acid-induced seizures [26,27]. Second, Nurr1 isinduced by focal brain injury and  FABP5  is up-regulated uponperipheral nerve injury [17,28]. Third, treatment with lipopolysac-charide induces  NR4A  and  FABP5  expression in macrophages[29,30]. NR4A NRs are believed to be transcriptional mediators of inflammatorysignalsinactivatedmacrophageswhereFABP5couldbearelevantNR4Atarget.Fourth, Nurr1isexpressedinepidermis,blood vessels and inflammatory infiltrates of psoriasis skin and FABP5  is dramatically up-regulated in psoriatic tissue [31,32].These dataprovideadditional evidence linkingbothgenes inregu-latory responses to inflammation.FABPs also have diverse roles in regulating the metabolismandactivities of their ligands [33]. A relevant aspect of FABP5 in thecontext of our data is its potential link to NR-mediated signalingpathways. FABP5 has been shown to relocate to the nucleus inresponse to a ligand binding PPAR  b / d , and enhances its transcrip-tional activity [34]. FABP5 was also shown to bind RA and deliverit from the cytosol to stimulate PPAR  b / d  transcriptional activity[19]. In this study we complement these observations by showingthat Nurr1 enhances RA-induced PPAR  b / d  signaling by inducingtheexpressionofFABP5andthusmayplayanindirectpro-survivalrole since RA-activation of PPAR  b / d  activates cellular pro-survivalpathways (Fig. 3) [19]. FABP5 has also been shown to bind DHA, which can bind to and activate RXR  [13,19,20]. We provide evi-dence showing that FABP5 can enhance DHA-induced RXR signal-ing presumably influenced by the ability of DHA to inducetranslocation of FABP5 from the cytoplasm to the nucleus(Fig.4).Thus,thepositiveeffectofFABP5onDHA-inducedRXRsig-naling might resemble how FABP5 facilitates PPAR  b / d  signaling bydirect delivery of cognate ligands to the nucleus.Nurr1 and NGFI-B can form heterodimers with RXR that areresponsive to ligands activating RXR. We show that Nurr1/NGFI-Binduction of FABP5 can serve to deliver DHA to the Nurr1/NGFI-B-RXR heterodimers and further potentiate DHA-mediated RXR acti-vation. Thus, it is interesting to consider the significance of thecross-regulatory relationships that might tie Nurr1/NGFI-B to RXR signaling via  FABP5  up-regulation [12]. Stressful stimuli, such asischemia,caninduceNurr1expressionandDHAreleasefromthecellmembrane[35,36].Nurr1-inducedFABP5expressioncouldbeenvi-sioned to positively influence the outcome after neuronal stress intwoways:First,directfattyacidFABP5bindingcouldservetoreducetoxic effects caused by fatty acid accumulation under stressfulinsults. Second, FABP5 can also bind to DHA and deliver it toNurr1-RXRheterodimersthathavebeenshowntomediatesurvivalsignalinginneurons[37].ItisintriguingtospeculatethatbothNurr1andDHAaremadeavailableundersituationsrequiringengagementbyacutelyinducedneuroprotectivemechanisms. Fig. 4.  Nurr1 enhances DHA-induced RXR signaling. (A, B) JEG-3 cells were transiently transfected with the luciferase reporter MH100-tk-luc, GAL4-Nurr1, RXR  a  and emptyvector (mock) or cmx-FABP5 (B) or cmx-Nurr1 dim and cmx-Nurr1 R334A (C). In (B) cells transfected with cmx-Nurr1 dim were pre-transfected for 24h with control siRNA orsiRNAagainstFABP5.DHAwasadded5h(A)or17h(B)posttransfectionandcellswereharvested20hpostDHAaddition.LG268wasaddeddirectlytothemedium5hposttransfection (A) and cells were harvested 20h post LG268 addition. (C). Steady-state protein levels of FABP5. JEG-3 cells were transfected pCMX-FABP5 and treated with200 l M DHA 23h post transfection for 1h. Cytoplasmic and nuclear extracts were prepared 1h after DHA addition.1190  N. Volakakis et al./Biochemical and Biophysical Research Communications 390 (2009) 1186–1191
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