ORIGINAL ARTICLE

Mechanical Stress Induces PrendashB-cell Colony-EnhancingFactorNAMPT Expression via Epigenetic Regulation bymiR-374a and miR-568 in Human Lung EndotheliumDjanybek M Adyshev Venkateswaran Ramamoorthi Elangovan Nurgul Moldobaeva Brandon MapesXiaoguang Sun and Joe G N Garcia

Institute for Personalized Respiratory Medicine Department of Medicine Section of Pulmonary Critical Care Sleep and AllergyUniversity of Illinois at Chicago Chicago Illinois

Abstract

Increased lung vascular permeability and alveolar edema are cardinalfeatures of inflammatory conditions such as acute respiratory distresssyndrome (ARDS) and ventilator-induced lung injury (VILI) Wepreviously demonstrated that prendashB-cell colony-enhancing factor(PBEF)NAMPT the proinflammatory cytokine encoded byNAMPTparticipates in ARDS and VILI inflammatory syndromes The presentstudy evaluated posttranscriptional regulation of PBEFNAMPTgene expression in human lung endothelium via 39-untranslatedregion (UTR) microRNA (miRNA) binding In silico analysisidentified hsa-miR-374a and hsa-miR-568 as potential miRNAcandidates Increased PBEFNAMPT transcription (by RT-PCR) andexpression (by Western blotting) induced by 18 cyclic stretch (CS)(2 h 346 006 mRNA fold increase (FI) 10 h 156 006 protein FI)and by LPS (4 h 386 02 mRNA FI 48 h 266 02 protein FI) weresignificantly attenuated by transfection with mimics of hsa-miR-374aor hsa-miR-568 (40ndash60 reductions each) LPS and 18CS increasedthe activity of a PBEFNAMPT 39-UTR luciferase reporter (24ndash325FI) with induction reduced by mimics of each miRNA (44ndash60reduction) Specific miRNA inhibitors (antagomirs) for each PBEFNAMPTmiRNA significantly increased the endogenous PBEF

NAMPTmRNA(14ndash34601FI) andprotein levels (12ndash14601FI)and 39-UTR luciferase activity (14ndash176 01 FI) compared withnegative antagomir controls Collectively these data demonstrate thatincreased PBEFNAMPT expression induced by bioactive agonists(ie excessive mechanical stress LPS) involves epigenetic regulationwith hsa-miR-374a and hsa-miR-568 representing novel therapeuticstrategies to reduce inflammatory lung injury

Keywords miRNA PBEFNAMPT ARDS ventilator-inducedlung injury endothelial cells

Clinical Relevance

Targeting of prendashB-cell colony-enhancing factorNAMPTa critical inflammatory biomarker in acute respiratory distresssyndrome and ventilator-induced lung injury via specificmicroRNAs is a potentially promising novel therapeuticstrategy to reduce lung vascular permeabilityedema andinflammation in acute respiratory distress syndrome andventilator-induced lung injury

Acute lung injury (ALI) acute respiratorydistress syndrome (ARDS) and ventilator-induced lung injury (VILI) are devastatingsyndromes characterized by profoundlung inflammation increased lung vascularpermeability and protein-rich alveolaredema (1 2) Despite improved

understanding of ARDS pathophysiologythe morbidity and mortality of ARDScontinue to be unacceptably high (3) andthe mechanisms underlying VILI inthe setting of ARDS remain unclearFurthermore effective pharmacotherapyfor ARDS or VILI has yet to emerge Using

genomic-intensive expression profiling incanine and murine models of humanALI we previously identified prendashB-cellcolony enhancing factor (PBEF)a proinflammatory cytokine andnicotinamide adenine dinucleotidebiosynthetic enzyme (nicotinamide

(Received in original form June 27 2013 accepted in final form September 12 2013 )

Joe G N Garcia is now employed at the University of Arizona

This work was supported by National Institutes of HealthHeart Lung Blood Institute grants P01 HL58064 P01 HL98050 and R01 HL94394

Correspondence and requests for reprints should be addressed to Joe GN Garcia MD Senior Vice President for Health Sciences University of ArizonaRoy P Drachman Hall Bldg B207 1295 North Martin Avenue PO Box 210202 Tucson AZ 85721-0202 E-mail skipgarciaemailarizonaedu

This article has an online supplement which is accessible from this issuersquos table of contents at wwwatsjournalsorg

Am J Respir Cell Mol Biol Vol 50 Iss 2 pp 409ndash418 Feb 2014

Copyright copy 2014 by the American Thoracic Society

Originally Published in Press as DOI 101165rcmb2013-0292OC on September 20 2013

Internet address wwwatsjournalsorg

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 409

phosphoribosyltransferase) (4ndash6) encodedby NAMPT as a novel biomarker in ARDSand as an ARDS and VILI candidate gene(7) PBEFNAMPT levels are increased invisceral fat leading to its renaming asvisfatin an adipokine regulating metabolicsyndrome and diabetes (8) PBEFNAMPTis up-regulated in murine canine andhuman ARDS with spatial localizationto lung leukocytes and the vascularendothelium (7) PBEFNAMPT is also aninflammatory mediator secreted in thedevelopment of VILI (9) and influencesARDS severity (10) Supporting the humanand animal ARDS data PBEFNAMPTalters vascular barrier integrity viaCa21ndashdependent cytoskeletal rearrangementand enhances the inflammatory response inexperimental and clinical sepsis throughregulation of neutrophil apoptosis andinflammatory cytokine secretion (11 12)A number of stimuli associated withinflammation and infection (LPS TNF-aoxidized low-density lipoprotein IL-1b IL-6and cyclic stretch) up-regulate PBEFNAMPT expression (4 13ndash15) Despite themultifunctionality of PBEF as an importantproinflammatory and immune systemregulator the mechanisms governing PBEFNAMPT expression are poorly understood

MicroRNAs (miRNAs) are smallconserved noncoding RNAs that regulategene expression posttranscriptionally bytargeting the 39-untranslated region (39-UTR)of specific messenger RNAs (mRNAs) fordegradation or translational repressionEmerging evidence supports miRNAs ascritical regulators of biological processes suchas cell differentiation proliferation apoptosismetabolism neuronal developmentbrain morphogenesis DNA repair andmethylation and stem cell division (16ndash18)Because miRNA deregulation is linked toinflammatory conditions (19 20) metabolicdisorders (21) cancer (22 23) cardiovascular(24 25) myeloproliferative (26) autoimmune(27) and neurodegenerative diseases (28)miRNAs have emerged as novel biomarkersand therapeutic targets In the present studywe explored the role of miRNAs inARDSVILI pathogenesis focusing onposttranscriptional PBEF gene regulationusing the PBEFNAMPT 39-UTR luciferasereporter overexpression of mature miRNAsand endogenous miRNA inhibition byantagomirs Preliminary in silico analysisidentified two miRNA candidates hsa-miR-374a and hsa-miR-568 as potentially bindingto the 39-UTR of PBEFNAMPT We

investigated whether these miRNAsparticipate in regulation of LPS- and 18cyclic stretch (CS)-induced NAMPT geneexpression in vitro

Materials and Methods

ReagentsLPS from Escherichia coli was obtained fromSigma-Aldrich (St Louis MO) Human PBEF39-UTR cloned downstream of a fireflyluciferase gene was obtained from SwitchGearGenomics (Menlo Park CA) Rabbitpolyclonal anti-PBEF antibodies (Ab) wereobtained from Bethyl Laboratories Inc(Montgomery TX) and mouse monoclonalAb against b-actin was obtained from Sigma-Aldrich Unless specified biochemicalreagents were obtained from Sigma-Aldrich

Cell CultureHuman pulmonary artery endothelial cells(ECs) were obtained from Lonza Inc(Allendale NJ) and cultured in endothelialbasal medium-2 with 10 FBS at 378C ina humidified incubator with 5 CO2 aspreviously described (29) ECs at passages 5through 9 were used for all experiments

Bioinformatics Analysis andmiRNA PredictionTo predict miRNAs that potentially regulatePBEFNAMPT mRNA three computationalalgorithms were used TargetScan 62 (httpwwwtargetscanorg) developed byWhitehead Institute for Biomedical Research(Cambridge MA) (30 31) MiRanda (httpwwwmicrornaorgmicrornahomedo)developed by Computational and SystemsBiology Center Memorial Sloan-KetteringCancer Center (New York NY) (32) andDIANA-microT-CDS (httpdianacslabecentuagrmicroT) developed by DIANA-LabInstitute of Molecular Oncology BiomedicalSciences Research Center ldquoAlexanderFlemingrdquo (Vari Greece) (33) Further detailsare provided in the online supplement

Transient Transfections andReporter AssaysTransfection of luciferase reporterconstructs and miRNA or miRNAinhibitors was performed according to themanufacturerrsquos protocol Luciferase activitywas measured using the Dual LuciferaseAssay System (Promega Fitchburg WI)following the manufacturerrsquos protocol

Further details are provided in the onlinesupplement

Cyclic Stretch ExperimentsAll CS experiments were performed using theFX-4000T Flexcell Tension Plus system(Flexcell International Hillsborough NC)equipped with a 25-mm BioFlex loadingstation designed to provide uniform radialand circumferential strain across a membranesurface along all radii as we describedpreviously (34 35) Further details areprovided in the online supplement

RNA Extraction and QuantitativeReal-Time PCR AnalysisTotal RNA from ECs was isolated usingTrizol and purified using miRNeasy columns(Qiagen Valencia CA) All procedures wereperformed according the manufacturerrsquosrecommendations for each assay as describedin detail in the online supplement

ImmunoblottingProtein extracts were separated by SDS-PAGE transferred to nitrocellulose orpolyvinylidene difluoride membranes(30 V for 18 h or 100 V for 15 h) andimmunoreacted with an Ab that recognizesPBEF or b-actin Immunoreactive proteinswere detected with the enhancedchemiluminescent detection systemaccording to the manufacturerrsquos directions(Amersham Little Chalfont UK) Intensitiesof immunoreactive protein bands werequantified using ImageQuant software(Molecular Dynamics Sunnyvale CA)

Statistical AnalysisResults are expressed as mean 6 SE ofthree to six independent experiments Weperformed statistical comparison amongtreatment groups by unpaired Studentrsquos t testor by randomized-design two-way ANOVAfollowed by the Newman-Keuls post hoc testfor multiple groups Results with P 005were considered statistically significant

Results

Inflammatory Agonists IncreasePBEFNAMPT Gene Expression and39-UTR Reporter Activity in HumanLung ECsLPS and excessive mechanical stress areimportant contributors to the developmentand severity of ARDS and VILI To elucidatethe effect of inflammatory agonists on PBEF

ORIGINAL ARTICLE

410 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 2 | February 2014

NAMPT gene expression we quantifiedPBEFNAMPT protein levels (0ndash48 h) PBEFNAMPT mRNA levels (0ndash8 h) and theactivity of a PBEFNAMPT 39-UTR luciferasereporter in ECs exposed to LPS (100 ngml)or to 18 CS LPS and 18 CS independently

induced sustained increased levels of PBEFNAMPT protein and PBEFNAMPT mRNACS at 18 induced early elevation in PBEFNAMPT mRNA (maximum 34 6 006 foldincrease [FI] at 2 h) and protein levels in ECs(maximum 15 6 006 FI at 10 h) with levels

remaining elevated for up to 24 hours Incontrast LPS augmented PBEFNAMPTtranscript levels that reached maximum levelsat 4 hours (38 6 02 FI) with PBEFNAMPTprotein levels reaching a maximum at48 hours (265 6 025 FI) (Figures 1Andash1D)

Figure 1 Effects of inflammatory agonists on prendashB-cell colony-enhancing factor (PBEF)NAMPT expression and 39-untranslated region (UTR) reporteractivity Total RNA was isolated from human pulmonary artery endothelial cells (ECs) challenged with control vehicle or LPS (100 ngml) or exposed to 18cyclic stretch (CS) for 0 to 8 hours PBEFNAMPT mRNA levels were detected via real-time PCR (A) Data are presented as fold change in mRNAlevel over vehicle-treated control and expressed as mean 6 SE from three independent experiments P 005 versus unstimulated control P 001versus unstimulated control Confluent ECs were treated with control vehicle or 18 CS (B) and LPS for the indicated times and endogenous PBEFwas detected via immunoblot The bar graphs represent relative densitometry (18 CS [C] LPS [D]) Data are presented as fold changes in PBEFNAMPT

over vehicle-treated control and expressed as means 6 SE from three independent experiments P 005 versus unstimulated control P 001versus unstimulated control ECs were cotransfected with PBEFNAMPT 39-UTR reporter together with phRL-TK a Renilla luciferase normalization controlvector and exposed to 18 CS (E) or treated with LPS (F) (24 h) and luciferase activity was measured using the Dual Luciferase Assay System accordingthe manufacturerrsquos protocol TNF-a luciferase reporter was used as a positive control The bar graph represents relative luciferase units Data arepresented as relative luciferase units over vehicle-treated control and expressed as means 6 SE from three independent experiments P 005 versusunstimulated control P 001 versus unstimulated control

ORIGINAL ARTICLE

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 411

Agonist-induced PBEFNAMPT increaseswere also observed using PBEFNAMPT 39-UTR luciferase reporter activity with a 32 6

03 FI after 18 CS exposure (Figure 1E)and a 24 6 012 FI after LPS challenge(Figure 1F) (24 h) compared with

unstimulated cells These data suggest thatPBEFNAMPT EC expression is altered byexposure to 18 CS or LPS

The miRNA miR-568 IsDown-Regulated in 18 CS- andLPS-Challenged Human ECsTo identify candidate miRNAs potentiallytargeting PBEFNAMPT mRNAs weused in silico prediction programs andidentified miR-374a and miR-568 aspotential miRNAs regulating PBEFNAMPT gene expression by 39-UTRbinding with miR-374a exhibiting twopotential 39-UTR binding sites Aspredicted the potential binding site onPBEFNAMPT 39-UTR for miR-374a isbroadly conserved among mammalsMoreover we noted that MYLK anARDS candidate gene (36) encodingmyosin light chain kinase is a potentialtarget of miR-374a (37) In recent studieswe noted that expression of the maturemiR-374a is significantly decreased by

Figure 2 Time-dependent effects of inflammatory agonists on mature miR-568 expression in human lungECs Total RNA was isolated from ECs and treated with control vehicle 18 CS or LPS (100 ngml) for0 to 24 hours and the level of miR-568 was determined via real-time PCR Data are presented as foldchange in microRNA level over vehicle-treated control and expressed as means 6 SE from threeindependent experiments P 005 versus unstimulated control P 001 versus unstimulated control

Figure 3 Effect of miR-568 mimic on LPS-induced human lung EC dysfunction ECs grown in chambers on gold microelectrodes were transfectedwith miR-568 mimic (A) or treated with nonspecific negative control mimic (nc) as described in MATERIALS AND METHODS and used for transendothelialelectrical resistance (TER) measurements At time = 0 cells were stimulated with LPS (100 ngml) or vehicle control Shown are pooled TER data of fiveindependent experiments (B) Maximal value of normalized TER in RCs with nc reagent (control) achieved within 12 hours was taken as 100 6 SESignificantly different from cells treated with nc reagent with LPS (P 001)

ORIGINAL ARTICLE

412 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 2 | February 2014

18 CS TNF-a or LPS with maximaldecreases by 4 hours (37) These resultssuggested that changes in miR-374aexpression may play a role in PBEFNAMPT posttranscriptional regulationTo investigate whether miR-568participates in PBEFNAMPT epigeneticregulation similar to miR-374a wedetermined whether 18 CS and LPSregulate mature miR-568 expression inECs Confluent human pulmonary ECswere exposed to 18 CS or LPS for 0 to24 hours and mature miRNA expressionwas assessed by quantitative real-timePCR analysis Figure 2 demonstrates thatmature miR-568 expression is significantlydecreased by these agonists with maximalreduction in expression at 8 hours theseresults were negatively correlated withagonist-mediated augmented PBEFNAMPTexpression (Figures 1Andash1D) and 39-UTRreporter activity (Figures 1E and 1F) Theseresults are consistent with the observation thatthe majority of miRNAs affect target geneexpression by translational repression andsubsequent mRNA degradation (38)Overall such inverse correlation betweenexpression levels of putative miRNA andmRNA andor protein increases thelikelihood that this interaction isfunctional in vivo (39) A limited numberof examples have been described showinga positive correlation between miRNAsand target mRNAs (40 41)

Mimics of miRNAs Attenuate LPS-Induced Human Lung EC PermeabilityOur previous in vitro studies demonstratedthat PBEFNAMPT-specific siRNAs (11) andmiR-374a (37) abolish PBEF expression andinflammatory agonistsrsquo thrombin- IL-1bndashand LPS-mediated hyperpermeability(43 6 29 attenuation for miR-374a)Furthermore intratracheal or intravenousdelivery of siRNA (unpublished data) orPBEFNAMPT neutralizing antibody (9)produce significant reduction in LPS-mediated lung injury and VILI To evaluatethe functional involvement of miR-568 inregulation of PBEFNAMPT gene expressionwe measured changes in LPS-induced lungEC barrier function reflected by increasedTER a highly sensitive in vitro assay ofvascular permeability ECs treated withsingle miR-568 mimics demonstratestatistically significant attenuation(388 6 18) of LPS-inducedhyperpermeability in ECs reflectedby reduced TER (P 001) (Figures 3A

and 3B) compared with agonist-stimulated ECs transfected with negativecontrol mimics These data together withour previously published data (37)indicate novel roles for miR-374a andmiR-568 in mediating LPS-induced lungEC permeability

miRNA Mimics and Antagomirs AlterInflammatory Agonist-Induced PBEFNAMPT Expression and 39-UTRReporter Activity in Human Lung ECsTo assess the potential role of miR-374a andmiR-568 in 18 CS- or LPS-inducedregulation of PBEFNAMPT expression weconducted gain- and loss-of-function

experiments by transfecting ECs withnegative control miRNA mimics orsynthetic mimics andor specific miRNAantagomirs for miR-374a or miR-568Figures 4A and 4B and Figures 5Andash5Cdepict the significant decreases in PBEFNAMPT mRNA and protein levels afterexposure of ECs to LPS or 18 CS that wasproduced by miR-374a and miR-568 mimictransfection However in cells transfectedwith miR-374a miR-568 and with thecombination of miR-374a and miR-568but not exposed to LPS or 18 CS thebasal level of PBEFNAMPT mRNA notaltered significantly (Figures 4A and 4B)suggesting that in static cells the

Figure 4 Effect of miRNA mimics on inflammatory agonistndashinduced PBEFNAMPT transcription inhuman lung ECs Total RNA was isolated from ECs transfected with nc or with the indicatedmiRNA mimics (48 h) and untreated (A and B) exposed to 18 CS (A 2 h) or treated with LPS(B 4 h) PBEFNAMPT mRNA levels were detected via real-time PCR Transfection was performedwith miR-1290 used as the second negative control Data are presented as fold change inmRNA level over vehicle-treated control and expressed as means 6 SE from four independentexperiments

ORIGINAL ARTICLE

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 413

steady-state level of mRNA is due to thesaturation of its 39-UTRs with endogenousmiRs Transfection of ECs with thecombination of miR-374a and miR-568enhanced the attenuation of LPS-inducedPBEFNAMPT transcript levels suggestingthat these miRNAs may have additive orsynergistic effects in the setting of LPS-induced EC responses Consistent withthese results overexpression of miR-374aand miR-568 mimics significantlyattenuated basal 39-UTR reporter activity(Figure 6A) (47 6 11 and 44 6 31respectively compared with stimulatedECs) as well as 39-UTR reporter activityafter exposure to 18 CS (Figure 6B)(30 6 125 and 312 6 10 respectively)and LPS (604 6 6 and 5045 6 44

respectively) (Figure 6C) Combinedoverexpression of miR-374a and miR-568mimics enhances the attenuation of 39-UTR reporter activity after EC exposure to18 CS (Figure 6B) again suggestingcooperative regulation by these miRNAsConversely Figures 7A and 7B demonstratethat miR-374a and miR-568 antagomirsenhance levels of endogenous PBEFNAMPT mRNA (338 6 008 FI for hsa-miR-374a antagomir 139 6 0028 FI forhsa-miR-568 antagomir) and protein levels(12 6 013 FI for hsa-miR-374a antagomir14 6 01 FI for hsa-miR-568 antagomir) inECs (compared with ECs transfected withnegative antagomir control) Furthermorethese antagomirs enhance PBEFNAMPT39-UTR reporter activity in ECs (14 6 01

FI for hsa-miR-374a antagomir 18 6 01 FIfor hsa-miR-568 antagomir) compared withnegative antagomir controls (Figure 7C)Together these data suggest that miR-374aand miR-568 strongly influence PBEFNAMPT gene and protein expression

Discussion

The pulmonary vascular endotheliumis a monolayer of ECs and serves asa semiselective barrier between circulatingblood and surrounding tissues with ECintegrity being critical to tissue and organfunction EC dysfunction disrupts thisvascular barrier and triggers loss of cellndashcelladhesion prooxidation and an increasein EC permeability and proliferationa process implicated in inflammatorydiseases such as ALI ARDS and VILI Themolecular mechanisms that facilitate thesephenotypic alterations are only partiallyunderstood In our previous studies (7)PBEFNAMPT was identified as a novelcandidate gene in human inflammatorylung injuries such as VILI and novelbiomarker of ARDS and sepsis withincreased PBEF expression in murinecanine and human ARDS models whichwas spatially localized to lung epitheliumleukocytes and lung vascular endotheliumHowever the exact regulatory mechanismsof PBEFNAMPT expression modulationin the pathogenesis of ARDS and VILIincluding epigenetic regulation via the39-UTR are unknown The 39-UTR isa versatile region that is enriched inregulatory elements modulating processessuch as mRNA stabilizationpolyadenylation transcript degradationtranslation and mRNA localization Thusthe 39-UTR is critical for integratedregulation of gene expression In silicoanalysis revealed that compared with thepromoter region 39-UTR regulatory motifsare preferentially conserved only on onestrand consistent with posttranscriptionalimpact (42) In addition 39-UTRs containhighly conserved sequences that arecomplementary to miRNA 8-mer motifswhich are believed to represent bindingsites for conserved miRNAs andconsequently 39-UTRs have emerged astarget sites for multiple miRNAs (43) The39-UTR length is a major determinant inmRNA expression with longer 39-UTRsbeing more likely to possess miRNA-binding sites that inhibit mRNA translation

Figure 5 Effect of microRNA (miRNA) mimics on inflammatory agonistndashinduced PBEFNAMPTexpression in human lung ECs ECs were transfected with nc or with the indicated miRNA mimics(48 h) and exposed to 18 CS (10 h) or LPS (B 48 h) and endogenous PBEFNAMPT protein wasdetected via immunoblots The bar graphs represent relative densitometry (18 CS [A] LPS [C])Data are presented as fold changes in PBEFNAMPT compared with nc or corresponding miRNApretreatment without agonist challenge and expressed as means 6 SE from three independentexperiments P 005 versus unstimulated control P 001 versus unstimulated control

ORIGINAL ARTICLE

414 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 2 | February 2014

than short 39-UTRs (43 44) In this regardthe PBEFNAMPT gene exhibits anextended 39-UTR (2809 base pairs) and ledto our speculation that PBEFNAMPTexpression may be influenced by VILI- andARDS-modulating miRNAs consistent

with the recognition that miRNAs areimportant modulators of endothelialfunction in vascular diseases and critical tothe regulation of multiple inflammatoryprocesses (45ndash48) Therefore miRNAsrepresent promising therapeutic targets and

biomarkers for ALIARDS and VILI andfacilitate the realization of personalizedmedicine for individuals with acuteinflammatory lung disease MiRNAsare inhibited by chemically stablecomplimentary oligonucleotides (or anti-miRs) or their activity is mimicked bymiRNA mimics Overall the successfulstudies in rodents and nonhuman primatesas well as the preliminary results on clinicaltrials demonstrate that inhibition andoverexpression of miRNAs is a promisingtherapeutic tool (reviewed in Reference 49)MiRNA studies will facilitate theconstruction of a comprehensive diseasemodel for ALIARDS or VILI by applyinga systems biology approach that integratesgenetic genomic epigenetic proteomicand environmental information Thesespeculations do not preclude thecontribution of additional regulatoryelements such as AU-rich elementsalternative 39-UTRs 59 and 39 ends ofmRNA interaction 39-UTR secondarystructure and 39-UTR binding regulatoryproteins in 39-UTRndashmediated control ofPBEFNAMPT gene expression Furtherinvestigation into the regulatory function ofthe PBEFNAMPT 39-UTR is needed

Because in silico bioinformatic analysesrevealed the 39-UTR of PBEF to containbinding sites for miR-374a and miR-568we sought to define their participationin the regulation of inflammatoryagonistndashinduced PBEFNAMPT geneexpression (18 CS and LPS) Previouslywe indicated that expression of the broadlyconserved mature miR-374a (50) issignificantly decreased by 18 CS TNF-aor LPS (37) We now demonstrate thatmiR-568 is expressed in ECs withsignificant down-regulation by 18 CS andLPS (Figure 2) In the present study wefound that PBEFNAMPT expression inECs is markedly up-regulated at mRNAand protein levels in response to 18 CSand LPS (Figure 1) Our combined data(37 39 51) demonstrate the negativecorrelation between expression levels ofputative miRNA and mRNAprotein andsuggest that down-regulation of thesemiRNAs may contribute to augmentedPBEFNAMPT gene expression in ECstreated with these proinflammatoryagonists To further evaluate whethermiR-374a and miR-568 specifically regulatePBEFNAMPT expression in ECstransfection with synthetic miRNA mimicsand antagomirs was used We previously

Figure 6 Effect of miRNA mimics on inflammatory agonistndashinduced PBEFNAMPT 39-UTR reporteractivity in human lung ECs ECs were cotransfected with PBEFNAMPT 39-UTR reporter togetherwith nc or with the indicated miRNA mimics and phRL-TK a Renilla luciferase normalization controlvector ECs were untreated and exposed to 18 CS (A) or treated with LPS (C) (24 h) (B) andluciferase activity was measured according the manufacturerrsquos protocol Data are presented asrelative luciferase units over vehicle-treated control and expressed as means 6 SE from fourindependent experiments Significantly different from control cells without stimulation (P 005)Significantly different from control cells stimulated with 18 CS or LPS (P 005) Significantlydifferent from control cells stimulated with 18 CS or LPS (P 001)

ORIGINAL ARTICLE

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 415

reported that targeting PBEFNAMPTby specific siRNAs (11) or by miR-374a(37) attenuates inflammatory agonist(thrombin IL-1b and LPS)-mediated EChyperpermeability consistent with the key

regulatory role of PBEF in inducing lunginflammation vascular permeability andpathobiology (11 13 14) Similarly ourdata demonstrate that miR-568 mimicsattenuate LPS-induced permeability in ECs

(Figures 3A and 3B) These mRNAs(miR-374a and miR-568) may modulateLPS-induced EC permeability by targetingthe PBEFNAMPT 39-UTR thus alteringexpression We found that transfection ofECs with mimics of miR-374a and miR-568significantly attenuated the 18 CSndash andLPS-induced augmented PBEFNAMPTmRNA and protein levels (Figures 4A and4B and 5Andash5C) Although the exactmechanism by which miR-374a andmiR-568 mimics alter PBEFNAMPTexpression is not known our data supportrecent evidence that the majority ofmammalian miRNAs directly target mRNAdown-regulation via cleavage anddegradation (52 53) or accelerateddeadenylation of mRNAs (54 55)

We further investigated the role ofmiRNAs in the regulation of PBEFNAMPTexpression by using a luciferase 39-UTRreporter gene system a sensitive methodfor measuring miRNA and the 39-UTReffects on gene regulation Our datademonstrated that EC exposure to thesemiRNAs significantly attenuates basalPBEFNAMPT 39-UTR reporter activity(Figure 6A) as well as 18 CS- andLPS-induced increases in luciferase activity(Figures 6B and 6C) Because miRNAoverexpression produces false-positiveresults by targeting genes not affected inphysiologic conditions (ie ldquooff-targeteffectsrdquo) we used the complementary ldquoloss-of-functionrdquo approach by inhibiting specificendogenous miRNA function viaantagomirs and demonstrated that specificmiRNA antagomirs increase PBEFNAMPTexpression (Figures 7A and 7B) and39-UTR reporter activity (Figure 7C) Thusour studies clearly demonstrate thatmiRNAs miR-374a and miR-568 stronglyinfluence PBEFNAMPT expression Ourdata indicate that these two miRNAscooperatively regulate PBEFNAMPTexpression in human pulmonary ECs(Figures 4B and 6B)

PBEFNAMPT is a key participantin VILI pathogenesis and is a criticalbiomarker of diverse inflammatorypathobiologies such as sepsis pneumoniaand ARDS (7 9) potentially via directeffects on innate immunity apoptosis andmitochondrial function Information islimited concerning the possible regulatorymechanisms of PBEFNAMPT expressionvia the 59 and 39 gene regions however ourin silico analysis of the 59 PBEFNAMPTpromoter region by using the Genomatix

Figure 7 Effects of miRNA antagomirs on inflammatory agonistndashinduced PBEFNAMPT expressionand 39-UTR reporter activity Total RNA was isolated from ECs and transfected with nc or with theindicated miRNA Anti- and PBEFNAMPTmRNA levels were detected via real-time PCR (A) Data arepresented as fold change in mRNA level over control and expressed as means 6 SE from fourindependent experiments Significantly different from control cells (P 005) Significantly differentfrom control cells (P 001) ECs transfected with nc or with the indicated miRNA anti- andendogenous PBEFNAMPT protein were detected via immunoblot The bar graph represents relativedensitometry (B) Data are presented as fold changes in endogenous PBEFNAMPT over ncpretreatment and expressed as means 6 SE from three independent experiments P 005 versuscontrol ECs were cotransfected with the PBEFNAMPT 39-UTR reporter together with phRL-TK ncor with the indicated miRNA (C) and luciferase activity was measured according the manufacturerrsquosprotocol Data are presented as relative luciferase units over control and expressed as means 6 SEfrom four independent experiments Significantly different from control cells (P 005)

ORIGINAL ARTICLE

416 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 2 | February 2014

genome analyzer revealed several putativecis regulatory elements including sites forbinding by transcription activators (egNF-kB SP1 STAT and GATA-1) andsequences of homology to antioxidant- shearstressndash and stretch-responsive elements(unpublished data) In addition we notednovel CpG islands in the PBEFNAMPTpromoter suggesting epigenetic regulationof PBEFNAMPT expression via DNAmethylation and 59 promoter interacting

elements The current study demonstratesthat mature expression of miR-374a andmiR-568 epigenetically regulates LPS and18 CSndashinduced PBEFNAMPT expressionin human pulmonary ECs via 39-UTRmodulation Future studies are needed todemonstrate whether miRNAs and otherregulatory elements synergistically participatein PBEFNAMPT gene regulation Targetingof PBEFNAMPT a critical inflammatorybiomarker in ARDS and VILI via specific

miRNAs is a potentially promising noveltherapeutic strategy to reduce lung vascularpermeabilityedema and inflammation inARDS and VILI n

Author disclosures are available with the textof this article at wwwatsjournalsorg

Acknowledgments The authors thank LakshmiNatarajan for providing assistance with theendothelial cell cultures

References

1 Levitt JE Matthay MA Treatment of acute lung injury historicalperspective and potential future therapies Semin Respir Crit CareMed 200627426ndash437

2 Leaver SK Evans TW Acute respiratory distress syndrome BMJ 2007335389ndash394

3 Rubenfeld GD Caldwell E Peabody E Weaver J Martin DP Neff MStern EJ Hudson LD Incidence and outcomes of acute lung injuryN Engl J Med 20053531685ndash1693

4 Ognjanovic S Bao S Yamamoto SY Garibay-Tupas J Samal B Bryant-Greenwood GD Genomic organization of the gene coding for humanpre-b-cell colony enhancing factor and expression in human fetalmembranes J Mol Endocrinol 200126107ndash117

5 Rongvaux A Shea RJ Mulks MH Gigot D Urbain J Leo O Andris FPre-b-cell colony-enhancing factor whose expression isup-regulated in activated lymphocytes is a nicotinamidephosphoribosyltransferase a cytosolic enzyme involved in NADbiosynthesis Eur J Immunol 2002323225ndash3234

6 Sommer G Garten A Petzold S Beck-Sickinger AG Bluher M Stumvoll MFasshauer M VisfatinPBEFNampt structure regulation and potentialfunction of a novel adipokine Clin Sci (Lond) 200811513ndash23

7 Ye SQ Simon BA Maloney JP Zambelli-Weiner A Gao L Grant AEasley RB McVerry BJ Tuder RM Standiford T et al Pre-b-cellcolony-enhancing factor as a potential novel biomarker in acute lunginjury Am J Respir Crit Care Med 2005171361ndash370

8 Hug C Lodish HF Medicine Visfatin a new adipokine Science 2005307366ndash367

9 Hong SB Huang Y Moreno-Vinasco L Sammani S Moitra J BarnardJW Ma SF Mirzapoiazova T Evenoski C Reeves RR et al Essentialrole of pre-b-cell colony enhancing factor in ventilator-induced lunginjury Am J Respir Crit Care Med 2008178605ndash617

10 Bajwa EK Yu CL Gong MN Thompson BT Christiani DC Pre-b-cellcolony-enhancing factor gene polymorphisms and risk of acuterespiratory distress syndrome Crit Care Med 2007351290ndash1295

11 Ye SQ Zhang LQ Adyshev D Usatyuk PV Garcia AN Lavoie TL VerinAD Natarajan V Garcia JG Pre-b-cell-colony-enhancing factor iscritically involved in thrombin-induced lung endothelial cell barrierdysregulation Microvasc Res 200570142ndash151

12 Jia SH Li Y Parodo J Kapus A Fan L Rotstein OD Marshall JCPre-b cell colony-enhancing factor inhibits neutrophil apoptosis inexperimental inflammation and clinical sepsis J Clin Invest 20041131318ndash1327

13 Liu P Li H Cepeda J Zhang LQ Cui X Garcia JG Ye SQ Critical roleof PBEF expression in pulmonary cell inflammation and permeabilityCell Biol Int 20093319ndash30

14 Li H Liu P Cepeda J Fang D Easley RB Simon BA Zhang LQ Ye SQAugmentation of pulmonary epithelial cell IL-8 expression andpermeability by pre-b-cell colony enhancing factor J Inflamm (Lond)2008515

15 Dahl TB Yndestad A Skjelland M Oie E Dahl A Michelsen A DamasJK Tunheim SH Ueland T Smith C et al Increased expression ofvisfatin in macrophages of human unstable carotid and coronaryatherosclerosis possible role in inflammation and plaquedestabilization Circulation 2007115972ndash980

16 Tomankova T Petrek M Kriegova E Involvement of microRNAs inphysiological and pathological processes in the lung Respir Res201011159

17 Sokolov MV Panyutin IV Neumann RD Unraveling the globalmicroRNAome responses to ionizing radiation in human embryonicstem cells PLoS ONE 20127e31028

18 Ji F Lv X Jiao J The role of micrornas in neural stem cells andneurogenesis J Genet Genomics 20134061ndash66

19 Zhou T Garcia JG Zhang W Integrating microRNAs into a systembiology approach to acute lung injury Transl Res 2011157180ndash190

20 Ranjha R Paul J Micro-RNAs in inflammatory diseases and asa link between inflammation and cancer Inflamm Res 201362343ndash355

21 Kornfeld JW Baitzel C Konner AC Nicholls HT Vogt MC HerrmannsK Scheja L Haumaitre C Wolf AM Knippschild U et al Obesity-induced overexpression of miR-802 impairs glucose metabolismthrough silencing of Hnf1b Nature 2013494111ndash115

22 Li N Zhong X Lin X Guo J Zou L Tanyi JL Shao Z Liang S Wang LPHwang WT et al Lin-28 homologue A (LIN28A) promotes cell cycleprogression via the regulation of cyclin-dependent kinase 2 (CDK2)cyclin D1 (CCND1) and cell division cycle 25 homolog A (CDC25A)expression in cancer J Biol Chem 201228717386ndash17397

23 Grosso S Doyen J Parks SK Bertero T Paye A Cardinaud B GounonP Lacas-Gervais S Noel A Pouyssegur J et al MiR-210 promotesa hypoxic phenotype and increases radioresistance in human lungcancer cell lines Cell Death Dis 20134e544

24 Creemers EE Tijsen AJ Pinto YM Circulating microRNAs Novelbiomarkers and extracellular communicators in cardiovasculardisease Circ Res 2012110483ndash495

25 Kohlstedt K Trouvain C Boettger T Shi L Fisslthaler B Fleming IThe AMP-activated protein kinase regulates endothelial cellangiotensin-converting enzyme expression via p53 and the post-transcriptional regulation of microRNA-143145 Circ Res 20131121150ndash1158

26 Zhao JL Rao DS Boldin MP Taganov KD OrsquoConnell RM Baltimore DNF-kappaB dysregulation in microRNA-146a-deficient mice drivesthe development of myeloid malignancies Proc Natl Acad Sci USA20111089184ndash9189

27 Persengiev SP MiRNAs at the crossroad between hematopoieticmalignancies and autoimmune pathogenesis Discov Med 201213211ndash221

28 Liu N Landreh M Cao K Abe M Hendriks GJ Kennerdell JR Zhu YWang LS Bonini NM The microRNA miR-34 modulates ageing andneurodegeneration in drosophila Nature 2011482519ndash523

29 Garcia JG Liu F Verin AD Birukova A Dechert MA Gerthoffer WTBamburg JR English D Sphingosine 1-phosphate promotesendothelial cell barrier integrity by Edg-dependent cytoskeletalrearrangement J Clin Invest 2001108689ndash701

30 Lewis BP Burge CB Bartel DP Conserved seed pairing often flankedby adenosines indicates that thousands of human genes aremicroRNA targets Cell 200512015ndash20

31 Grimson A Farh KK Johnston WK Garrett-Engele P Lim LP BartelDP MicroRNA targeting specificity in mammals determinantsbeyond seed pairing Mol Cell 20072791ndash105

ORIGINAL ARTICLE

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 417

Agonist-induced PBEFNAMPT increaseswere also observed using PBEFNAMPT 39-UTR luciferase reporter activity with a 32 6

03 FI after 18 CS exposure (Figure 1E)and a 24 6 012 FI after LPS challenge(Figure 1F) (24 h) compared with

unstimulated cells These data suggest thatPBEFNAMPT EC expression is altered byexposure to 18 CS or LPS

The miRNA miR-568 IsDown-Regulated in 18 CS- andLPS-Challenged Human ECsTo identify candidate miRNAs potentiallytargeting PBEFNAMPT mRNAs weused in silico prediction programs andidentified miR-374a and miR-568 aspotential miRNAs regulating PBEFNAMPT gene expression by 39-UTRbinding with miR-374a exhibiting twopotential 39-UTR binding sites Aspredicted the potential binding site onPBEFNAMPT 39-UTR for miR-374a isbroadly conserved among mammalsMoreover we noted that MYLK anARDS candidate gene (36) encodingmyosin light chain kinase is a potentialtarget of miR-374a (37) In recent studieswe noted that expression of the maturemiR-374a is significantly decreased by

Figure 2 Time-dependent effects of inflammatory agonists on mature miR-568 expression in human lungECs Total RNA was isolated from ECs and treated with control vehicle 18 CS or LPS (100 ngml) for0 to 24 hours and the level of miR-568 was determined via real-time PCR Data are presented as foldchange in microRNA level over vehicle-treated control and expressed as means 6 SE from threeindependent experiments P 005 versus unstimulated control P 001 versus unstimulated control

Figure 3 Effect of miR-568 mimic on LPS-induced human lung EC dysfunction ECs grown in chambers on gold microelectrodes were transfectedwith miR-568 mimic (A) or treated with nonspecific negative control mimic (nc) as described in MATERIALS AND METHODS and used for transendothelialelectrical resistance (TER) measurements At time = 0 cells were stimulated with LPS (100 ngml) or vehicle control Shown are pooled TER data of fiveindependent experiments (B) Maximal value of normalized TER in RCs with nc reagent (control) achieved within 12 hours was taken as 100 6 SESignificantly different from cells treated with nc reagent with LPS (P 001)

ORIGINAL ARTICLE

412 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 2 | February 2014

18 CS TNF-a or LPS with maximaldecreases by 4 hours (37) These resultssuggested that changes in miR-374aexpression may play a role in PBEFNAMPT posttranscriptional regulationTo investigate whether miR-568participates in PBEFNAMPT epigeneticregulation similar to miR-374a wedetermined whether 18 CS and LPSregulate mature miR-568 expression inECs Confluent human pulmonary ECswere exposed to 18 CS or LPS for 0 to24 hours and mature miRNA expressionwas assessed by quantitative real-timePCR analysis Figure 2 demonstrates thatmature miR-568 expression is significantlydecreased by these agonists with maximalreduction in expression at 8 hours theseresults were negatively correlated withagonist-mediated augmented PBEFNAMPTexpression (Figures 1Andash1D) and 39-UTRreporter activity (Figures 1E and 1F) Theseresults are consistent with the observation thatthe majority of miRNAs affect target geneexpression by translational repression andsubsequent mRNA degradation (38)Overall such inverse correlation betweenexpression levels of putative miRNA andmRNA andor protein increases thelikelihood that this interaction isfunctional in vivo (39) A limited numberof examples have been described showinga positive correlation between miRNAsand target mRNAs (40 41)

Mimics of miRNAs Attenuate LPS-Induced Human Lung EC PermeabilityOur previous in vitro studies demonstratedthat PBEFNAMPT-specific siRNAs (11) andmiR-374a (37) abolish PBEF expression andinflammatory agonistsrsquo thrombin- IL-1bndashand LPS-mediated hyperpermeability(43 6 29 attenuation for miR-374a)Furthermore intratracheal or intravenousdelivery of siRNA (unpublished data) orPBEFNAMPT neutralizing antibody (9)produce significant reduction in LPS-mediated lung injury and VILI To evaluatethe functional involvement of miR-568 inregulation of PBEFNAMPT gene expressionwe measured changes in LPS-induced lungEC barrier function reflected by increasedTER a highly sensitive in vitro assay ofvascular permeability ECs treated withsingle miR-568 mimics demonstratestatistically significant attenuation(388 6 18) of LPS-inducedhyperpermeability in ECs reflectedby reduced TER (P 001) (Figures 3A

and 3B) compared with agonist-stimulated ECs transfected with negativecontrol mimics These data together withour previously published data (37)indicate novel roles for miR-374a andmiR-568 in mediating LPS-induced lungEC permeability

miRNA Mimics and Antagomirs AlterInflammatory Agonist-Induced PBEFNAMPT Expression and 39-UTRReporter Activity in Human Lung ECsTo assess the potential role of miR-374a andmiR-568 in 18 CS- or LPS-inducedregulation of PBEFNAMPT expression weconducted gain- and loss-of-function

experiments by transfecting ECs withnegative control miRNA mimics orsynthetic mimics andor specific miRNAantagomirs for miR-374a or miR-568Figures 4A and 4B and Figures 5Andash5Cdepict the significant decreases in PBEFNAMPT mRNA and protein levels afterexposure of ECs to LPS or 18 CS that wasproduced by miR-374a and miR-568 mimictransfection However in cells transfectedwith miR-374a miR-568 and with thecombination of miR-374a and miR-568but not exposed to LPS or 18 CS thebasal level of PBEFNAMPT mRNA notaltered significantly (Figures 4A and 4B)suggesting that in static cells the

Figure 4 Effect of miRNA mimics on inflammatory agonistndashinduced PBEFNAMPT transcription inhuman lung ECs Total RNA was isolated from ECs transfected with nc or with the indicatedmiRNA mimics (48 h) and untreated (A and B) exposed to 18 CS (A 2 h) or treated with LPS(B 4 h) PBEFNAMPT mRNA levels were detected via real-time PCR Transfection was performedwith miR-1290 used as the second negative control Data are presented as fold change inmRNA level over vehicle-treated control and expressed as means 6 SE from four independentexperiments

ORIGINAL ARTICLE

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 413

steady-state level of mRNA is due to thesaturation of its 39-UTRs with endogenousmiRs Transfection of ECs with thecombination of miR-374a and miR-568enhanced the attenuation of LPS-inducedPBEFNAMPT transcript levels suggestingthat these miRNAs may have additive orsynergistic effects in the setting of LPS-induced EC responses Consistent withthese results overexpression of miR-374aand miR-568 mimics significantlyattenuated basal 39-UTR reporter activity(Figure 6A) (47 6 11 and 44 6 31respectively compared with stimulatedECs) as well as 39-UTR reporter activityafter exposure to 18 CS (Figure 6B)(30 6 125 and 312 6 10 respectively)and LPS (604 6 6 and 5045 6 44

respectively) (Figure 6C) Combinedoverexpression of miR-374a and miR-568mimics enhances the attenuation of 39-UTR reporter activity after EC exposure to18 CS (Figure 6B) again suggestingcooperative regulation by these miRNAsConversely Figures 7A and 7B demonstratethat miR-374a and miR-568 antagomirsenhance levels of endogenous PBEFNAMPT mRNA (338 6 008 FI for hsa-miR-374a antagomir 139 6 0028 FI forhsa-miR-568 antagomir) and protein levels(12 6 013 FI for hsa-miR-374a antagomir14 6 01 FI for hsa-miR-568 antagomir) inECs (compared with ECs transfected withnegative antagomir control) Furthermorethese antagomirs enhance PBEFNAMPT39-UTR reporter activity in ECs (14 6 01

FI for hsa-miR-374a antagomir 18 6 01 FIfor hsa-miR-568 antagomir) compared withnegative antagomir controls (Figure 7C)Together these data suggest that miR-374aand miR-568 strongly influence PBEFNAMPT gene and protein expression

Discussion

The pulmonary vascular endotheliumis a monolayer of ECs and serves asa semiselective barrier between circulatingblood and surrounding tissues with ECintegrity being critical to tissue and organfunction EC dysfunction disrupts thisvascular barrier and triggers loss of cellndashcelladhesion prooxidation and an increasein EC permeability and proliferationa process implicated in inflammatorydiseases such as ALI ARDS and VILI Themolecular mechanisms that facilitate thesephenotypic alterations are only partiallyunderstood In our previous studies (7)PBEFNAMPT was identified as a novelcandidate gene in human inflammatorylung injuries such as VILI and novelbiomarker of ARDS and sepsis withincreased PBEF expression in murinecanine and human ARDS models whichwas spatially localized to lung epitheliumleukocytes and lung vascular endotheliumHowever the exact regulatory mechanismsof PBEFNAMPT expression modulationin the pathogenesis of ARDS and VILIincluding epigenetic regulation via the39-UTR are unknown The 39-UTR isa versatile region that is enriched inregulatory elements modulating processessuch as mRNA stabilizationpolyadenylation transcript degradationtranslation and mRNA localization Thusthe 39-UTR is critical for integratedregulation of gene expression In silicoanalysis revealed that compared with thepromoter region 39-UTR regulatory motifsare preferentially conserved only on onestrand consistent with posttranscriptionalimpact (42) In addition 39-UTRs containhighly conserved sequences that arecomplementary to miRNA 8-mer motifswhich are believed to represent bindingsites for conserved miRNAs andconsequently 39-UTRs have emerged astarget sites for multiple miRNAs (43) The39-UTR length is a major determinant inmRNA expression with longer 39-UTRsbeing more likely to possess miRNA-binding sites that inhibit mRNA translation

Figure 5 Effect of microRNA (miRNA) mimics on inflammatory agonistndashinduced PBEFNAMPTexpression in human lung ECs ECs were transfected with nc or with the indicated miRNA mimics(48 h) and exposed to 18 CS (10 h) or LPS (B 48 h) and endogenous PBEFNAMPT protein wasdetected via immunoblots The bar graphs represent relative densitometry (18 CS [A] LPS [C])Data are presented as fold changes in PBEFNAMPT compared with nc or corresponding miRNApretreatment without agonist challenge and expressed as means 6 SE from three independentexperiments P 005 versus unstimulated control P 001 versus unstimulated control

ORIGINAL ARTICLE

414 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 2 | February 2014

than short 39-UTRs (43 44) In this regardthe PBEFNAMPT gene exhibits anextended 39-UTR (2809 base pairs) and ledto our speculation that PBEFNAMPTexpression may be influenced by VILI- andARDS-modulating miRNAs consistent

with the recognition that miRNAs areimportant modulators of endothelialfunction in vascular diseases and critical tothe regulation of multiple inflammatoryprocesses (45ndash48) Therefore miRNAsrepresent promising therapeutic targets and

biomarkers for ALIARDS and VILI andfacilitate the realization of personalizedmedicine for individuals with acuteinflammatory lung disease MiRNAsare inhibited by chemically stablecomplimentary oligonucleotides (or anti-miRs) or their activity is mimicked bymiRNA mimics Overall the successfulstudies in rodents and nonhuman primatesas well as the preliminary results on clinicaltrials demonstrate that inhibition andoverexpression of miRNAs is a promisingtherapeutic tool (reviewed in Reference 49)MiRNA studies will facilitate theconstruction of a comprehensive diseasemodel for ALIARDS or VILI by applyinga systems biology approach that integratesgenetic genomic epigenetic proteomicand environmental information Thesespeculations do not preclude thecontribution of additional regulatoryelements such as AU-rich elementsalternative 39-UTRs 59 and 39 ends ofmRNA interaction 39-UTR secondarystructure and 39-UTR binding regulatoryproteins in 39-UTRndashmediated control ofPBEFNAMPT gene expression Furtherinvestigation into the regulatory function ofthe PBEFNAMPT 39-UTR is needed

Because in silico bioinformatic analysesrevealed the 39-UTR of PBEF to containbinding sites for miR-374a and miR-568we sought to define their participationin the regulation of inflammatoryagonistndashinduced PBEFNAMPT geneexpression (18 CS and LPS) Previouslywe indicated that expression of the broadlyconserved mature miR-374a (50) issignificantly decreased by 18 CS TNF-aor LPS (37) We now demonstrate thatmiR-568 is expressed in ECs withsignificant down-regulation by 18 CS andLPS (Figure 2) In the present study wefound that PBEFNAMPT expression inECs is markedly up-regulated at mRNAand protein levels in response to 18 CSand LPS (Figure 1) Our combined data(37 39 51) demonstrate the negativecorrelation between expression levels ofputative miRNA and mRNAprotein andsuggest that down-regulation of thesemiRNAs may contribute to augmentedPBEFNAMPT gene expression in ECstreated with these proinflammatoryagonists To further evaluate whethermiR-374a and miR-568 specifically regulatePBEFNAMPT expression in ECstransfection with synthetic miRNA mimicsand antagomirs was used We previously

Figure 6 Effect of miRNA mimics on inflammatory agonistndashinduced PBEFNAMPT 39-UTR reporteractivity in human lung ECs ECs were cotransfected with PBEFNAMPT 39-UTR reporter togetherwith nc or with the indicated miRNA mimics and phRL-TK a Renilla luciferase normalization controlvector ECs were untreated and exposed to 18 CS (A) or treated with LPS (C) (24 h) (B) andluciferase activity was measured according the manufacturerrsquos protocol Data are presented asrelative luciferase units over vehicle-treated control and expressed as means 6 SE from fourindependent experiments Significantly different from control cells without stimulation (P 005)Significantly different from control cells stimulated with 18 CS or LPS (P 005) Significantlydifferent from control cells stimulated with 18 CS or LPS (P 001)

ORIGINAL ARTICLE

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 415

reported that targeting PBEFNAMPTby specific siRNAs (11) or by miR-374a(37) attenuates inflammatory agonist(thrombin IL-1b and LPS)-mediated EChyperpermeability consistent with the key

regulatory role of PBEF in inducing lunginflammation vascular permeability andpathobiology (11 13 14) Similarly ourdata demonstrate that miR-568 mimicsattenuate LPS-induced permeability in ECs

(Figures 3A and 3B) These mRNAs(miR-374a and miR-568) may modulateLPS-induced EC permeability by targetingthe PBEFNAMPT 39-UTR thus alteringexpression We found that transfection ofECs with mimics of miR-374a and miR-568significantly attenuated the 18 CSndash andLPS-induced augmented PBEFNAMPTmRNA and protein levels (Figures 4A and4B and 5Andash5C) Although the exactmechanism by which miR-374a andmiR-568 mimics alter PBEFNAMPTexpression is not known our data supportrecent evidence that the majority ofmammalian miRNAs directly target mRNAdown-regulation via cleavage anddegradation (52 53) or accelerateddeadenylation of mRNAs (54 55)

We further investigated the role ofmiRNAs in the regulation of PBEFNAMPTexpression by using a luciferase 39-UTRreporter gene system a sensitive methodfor measuring miRNA and the 39-UTReffects on gene regulation Our datademonstrated that EC exposure to thesemiRNAs significantly attenuates basalPBEFNAMPT 39-UTR reporter activity(Figure 6A) as well as 18 CS- andLPS-induced increases in luciferase activity(Figures 6B and 6C) Because miRNAoverexpression produces false-positiveresults by targeting genes not affected inphysiologic conditions (ie ldquooff-targeteffectsrdquo) we used the complementary ldquoloss-of-functionrdquo approach by inhibiting specificendogenous miRNA function viaantagomirs and demonstrated that specificmiRNA antagomirs increase PBEFNAMPTexpression (Figures 7A and 7B) and39-UTR reporter activity (Figure 7C) Thusour studies clearly demonstrate thatmiRNAs miR-374a and miR-568 stronglyinfluence PBEFNAMPT expression Ourdata indicate that these two miRNAscooperatively regulate PBEFNAMPTexpression in human pulmonary ECs(Figures 4B and 6B)

PBEFNAMPT is a key participantin VILI pathogenesis and is a criticalbiomarker of diverse inflammatorypathobiologies such as sepsis pneumoniaand ARDS (7 9) potentially via directeffects on innate immunity apoptosis andmitochondrial function Information islimited concerning the possible regulatorymechanisms of PBEFNAMPT expressionvia the 59 and 39 gene regions however ourin silico analysis of the 59 PBEFNAMPTpromoter region by using the Genomatix

Figure 7 Effects of miRNA antagomirs on inflammatory agonistndashinduced PBEFNAMPT expressionand 39-UTR reporter activity Total RNA was isolated from ECs and transfected with nc or with theindicated miRNA Anti- and PBEFNAMPTmRNA levels were detected via real-time PCR (A) Data arepresented as fold change in mRNA level over control and expressed as means 6 SE from fourindependent experiments Significantly different from control cells (P 005) Significantly differentfrom control cells (P 001) ECs transfected with nc or with the indicated miRNA anti- andendogenous PBEFNAMPT protein were detected via immunoblot The bar graph represents relativedensitometry (B) Data are presented as fold changes in endogenous PBEFNAMPT over ncpretreatment and expressed as means 6 SE from three independent experiments P 005 versuscontrol ECs were cotransfected with the PBEFNAMPT 39-UTR reporter together with phRL-TK ncor with the indicated miRNA (C) and luciferase activity was measured according the manufacturerrsquosprotocol Data are presented as relative luciferase units over control and expressed as means 6 SEfrom four independent experiments Significantly different from control cells (P 005)

ORIGINAL ARTICLE

416 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 2 | February 2014

genome analyzer revealed several putativecis regulatory elements including sites forbinding by transcription activators (egNF-kB SP1 STAT and GATA-1) andsequences of homology to antioxidant- shearstressndash and stretch-responsive elements(unpublished data) In addition we notednovel CpG islands in the PBEFNAMPTpromoter suggesting epigenetic regulationof PBEFNAMPT expression via DNAmethylation and 59 promoter interacting

elements The current study demonstratesthat mature expression of miR-374a andmiR-568 epigenetically regulates LPS and18 CSndashinduced PBEFNAMPT expressionin human pulmonary ECs via 39-UTRmodulation Future studies are needed todemonstrate whether miRNAs and otherregulatory elements synergistically participatein PBEFNAMPT gene regulation Targetingof PBEFNAMPT a critical inflammatorybiomarker in ARDS and VILI via specific

miRNAs is a potentially promising noveltherapeutic strategy to reduce lung vascularpermeabilityedema and inflammation inARDS and VILI n

Author disclosures are available with the textof this article at wwwatsjournalsorg

Acknowledgments The authors thank LakshmiNatarajan for providing assistance with theendothelial cell cultures

References

1 Levitt JE Matthay MA Treatment of acute lung injury historicalperspective and potential future therapies Semin Respir Crit CareMed 200627426ndash437

2 Leaver SK Evans TW Acute respiratory distress syndrome BMJ 2007335389ndash394

3 Rubenfeld GD Caldwell E Peabody E Weaver J Martin DP Neff MStern EJ Hudson LD Incidence and outcomes of acute lung injuryN Engl J Med 20053531685ndash1693

4 Ognjanovic S Bao S Yamamoto SY Garibay-Tupas J Samal B Bryant-Greenwood GD Genomic organization of the gene coding for humanpre-b-cell colony enhancing factor and expression in human fetalmembranes J Mol Endocrinol 200126107ndash117

5 Rongvaux A Shea RJ Mulks MH Gigot D Urbain J Leo O Andris FPre-b-cell colony-enhancing factor whose expression isup-regulated in activated lymphocytes is a nicotinamidephosphoribosyltransferase a cytosolic enzyme involved in NADbiosynthesis Eur J Immunol 2002323225ndash3234

6 Sommer G Garten A Petzold S Beck-Sickinger AG Bluher M Stumvoll MFasshauer M VisfatinPBEFNampt structure regulation and potentialfunction of a novel adipokine Clin Sci (Lond) 200811513ndash23

7 Ye SQ Simon BA Maloney JP Zambelli-Weiner A Gao L Grant AEasley RB McVerry BJ Tuder RM Standiford T et al Pre-b-cellcolony-enhancing factor as a potential novel biomarker in acute lunginjury Am J Respir Crit Care Med 2005171361ndash370

8 Hug C Lodish HF Medicine Visfatin a new adipokine Science 2005307366ndash367

9 Hong SB Huang Y Moreno-Vinasco L Sammani S Moitra J BarnardJW Ma SF Mirzapoiazova T Evenoski C Reeves RR et al Essentialrole of pre-b-cell colony enhancing factor in ventilator-induced lunginjury Am J Respir Crit Care Med 2008178605ndash617

10 Bajwa EK Yu CL Gong MN Thompson BT Christiani DC Pre-b-cellcolony-enhancing factor gene polymorphisms and risk of acuterespiratory distress syndrome Crit Care Med 2007351290ndash1295

11 Ye SQ Zhang LQ Adyshev D Usatyuk PV Garcia AN Lavoie TL VerinAD Natarajan V Garcia JG Pre-b-cell-colony-enhancing factor iscritically involved in thrombin-induced lung endothelial cell barrierdysregulation Microvasc Res 200570142ndash151

12 Jia SH Li Y Parodo J Kapus A Fan L Rotstein OD Marshall JCPre-b cell colony-enhancing factor inhibits neutrophil apoptosis inexperimental inflammation and clinical sepsis J Clin Invest 20041131318ndash1327

13 Liu P Li H Cepeda J Zhang LQ Cui X Garcia JG Ye SQ Critical roleof PBEF expression in pulmonary cell inflammation and permeabilityCell Biol Int 20093319ndash30

14 Li H Liu P Cepeda J Fang D Easley RB Simon BA Zhang LQ Ye SQAugmentation of pulmonary epithelial cell IL-8 expression andpermeability by pre-b-cell colony enhancing factor J Inflamm (Lond)2008515

15 Dahl TB Yndestad A Skjelland M Oie E Dahl A Michelsen A DamasJK Tunheim SH Ueland T Smith C et al Increased expression ofvisfatin in macrophages of human unstable carotid and coronaryatherosclerosis possible role in inflammation and plaquedestabilization Circulation 2007115972ndash980

16 Tomankova T Petrek M Kriegova E Involvement of microRNAs inphysiological and pathological processes in the lung Respir Res201011159

17 Sokolov MV Panyutin IV Neumann RD Unraveling the globalmicroRNAome responses to ionizing radiation in human embryonicstem cells PLoS ONE 20127e31028

18 Ji F Lv X Jiao J The role of micrornas in neural stem cells andneurogenesis J Genet Genomics 20134061ndash66

19 Zhou T Garcia JG Zhang W Integrating microRNAs into a systembiology approach to acute lung injury Transl Res 2011157180ndash190

20 Ranjha R Paul J Micro-RNAs in inflammatory diseases and asa link between inflammation and cancer Inflamm Res 201362343ndash355

21 Kornfeld JW Baitzel C Konner AC Nicholls HT Vogt MC HerrmannsK Scheja L Haumaitre C Wolf AM Knippschild U et al Obesity-induced overexpression of miR-802 impairs glucose metabolismthrough silencing of Hnf1b Nature 2013494111ndash115

22 Li N Zhong X Lin X Guo J Zou L Tanyi JL Shao Z Liang S Wang LPHwang WT et al Lin-28 homologue A (LIN28A) promotes cell cycleprogression via the regulation of cyclin-dependent kinase 2 (CDK2)cyclin D1 (CCND1) and cell division cycle 25 homolog A (CDC25A)expression in cancer J Biol Chem 201228717386ndash17397

23 Grosso S Doyen J Parks SK Bertero T Paye A Cardinaud B GounonP Lacas-Gervais S Noel A Pouyssegur J et al MiR-210 promotesa hypoxic phenotype and increases radioresistance in human lungcancer cell lines Cell Death Dis 20134e544

24 Creemers EE Tijsen AJ Pinto YM Circulating microRNAs Novelbiomarkers and extracellular communicators in cardiovasculardisease Circ Res 2012110483ndash495

25 Kohlstedt K Trouvain C Boettger T Shi L Fisslthaler B Fleming IThe AMP-activated protein kinase regulates endothelial cellangiotensin-converting enzyme expression via p53 and the post-transcriptional regulation of microRNA-143145 Circ Res 20131121150ndash1158

26 Zhao JL Rao DS Boldin MP Taganov KD OrsquoConnell RM Baltimore DNF-kappaB dysregulation in microRNA-146a-deficient mice drivesthe development of myeloid malignancies Proc Natl Acad Sci USA20111089184ndash9189

27 Persengiev SP MiRNAs at the crossroad between hematopoieticmalignancies and autoimmune pathogenesis Discov Med 201213211ndash221

28 Liu N Landreh M Cao K Abe M Hendriks GJ Kennerdell JR Zhu YWang LS Bonini NM The microRNA miR-34 modulates ageing andneurodegeneration in drosophila Nature 2011482519ndash523

29 Garcia JG Liu F Verin AD Birukova A Dechert MA Gerthoffer WTBamburg JR English D Sphingosine 1-phosphate promotesendothelial cell barrier integrity by Edg-dependent cytoskeletalrearrangement J Clin Invest 2001108689ndash701

30 Lewis BP Burge CB Bartel DP Conserved seed pairing often flankedby adenosines indicates that thousands of human genes aremicroRNA targets Cell 200512015ndash20

31 Grimson A Farh KK Johnston WK Garrett-Engele P Lim LP BartelDP MicroRNA targeting specificity in mammals determinantsbeyond seed pairing Mol Cell 20072791ndash105

ORIGINAL ARTICLE

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 417

18 CS TNF-a or LPS with maximaldecreases by 4 hours (37) These resultssuggested that changes in miR-374aexpression may play a role in PBEFNAMPT posttranscriptional regulationTo investigate whether miR-568participates in PBEFNAMPT epigeneticregulation similar to miR-374a wedetermined whether 18 CS and LPSregulate mature miR-568 expression inECs Confluent human pulmonary ECswere exposed to 18 CS or LPS for 0 to24 hours and mature miRNA expressionwas assessed by quantitative real-timePCR analysis Figure 2 demonstrates thatmature miR-568 expression is significantlydecreased by these agonists with maximalreduction in expression at 8 hours theseresults were negatively correlated withagonist-mediated augmented PBEFNAMPTexpression (Figures 1Andash1D) and 39-UTRreporter activity (Figures 1E and 1F) Theseresults are consistent with the observation thatthe majority of miRNAs affect target geneexpression by translational repression andsubsequent mRNA degradation (38)Overall such inverse correlation betweenexpression levels of putative miRNA andmRNA andor protein increases thelikelihood that this interaction isfunctional in vivo (39) A limited numberof examples have been described showinga positive correlation between miRNAsand target mRNAs (40 41)

Mimics of miRNAs Attenuate LPS-Induced Human Lung EC PermeabilityOur previous in vitro studies demonstratedthat PBEFNAMPT-specific siRNAs (11) andmiR-374a (37) abolish PBEF expression andinflammatory agonistsrsquo thrombin- IL-1bndashand LPS-mediated hyperpermeability(43 6 29 attenuation for miR-374a)Furthermore intratracheal or intravenousdelivery of siRNA (unpublished data) orPBEFNAMPT neutralizing antibody (9)produce significant reduction in LPS-mediated lung injury and VILI To evaluatethe functional involvement of miR-568 inregulation of PBEFNAMPT gene expressionwe measured changes in LPS-induced lungEC barrier function reflected by increasedTER a highly sensitive in vitro assay ofvascular permeability ECs treated withsingle miR-568 mimics demonstratestatistically significant attenuation(388 6 18) of LPS-inducedhyperpermeability in ECs reflectedby reduced TER (P 001) (Figures 3A

and 3B) compared with agonist-stimulated ECs transfected with negativecontrol mimics These data together withour previously published data (37)indicate novel roles for miR-374a andmiR-568 in mediating LPS-induced lungEC permeability

miRNA Mimics and Antagomirs AlterInflammatory Agonist-Induced PBEFNAMPT Expression and 39-UTRReporter Activity in Human Lung ECsTo assess the potential role of miR-374a andmiR-568 in 18 CS- or LPS-inducedregulation of PBEFNAMPT expression weconducted gain- and loss-of-function

experiments by transfecting ECs withnegative control miRNA mimics orsynthetic mimics andor specific miRNAantagomirs for miR-374a or miR-568Figures 4A and 4B and Figures 5Andash5Cdepict the significant decreases in PBEFNAMPT mRNA and protein levels afterexposure of ECs to LPS or 18 CS that wasproduced by miR-374a and miR-568 mimictransfection However in cells transfectedwith miR-374a miR-568 and with thecombination of miR-374a and miR-568but not exposed to LPS or 18 CS thebasal level of PBEFNAMPT mRNA notaltered significantly (Figures 4A and 4B)suggesting that in static cells the

Figure 4 Effect of miRNA mimics on inflammatory agonistndashinduced PBEFNAMPT transcription inhuman lung ECs Total RNA was isolated from ECs transfected with nc or with the indicatedmiRNA mimics (48 h) and untreated (A and B) exposed to 18 CS (A 2 h) or treated with LPS(B 4 h) PBEFNAMPT mRNA levels were detected via real-time PCR Transfection was performedwith miR-1290 used as the second negative control Data are presented as fold change inmRNA level over vehicle-treated control and expressed as means 6 SE from four independentexperiments

ORIGINAL ARTICLE

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 413

steady-state level of mRNA is due to thesaturation of its 39-UTRs with endogenousmiRs Transfection of ECs with thecombination of miR-374a and miR-568enhanced the attenuation of LPS-inducedPBEFNAMPT transcript levels suggestingthat these miRNAs may have additive orsynergistic effects in the setting of LPS-induced EC responses Consistent withthese results overexpression of miR-374aand miR-568 mimics significantlyattenuated basal 39-UTR reporter activity(Figure 6A) (47 6 11 and 44 6 31respectively compared with stimulatedECs) as well as 39-UTR reporter activityafter exposure to 18 CS (Figure 6B)(30 6 125 and 312 6 10 respectively)and LPS (604 6 6 and 5045 6 44

respectively) (Figure 6C) Combinedoverexpression of miR-374a and miR-568mimics enhances the attenuation of 39-UTR reporter activity after EC exposure to18 CS (Figure 6B) again suggestingcooperative regulation by these miRNAsConversely Figures 7A and 7B demonstratethat miR-374a and miR-568 antagomirsenhance levels of endogenous PBEFNAMPT mRNA (338 6 008 FI for hsa-miR-374a antagomir 139 6 0028 FI forhsa-miR-568 antagomir) and protein levels(12 6 013 FI for hsa-miR-374a antagomir14 6 01 FI for hsa-miR-568 antagomir) inECs (compared with ECs transfected withnegative antagomir control) Furthermorethese antagomirs enhance PBEFNAMPT39-UTR reporter activity in ECs (14 6 01

FI for hsa-miR-374a antagomir 18 6 01 FIfor hsa-miR-568 antagomir) compared withnegative antagomir controls (Figure 7C)Together these data suggest that miR-374aand miR-568 strongly influence PBEFNAMPT gene and protein expression

Discussion

The pulmonary vascular endotheliumis a monolayer of ECs and serves asa semiselective barrier between circulatingblood and surrounding tissues with ECintegrity being critical to tissue and organfunction EC dysfunction disrupts thisvascular barrier and triggers loss of cellndashcelladhesion prooxidation and an increasein EC permeability and proliferationa process implicated in inflammatorydiseases such as ALI ARDS and VILI Themolecular mechanisms that facilitate thesephenotypic alterations are only partiallyunderstood In our previous studies (7)PBEFNAMPT was identified as a novelcandidate gene in human inflammatorylung injuries such as VILI and novelbiomarker of ARDS and sepsis withincreased PBEF expression in murinecanine and human ARDS models whichwas spatially localized to lung epitheliumleukocytes and lung vascular endotheliumHowever the exact regulatory mechanismsof PBEFNAMPT expression modulationin the pathogenesis of ARDS and VILIincluding epigenetic regulation via the39-UTR are unknown The 39-UTR isa versatile region that is enriched inregulatory elements modulating processessuch as mRNA stabilizationpolyadenylation transcript degradationtranslation and mRNA localization Thusthe 39-UTR is critical for integratedregulation of gene expression In silicoanalysis revealed that compared with thepromoter region 39-UTR regulatory motifsare preferentially conserved only on onestrand consistent with posttranscriptionalimpact (42) In addition 39-UTRs containhighly conserved sequences that arecomplementary to miRNA 8-mer motifswhich are believed to represent bindingsites for conserved miRNAs andconsequently 39-UTRs have emerged astarget sites for multiple miRNAs (43) The39-UTR length is a major determinant inmRNA expression with longer 39-UTRsbeing more likely to possess miRNA-binding sites that inhibit mRNA translation

Figure 5 Effect of microRNA (miRNA) mimics on inflammatory agonistndashinduced PBEFNAMPTexpression in human lung ECs ECs were transfected with nc or with the indicated miRNA mimics(48 h) and exposed to 18 CS (10 h) or LPS (B 48 h) and endogenous PBEFNAMPT protein wasdetected via immunoblots The bar graphs represent relative densitometry (18 CS [A] LPS [C])Data are presented as fold changes in PBEFNAMPT compared with nc or corresponding miRNApretreatment without agonist challenge and expressed as means 6 SE from three independentexperiments P 005 versus unstimulated control P 001 versus unstimulated control

ORIGINAL ARTICLE

414 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 2 | February 2014

than short 39-UTRs (43 44) In this regardthe PBEFNAMPT gene exhibits anextended 39-UTR (2809 base pairs) and ledto our speculation that PBEFNAMPTexpression may be influenced by VILI- andARDS-modulating miRNAs consistent

with the recognition that miRNAs areimportant modulators of endothelialfunction in vascular diseases and critical tothe regulation of multiple inflammatoryprocesses (45ndash48) Therefore miRNAsrepresent promising therapeutic targets and

biomarkers for ALIARDS and VILI andfacilitate the realization of personalizedmedicine for individuals with acuteinflammatory lung disease MiRNAsare inhibited by chemically stablecomplimentary oligonucleotides (or anti-miRs) or their activity is mimicked bymiRNA mimics Overall the successfulstudies in rodents and nonhuman primatesas well as the preliminary results on clinicaltrials demonstrate that inhibition andoverexpression of miRNAs is a promisingtherapeutic tool (reviewed in Reference 49)MiRNA studies will facilitate theconstruction of a comprehensive diseasemodel for ALIARDS or VILI by applyinga systems biology approach that integratesgenetic genomic epigenetic proteomicand environmental information Thesespeculations do not preclude thecontribution of additional regulatoryelements such as AU-rich elementsalternative 39-UTRs 59 and 39 ends ofmRNA interaction 39-UTR secondarystructure and 39-UTR binding regulatoryproteins in 39-UTRndashmediated control ofPBEFNAMPT gene expression Furtherinvestigation into the regulatory function ofthe PBEFNAMPT 39-UTR is needed

Because in silico bioinformatic analysesrevealed the 39-UTR of PBEF to containbinding sites for miR-374a and miR-568we sought to define their participationin the regulation of inflammatoryagonistndashinduced PBEFNAMPT geneexpression (18 CS and LPS) Previouslywe indicated that expression of the broadlyconserved mature miR-374a (50) issignificantly decreased by 18 CS TNF-aor LPS (37) We now demonstrate thatmiR-568 is expressed in ECs withsignificant down-regulation by 18 CS andLPS (Figure 2) In the present study wefound that PBEFNAMPT expression inECs is markedly up-regulated at mRNAand protein levels in response to 18 CSand LPS (Figure 1) Our combined data(37 39 51) demonstrate the negativecorrelation between expression levels ofputative miRNA and mRNAprotein andsuggest that down-regulation of thesemiRNAs may contribute to augmentedPBEFNAMPT gene expression in ECstreated with these proinflammatoryagonists To further evaluate whethermiR-374a and miR-568 specifically regulatePBEFNAMPT expression in ECstransfection with synthetic miRNA mimicsand antagomirs was used We previously

Figure 6 Effect of miRNA mimics on inflammatory agonistndashinduced PBEFNAMPT 39-UTR reporteractivity in human lung ECs ECs were cotransfected with PBEFNAMPT 39-UTR reporter togetherwith nc or with the indicated miRNA mimics and phRL-TK a Renilla luciferase normalization controlvector ECs were untreated and exposed to 18 CS (A) or treated with LPS (C) (24 h) (B) andluciferase activity was measured according the manufacturerrsquos protocol Data are presented asrelative luciferase units over vehicle-treated control and expressed as means 6 SE from fourindependent experiments Significantly different from control cells without stimulation (P 005)Significantly different from control cells stimulated with 18 CS or LPS (P 005) Significantlydifferent from control cells stimulated with 18 CS or LPS (P 001)

ORIGINAL ARTICLE

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 415

reported that targeting PBEFNAMPTby specific siRNAs (11) or by miR-374a(37) attenuates inflammatory agonist(thrombin IL-1b and LPS)-mediated EChyperpermeability consistent with the key

regulatory role of PBEF in inducing lunginflammation vascular permeability andpathobiology (11 13 14) Similarly ourdata demonstrate that miR-568 mimicsattenuate LPS-induced permeability in ECs

(Figures 3A and 3B) These mRNAs(miR-374a and miR-568) may modulateLPS-induced EC permeability by targetingthe PBEFNAMPT 39-UTR thus alteringexpression We found that transfection ofECs with mimics of miR-374a and miR-568significantly attenuated the 18 CSndash andLPS-induced augmented PBEFNAMPTmRNA and protein levels (Figures 4A and4B and 5Andash5C) Although the exactmechanism by which miR-374a andmiR-568 mimics alter PBEFNAMPTexpression is not known our data supportrecent evidence that the majority ofmammalian miRNAs directly target mRNAdown-regulation via cleavage anddegradation (52 53) or accelerateddeadenylation of mRNAs (54 55)

We further investigated the role ofmiRNAs in the regulation of PBEFNAMPTexpression by using a luciferase 39-UTRreporter gene system a sensitive methodfor measuring miRNA and the 39-UTReffects on gene regulation Our datademonstrated that EC exposure to thesemiRNAs significantly attenuates basalPBEFNAMPT 39-UTR reporter activity(Figure 6A) as well as 18 CS- andLPS-induced increases in luciferase activity(Figures 6B and 6C) Because miRNAoverexpression produces false-positiveresults by targeting genes not affected inphysiologic conditions (ie ldquooff-targeteffectsrdquo) we used the complementary ldquoloss-of-functionrdquo approach by inhibiting specificendogenous miRNA function viaantagomirs and demonstrated that specificmiRNA antagomirs increase PBEFNAMPTexpression (Figures 7A and 7B) and39-UTR reporter activity (Figure 7C) Thusour studies clearly demonstrate thatmiRNAs miR-374a and miR-568 stronglyinfluence PBEFNAMPT expression Ourdata indicate that these two miRNAscooperatively regulate PBEFNAMPTexpression in human pulmonary ECs(Figures 4B and 6B)

PBEFNAMPT is a key participantin VILI pathogenesis and is a criticalbiomarker of diverse inflammatorypathobiologies such as sepsis pneumoniaand ARDS (7 9) potentially via directeffects on innate immunity apoptosis andmitochondrial function Information islimited concerning the possible regulatorymechanisms of PBEFNAMPT expressionvia the 59 and 39 gene regions however ourin silico analysis of the 59 PBEFNAMPTpromoter region by using the Genomatix

Figure 7 Effects of miRNA antagomirs on inflammatory agonistndashinduced PBEFNAMPT expressionand 39-UTR reporter activity Total RNA was isolated from ECs and transfected with nc or with theindicated miRNA Anti- and PBEFNAMPTmRNA levels were detected via real-time PCR (A) Data arepresented as fold change in mRNA level over control and expressed as means 6 SE from fourindependent experiments Significantly different from control cells (P 005) Significantly differentfrom control cells (P 001) ECs transfected with nc or with the indicated miRNA anti- andendogenous PBEFNAMPT protein were detected via immunoblot The bar graph represents relativedensitometry (B) Data are presented as fold changes in endogenous PBEFNAMPT over ncpretreatment and expressed as means 6 SE from three independent experiments P 005 versuscontrol ECs were cotransfected with the PBEFNAMPT 39-UTR reporter together with phRL-TK ncor with the indicated miRNA (C) and luciferase activity was measured according the manufacturerrsquosprotocol Data are presented as relative luciferase units over control and expressed as means 6 SEfrom four independent experiments Significantly different from control cells (P 005)

ORIGINAL ARTICLE

416 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 2 | February 2014

genome analyzer revealed several putativecis regulatory elements including sites forbinding by transcription activators (egNF-kB SP1 STAT and GATA-1) andsequences of homology to antioxidant- shearstressndash and stretch-responsive elements(unpublished data) In addition we notednovel CpG islands in the PBEFNAMPTpromoter suggesting epigenetic regulationof PBEFNAMPT expression via DNAmethylation and 59 promoter interacting

elements The current study demonstratesthat mature expression of miR-374a andmiR-568 epigenetically regulates LPS and18 CSndashinduced PBEFNAMPT expressionin human pulmonary ECs via 39-UTRmodulation Future studies are needed todemonstrate whether miRNAs and otherregulatory elements synergistically participatein PBEFNAMPT gene regulation Targetingof PBEFNAMPT a critical inflammatorybiomarker in ARDS and VILI via specific

miRNAs is a potentially promising noveltherapeutic strategy to reduce lung vascularpermeabilityedema and inflammation inARDS and VILI n

Author disclosures are available with the textof this article at wwwatsjournalsorg

Acknowledgments The authors thank LakshmiNatarajan for providing assistance with theendothelial cell cultures

References

1 Levitt JE Matthay MA Treatment of acute lung injury historicalperspective and potential future therapies Semin Respir Crit CareMed 200627426ndash437

2 Leaver SK Evans TW Acute respiratory distress syndrome BMJ 2007335389ndash394

3 Rubenfeld GD Caldwell E Peabody E Weaver J Martin DP Neff MStern EJ Hudson LD Incidence and outcomes of acute lung injuryN Engl J Med 20053531685ndash1693

4 Ognjanovic S Bao S Yamamoto SY Garibay-Tupas J Samal B Bryant-Greenwood GD Genomic organization of the gene coding for humanpre-b-cell colony enhancing factor and expression in human fetalmembranes J Mol Endocrinol 200126107ndash117

5 Rongvaux A Shea RJ Mulks MH Gigot D Urbain J Leo O Andris FPre-b-cell colony-enhancing factor whose expression isup-regulated in activated lymphocytes is a nicotinamidephosphoribosyltransferase a cytosolic enzyme involved in NADbiosynthesis Eur J Immunol 2002323225ndash3234

6 Sommer G Garten A Petzold S Beck-Sickinger AG Bluher M Stumvoll MFasshauer M VisfatinPBEFNampt structure regulation and potentialfunction of a novel adipokine Clin Sci (Lond) 200811513ndash23

7 Ye SQ Simon BA Maloney JP Zambelli-Weiner A Gao L Grant AEasley RB McVerry BJ Tuder RM Standiford T et al Pre-b-cellcolony-enhancing factor as a potential novel biomarker in acute lunginjury Am J Respir Crit Care Med 2005171361ndash370

8 Hug C Lodish HF Medicine Visfatin a new adipokine Science 2005307366ndash367

9 Hong SB Huang Y Moreno-Vinasco L Sammani S Moitra J BarnardJW Ma SF Mirzapoiazova T Evenoski C Reeves RR et al Essentialrole of pre-b-cell colony enhancing factor in ventilator-induced lunginjury Am J Respir Crit Care Med 2008178605ndash617

10 Bajwa EK Yu CL Gong MN Thompson BT Christiani DC Pre-b-cellcolony-enhancing factor gene polymorphisms and risk of acuterespiratory distress syndrome Crit Care Med 2007351290ndash1295

11 Ye SQ Zhang LQ Adyshev D Usatyuk PV Garcia AN Lavoie TL VerinAD Natarajan V Garcia JG Pre-b-cell-colony-enhancing factor iscritically involved in thrombin-induced lung endothelial cell barrierdysregulation Microvasc Res 200570142ndash151

12 Jia SH Li Y Parodo J Kapus A Fan L Rotstein OD Marshall JCPre-b cell colony-enhancing factor inhibits neutrophil apoptosis inexperimental inflammation and clinical sepsis J Clin Invest 20041131318ndash1327

13 Liu P Li H Cepeda J Zhang LQ Cui X Garcia JG Ye SQ Critical roleof PBEF expression in pulmonary cell inflammation and permeabilityCell Biol Int 20093319ndash30

14 Li H Liu P Cepeda J Fang D Easley RB Simon BA Zhang LQ Ye SQAugmentation of pulmonary epithelial cell IL-8 expression andpermeability by pre-b-cell colony enhancing factor J Inflamm (Lond)2008515

15 Dahl TB Yndestad A Skjelland M Oie E Dahl A Michelsen A DamasJK Tunheim SH Ueland T Smith C et al Increased expression ofvisfatin in macrophages of human unstable carotid and coronaryatherosclerosis possible role in inflammation and plaquedestabilization Circulation 2007115972ndash980

16 Tomankova T Petrek M Kriegova E Involvement of microRNAs inphysiological and pathological processes in the lung Respir Res201011159

17 Sokolov MV Panyutin IV Neumann RD Unraveling the globalmicroRNAome responses to ionizing radiation in human embryonicstem cells PLoS ONE 20127e31028

18 Ji F Lv X Jiao J The role of micrornas in neural stem cells andneurogenesis J Genet Genomics 20134061ndash66

19 Zhou T Garcia JG Zhang W Integrating microRNAs into a systembiology approach to acute lung injury Transl Res 2011157180ndash190

20 Ranjha R Paul J Micro-RNAs in inflammatory diseases and asa link between inflammation and cancer Inflamm Res 201362343ndash355

21 Kornfeld JW Baitzel C Konner AC Nicholls HT Vogt MC HerrmannsK Scheja L Haumaitre C Wolf AM Knippschild U et al Obesity-induced overexpression of miR-802 impairs glucose metabolismthrough silencing of Hnf1b Nature 2013494111ndash115

22 Li N Zhong X Lin X Guo J Zou L Tanyi JL Shao Z Liang S Wang LPHwang WT et al Lin-28 homologue A (LIN28A) promotes cell cycleprogression via the regulation of cyclin-dependent kinase 2 (CDK2)cyclin D1 (CCND1) and cell division cycle 25 homolog A (CDC25A)expression in cancer J Biol Chem 201228717386ndash17397

23 Grosso S Doyen J Parks SK Bertero T Paye A Cardinaud B GounonP Lacas-Gervais S Noel A Pouyssegur J et al MiR-210 promotesa hypoxic phenotype and increases radioresistance in human lungcancer cell lines Cell Death Dis 20134e544

24 Creemers EE Tijsen AJ Pinto YM Circulating microRNAs Novelbiomarkers and extracellular communicators in cardiovasculardisease Circ Res 2012110483ndash495

25 Kohlstedt K Trouvain C Boettger T Shi L Fisslthaler B Fleming IThe AMP-activated protein kinase regulates endothelial cellangiotensin-converting enzyme expression via p53 and the post-transcriptional regulation of microRNA-143145 Circ Res 20131121150ndash1158

26 Zhao JL Rao DS Boldin MP Taganov KD OrsquoConnell RM Baltimore DNF-kappaB dysregulation in microRNA-146a-deficient mice drivesthe development of myeloid malignancies Proc Natl Acad Sci USA20111089184ndash9189

27 Persengiev SP MiRNAs at the crossroad between hematopoieticmalignancies and autoimmune pathogenesis Discov Med 201213211ndash221

28 Liu N Landreh M Cao K Abe M Hendriks GJ Kennerdell JR Zhu YWang LS Bonini NM The microRNA miR-34 modulates ageing andneurodegeneration in drosophila Nature 2011482519ndash523

29 Garcia JG Liu F Verin AD Birukova A Dechert MA Gerthoffer WTBamburg JR English D Sphingosine 1-phosphate promotesendothelial cell barrier integrity by Edg-dependent cytoskeletalrearrangement J Clin Invest 2001108689ndash701

30 Lewis BP Burge CB Bartel DP Conserved seed pairing often flankedby adenosines indicates that thousands of human genes aremicroRNA targets Cell 200512015ndash20

31 Grimson A Farh KK Johnston WK Garrett-Engele P Lim LP BartelDP MicroRNA targeting specificity in mammals determinantsbeyond seed pairing Mol Cell 20072791ndash105

ORIGINAL ARTICLE

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 417

steady-state level of mRNA is due to thesaturation of its 39-UTRs with endogenousmiRs Transfection of ECs with thecombination of miR-374a and miR-568enhanced the attenuation of LPS-inducedPBEFNAMPT transcript levels suggestingthat these miRNAs may have additive orsynergistic effects in the setting of LPS-induced EC responses Consistent withthese results overexpression of miR-374aand miR-568 mimics significantlyattenuated basal 39-UTR reporter activity(Figure 6A) (47 6 11 and 44 6 31respectively compared with stimulatedECs) as well as 39-UTR reporter activityafter exposure to 18 CS (Figure 6B)(30 6 125 and 312 6 10 respectively)and LPS (604 6 6 and 5045 6 44

respectively) (Figure 6C) Combinedoverexpression of miR-374a and miR-568mimics enhances the attenuation of 39-UTR reporter activity after EC exposure to18 CS (Figure 6B) again suggestingcooperative regulation by these miRNAsConversely Figures 7A and 7B demonstratethat miR-374a and miR-568 antagomirsenhance levels of endogenous PBEFNAMPT mRNA (338 6 008 FI for hsa-miR-374a antagomir 139 6 0028 FI forhsa-miR-568 antagomir) and protein levels(12 6 013 FI for hsa-miR-374a antagomir14 6 01 FI for hsa-miR-568 antagomir) inECs (compared with ECs transfected withnegative antagomir control) Furthermorethese antagomirs enhance PBEFNAMPT39-UTR reporter activity in ECs (14 6 01

FI for hsa-miR-374a antagomir 18 6 01 FIfor hsa-miR-568 antagomir) compared withnegative antagomir controls (Figure 7C)Together these data suggest that miR-374aand miR-568 strongly influence PBEFNAMPT gene and protein expression

Discussion

The pulmonary vascular endotheliumis a monolayer of ECs and serves asa semiselective barrier between circulatingblood and surrounding tissues with ECintegrity being critical to tissue and organfunction EC dysfunction disrupts thisvascular barrier and triggers loss of cellndashcelladhesion prooxidation and an increasein EC permeability and proliferationa process implicated in inflammatorydiseases such as ALI ARDS and VILI Themolecular mechanisms that facilitate thesephenotypic alterations are only partiallyunderstood In our previous studies (7)PBEFNAMPT was identified as a novelcandidate gene in human inflammatorylung injuries such as VILI and novelbiomarker of ARDS and sepsis withincreased PBEF expression in murinecanine and human ARDS models whichwas spatially localized to lung epitheliumleukocytes and lung vascular endotheliumHowever the exact regulatory mechanismsof PBEFNAMPT expression modulationin the pathogenesis of ARDS and VILIincluding epigenetic regulation via the39-UTR are unknown The 39-UTR isa versatile region that is enriched inregulatory elements modulating processessuch as mRNA stabilizationpolyadenylation transcript degradationtranslation and mRNA localization Thusthe 39-UTR is critical for integratedregulation of gene expression In silicoanalysis revealed that compared with thepromoter region 39-UTR regulatory motifsare preferentially conserved only on onestrand consistent with posttranscriptionalimpact (42) In addition 39-UTRs containhighly conserved sequences that arecomplementary to miRNA 8-mer motifswhich are believed to represent bindingsites for conserved miRNAs andconsequently 39-UTRs have emerged astarget sites for multiple miRNAs (43) The39-UTR length is a major determinant inmRNA expression with longer 39-UTRsbeing more likely to possess miRNA-binding sites that inhibit mRNA translation

Figure 5 Effect of microRNA (miRNA) mimics on inflammatory agonistndashinduced PBEFNAMPTexpression in human lung ECs ECs were transfected with nc or with the indicated miRNA mimics(48 h) and exposed to 18 CS (10 h) or LPS (B 48 h) and endogenous PBEFNAMPT protein wasdetected via immunoblots The bar graphs represent relative densitometry (18 CS [A] LPS [C])Data are presented as fold changes in PBEFNAMPT compared with nc or corresponding miRNApretreatment without agonist challenge and expressed as means 6 SE from three independentexperiments P 005 versus unstimulated control P 001 versus unstimulated control

ORIGINAL ARTICLE

414 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 2 | February 2014

than short 39-UTRs (43 44) In this regardthe PBEFNAMPT gene exhibits anextended 39-UTR (2809 base pairs) and ledto our speculation that PBEFNAMPTexpression may be influenced by VILI- andARDS-modulating miRNAs consistent

with the recognition that miRNAs areimportant modulators of endothelialfunction in vascular diseases and critical tothe regulation of multiple inflammatoryprocesses (45ndash48) Therefore miRNAsrepresent promising therapeutic targets and

biomarkers for ALIARDS and VILI andfacilitate the realization of personalizedmedicine for individuals with acuteinflammatory lung disease MiRNAsare inhibited by chemically stablecomplimentary oligonucleotides (or anti-miRs) or their activity is mimicked bymiRNA mimics Overall the successfulstudies in rodents and nonhuman primatesas well as the preliminary results on clinicaltrials demonstrate that inhibition andoverexpression of miRNAs is a promisingtherapeutic tool (reviewed in Reference 49)MiRNA studies will facilitate theconstruction of a comprehensive diseasemodel for ALIARDS or VILI by applyinga systems biology approach that integratesgenetic genomic epigenetic proteomicand environmental information Thesespeculations do not preclude thecontribution of additional regulatoryelements such as AU-rich elementsalternative 39-UTRs 59 and 39 ends ofmRNA interaction 39-UTR secondarystructure and 39-UTR binding regulatoryproteins in 39-UTRndashmediated control ofPBEFNAMPT gene expression Furtherinvestigation into the regulatory function ofthe PBEFNAMPT 39-UTR is needed

Because in silico bioinformatic analysesrevealed the 39-UTR of PBEF to containbinding sites for miR-374a and miR-568we sought to define their participationin the regulation of inflammatoryagonistndashinduced PBEFNAMPT geneexpression (18 CS and LPS) Previouslywe indicated that expression of the broadlyconserved mature miR-374a (50) issignificantly decreased by 18 CS TNF-aor LPS (37) We now demonstrate thatmiR-568 is expressed in ECs withsignificant down-regulation by 18 CS andLPS (Figure 2) In the present study wefound that PBEFNAMPT expression inECs is markedly up-regulated at mRNAand protein levels in response to 18 CSand LPS (Figure 1) Our combined data(37 39 51) demonstrate the negativecorrelation between expression levels ofputative miRNA and mRNAprotein andsuggest that down-regulation of thesemiRNAs may contribute to augmentedPBEFNAMPT gene expression in ECstreated with these proinflammatoryagonists To further evaluate whethermiR-374a and miR-568 specifically regulatePBEFNAMPT expression in ECstransfection with synthetic miRNA mimicsand antagomirs was used We previously

Figure 6 Effect of miRNA mimics on inflammatory agonistndashinduced PBEFNAMPT 39-UTR reporteractivity in human lung ECs ECs were cotransfected with PBEFNAMPT 39-UTR reporter togetherwith nc or with the indicated miRNA mimics and phRL-TK a Renilla luciferase normalization controlvector ECs were untreated and exposed to 18 CS (A) or treated with LPS (C) (24 h) (B) andluciferase activity was measured according the manufacturerrsquos protocol Data are presented asrelative luciferase units over vehicle-treated control and expressed as means 6 SE from fourindependent experiments Significantly different from control cells without stimulation (P 005)Significantly different from control cells stimulated with 18 CS or LPS (P 005) Significantlydifferent from control cells stimulated with 18 CS or LPS (P 001)

ORIGINAL ARTICLE

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 415

reported that targeting PBEFNAMPTby specific siRNAs (11) or by miR-374a(37) attenuates inflammatory agonist(thrombin IL-1b and LPS)-mediated EChyperpermeability consistent with the key

regulatory role of PBEF in inducing lunginflammation vascular permeability andpathobiology (11 13 14) Similarly ourdata demonstrate that miR-568 mimicsattenuate LPS-induced permeability in ECs

(Figures 3A and 3B) These mRNAs(miR-374a and miR-568) may modulateLPS-induced EC permeability by targetingthe PBEFNAMPT 39-UTR thus alteringexpression We found that transfection ofECs with mimics of miR-374a and miR-568significantly attenuated the 18 CSndash andLPS-induced augmented PBEFNAMPTmRNA and protein levels (Figures 4A and4B and 5Andash5C) Although the exactmechanism by which miR-374a andmiR-568 mimics alter PBEFNAMPTexpression is not known our data supportrecent evidence that the majority ofmammalian miRNAs directly target mRNAdown-regulation via cleavage anddegradation (52 53) or accelerateddeadenylation of mRNAs (54 55)

We further investigated the role ofmiRNAs in the regulation of PBEFNAMPTexpression by using a luciferase 39-UTRreporter gene system a sensitive methodfor measuring miRNA and the 39-UTReffects on gene regulation Our datademonstrated that EC exposure to thesemiRNAs significantly attenuates basalPBEFNAMPT 39-UTR reporter activity(Figure 6A) as well as 18 CS- andLPS-induced increases in luciferase activity(Figures 6B and 6C) Because miRNAoverexpression produces false-positiveresults by targeting genes not affected inphysiologic conditions (ie ldquooff-targeteffectsrdquo) we used the complementary ldquoloss-of-functionrdquo approach by inhibiting specificendogenous miRNA function viaantagomirs and demonstrated that specificmiRNA antagomirs increase PBEFNAMPTexpression (Figures 7A and 7B) and39-UTR reporter activity (Figure 7C) Thusour studies clearly demonstrate thatmiRNAs miR-374a and miR-568 stronglyinfluence PBEFNAMPT expression Ourdata indicate that these two miRNAscooperatively regulate PBEFNAMPTexpression in human pulmonary ECs(Figures 4B and 6B)

PBEFNAMPT is a key participantin VILI pathogenesis and is a criticalbiomarker of diverse inflammatorypathobiologies such as sepsis pneumoniaand ARDS (7 9) potentially via directeffects on innate immunity apoptosis andmitochondrial function Information islimited concerning the possible regulatorymechanisms of PBEFNAMPT expressionvia the 59 and 39 gene regions however ourin silico analysis of the 59 PBEFNAMPTpromoter region by using the Genomatix

Figure 7 Effects of miRNA antagomirs on inflammatory agonistndashinduced PBEFNAMPT expressionand 39-UTR reporter activity Total RNA was isolated from ECs and transfected with nc or with theindicated miRNA Anti- and PBEFNAMPTmRNA levels were detected via real-time PCR (A) Data arepresented as fold change in mRNA level over control and expressed as means 6 SE from fourindependent experiments Significantly different from control cells (P 005) Significantly differentfrom control cells (P 001) ECs transfected with nc or with the indicated miRNA anti- andendogenous PBEFNAMPT protein were detected via immunoblot The bar graph represents relativedensitometry (B) Data are presented as fold changes in endogenous PBEFNAMPT over ncpretreatment and expressed as means 6 SE from three independent experiments P 005 versuscontrol ECs were cotransfected with the PBEFNAMPT 39-UTR reporter together with phRL-TK ncor with the indicated miRNA (C) and luciferase activity was measured according the manufacturerrsquosprotocol Data are presented as relative luciferase units over control and expressed as means 6 SEfrom four independent experiments Significantly different from control cells (P 005)

ORIGINAL ARTICLE

416 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 2 | February 2014

genome analyzer revealed several putativecis regulatory elements including sites forbinding by transcription activators (egNF-kB SP1 STAT and GATA-1) andsequences of homology to antioxidant- shearstressndash and stretch-responsive elements(unpublished data) In addition we notednovel CpG islands in the PBEFNAMPTpromoter suggesting epigenetic regulationof PBEFNAMPT expression via DNAmethylation and 59 promoter interacting

elements The current study demonstratesthat mature expression of miR-374a andmiR-568 epigenetically regulates LPS and18 CSndashinduced PBEFNAMPT expressionin human pulmonary ECs via 39-UTRmodulation Future studies are needed todemonstrate whether miRNAs and otherregulatory elements synergistically participatein PBEFNAMPT gene regulation Targetingof PBEFNAMPT a critical inflammatorybiomarker in ARDS and VILI via specific

miRNAs is a potentially promising noveltherapeutic strategy to reduce lung vascularpermeabilityedema and inflammation inARDS and VILI n

Author disclosures are available with the textof this article at wwwatsjournalsorg

Acknowledgments The authors thank LakshmiNatarajan for providing assistance with theendothelial cell cultures

References

1 Levitt JE Matthay MA Treatment of acute lung injury historicalperspective and potential future therapies Semin Respir Crit CareMed 200627426ndash437

2 Leaver SK Evans TW Acute respiratory distress syndrome BMJ 2007335389ndash394

3 Rubenfeld GD Caldwell E Peabody E Weaver J Martin DP Neff MStern EJ Hudson LD Incidence and outcomes of acute lung injuryN Engl J Med 20053531685ndash1693

4 Ognjanovic S Bao S Yamamoto SY Garibay-Tupas J Samal B Bryant-Greenwood GD Genomic organization of the gene coding for humanpre-b-cell colony enhancing factor and expression in human fetalmembranes J Mol Endocrinol 200126107ndash117

5 Rongvaux A Shea RJ Mulks MH Gigot D Urbain J Leo O Andris FPre-b-cell colony-enhancing factor whose expression isup-regulated in activated lymphocytes is a nicotinamidephosphoribosyltransferase a cytosolic enzyme involved in NADbiosynthesis Eur J Immunol 2002323225ndash3234

6 Sommer G Garten A Petzold S Beck-Sickinger AG Bluher M Stumvoll MFasshauer M VisfatinPBEFNampt structure regulation and potentialfunction of a novel adipokine Clin Sci (Lond) 200811513ndash23

7 Ye SQ Simon BA Maloney JP Zambelli-Weiner A Gao L Grant AEasley RB McVerry BJ Tuder RM Standiford T et al Pre-b-cellcolony-enhancing factor as a potential novel biomarker in acute lunginjury Am J Respir Crit Care Med 2005171361ndash370

8 Hug C Lodish HF Medicine Visfatin a new adipokine Science 2005307366ndash367

9 Hong SB Huang Y Moreno-Vinasco L Sammani S Moitra J BarnardJW Ma SF Mirzapoiazova T Evenoski C Reeves RR et al Essentialrole of pre-b-cell colony enhancing factor in ventilator-induced lunginjury Am J Respir Crit Care Med 2008178605ndash617

10 Bajwa EK Yu CL Gong MN Thompson BT Christiani DC Pre-b-cellcolony-enhancing factor gene polymorphisms and risk of acuterespiratory distress syndrome Crit Care Med 2007351290ndash1295

11 Ye SQ Zhang LQ Adyshev D Usatyuk PV Garcia AN Lavoie TL VerinAD Natarajan V Garcia JG Pre-b-cell-colony-enhancing factor iscritically involved in thrombin-induced lung endothelial cell barrierdysregulation Microvasc Res 200570142ndash151

12 Jia SH Li Y Parodo J Kapus A Fan L Rotstein OD Marshall JCPre-b cell colony-enhancing factor inhibits neutrophil apoptosis inexperimental inflammation and clinical sepsis J Clin Invest 20041131318ndash1327

13 Liu P Li H Cepeda J Zhang LQ Cui X Garcia JG Ye SQ Critical roleof PBEF expression in pulmonary cell inflammation and permeabilityCell Biol Int 20093319ndash30

14 Li H Liu P Cepeda J Fang D Easley RB Simon BA Zhang LQ Ye SQAugmentation of pulmonary epithelial cell IL-8 expression andpermeability by pre-b-cell colony enhancing factor J Inflamm (Lond)2008515

15 Dahl TB Yndestad A Skjelland M Oie E Dahl A Michelsen A DamasJK Tunheim SH Ueland T Smith C et al Increased expression ofvisfatin in macrophages of human unstable carotid and coronaryatherosclerosis possible role in inflammation and plaquedestabilization Circulation 2007115972ndash980

16 Tomankova T Petrek M Kriegova E Involvement of microRNAs inphysiological and pathological processes in the lung Respir Res201011159

17 Sokolov MV Panyutin IV Neumann RD Unraveling the globalmicroRNAome responses to ionizing radiation in human embryonicstem cells PLoS ONE 20127e31028

18 Ji F Lv X Jiao J The role of micrornas in neural stem cells andneurogenesis J Genet Genomics 20134061ndash66

19 Zhou T Garcia JG Zhang W Integrating microRNAs into a systembiology approach to acute lung injury Transl Res 2011157180ndash190

20 Ranjha R Paul J Micro-RNAs in inflammatory diseases and asa link between inflammation and cancer Inflamm Res 201362343ndash355

21 Kornfeld JW Baitzel C Konner AC Nicholls HT Vogt MC HerrmannsK Scheja L Haumaitre C Wolf AM Knippschild U et al Obesity-induced overexpression of miR-802 impairs glucose metabolismthrough silencing of Hnf1b Nature 2013494111ndash115

22 Li N Zhong X Lin X Guo J Zou L Tanyi JL Shao Z Liang S Wang LPHwang WT et al Lin-28 homologue A (LIN28A) promotes cell cycleprogression via the regulation of cyclin-dependent kinase 2 (CDK2)cyclin D1 (CCND1) and cell division cycle 25 homolog A (CDC25A)expression in cancer J Biol Chem 201228717386ndash17397

23 Grosso S Doyen J Parks SK Bertero T Paye A Cardinaud B GounonP Lacas-Gervais S Noel A Pouyssegur J et al MiR-210 promotesa hypoxic phenotype and increases radioresistance in human lungcancer cell lines Cell Death Dis 20134e544

24 Creemers EE Tijsen AJ Pinto YM Circulating microRNAs Novelbiomarkers and extracellular communicators in cardiovasculardisease Circ Res 2012110483ndash495

25 Kohlstedt K Trouvain C Boettger T Shi L Fisslthaler B Fleming IThe AMP-activated protein kinase regulates endothelial cellangiotensin-converting enzyme expression via p53 and the post-transcriptional regulation of microRNA-143145 Circ Res 20131121150ndash1158

26 Zhao JL Rao DS Boldin MP Taganov KD OrsquoConnell RM Baltimore DNF-kappaB dysregulation in microRNA-146a-deficient mice drivesthe development of myeloid malignancies Proc Natl Acad Sci USA20111089184ndash9189

27 Persengiev SP MiRNAs at the crossroad between hematopoieticmalignancies and autoimmune pathogenesis Discov Med 201213211ndash221

28 Liu N Landreh M Cao K Abe M Hendriks GJ Kennerdell JR Zhu YWang LS Bonini NM The microRNA miR-34 modulates ageing andneurodegeneration in drosophila Nature 2011482519ndash523

29 Garcia JG Liu F Verin AD Birukova A Dechert MA Gerthoffer WTBamburg JR English D Sphingosine 1-phosphate promotesendothelial cell barrier integrity by Edg-dependent cytoskeletalrearrangement J Clin Invest 2001108689ndash701

30 Lewis BP Burge CB Bartel DP Conserved seed pairing often flankedby adenosines indicates that thousands of human genes aremicroRNA targets Cell 200512015ndash20

31 Grimson A Farh KK Johnston WK Garrett-Engele P Lim LP BartelDP MicroRNA targeting specificity in mammals determinantsbeyond seed pairing Mol Cell 20072791ndash105

ORIGINAL ARTICLE

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 417

than short 39-UTRs (43 44) In this regardthe PBEFNAMPT gene exhibits anextended 39-UTR (2809 base pairs) and ledto our speculation that PBEFNAMPTexpression may be influenced by VILI- andARDS-modulating miRNAs consistent

with the recognition that miRNAs areimportant modulators of endothelialfunction in vascular diseases and critical tothe regulation of multiple inflammatoryprocesses (45ndash48) Therefore miRNAsrepresent promising therapeutic targets and

biomarkers for ALIARDS and VILI andfacilitate the realization of personalizedmedicine for individuals with acuteinflammatory lung disease MiRNAsare inhibited by chemically stablecomplimentary oligonucleotides (or anti-miRs) or their activity is mimicked bymiRNA mimics Overall the successfulstudies in rodents and nonhuman primatesas well as the preliminary results on clinicaltrials demonstrate that inhibition andoverexpression of miRNAs is a promisingtherapeutic tool (reviewed in Reference 49)MiRNA studies will facilitate theconstruction of a comprehensive diseasemodel for ALIARDS or VILI by applyinga systems biology approach that integratesgenetic genomic epigenetic proteomicand environmental information Thesespeculations do not preclude thecontribution of additional regulatoryelements such as AU-rich elementsalternative 39-UTRs 59 and 39 ends ofmRNA interaction 39-UTR secondarystructure and 39-UTR binding regulatoryproteins in 39-UTRndashmediated control ofPBEFNAMPT gene expression Furtherinvestigation into the regulatory function ofthe PBEFNAMPT 39-UTR is needed

Because in silico bioinformatic analysesrevealed the 39-UTR of PBEF to containbinding sites for miR-374a and miR-568we sought to define their participationin the regulation of inflammatoryagonistndashinduced PBEFNAMPT geneexpression (18 CS and LPS) Previouslywe indicated that expression of the broadlyconserved mature miR-374a (50) issignificantly decreased by 18 CS TNF-aor LPS (37) We now demonstrate thatmiR-568 is expressed in ECs withsignificant down-regulation by 18 CS andLPS (Figure 2) In the present study wefound that PBEFNAMPT expression inECs is markedly up-regulated at mRNAand protein levels in response to 18 CSand LPS (Figure 1) Our combined data(37 39 51) demonstrate the negativecorrelation between expression levels ofputative miRNA and mRNAprotein andsuggest that down-regulation of thesemiRNAs may contribute to augmentedPBEFNAMPT gene expression in ECstreated with these proinflammatoryagonists To further evaluate whethermiR-374a and miR-568 specifically regulatePBEFNAMPT expression in ECstransfection with synthetic miRNA mimicsand antagomirs was used We previously

Figure 6 Effect of miRNA mimics on inflammatory agonistndashinduced PBEFNAMPT 39-UTR reporteractivity in human lung ECs ECs were cotransfected with PBEFNAMPT 39-UTR reporter togetherwith nc or with the indicated miRNA mimics and phRL-TK a Renilla luciferase normalization controlvector ECs were untreated and exposed to 18 CS (A) or treated with LPS (C) (24 h) (B) andluciferase activity was measured according the manufacturerrsquos protocol Data are presented asrelative luciferase units over vehicle-treated control and expressed as means 6 SE from fourindependent experiments Significantly different from control cells without stimulation (P 005)Significantly different from control cells stimulated with 18 CS or LPS (P 005) Significantlydifferent from control cells stimulated with 18 CS or LPS (P 001)

ORIGINAL ARTICLE

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 415

reported that targeting PBEFNAMPTby specific siRNAs (11) or by miR-374a(37) attenuates inflammatory agonist(thrombin IL-1b and LPS)-mediated EChyperpermeability consistent with the key

regulatory role of PBEF in inducing lunginflammation vascular permeability andpathobiology (11 13 14) Similarly ourdata demonstrate that miR-568 mimicsattenuate LPS-induced permeability in ECs

(Figures 3A and 3B) These mRNAs(miR-374a and miR-568) may modulateLPS-induced EC permeability by targetingthe PBEFNAMPT 39-UTR thus alteringexpression We found that transfection ofECs with mimics of miR-374a and miR-568significantly attenuated the 18 CSndash andLPS-induced augmented PBEFNAMPTmRNA and protein levels (Figures 4A and4B and 5Andash5C) Although the exactmechanism by which miR-374a andmiR-568 mimics alter PBEFNAMPTexpression is not known our data supportrecent evidence that the majority ofmammalian miRNAs directly target mRNAdown-regulation via cleavage anddegradation (52 53) or accelerateddeadenylation of mRNAs (54 55)

We further investigated the role ofmiRNAs in the regulation of PBEFNAMPTexpression by using a luciferase 39-UTRreporter gene system a sensitive methodfor measuring miRNA and the 39-UTReffects on gene regulation Our datademonstrated that EC exposure to thesemiRNAs significantly attenuates basalPBEFNAMPT 39-UTR reporter activity(Figure 6A) as well as 18 CS- andLPS-induced increases in luciferase activity(Figures 6B and 6C) Because miRNAoverexpression produces false-positiveresults by targeting genes not affected inphysiologic conditions (ie ldquooff-targeteffectsrdquo) we used the complementary ldquoloss-of-functionrdquo approach by inhibiting specificendogenous miRNA function viaantagomirs and demonstrated that specificmiRNA antagomirs increase PBEFNAMPTexpression (Figures 7A and 7B) and39-UTR reporter activity (Figure 7C) Thusour studies clearly demonstrate thatmiRNAs miR-374a and miR-568 stronglyinfluence PBEFNAMPT expression Ourdata indicate that these two miRNAscooperatively regulate PBEFNAMPTexpression in human pulmonary ECs(Figures 4B and 6B)

PBEFNAMPT is a key participantin VILI pathogenesis and is a criticalbiomarker of diverse inflammatorypathobiologies such as sepsis pneumoniaand ARDS (7 9) potentially via directeffects on innate immunity apoptosis andmitochondrial function Information islimited concerning the possible regulatorymechanisms of PBEFNAMPT expressionvia the 59 and 39 gene regions however ourin silico analysis of the 59 PBEFNAMPTpromoter region by using the Genomatix

Figure 7 Effects of miRNA antagomirs on inflammatory agonistndashinduced PBEFNAMPT expressionand 39-UTR reporter activity Total RNA was isolated from ECs and transfected with nc or with theindicated miRNA Anti- and PBEFNAMPTmRNA levels were detected via real-time PCR (A) Data arepresented as fold change in mRNA level over control and expressed as means 6 SE from fourindependent experiments Significantly different from control cells (P 005) Significantly differentfrom control cells (P 001) ECs transfected with nc or with the indicated miRNA anti- andendogenous PBEFNAMPT protein were detected via immunoblot The bar graph represents relativedensitometry (B) Data are presented as fold changes in endogenous PBEFNAMPT over ncpretreatment and expressed as means 6 SE from three independent experiments P 005 versuscontrol ECs were cotransfected with the PBEFNAMPT 39-UTR reporter together with phRL-TK ncor with the indicated miRNA (C) and luciferase activity was measured according the manufacturerrsquosprotocol Data are presented as relative luciferase units over control and expressed as means 6 SEfrom four independent experiments Significantly different from control cells (P 005)

ORIGINAL ARTICLE

416 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 2 | February 2014

genome analyzer revealed several putativecis regulatory elements including sites forbinding by transcription activators (egNF-kB SP1 STAT and GATA-1) andsequences of homology to antioxidant- shearstressndash and stretch-responsive elements(unpublished data) In addition we notednovel CpG islands in the PBEFNAMPTpromoter suggesting epigenetic regulationof PBEFNAMPT expression via DNAmethylation and 59 promoter interacting

elements The current study demonstratesthat mature expression of miR-374a andmiR-568 epigenetically regulates LPS and18 CSndashinduced PBEFNAMPT expressionin human pulmonary ECs via 39-UTRmodulation Future studies are needed todemonstrate whether miRNAs and otherregulatory elements synergistically participatein PBEFNAMPT gene regulation Targetingof PBEFNAMPT a critical inflammatorybiomarker in ARDS and VILI via specific

miRNAs is a potentially promising noveltherapeutic strategy to reduce lung vascularpermeabilityedema and inflammation inARDS and VILI n

Author disclosures are available with the textof this article at wwwatsjournalsorg

Acknowledgments The authors thank LakshmiNatarajan for providing assistance with theendothelial cell cultures

References

1 Levitt JE Matthay MA Treatment of acute lung injury historicalperspective and potential future therapies Semin Respir Crit CareMed 200627426ndash437

2 Leaver SK Evans TW Acute respiratory distress syndrome BMJ 2007335389ndash394

3 Rubenfeld GD Caldwell E Peabody E Weaver J Martin DP Neff MStern EJ Hudson LD Incidence and outcomes of acute lung injuryN Engl J Med 20053531685ndash1693

4 Ognjanovic S Bao S Yamamoto SY Garibay-Tupas J Samal B Bryant-Greenwood GD Genomic organization of the gene coding for humanpre-b-cell colony enhancing factor and expression in human fetalmembranes J Mol Endocrinol 200126107ndash117

5 Rongvaux A Shea RJ Mulks MH Gigot D Urbain J Leo O Andris FPre-b-cell colony-enhancing factor whose expression isup-regulated in activated lymphocytes is a nicotinamidephosphoribosyltransferase a cytosolic enzyme involved in NADbiosynthesis Eur J Immunol 2002323225ndash3234

6 Sommer G Garten A Petzold S Beck-Sickinger AG Bluher M Stumvoll MFasshauer M VisfatinPBEFNampt structure regulation and potentialfunction of a novel adipokine Clin Sci (Lond) 200811513ndash23

7 Ye SQ Simon BA Maloney JP Zambelli-Weiner A Gao L Grant AEasley RB McVerry BJ Tuder RM Standiford T et al Pre-b-cellcolony-enhancing factor as a potential novel biomarker in acute lunginjury Am J Respir Crit Care Med 2005171361ndash370

8 Hug C Lodish HF Medicine Visfatin a new adipokine Science 2005307366ndash367

9 Hong SB Huang Y Moreno-Vinasco L Sammani S Moitra J BarnardJW Ma SF Mirzapoiazova T Evenoski C Reeves RR et al Essentialrole of pre-b-cell colony enhancing factor in ventilator-induced lunginjury Am J Respir Crit Care Med 2008178605ndash617

10 Bajwa EK Yu CL Gong MN Thompson BT Christiani DC Pre-b-cellcolony-enhancing factor gene polymorphisms and risk of acuterespiratory distress syndrome Crit Care Med 2007351290ndash1295

11 Ye SQ Zhang LQ Adyshev D Usatyuk PV Garcia AN Lavoie TL VerinAD Natarajan V Garcia JG Pre-b-cell-colony-enhancing factor iscritically involved in thrombin-induced lung endothelial cell barrierdysregulation Microvasc Res 200570142ndash151

12 Jia SH Li Y Parodo J Kapus A Fan L Rotstein OD Marshall JCPre-b cell colony-enhancing factor inhibits neutrophil apoptosis inexperimental inflammation and clinical sepsis J Clin Invest 20041131318ndash1327

13 Liu P Li H Cepeda J Zhang LQ Cui X Garcia JG Ye SQ Critical roleof PBEF expression in pulmonary cell inflammation and permeabilityCell Biol Int 20093319ndash30

14 Li H Liu P Cepeda J Fang D Easley RB Simon BA Zhang LQ Ye SQAugmentation of pulmonary epithelial cell IL-8 expression andpermeability by pre-b-cell colony enhancing factor J Inflamm (Lond)2008515

15 Dahl TB Yndestad A Skjelland M Oie E Dahl A Michelsen A DamasJK Tunheim SH Ueland T Smith C et al Increased expression ofvisfatin in macrophages of human unstable carotid and coronaryatherosclerosis possible role in inflammation and plaquedestabilization Circulation 2007115972ndash980

16 Tomankova T Petrek M Kriegova E Involvement of microRNAs inphysiological and pathological processes in the lung Respir Res201011159

17 Sokolov MV Panyutin IV Neumann RD Unraveling the globalmicroRNAome responses to ionizing radiation in human embryonicstem cells PLoS ONE 20127e31028

18 Ji F Lv X Jiao J The role of micrornas in neural stem cells andneurogenesis J Genet Genomics 20134061ndash66

19 Zhou T Garcia JG Zhang W Integrating microRNAs into a systembiology approach to acute lung injury Transl Res 2011157180ndash190

20 Ranjha R Paul J Micro-RNAs in inflammatory diseases and asa link between inflammation and cancer Inflamm Res 201362343ndash355

21 Kornfeld JW Baitzel C Konner AC Nicholls HT Vogt MC HerrmannsK Scheja L Haumaitre C Wolf AM Knippschild U et al Obesity-induced overexpression of miR-802 impairs glucose metabolismthrough silencing of Hnf1b Nature 2013494111ndash115

22 Li N Zhong X Lin X Guo J Zou L Tanyi JL Shao Z Liang S Wang LPHwang WT et al Lin-28 homologue A (LIN28A) promotes cell cycleprogression via the regulation of cyclin-dependent kinase 2 (CDK2)cyclin D1 (CCND1) and cell division cycle 25 homolog A (CDC25A)expression in cancer J Biol Chem 201228717386ndash17397

23 Grosso S Doyen J Parks SK Bertero T Paye A Cardinaud B GounonP Lacas-Gervais S Noel A Pouyssegur J et al MiR-210 promotesa hypoxic phenotype and increases radioresistance in human lungcancer cell lines Cell Death Dis 20134e544

24 Creemers EE Tijsen AJ Pinto YM Circulating microRNAs Novelbiomarkers and extracellular communicators in cardiovasculardisease Circ Res 2012110483ndash495

25 Kohlstedt K Trouvain C Boettger T Shi L Fisslthaler B Fleming IThe AMP-activated protein kinase regulates endothelial cellangiotensin-converting enzyme expression via p53 and the post-transcriptional regulation of microRNA-143145 Circ Res 20131121150ndash1158

26 Zhao JL Rao DS Boldin MP Taganov KD OrsquoConnell RM Baltimore DNF-kappaB dysregulation in microRNA-146a-deficient mice drivesthe development of myeloid malignancies Proc Natl Acad Sci USA20111089184ndash9189

27 Persengiev SP MiRNAs at the crossroad between hematopoieticmalignancies and autoimmune pathogenesis Discov Med 201213211ndash221

28 Liu N Landreh M Cao K Abe M Hendriks GJ Kennerdell JR Zhu YWang LS Bonini NM The microRNA miR-34 modulates ageing andneurodegeneration in drosophila Nature 2011482519ndash523

29 Garcia JG Liu F Verin AD Birukova A Dechert MA Gerthoffer WTBamburg JR English D Sphingosine 1-phosphate promotesendothelial cell barrier integrity by Edg-dependent cytoskeletalrearrangement J Clin Invest 2001108689ndash701

30 Lewis BP Burge CB Bartel DP Conserved seed pairing often flankedby adenosines indicates that thousands of human genes aremicroRNA targets Cell 200512015ndash20

31 Grimson A Farh KK Johnston WK Garrett-Engele P Lim LP BartelDP MicroRNA targeting specificity in mammals determinantsbeyond seed pairing Mol Cell 20072791ndash105

ORIGINAL ARTICLE

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 417

reported that targeting PBEFNAMPTby specific siRNAs (11) or by miR-374a(37) attenuates inflammatory agonist(thrombin IL-1b and LPS)-mediated EChyperpermeability consistent with the key

regulatory role of PBEF in inducing lunginflammation vascular permeability andpathobiology (11 13 14) Similarly ourdata demonstrate that miR-568 mimicsattenuate LPS-induced permeability in ECs

(Figures 3A and 3B) These mRNAs(miR-374a and miR-568) may modulateLPS-induced EC permeability by targetingthe PBEFNAMPT 39-UTR thus alteringexpression We found that transfection ofECs with mimics of miR-374a and miR-568significantly attenuated the 18 CSndash andLPS-induced augmented PBEFNAMPTmRNA and protein levels (Figures 4A and4B and 5Andash5C) Although the exactmechanism by which miR-374a andmiR-568 mimics alter PBEFNAMPTexpression is not known our data supportrecent evidence that the majority ofmammalian miRNAs directly target mRNAdown-regulation via cleavage anddegradation (52 53) or accelerateddeadenylation of mRNAs (54 55)

We further investigated the role ofmiRNAs in the regulation of PBEFNAMPTexpression by using a luciferase 39-UTRreporter gene system a sensitive methodfor measuring miRNA and the 39-UTReffects on gene regulation Our datademonstrated that EC exposure to thesemiRNAs significantly attenuates basalPBEFNAMPT 39-UTR reporter activity(Figure 6A) as well as 18 CS- andLPS-induced increases in luciferase activity(Figures 6B and 6C) Because miRNAoverexpression produces false-positiveresults by targeting genes not affected inphysiologic conditions (ie ldquooff-targeteffectsrdquo) we used the complementary ldquoloss-of-functionrdquo approach by inhibiting specificendogenous miRNA function viaantagomirs and demonstrated that specificmiRNA antagomirs increase PBEFNAMPTexpression (Figures 7A and 7B) and39-UTR reporter activity (Figure 7C) Thusour studies clearly demonstrate thatmiRNAs miR-374a and miR-568 stronglyinfluence PBEFNAMPT expression Ourdata indicate that these two miRNAscooperatively regulate PBEFNAMPTexpression in human pulmonary ECs(Figures 4B and 6B)

PBEFNAMPT is a key participantin VILI pathogenesis and is a criticalbiomarker of diverse inflammatorypathobiologies such as sepsis pneumoniaand ARDS (7 9) potentially via directeffects on innate immunity apoptosis andmitochondrial function Information islimited concerning the possible regulatorymechanisms of PBEFNAMPT expressionvia the 59 and 39 gene regions however ourin silico analysis of the 59 PBEFNAMPTpromoter region by using the Genomatix

Figure 7 Effects of miRNA antagomirs on inflammatory agonistndashinduced PBEFNAMPT expressionand 39-UTR reporter activity Total RNA was isolated from ECs and transfected with nc or with theindicated miRNA Anti- and PBEFNAMPTmRNA levels were detected via real-time PCR (A) Data arepresented as fold change in mRNA level over control and expressed as means 6 SE from fourindependent experiments Significantly different from control cells (P 005) Significantly differentfrom control cells (P 001) ECs transfected with nc or with the indicated miRNA anti- andendogenous PBEFNAMPT protein were detected via immunoblot The bar graph represents relativedensitometry (B) Data are presented as fold changes in endogenous PBEFNAMPT over ncpretreatment and expressed as means 6 SE from three independent experiments P 005 versuscontrol ECs were cotransfected with the PBEFNAMPT 39-UTR reporter together with phRL-TK ncor with the indicated miRNA (C) and luciferase activity was measured according the manufacturerrsquosprotocol Data are presented as relative luciferase units over control and expressed as means 6 SEfrom four independent experiments Significantly different from control cells (P 005)

ORIGINAL ARTICLE

416 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 2 | February 2014

genome analyzer revealed several putativecis regulatory elements including sites forbinding by transcription activators (egNF-kB SP1 STAT and GATA-1) andsequences of homology to antioxidant- shearstressndash and stretch-responsive elements(unpublished data) In addition we notednovel CpG islands in the PBEFNAMPTpromoter suggesting epigenetic regulationof PBEFNAMPT expression via DNAmethylation and 59 promoter interacting

elements The current study demonstratesthat mature expression of miR-374a andmiR-568 epigenetically regulates LPS and18 CSndashinduced PBEFNAMPT expressionin human pulmonary ECs via 39-UTRmodulation Future studies are needed todemonstrate whether miRNAs and otherregulatory elements synergistically participatein PBEFNAMPT gene regulation Targetingof PBEFNAMPT a critical inflammatorybiomarker in ARDS and VILI via specific

miRNAs is a potentially promising noveltherapeutic strategy to reduce lung vascularpermeabilityedema and inflammation inARDS and VILI n

Author disclosures are available with the textof this article at wwwatsjournalsorg

Acknowledgments The authors thank LakshmiNatarajan for providing assistance with theendothelial cell cultures

References

1 Levitt JE Matthay MA Treatment of acute lung injury historicalperspective and potential future therapies Semin Respir Crit CareMed 200627426ndash437

2 Leaver SK Evans TW Acute respiratory distress syndrome BMJ 2007335389ndash394

3 Rubenfeld GD Caldwell E Peabody E Weaver J Martin DP Neff MStern EJ Hudson LD Incidence and outcomes of acute lung injuryN Engl J Med 20053531685ndash1693

4 Ognjanovic S Bao S Yamamoto SY Garibay-Tupas J Samal B Bryant-Greenwood GD Genomic organization of the gene coding for humanpre-b-cell colony enhancing factor and expression in human fetalmembranes J Mol Endocrinol 200126107ndash117

5 Rongvaux A Shea RJ Mulks MH Gigot D Urbain J Leo O Andris FPre-b-cell colony-enhancing factor whose expression isup-regulated in activated lymphocytes is a nicotinamidephosphoribosyltransferase a cytosolic enzyme involved in NADbiosynthesis Eur J Immunol 2002323225ndash3234

6 Sommer G Garten A Petzold S Beck-Sickinger AG Bluher M Stumvoll MFasshauer M VisfatinPBEFNampt structure regulation and potentialfunction of a novel adipokine Clin Sci (Lond) 200811513ndash23

7 Ye SQ Simon BA Maloney JP Zambelli-Weiner A Gao L Grant AEasley RB McVerry BJ Tuder RM Standiford T et al Pre-b-cellcolony-enhancing factor as a potential novel biomarker in acute lunginjury Am J Respir Crit Care Med 2005171361ndash370

8 Hug C Lodish HF Medicine Visfatin a new adipokine Science 2005307366ndash367

9 Hong SB Huang Y Moreno-Vinasco L Sammani S Moitra J BarnardJW Ma SF Mirzapoiazova T Evenoski C Reeves RR et al Essentialrole of pre-b-cell colony enhancing factor in ventilator-induced lunginjury Am J Respir Crit Care Med 2008178605ndash617

10 Bajwa EK Yu CL Gong MN Thompson BT Christiani DC Pre-b-cellcolony-enhancing factor gene polymorphisms and risk of acuterespiratory distress syndrome Crit Care Med 2007351290ndash1295

11 Ye SQ Zhang LQ Adyshev D Usatyuk PV Garcia AN Lavoie TL VerinAD Natarajan V Garcia JG Pre-b-cell-colony-enhancing factor iscritically involved in thrombin-induced lung endothelial cell barrierdysregulation Microvasc Res 200570142ndash151

12 Jia SH Li Y Parodo J Kapus A Fan L Rotstein OD Marshall JCPre-b cell colony-enhancing factor inhibits neutrophil apoptosis inexperimental inflammation and clinical sepsis J Clin Invest 20041131318ndash1327

13 Liu P Li H Cepeda J Zhang LQ Cui X Garcia JG Ye SQ Critical roleof PBEF expression in pulmonary cell inflammation and permeabilityCell Biol Int 20093319ndash30

14 Li H Liu P Cepeda J Fang D Easley RB Simon BA Zhang LQ Ye SQAugmentation of pulmonary epithelial cell IL-8 expression andpermeability by pre-b-cell colony enhancing factor J Inflamm (Lond)2008515

15 Dahl TB Yndestad A Skjelland M Oie E Dahl A Michelsen A DamasJK Tunheim SH Ueland T Smith C et al Increased expression ofvisfatin in macrophages of human unstable carotid and coronaryatherosclerosis possible role in inflammation and plaquedestabilization Circulation 2007115972ndash980

16 Tomankova T Petrek M Kriegova E Involvement of microRNAs inphysiological and pathological processes in the lung Respir Res201011159

17 Sokolov MV Panyutin IV Neumann RD Unraveling the globalmicroRNAome responses to ionizing radiation in human embryonicstem cells PLoS ONE 20127e31028

18 Ji F Lv X Jiao J The role of micrornas in neural stem cells andneurogenesis J Genet Genomics 20134061ndash66

19 Zhou T Garcia JG Zhang W Integrating microRNAs into a systembiology approach to acute lung injury Transl Res 2011157180ndash190

20 Ranjha R Paul J Micro-RNAs in inflammatory diseases and asa link between inflammation and cancer Inflamm Res 201362343ndash355

21 Kornfeld JW Baitzel C Konner AC Nicholls HT Vogt MC HerrmannsK Scheja L Haumaitre C Wolf AM Knippschild U et al Obesity-induced overexpression of miR-802 impairs glucose metabolismthrough silencing of Hnf1b Nature 2013494111ndash115

22 Li N Zhong X Lin X Guo J Zou L Tanyi JL Shao Z Liang S Wang LPHwang WT et al Lin-28 homologue A (LIN28A) promotes cell cycleprogression via the regulation of cyclin-dependent kinase 2 (CDK2)cyclin D1 (CCND1) and cell division cycle 25 homolog A (CDC25A)expression in cancer J Biol Chem 201228717386ndash17397

23 Grosso S Doyen J Parks SK Bertero T Paye A Cardinaud B GounonP Lacas-Gervais S Noel A Pouyssegur J et al MiR-210 promotesa hypoxic phenotype and increases radioresistance in human lungcancer cell lines Cell Death Dis 20134e544

24 Creemers EE Tijsen AJ Pinto YM Circulating microRNAs Novelbiomarkers and extracellular communicators in cardiovasculardisease Circ Res 2012110483ndash495

25 Kohlstedt K Trouvain C Boettger T Shi L Fisslthaler B Fleming IThe AMP-activated protein kinase regulates endothelial cellangiotensin-converting enzyme expression via p53 and the post-transcriptional regulation of microRNA-143145 Circ Res 20131121150ndash1158

26 Zhao JL Rao DS Boldin MP Taganov KD OrsquoConnell RM Baltimore DNF-kappaB dysregulation in microRNA-146a-deficient mice drivesthe development of myeloid malignancies Proc Natl Acad Sci USA20111089184ndash9189

27 Persengiev SP MiRNAs at the crossroad between hematopoieticmalignancies and autoimmune pathogenesis Discov Med 201213211ndash221

28 Liu N Landreh M Cao K Abe M Hendriks GJ Kennerdell JR Zhu YWang LS Bonini NM The microRNA miR-34 modulates ageing andneurodegeneration in drosophila Nature 2011482519ndash523

29 Garcia JG Liu F Verin AD Birukova A Dechert MA Gerthoffer WTBamburg JR English D Sphingosine 1-phosphate promotesendothelial cell barrier integrity by Edg-dependent cytoskeletalrearrangement J Clin Invest 2001108689ndash701

30 Lewis BP Burge CB Bartel DP Conserved seed pairing often flankedby adenosines indicates that thousands of human genes aremicroRNA targets Cell 200512015ndash20

31 Grimson A Farh KK Johnston WK Garrett-Engele P Lim LP BartelDP MicroRNA targeting specificity in mammals determinantsbeyond seed pairing Mol Cell 20072791ndash105

ORIGINAL ARTICLE

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 417

genome analyzer revealed several putativecis regulatory elements including sites forbinding by transcription activators (egNF-kB SP1 STAT and GATA-1) andsequences of homology to antioxidant- shearstressndash and stretch-responsive elements(unpublished data) In addition we notednovel CpG islands in the PBEFNAMPTpromoter suggesting epigenetic regulationof PBEFNAMPT expression via DNAmethylation and 59 promoter interacting

elements The current study demonstratesthat mature expression of miR-374a andmiR-568 epigenetically regulates LPS and18 CSndashinduced PBEFNAMPT expressionin human pulmonary ECs via 39-UTRmodulation Future studies are needed todemonstrate whether miRNAs and otherregulatory elements synergistically participatein PBEFNAMPT gene regulation Targetingof PBEFNAMPT a critical inflammatorybiomarker in ARDS and VILI via specific

miRNAs is a potentially promising noveltherapeutic strategy to reduce lung vascularpermeabilityedema and inflammation inARDS and VILI n

Author disclosures are available with the textof this article at wwwatsjournalsorg

Acknowledgments The authors thank LakshmiNatarajan for providing assistance with theendothelial cell cultures

References

1 Levitt JE Matthay MA Treatment of acute lung injury historicalperspective and potential future therapies Semin Respir Crit CareMed 200627426ndash437

2 Leaver SK Evans TW Acute respiratory distress syndrome BMJ 2007335389ndash394

3 Rubenfeld GD Caldwell E Peabody E Weaver J Martin DP Neff MStern EJ Hudson LD Incidence and outcomes of acute lung injuryN Engl J Med 20053531685ndash1693

4 Ognjanovic S Bao S Yamamoto SY Garibay-Tupas J Samal B Bryant-Greenwood GD Genomic organization of the gene coding for humanpre-b-cell colony enhancing factor and expression in human fetalmembranes J Mol Endocrinol 200126107ndash117

5 Rongvaux A Shea RJ Mulks MH Gigot D Urbain J Leo O Andris FPre-b-cell colony-enhancing factor whose expression isup-regulated in activated lymphocytes is a nicotinamidephosphoribosyltransferase a cytosolic enzyme involved in NADbiosynthesis Eur J Immunol 2002323225ndash3234

6 Sommer G Garten A Petzold S Beck-Sickinger AG Bluher M Stumvoll MFasshauer M VisfatinPBEFNampt structure regulation and potentialfunction of a novel adipokine Clin Sci (Lond) 200811513ndash23

7 Ye SQ Simon BA Maloney JP Zambelli-Weiner A Gao L Grant AEasley RB McVerry BJ Tuder RM Standiford T et al Pre-b-cellcolony-enhancing factor as a potential novel biomarker in acute lunginjury Am J Respir Crit Care Med 2005171361ndash370

8 Hug C Lodish HF Medicine Visfatin a new adipokine Science 2005307366ndash367

9 Hong SB Huang Y Moreno-Vinasco L Sammani S Moitra J BarnardJW Ma SF Mirzapoiazova T Evenoski C Reeves RR et al Essentialrole of pre-b-cell colony enhancing factor in ventilator-induced lunginjury Am J Respir Crit Care Med 2008178605ndash617

10 Bajwa EK Yu CL Gong MN Thompson BT Christiani DC Pre-b-cellcolony-enhancing factor gene polymorphisms and risk of acuterespiratory distress syndrome Crit Care Med 2007351290ndash1295

11 Ye SQ Zhang LQ Adyshev D Usatyuk PV Garcia AN Lavoie TL VerinAD Natarajan V Garcia JG Pre-b-cell-colony-enhancing factor iscritically involved in thrombin-induced lung endothelial cell barrierdysregulation Microvasc Res 200570142ndash151

12 Jia SH Li Y Parodo J Kapus A Fan L Rotstein OD Marshall JCPre-b cell colony-enhancing factor inhibits neutrophil apoptosis inexperimental inflammation and clinical sepsis J Clin Invest 20041131318ndash1327

13 Liu P Li H Cepeda J Zhang LQ Cui X Garcia JG Ye SQ Critical roleof PBEF expression in pulmonary cell inflammation and permeabilityCell Biol Int 20093319ndash30

14 Li H Liu P Cepeda J Fang D Easley RB Simon BA Zhang LQ Ye SQAugmentation of pulmonary epithelial cell IL-8 expression andpermeability by pre-b-cell colony enhancing factor J Inflamm (Lond)2008515

15 Dahl TB Yndestad A Skjelland M Oie E Dahl A Michelsen A DamasJK Tunheim SH Ueland T Smith C et al Increased expression ofvisfatin in macrophages of human unstable carotid and coronaryatherosclerosis possible role in inflammation and plaquedestabilization Circulation 2007115972ndash980

16 Tomankova T Petrek M Kriegova E Involvement of microRNAs inphysiological and pathological processes in the lung Respir Res201011159

17 Sokolov MV Panyutin IV Neumann RD Unraveling the globalmicroRNAome responses to ionizing radiation in human embryonicstem cells PLoS ONE 20127e31028

18 Ji F Lv X Jiao J The role of micrornas in neural stem cells andneurogenesis J Genet Genomics 20134061ndash66

19 Zhou T Garcia JG Zhang W Integrating microRNAs into a systembiology approach to acute lung injury Transl Res 2011157180ndash190

20 Ranjha R Paul J Micro-RNAs in inflammatory diseases and asa link between inflammation and cancer Inflamm Res 201362343ndash355

21 Kornfeld JW Baitzel C Konner AC Nicholls HT Vogt MC HerrmannsK Scheja L Haumaitre C Wolf AM Knippschild U et al Obesity-induced overexpression of miR-802 impairs glucose metabolismthrough silencing of Hnf1b Nature 2013494111ndash115

22 Li N Zhong X Lin X Guo J Zou L Tanyi JL Shao Z Liang S Wang LPHwang WT et al Lin-28 homologue A (LIN28A) promotes cell cycleprogression via the regulation of cyclin-dependent kinase 2 (CDK2)cyclin D1 (CCND1) and cell division cycle 25 homolog A (CDC25A)expression in cancer J Biol Chem 201228717386ndash17397

23 Grosso S Doyen J Parks SK Bertero T Paye A Cardinaud B GounonP Lacas-Gervais S Noel A Pouyssegur J et al MiR-210 promotesa hypoxic phenotype and increases radioresistance in human lungcancer cell lines Cell Death Dis 20134e544

24 Creemers EE Tijsen AJ Pinto YM Circulating microRNAs Novelbiomarkers and extracellular communicators in cardiovasculardisease Circ Res 2012110483ndash495

25 Kohlstedt K Trouvain C Boettger T Shi L Fisslthaler B Fleming IThe AMP-activated protein kinase regulates endothelial cellangiotensin-converting enzyme expression via p53 and the post-transcriptional regulation of microRNA-143145 Circ Res 20131121150ndash1158

26 Zhao JL Rao DS Boldin MP Taganov KD OrsquoConnell RM Baltimore DNF-kappaB dysregulation in microRNA-146a-deficient mice drivesthe development of myeloid malignancies Proc Natl Acad Sci USA20111089184ndash9189

27 Persengiev SP MiRNAs at the crossroad between hematopoieticmalignancies and autoimmune pathogenesis Discov Med 201213211ndash221

28 Liu N Landreh M Cao K Abe M Hendriks GJ Kennerdell JR Zhu YWang LS Bonini NM The microRNA miR-34 modulates ageing andneurodegeneration in drosophila Nature 2011482519ndash523

29 Garcia JG Liu F Verin AD Birukova A Dechert MA Gerthoffer WTBamburg JR English D Sphingosine 1-phosphate promotesendothelial cell barrier integrity by Edg-dependent cytoskeletalrearrangement J Clin Invest 2001108689ndash701

30 Lewis BP Burge CB Bartel DP Conserved seed pairing often flankedby adenosines indicates that thousands of human genes aremicroRNA targets Cell 200512015ndash20

31 Grimson A Farh KK Johnston WK Garrett-Engele P Lim LP BartelDP MicroRNA targeting specificity in mammals determinantsbeyond seed pairing Mol Cell 20072791ndash105

ORIGINAL ARTICLE

Adyshev Elangovan Moldobaeva et al MicroRNAs Regulate PBEFNAMPT Expression in ECs 417

32 Betel D Wilson M Gabow A Marks DS Sander C The microRNAOrgresource targets and expression Nucleic Acids Res 200836D149ndashD153

33 Maragkakis M Reczko M Simossis VA Alexiou P Papadopoulos GLDalamagas T Giannopoulos G Goumas G Koukis E Kourtis K et alDiana-micro t web server elucidating microrna functions throughtarget prediction Nucleic Acids Res 200937W273ndashW276

34 Adyshev DM Moldobaeva NK Elangovan VR Garcia JG Dudek SMDifferential involvement of ezrinradixinmoesin proteins insphingosine 1-phosphate-induced human pulmonary endothelial cellbarrier enhancement Cell Signal 2011232086ndash2096

35 Mitra S Sammani S Wang T Boone DL Meyer NJ Dudek SMMoreno-Vinasco L Garcia JG Jacobson JR Role of growth arrestand DNA damage-inducible alpha in Akt phosphorylation andubiquitination after mechanical stress-induced vascular injury AmJ Respir Crit Care Med 20111841030ndash1040

36 Gao L Grant A Halder I Brower R Sevransky J Maloney JP Moss MShanholtz C Yates CR Meduri GU et al Novel polymorphisms inthe myosin light chain kinase gene confer risk for acute lung injuryAm J Respir Cell Mol Biol 200634487ndash495

37 Adyshev DM Moldobaeva N Mapes B Elangovan V Garcia JGMicroRNA regulation of non-muscle myosin light chain kinase(nmmlck) expression in human lung endothelium Am J Respir CellMol Biol 20134958ndash66

38 Bartel DP Micrornas target recognition and regulatory functions Cell2009136215ndash233

39 Witkos TM Koscianska E Krzyzosiak WJ Practical aspects ofmicroRNA target prediction Curr Mol Med 20111193ndash109

40 Place RF Li LC Pookot D Noonan EJ Dahiya R MicroRNA-373induces expression of genes with complementary promotersequences Proc Natl Acad Sci USA 20081051608ndash1613

41 Vasudevan S Tong Y Steitz JA Switching from repression toactivation micrornas can up-regulate translation Science 20073181931ndash1934

42 Xie X Lu J Kulbokas EJ Golub TR Mootha V Lindblad-Toh K LanderES Kellis M Systematic discovery of regulatory motifs in humanpromoters and 39 UTRs by comparison of several mammals Nature2005434338ndash345

43 Barrett LW Fletcher S Wilton SD Regulation of eukaryotic geneexpression by the untranslated gene regions and other non-codingelements Cell Mol Life Sci 2012693613ndash3634

44 Stark A Brennecke J Bushati N Russell RB Cohen SM AnimalMicroRNAs confer robustness to gene expression and havea significant impact on 39UTR evolution Cell 20051231133ndash1146

45 Taganov KD Boldin MP Chang KJ Baltimore D NF-kappaB-dependent induction of microRNA miR-146 an inhibitor targeted tosignaling proteins of innate immune responses Proc Natl Acad SciUSA 200610312481ndash12486

46 OrsquoConnell RM Chaudhuri AA Rao DS Baltimore D Inositolphosphatase SHIP1 is a primary target of miR-155 Proc Natl AcadSci USA 20091067113ndash7118

47 Bazzoni F Rossato M Fabbri M Gaudiosi D Mirolo M Mori LTamassia N Mantovani A Cassatella MA Locati M Induction andregulatory function of miR-9 in human monocytes and neutrophilsexposed to proinflammatory signals Proc Natl Acad Sci USA 20091065282ndash5287

48 Moschos SA Williams AE Perry MM Birrell MA Belvisi MG LindsayMA Expression profiling in vivo demonstrates rapid changes in lungmicroRNA levels following lipopolysaccharide-induced inflammationbut not in the anti-inflammatory action of glucocorticoids BMCGenomics 20078240

49 Chamorro-Jorganes A Araldi E Suarez Y MicroRNAs aspharmacological targets in endothelial cell function and dysfunctionPharmacol Res 20137515ndash27

50 Zhang R Su B Small but influential the role of micrornas on generegulatory network and 3rsquoutr evolution J Genet Genomics 2009361ndash6

51 Nielsen JA Lau P Maric D Barker JL Hudson LD IntegratingmicroRNA and mRNA expression profiles of neuronal progenitorsto identify regulatory networks underlying the onset of corticalneurogenesis BMC Neurosci 20091098

52 Bagga S Bracht J Hunter S Massirer K Holtz J Eachus R PasquinelliAE Regulation by let-7 and lin-4 miRNAs results in target mRNAdegradation Cell 2005122553ndash563

53 Liu Z Wang D Hu Y Zhou G Zhu C Yu Q Chi Y Cao Y Jia C Zou QMicroRNA-146a negatively regulates PTGS2 expression induced byhelicobacter pylori in human gastric epithelial cells J Gastroenterol20134886ndash92

54 Giraldez AJ Mishima Y Rihel J Grocock RJ Van Dongen S Inoue KEnright AJ Schier AF Zebrafish mir-430 promotes deadenylationand clearance of maternal mrnas Science 200631275ndash79

55 Wu L Fan J Belasco JG MicroRNAs direct rapid deadenylation ofmRNA Proc Natl Acad Sci USA 20061034034ndash4039

ORIGINAL ARTICLE

418 American Journal of Respiratory Cell and Molecular Biology Volume 50 Number 2 | February 2014