REVIEW�ARTICLE

ABSTRACTEpidermalgrowthfactor-likedomain-8(EGFL8)alsoknownasvascularendothelialstatin-2(VE-statin-2).Itwasidentified by using retroviral gene entrapment vectors, expressed in endothelial cells. It is located onchromosome6inhumansandchromosome17inthemouse.EGFL8codesaproteinof293aminoacidswithanamino-terminalsignalpeptideandhastwoEGF-likedomains.EGFL8playsaveryimportantregulatoryroleinthymopoiesis,cellmigrationandinvasionthroughthemodulationofNotchsignaling.AlthoughthesignalingregulatoryfactorsofEGFL8needtobeexploredbutrecentscientificadvanceshaverevealedsomeimportantaspectofitsregulation.ThisreviewsummarizesthecurrentknowledgeaboutallaspectsofEGFL8sinceitsdiscovery.

KeyWords:Endothelial, EGFL, EGFL8, Notch, Thymopoiesis.

How to cite this: Subhan F, Naeem M, Sajjad W, Ali L, Tauseef I, Haleem SK. Epidermal Growth Factor-Like Domain-8 (EGFL8) in Mammals: The Story so far. 2020; 1(3): 119-121. doi: http://doi.org/10.37185/LnS.1.1.59

436-EGFL repeatswithin theextracellulardomain. TheinteractionsofNotchandEGFLfamilymembershavebeen identified in recent studies.Oneof thestudiesshowedthebindingofEGFL7toNotchfamilyreceptorsactsasan oftheNotchsignalingantagonist

6pathway inculturedneuralstemcells. Oneof therecentlyidentifiedmembersofEGFLfamilyincludesEGFL8,whichsharessequenceaswellasfunctionalsimilaritytotheEGFLproteinsEGFL8isreportedtoregulate T cells, thymic epithelial cells, embryonicdevelopmentandshowamodulatedexpression incanceroustissues.1. Discovery,expressionandtissuedistributionof

EGFL8EGFL8 was first recognized in 2004 and wascharacterizedforitshighresemblancewithEGFL7.Itwasidentifiedinthemousegenomeforthefirsttimewith a Basic Local Alignment Search Tool (BLAST)search technique using the EGFL7 amino acidsequence.ItshowedaparalogofEGFL7encodingaproteinof293aminoacidswhich ispositionedonmousechromosome17(GenBankaccessionno.NM152922). EGFL8 shares about 35% identity withEGFL7andbothproteinsconsistofCa2+binding,aN-

7terminalsignalpeptideandtwoEGF-likedomains. The EGFL family members are classified into twogroups. B io informat ics analyses showedtransmembrane domainswithin the EGFL2, EGFL5andEGFL9,whereasEGFL3,EGFL6,EGFL7andEGFL8

IntroductionEpidermal Growth Factor-like (EGFL) proteinscontain various repeats. Their receptor bindingmodulates a variety of life functions, includingproliferation, differentiation, apoptosis, adhesion

1,2andmigration. TheexpressionofEGFreceptorshasbeen found in many types of cells such asosteoblasts,osteoclasts,andendothelialcells.TherearetwotypesofEGF-likefamilymembersthataremembrane-bound (EGFL2, EGFL5 and EGFL9) orsecreted proteins (EGFL3, EGFL6, EGFL7 and

3,4EGFL8). These proteins are involved in the5

regulationofangiogenesis.Notch genes, code for transmembrane receptors,whichregulatecell fateandcontrolcelldivision inbothmammalsandlowerorganisms.Inmammals,therearefourdifferentNotchreceptorsfromNotch-1toNotch-4.TheNotchreceptorscontainasingletransmembraneprotein,whichiscomposedof29to

EpidermalGrowthFactor-LikeDomain-8(EGFL8)inMammals:TheStorysofar1 1 1 1 2 2FazliSubhan ,MuhammadNaeem ,WasimSajjad ,LiaqatAli ,IsfahanTauseef ,SyedKashifHaleem

Correspondence:Dr. Fazli SubhanAssistant Professor, Biological Sciences National University of Medical Sciences, RawalpindiE-mail: fazli.subhan@numspak.edu.pk

1Department of Biological SciencesNational University of Medical Sciences, Rawalpindi2Department of MicrobiologyHazara University, Mansehra

Life&Science2020Vol.1,No.3 EGFL8FunctionalStory

Funding Source: NIL; Conflict of Interest: NILReceived: Jul 24, 2019; Revised: Oct 20, 2019Accepted: Jun 25, 2020

This is an Open Access article distributed under the terms of the Creative Commons Attribution License(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,providedtheoriginalworkisproperlycited.

119

8are without transmembrane domains. TheexpressionlevelofEGFL8wasidentifiedinanumberofadultmousetissuesincludingskin,adrenalglands,urinarybladder,ductus,lymphnodes,testis,ovary,penis, epididymis, spleen, liver, intestine, kidney,brain,thymus,lung,muscle,ovary,heartandalsoanincreasing expression level during embryonic

7development. AnotherstudyshowedexpressionofEGFL8bothinosteoclasticandosteoblastic-likecellsby usingmicroarray and semi quantitative RT-PCRanalysis.ItisidentifiedthathighexpressionofEGFL8in both osteoblastic and osteoclastic like cells iscomparable with other EGF-like family memberstermedasEGFL2,EGFL3,EGFL5,EGFL6,EGFL7,and

8EGFL9. This wide range expression of EGFL8suggestingthatitplaysavitalroleduringphysiologicandpathologicregulation.ForamoredetailedstudyEGFL8wasoverexpressedand knockdown in thymic epithelial cell line with

9,10siRNA(smallinterferingRNA). Theoverexpressionand knockdown of EGFL8 in TEC play a moreregulatory function in thymicepithelialcellsandTcelldevelopmentinthethymus.2. FunctionalBackgroundofEGFL8ThemultipleknowntargetsofEGFL8areshowninFigure 1. EGFL8 expression has been observed ingastriccancerandiscorrelatedwithelevatedtumor,node,metastasisstage,andpoorprognosisingastric

11,12,13aswellasincolorectalcancer. Thethymusisthefundamental lymphoid organ that provides asuitablemicroenvironment for T-cell development

14andmaturation. Therequireddifferentiationand

organization of different TECs (thymic epithelialcells)iscriticalforboththymocytesexpansionandT-

15cell repertoire selection. The negative regulatoryeffectofEGFL8onmousethymicepithelialcellsandthymocytes,andT-cellregulationhasbeenshown,byconstructionofbothEGFL8overexpressedstablecelllineaswellasproductionofmouserecombinantprotein.EGFL8downregulatesthethymicepithelialexpressionof ICAM-1 aswell as IL-7,GM-CSF andTECK.TheinhibitoryeffectofEGFL8wasrecoveredwithknockdownofEGFL8withEGFL8targetedsiRNAinepithelialcellsofmousethymus.EGFL8whichisaparalog of EGFL7 has an inhibitory effect ondownstream target of Notch receptors, Hes1 and

9,10Hey1. It is well known that Notch signalingmediates cell fate determination through theregulatory, inhibitory and inductive action. Notchsignalingplayakeyroleinembryogenesisofmanyorgan systems, which was verified in Notch1,

16Notch2,Jagged2,andDelta1knockoutmice. Otherthan normal cells or developmental regulation,Notch signaling regulates the balance among cellproliferation,differentiation,andapoptosis. Itmay

17 18belinkedwithpancreaticcancer, melanomacells

19andovariancancer. Theγ-SecretaseinhibitsNotchby cleaving the Notch intracellular domain (NICD)that prevents the generation of the intracellulardomainofNotchmolecules.However, the currentchallenge of the γ-secretase inhibitors are itscytotoxicsideeffects,whichneedtobeaddressedbystudies that can help design targeted therapiesagainstspecificmutations intheNotchgenewhile

20reducing adverse side effects. Recent data hasshown that secretory EGFL7 is a functionalantagonistofNotchreceptorsandinteractswithall

6four subtypes of Notch receptors. The effect ofEGFL8 on γ-Secretase activation is still to bedetermined in the context ofNotch downstreamsignalinginhibition.Another study described EGFL8 as a consistentmethylation marker in different human tissuessamples. A bioinformatics approach generated 27genesshowingconsistentmethylationlevelsacrossall samples. Among them, the peak five genes(N4BP2,EGFL8,CTRB1,TSPAN3,andZNF690)wereanalyzedusingmassspectrometryin24humancelllines. Thus, EGFL8 was highly methylated both intissues samples and 24 cell lines across 13 tissue

21typesexaminedbymassspectrometry. Epigenetic

Fig1:DiversefunctionaltargetofEGFL8.TheEGFL8areknowntoinfluencetheTcelldevelopment,thymicepithelialregulation,Cancerregulation,Vascularizationandidentifiedasmethylationmarker

120

processes such as hypermethylation play aregulatoryroleinthecelldifferentiation,chromatinstructure; however the abnormal methylation ingenes can affect the mechanisms of various vitalbiological functions. It is hypothesized thathypermethylat ion might regulate tumor

22development and progression. EGFL8 can be apotential new target gene for regulating tumordevelopmentandprogression.Furtherexploratorystudies on regulatory functions of EGFL8methylation status in cancer are required, ascurrently little information is available on its

23molecularcharacterization. 3. FutureProspectsThe involvement of EGFL8 cannot be ignored inmultiplesignalingpathwayssuchasWnt/β-catenin,Ras,NFkB,Notch.Theliteraturereviewhasshownthat regulating the functionof EGFL8, canplayanimportant role in various important biologicalfunctions such as, immunity, inflammation, Graftversus host disease (GvHD), regeneration, woundhealingandembryonicdevelopment.EGFL8maybeafuturetherapeutictargetforvariouspathologicalconditions.

REFERENCES1. Singh AB, Harris RC. Autocrine, paracrine and juxtacrine

signaling by EGFR ligands. Cellular signalling. 2005; 17:1183-93.

2. Sánchez-SolanaB,NuedaML,RuviraMD,Ruiz-HidalgoMJ,MonsalveEM,RiveroS,etal.TheEGF-likeproteinsDLK1andDLK2 function as inhibitory non-canonical ligandsofNotch1 receptor that modulate each other's activities.Biochimica et Biophysica Acta (BBA)-Molecular CellResearch.2011;1813:1153-64.

3. Mehta VB, Besner GE, Mehta VB, Besner GE. HB-EGFpromotesangiogenesis inendothelial cellsviaPI3-kinaseandMAPKsignalingpathways.Growth factors.2007;25:253-63.

4. ChimSM,TicknerJ,ChowST,KuekV,GuoB,ZhangG,etal.Angiogenicfactorsinbonelocalenvironment.Cytokine&growthfactorreviews.2013;24:297-310.

5. ParkerLH,SchmidtM,JinSW,GrayAM,BeisD,PhamT,etal.Theendothelial-cell-derivedsecretedfactorEgfl7regulatesvasculartubeformation.Nature.2004;428:754-8.

6. SchmidtMH,BickerF,NikolicI,MeisterJ,BabukeT,PicuricS,et al. Epidermal growth factor-like domain 7 (EGFL7)modulates Notch signalling and affects neural stem cellrenewal.Naturecellbiology.2009;11:873-80.

7. FitchMJ,CampagnoloL,KuhnertF,StuhlmannH.Egfl7,anovelepidermalgrowthfactor-domaingeneexpressedinendothelial cells. Developmental dynamics: an officialpublication of the American Association of Anatomists.

2004;230:316-24.8. ChimSM,QinA,TicknerJ,PavlosN,DaveyT,WangH,etal.

EGFL6 promotes endothelial cell migration andangiogenesisthroughtheactivationofextracellularsignal-regulated kinase. Journal of Biological Chemistry. 2011;286:22035-46.

9. ChoiHJ,YoonTD,MuhammadI,JeongMH,LeeJ,BaekSY,etal.Regulatoryroleofmouseepidermalgrowthfactor-likeprotein 8 in thymic epithelial cells. Biochemical andbiophysicalresearchcommunications.2012;425:250-5.

10. SubhanF,YoonTD,ChoiHJ,MuhammadI,LeeJ,HongC,etal. Epidermal growth factor-like domain 8 inhibits thesurvival and proliferation of mouse thymocytes.Internationaljournalofmolecularmedicine.2013;32:952-8.

11. SongIJ,IkramM,SubhanF,ChoiDJ,LeeJR,KimHS,etal.Molecular characterization and expression analysis ofmouse epidermal growth factor-like domain 8.Internationaljournalofmolecularmedicine.2015;36:541-50.

12. WuF,ShirahataA,SakurabaK,KitamuraY,GotoT,SaitoM,et al. Down-regulation of EGFL8: a novel prognosticbiomarker forpatientswithcolorectal cancer.Anticancerresearch.2011;31:2249-54.

13. WuF,ShirahataA,SakurabaK,KitamuraY,GotoT,SaitoM,et al. Down-regulation of EGFL8: a novel biomarker foradvanced gastric cancer. Anticancer research. 2011; 31:3377-80.

14. Anderson G, Harman BC, Hare KJ, Jenkinson EJ.MicroenvironmentalregulationofTcelldevelopmentinthethymus.InSeminarsinimmunology.2000;12:457-64.

15. RomanoR,PalamaroL,FuscoA,IannaceL,MaioS,ViglianoI,etal.Frommurinetohumannude/SCID:thethymus,T-cell development and the missing link. Clinical andDevelopmentalImmunology.2012;5:2012.

16. Olsauskas-KuprysR,ZlobinA,OsipoC.Gammasecretaseinhibitors of Notch signaling. OncoTargets and therapy.2013;6:943.

17. WangZ,ZhangY,LiY,BanerjeeS,LiaoJ,SarkarFH.Down-regulationofNotch-1contributestocellgrowthinhibitionandapoptosisinpancreaticcancercells.Molecularcancertherapeutics.2006;5:483-93.

18. BalintK,XiaoM,PinnixCC,SomaA,VeresI,JuhaszI,etal.Activation of Notch1 signaling is required for β-catenin–mediatedhumanprimarymelanomaprogression.TheJournalofclinicalinvestigation.2005;115:3166-76.

19. HopferO,ZwahlenD,FeyM,AebiS.TheNotchpathwayinovarian carcinomas and adenomas. British journal ofcancer.2005;93:709-18.

20. ShihIM,WangTL.Notchsignaling,γ-secretaseinhibitors,andcancertherapy.Cancerresearch.2007;67:1879-82.

21. ShihIM,WangTL.Notchsignaling,γ-secretaseinhibitors,andcancertherapy.Cancerresearch.2007;67:1879-82.

22. Lu TP, Chen KT, TsaiMH, Kuo KT,Hsiao CK, Lai LC, et al.Identificationofgeneswithconsistentmethylation levelsacrossdifferenthumantissues.Scientificreports.2014;4:4351.

23. BaylinSB.DNAmethylationandgenesilencing incancer.NatureReviewsClinicalOncology.2005;2:4-11

121

Page 33Page 34Page 35