Structural and functional analysis of the endometrial gene encoding insulin-like growth factor-binding protein-2

193 Pages·1996·5 MB·English

by  Song, Sihong

STRUCTURALANDFUNCTIONALANALYSISOFTHE
ENDOMETRIALGENEENCODINGINSULIN-LIKE
GROWTHFACTOR-BINDINGPROTEIN-2
By
SIHONGSONG
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
1996
Tomyparentswithdeepestappreciation.
ACKNOWLEDGMENTS
Firstofall,IwouldliketothankDr.FrankA.Simmenforhissupport,advice,
encouragementandpatience.Asanoutstandingadvisorheledmeintomolecularandcell
biologystepbystep.Itwaswonderfultoenjoythepursuitofscienceunderhisguidance.I
alsowouldliketoespeciallythankDr.RosaliaCM.Simmenforhercontinuoussupportand
manyhelpfulsuggestionsthroughoutmyPh.Dstudies.IwouldliketothankDrs.William
W.Thatcher,ThomasP.YangandMichaelJ.Fieldsforalltheirhelpfuldiscussionsand
suggestions.Inaddition,IwanttothankDr.WilliamC.BuhiforsharingtissuesandDr.
DavidR.ClemmonsforcollaborativeworkonIGFBP-2cDNA.
IalsothankallmycolleaguesinDrs.FrankA.SimmenandRosaliaCM.Simmen's
groups,especiallyDrs.Chul-YoungLeeforhishelpininitiatingtheresearchproject;Drs.
LokengaBadinga,MichaelL.Green,andOmaththageP.Pereraforhelpfuldiscussions;
WernerCollante,FrankJ.Michel,andTriciaE.Chungfortheirtechnicalassistance;Karen
L.Reedfortransferringtheprimarycellculturetechnique;andDrs.InhoChoiandWenjun
Liu,InseokKwakandYangWangforsharingexperiences,ideasandfriendshipthroughout
mygraduatestudies.MyspecialthanksalsogotoMaryEllenHissem,JoyceHayenandall
othermembersofthedepartmentforthewarmandfriendlyenvironmenttheytrytocreate.
IwouldliketoexpressmyappreciationtoDr.ChangzhengWangandallthefriends
1madeinAmerica,whohelpedmetostandupinthiscountry,whichisalsoimportantfor
iii
scientificaccomplishments.Ialsowanttoexpressmyappreciationtoalltheteacherswho
contributedtomyearlyeducation.
Iwouldliketogivemydeepestappreciationtomymotherandfatherfortheirlove
andallthattheydoforme,especiallyinmyearlyage,whentheytookmyandmybrother's
educationsastheirfirstconcern.Myspecialappreciationalsogoestomybrotherforhislove,
helpandencouragement.Allmyaccomplishmentsrepresentthecontributionsfrommy
lovely,warmfamily.Ialsowanttoexpressmyappreciationtomyparents-in-lawsfortheir
loveandencouragement.
Mylovelydaughter,AnnieSong,andsonAlexanderSonghavegivenmewonderful
timeswhichhelpedreleasemefromtiredness,regainenergyandhope;thereforetheyshould
beacknowledged.
Lastly,Iwouldlikegiveveryspecialthanksanddeepestappreciationtomywife,
YufeiTang,forherlove,encouragement,support,self-sacrificeandpatience,especiallyfor
thoseyears,months,daysandnightswhenshewastakingcareofthefamilyandwaitingfor
me.Thisworkcouldnothavebeendonewithouthersupportinsomanyaspects.
TABLEOFCONTENTS
ACKNOWLEDGMENTS iii
LISTOFTABLES vii
LISTOFFIGURES viii
ABSTRACT xi
CHAPTERS
1. INTRODUCTION 1
2 LITERATUREREVIEW 4
Insulin-like-GrowthFactorsandTheirBindingProteinFamily 4
IGFBP-2GeneandPhysiology 14
UterineIGFSystem 25
3 TORHGEAUNNIIZQAUTEIOENNDOOFMTEHTERIPAORLCEIXNPERIEGSFSBIPO-N2GAENNDEGENOMIC 31
Introduction 31
MaterialsandMethods 33
Results 37
Discussion 50
Summary 56
4. IADCETNITVIAFTIICNAGTIEOGNIOOFNOOFS-TEHLEEMIEGFNBTPS-2INGETNHEEUPSTREAM 57
Introduction 57
MaterialsandMethods 59
Results 66
Discussion 84
Summary 89
v
5. ISGTFEBRPO-I2DGAENNEDTPREPATNISDCERIHPOTRIMOONNALREGULATIONOFUTERINE 91
Introduction 91
MaterialsandMethods 93
Results 98
Discussion Ill
Summary 120
6. DTHNEAPMREOTMHOYTLEARTIOOFNTOHFETPHIGEICGpFGBPI-S2LGAENNDEEANCNODMIPTASSSING
EFFECTONTRANSCRIPTIONALINITIATION 122
Introduction 122
MaterialsandMethods 123
Results 124
Discussion 131
Summary 133
7. EGXRPORWETSSHIFOANCOTFORR-EBCIONMDBIINGNAPNRTOTPEOIRNC-2IN(ErpIINGFSBUPL-I2N)-LIKE 135
Introduction 135
MaterialsandMethods 137
Results 139
Discussion 147
Summary 149
8. SUMMARYANDCONCLUSIONS 150
REFERENCES 154
BIOGRAPHICALSKETCH 177
LISTOFTABLES
Table page
3-1. Thesequencesofexon-intronboundariesandthe3'endoftheIGFBP-2gene..45
3-2. IdentityofporcineIGFBP-2aminoacidsequencewithIGFBP-2proteinsof
otherspecies 47
LISTOFFIGURES
Figure page
3-1. NorthernanalysisofIGFBP-2mRNAinpregnantpiguterus 38
3-2. StructuralorganizationoftheporcineIGFBP-2chromosomallocus 40
3-3. DNAsequenceanddeducedaminoacidsequenceofporcineIGFBP-2
chromosomalgeneandprotein 41
3-4. Deducedaminoacidsequencesofpig(P),human(H),rat(R)andmouse(M)
IGFBP-2proteins 46
3-5. Identificationof5'-transcriptionalstartsites 49
3-6. Summaryoftheresultsofthelocalizationofthe5'-endsofIGFBP-2
mRNAs 51
3-7. Conservedsequencemotifsintheregionupstreamofthetranslational
initiationcodonoftheIGFBP-2gene 52
4-1. tDoNthAeclouncsitfreruacstesrceopnotratienrigngenteheupstreamregionoftheIGFBP-2genefused 67
4-2. ReportergeneactivitiesoftheHind-LUCe,Sac-LUCe,Bgl-LUCeand
Sma-LUCeplasmidstransfectedindifferentcelllinesanduterinecelltypes...68
4-3. Transcriptionalactivationbythe110bpupstreamregion 70
4-4. MapoftheGRAprobesderivedfromthe110bpregion 72
4-5. UintdeirviindeuaelndAo.meBtrainadlCnusculbearregpiroontepirnosbbeisndthe11 bpfragment,andthe 73
4-6. Localizationofnuclearprotein-bindingsiteswithinA,BandCsubregions
ofthe110bpfragment 75
via
4-7. Gelretardationassayinvolvingnineoverlappingprobes 76
4-8. Localizationofspecificandnon-specificprotein-bindingelements
usingGRA 77
4-9. Proteinsofthesamemolecularweightbindacommonsequencewithin
theA2andB3oligonucleotides 78
4-10. CharacterizationoftheA2elementDNAbindingprotein 81
4-11. SizedeterminationoftheA2DNAbindingprotein 83
5-1. EafbfuencdtasnocfeeisntrvoigtreonaannddipnrovgievsoteroneonIGFBP-2steady-statemRNA 100
5-2. E(f-f8e7c4t/s+7o3f)esatnrdogBegnla-nLdUCpero(g-e7s6t4e/r+o7n3e)onDtNheApcroonmsottreucrtasctiinviptriiemsaorfyScaulct-uLreUsCeof
transfectedendometrialglandularepithelialcellsfromDay18pregnantpigs.101
5-3. Effectsofestrogenandprogesteroneonreportergeneactivityofthe
Sac-LUCe(-874/+73)DNAconstructinatime-courseexperiment 102
5-4. EffectsofestrogenandprogesteroneonpromoteractivityofSaB(110)-
pLUC(-874/+764)DNAconstructintransfectedDay18GEcells 103
5-5. PeptidehormonaleffectsonIGFBP-2steady-statemRNAabundancein
endometrialglandularepithelialcellsfromDay18pregnantpigs(D18GE)...105
5-6. DmoRseN-AdeapbeunnddeanntceeffectsofIGF-II,cAMPandPMAonIGFBP-2 106
5-7. EffectsofIGF-IIonpromoteractivitiesofHind-LUCe(-1397/+73),
Sac-LUCe(-874/+73),Bgl-LUCe(-764/+73)andSma-LUCe
(-305/+73)DNAconstructstransfectedintoDI8GEcells 108
5-8. EffectsofIGF-IIonreportergeneactivitiesofSaB(l10)-pLUCand
SmB(459)-pLUCDNAconstructstransfectedintoD18GEcells 109
5-9. IGF-IIreceptordensityonendometrialcellmembranes 110
5-10. DifferentialexpressionofIGFBP-2geneandproteininendometrialcells....112
5-11. SpeculativemodelfortrophicregulationoftheIGFBP-2gene 119
6-1. Ailmlmeedxioantseo5f'tfhleanIkGiFnBgPr-e2giconhrcoomnsotsiotmutaelsgaeCnpeGariesGla/nCdrichandexon1and 125
6-2. GenomicSouthernBlotAssayofmethylatedIGFBP-2DNA 127
6-3. SchematicsummaryoftheresultsofSouthenblotanalysis 129
6-4. InvitroactivityofamethylatedpromoteroftheIGFBP-2gene 130
7-1. SchematicdiagramofIGFBP-2cDNAandgenomicDNA 141
7-2. PCRamplificationofthecDNAfragmentencodingmatureIGFBP-2 142
7-3. Schemeusedforthegenerationofanexpressionconstructencodingfor
recombinantporcineIGFBP-2 143
7-4. EnrichmentofrecombinantIGFBP-2 145
7-5. RecombinantIGFBP-2bindsIGF-I 146
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