Regulation of uterine endometrial gene expression and growth by the Sp/Kruppel-like family member, basic transcription element binding protein 1

200 Pages·2002·7.2 MB·English

by  Zhang, Daying

REGULATIONOFUTERINEENDOMETRIALGENEEXPRESSIONAND
GROWTHBYTHESP/KRUPPEL-LIKEFAMILYMEMBER,BASIC
TRANSCRIPTIONELEMENTBINDINGPROTEINI
By
DAYINGZHANG
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
2002
Thisdissertationisdedicatedtomyparentswithdeepestappreciationandtomyhusband
withloveandthanksfrommyheart.
ACKNOWLEDGMENTS
Iamdeeplygratefultomyadvisors.Dr.FrankA.SimmenandDr.RosaliaCM.
Simmen,forprovidingthehappiestandmostproductiveyearsofmyscientificcareer,for
introducingmetotheworldofmolecularandcellbiology,fortheirinvaluableadvice,
guidanceandencouragementinresearch,andfortheintellectualandscientifictrainingI
receivedintheirlaboratorieswhichprovidedmewithasolidbaseofscientificskills.
Memoriesoftheseyearswillremainwithmeforalifetime.
IwouldalsoliketoextendmydeepestthankstoDr.WilliamC.BuhiandDr.
ThomasP.Yangforservingasmembersofmycommittee,aswellasfortheirsupport,
suggestions, discussions and important insights into experimental design and
methodologicalissues,allofwhichhelpedensurethesoundnessofthisstudy.
IwouldliketogivespecialthankstoDr.InseokKwak,whoprovidedanexample
ofadevotedscientist,sharinghisexperiencesandknowledgeintheareaofmolecularand
cellbiology,andteachingmethebasictechniquesinthisfield.Igivesincerethanksto
Dr.Xue-LianZhang,whosharedherexperiences,knowledgeandideaswithmeandgave
meeverykindofhelpandsupportduringthisstudy.Mr.JasonBlumisappreciatedfor
hisassistancewithcomputeranalysis,effortstowardsimprovingmywrittenEnglish,and
hishelpwiththegenerallaboratorywork.IwouldalsoliketothankMr.FrankJ.Michel
andMr.GeZhaofortheirexcellenttechnicalassistance,especiallyfortheirhelpinthe
differential display-reverse transcriptase-polymerase chain reaction experiments. Dr.
iii
LokengaBadingaisalsoappreciatedforintroducingmetocellculturetechniques.Ialso
wouldliketothankmanyoftheformerandallofthecurrentmembersofbothSimmen
laboratories, especially Dr. Andres Kowalski, Dr. Seok-Hong Min, Miss Jennelle R.
McQuown, and Dr. Marcela von Reitzenstein for their friendship and sharing their
wisdom.
Iwanttoacknowledgemydeepestappreciationtomyfatherandmotherfortheir
endless love, for providing an academic environment and for their guidance in my
lifetime.Mostofall,Iwishtoexpressmydeepestappreciationandlovetomyhusband,
FuwangChen,forhisincrediblesupport,encouragement,aswellashislove.Iwouldalso
liketothankmyunbornbabyforprovidingtheincredibleexcitement,joyandhis/her
understandingduringtheprocessofwritingthisdissertation.
iv
TABLEOFCONTENTS
page
ACKNOWLEDGMENTS Hi
LISTOFFIGURES vii
ABSTRACT x
CHAPTERS
1 INTRODUCTION 1
2 REVIEWOFLITERATURE 6
Sp/Kriippel-likeFamilyofTranscriptionFactors 6
SteroidHormoneReceptors:ProgesteroneReceptor(PRs)andEstrogenReceptor
(ERs) 17
Implantation 32
UterineProteasesandTheirInhibitors 37
Summary 42
3 STEROIDHORMONEREGULATIONOFSplANDBTEBlEXPRESSION
INUTERINEENDOMETRL\LEPITHELIALCELLS 44
Introduction 44
MaterialsandMethods 46
Results 52
Discussion 63
4 INTERACTIONBETWEENBTEBlANDPROGESTERONERECEPTOR
MEDIATESPROGESTERONE-RESPONSIVEGENEEXPRESSIONIN
ENDOMETRL\LEPITHELIALCELLS 69
Introduction 69
MaterialsandMethods 72
Results 74
Discussion 87
V
5 APREGNANCY-ASSOCIATED,BTEBlDOWNSTREAMGENEPRODUCT,
SECRETORYLEUKOCYTEPROTEASEINHIBITOR,MEDIATES
PROLIFERATIONOFHUMANENDOMETRIALGLANDULAR
EPITHELIALCELLSBYPOSITIVEANDNEGATIVEREGULATIONOF
GROWTH-ASSOCIATEDGENES 98
Introduction 98
MaterialsandMethods 101
Results 108
Discussion 121
6 SUMMARYANDCONCLUSIONS 147
REFERENCES 156
BIOGRAPHICALSKETCH 188
vi
LISTOFFIGURES
Figure ^ eage
3-1. OptimizationofPGRconditions 50
3-2. EffectofestrogenonPRmRNAabundanceinGEcells 54
3-3. EffectofprogesteroneonUFmRNAabundanceinGEcells 57
3-4. EstrogenandprogesteroneeffectsonendometrialGEcellBTEBandSplmRNA
levels 59
3-5. BTEBlandSplmRNAlevelsinuterineendometrialGEcellsasafunctionofday
ofpregnancy 62
3-6. EstrogenandprogesteroneeffectsonBTEBlandSplproteinlevelsinDIO
pregnantpiguterineendometrialGEcells 65
4-1. Porcineuteroferrin(UF)promoter(-1143)5'flankingregion 77
4-2. PorcineIGFBP-2promoter(-1308)5'flankingregion 79
4-3. EffectofBTEBlonthetransactivationofUFgenepromoterbyPR-Binthe
presenceandabsenceofR5020 81
4-4. EffectofBTEBlonthetransactivationofIGFBP-2genepromoter-1397construct
byPR-BinthepresenceandabsenceofR5020 83
4-5. EffectofBTEBlonthetransactivationofIGFBP-2genepromoter-305construct
byPR-BinthepresenceandabsenceofR5020 84
4-6. EffectofBTEBlonPR-B/P-mediatedtransactivationoftheMMTV-LTRgene
promoter 86
4-7. EffectofBTEBlonPR-AandPR-BmediatedtransactivationoftheUFgene
promoterinthepresenceorabsenceofP 88
4-8. EffectofBTEBlandBTEB3onPR-B/P-mediatedtransactivationoftheUFgene
promoter 89
vn
1
4-9. ModelforBTEBl,PR-AandPR-BinteractionswithinaP-responsivegene
promoter 97
5-1. RegulationofSLPIgeneexpressionbyBTEBl 11
5-2. ExpressionofSLPIinstablytransfectedhumanIshikawaclonallines 114
5-3. DistinctgrowthparametersoftheIshikawasublinesaredependentoncellularSLPI
content 117
5-4. Expressionofcellcycle-associatedgenesinparentalIshikawaandderivedclonal
lineswithdistinctSLPIexpression 123
5-5. AbundanceofcyclinDlproteinsincontrol(13pIND)andSLPI-deficient(8As)
sublines 125
5-6. AbundanceofPCNAproteinsincontrol(13pIND)andSLPI-deficient(8As)
sublines 126
5-7. Differentialexpressionofgrowth-associatedgenesinstablytransfectedIshikawa
sublines 127
5-8. A-16transcriptsequence 128
5-9. ThesequencecomparisonbetweenA-16cloneandhumanlysyloxidasegenepartial
cdsand3'UTR 129
5-10. A-10transcriptsequence 130
5-11. DifferentabundanceofIGFBP-3proteininstablytransfectedIshikawasublinesl31
5-12. EffectsofexogenouslyaddedrhSLPIongeneexpressionbycontrolandSLPI-
deficientclonallines 133
5-13. EffectsofendogenousSLPIongeneexpressionbySLPI-deficientclonallines..135
5-14. EffectofSLPIoncyclinDlpromoteractivity 136
5-15. EffectsofTGF-pionIshikawacellgeneexpression(NorthernImage) 137
5-16. EffectsofTGF-pionIshikawacellgeneexpression(numericalanalysis) 139
5-17. ModelfortheregulationofendometrialepithelialcellproliferationbyBTEBl
throughtheSLPIpathway 146
vni
AmodelfortheroleofBTEBlinuterineendometrialepithelialcelldifferentiation
andproHferation 155
IX
AbstractofDissertationPresentedtotheGraduateSchool
oftheUniversityofFloridainPartialFulfillmentofthe
RequirementsfortheDegreeofDoctorofPhilosophy
REGULATIONOFUTERINEENDOMETRIALGENEEXPRESSIONAND
GROWTHBYTHESP/KRUPPEL-LIKEFAMILYMEMBER,BASIC
TRANSCRIPTIONELEMENTBINDINGPROTEIN1
By
DayingZhang
August2002
Chair: Dr.FrankA.Simmen
Department: AnimalSciences
Basictranscriptionelementbindingprotein(BTEBl)isatranscriptionfactorthat
regulatesgeneexpressionthroughinteractionswithregulatoryDNAelementsoftarget
genes. This dissertation examined the molecular mechanisms underlying pregnancy-
associatedgeneexpression,andtheconsequenteffectsonuterineendometrialepithelial
cellproliferationanddifferentiationmediatedbyBTEBl.
Usingthepregnantpigasananimalmodel,mRNAandproteinlevelsofBTEBl
anditswell-knownKriippel-likefamilymember,Spl,wereexaminedinprimarycultures
ofuterineendometrialglandularepithelialcellstreatedwithestrogenandprogesterone.
TheresultsshowedthatwhiletheproteinabundanceofbothBTEBl andSpl werenot
affected by either steroid hormone, their mRNA levels were modestly increased by
estrogeninglandularepithelialcellsisolatedfromuterineendometriumofanimalswith
lowerestrogenbackground.
X