Osteogenic hormones acting on the environment of the hematopoietic stem cell niche [Elektronische Ressource] / von Anett Illing

Anett Krieger

Description

Osteogenic hormones acting on the environment of the hematopoietic stem cell niche Dissertation Zur Erlangung des akademischen Grades Doktor der Naturwissenschaften doctor rerum naturalium angefertigt am Leibniz-Institut für Altersforschung - Fritz Lipmann Institut vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät der Friedrich Schiller Universität Jena von Diplom-Biologin Anett Illing geboren am 05. März 1980 in Erlabrunn Gutachter: Professor Dr. Peter Herrlich, Leibniz-Institute for Age Research, Jena Professor Dr. Falk Weih, Leibniz-Institute for Age Research, Jena Professor Dr. Thomas Schüler, Institut für Immunologie CBF, Charité Universitätsmedizin, Berlin Tag der Verteidigung: 04. Mai 2009 - 2 - ACKNOWLEDGEMENT First of all I would like to thank Dr. Jan Peter Tuckermann, for giving me the great oppurtunity to work on this challenging project and for supporting me in every step of this thesis. He was always helping me with fruitful discussions, important instructions, developing new ideas and motivating esprit – I could not have been in a better place for my PhD. I would like to thank Prof. Dr. Peter Herrlich as member of my thesis comittee and as my official supervisor for helpful discussions und ongoing support during these four years. In this regard I thank also Prof. Dr.

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Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2009
• Nombre de lectures: 32
• Langue: English
• Poids de l'ouvrage: 37 Mo

Extrait

Osteogenic hormones acting on the
environment of the hematopoietic stem cell
niche
Dissertation

Zur Erlangung des akademischen Grades
Doktor der Naturwissenschaften
doctor rerum naturalium
angefertigt am
Leibniz-Institut fr Altersforschung - Fritz Lipmann Institut
vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultt
der Friedrich Schiller Universitt Jena
von Diplom-Biologin Anett Illing
geboren am 05. Mrz 1980 in Erlabrunn

Gutachter:
Professor Dr. Peter Herrlich, Leibniz-Institute for Age Research, Jena
Professor Dr. Falk Weih, Leibniz-Institute for Age Research, Jena
Professor Dr. Thomas Schler, Institut fr Immunologie CBF, Charit
Universittsmedizin, Berlin
Tag der Verteidigung: 04. Mai 2009

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ACKNOWLEDGEMENT
First of all I would like to thank Dr. Jan Peter Tuckermann, for giving me the great
oppurtunity to work on this challenging project and for supporting me in every step of
this thesis. He was always helping me with fruitful discussions, important instructions,
developing new ideas and motivating esprit Ð I could not have been in a better place
for my PhD.
I would like to thank Prof. Dr. Peter Herrlich as member of my thesis comittee and as
my official supervisor for helpful discussions und ongoing support during these four
.earsyIn this regard I thank also Prof. Dr. Falk Weih as a member of my PhD-committee for
supporting discussions and good ideas for the ongoing project. Last but not least I
would like to thank Dr. Helen Morrison for participating in my PhD-committee, helping
with developing new strategies and giving adjuvant advice for the thesis, but also as
a good friend, from whom you could learn how to survive in science.
Furthermore I am really grateful for the help and support of my patient boyfriend
Ralph Gruber. He was always trying to help and to pull me up in hard times. He
knows how it is to go for a PhD and always felt with me.
Not to forget the best colleagues and friends a PhD-student can have Ð Uli Merkel,
Susanne Ostermay and Alexander Rauch Ð thank you for all your patient help and a
very nice working and private atmosphere.
Thanks to the complete Tuckermann, Morrison and Herrlich - laboratories for being
great colleagues and for fruitful discussions during these four years and special
thanks to Dr. Anna Kleyman who was a great teacher in the first time.
Finally I would like to thank my family Ð without them I would not be where I am.

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Meiner Familie gewidmet

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Table of contents

1 ABSTRACT / ZUSAMMENFASSUNG 8
1.1 Abstract 8
1.2 Zusammenfassung 9
2 INTRODUCTION 10
2.1 Adult stem cells in tissue homeostasis 10
2.2 Hematopoietic stem cells (HSCs) in hematopoiesis 10
2.2.2.2.21 HProSCpe rtdiviesisi ofon the HSC 1112
2.2.3 Intrinsic HSC regulation 13
2.3 The HSC niche 15
2.2.3.3.12 TThhe e evasndcosutlar eal HHSSC C nicnichehe 1618
2.4 Osteogenic hormones presumably affecting the endosteal HSC niche 21
2.2.4.4.12 GEsHt rogin ben onin e bmetone abometlisam bolism 2123
2.5 Aims of this study 26
3 RESULTS 27
3.1 Effects of estrogens on the HSC niche 27
3.1.1 Long-term treatment of mice with 17-β-E2 increases the bone mass but not
the bone-adhered HSCs 27
3.1.2 Long-term treatment of mice with 17-β-E2 leads to an increase in vascular
HSCs 31
3.1.3 17-β-E2 increases the multipotent long-term repopulating HSCs 33
3.1.4 The role of ERα is dispensable for 17-β-E2-induced increase of HSC-
numbers
3.1.5 Absence of ERβ does not impair the 17-β-E2-induced increase of HSC-
numbers
3.1.6 17-β-E2 treatment affects the niche cells and not the HSCs directly 39
3.1.7 17-β-E2 leads to lower numbers of HSCs in the peripheral blood 41
3.1.8 17-β-E2 regulates the mRNA levels of different adhesion molecules in HSC-
supporting FBMD1 cells 42
3.2 GH signaling in OBs increases HSC numbers and is modulated by STAT5 45
3.2.1 GH increases the number of HSCs in the vascular and endosteal niche of
wildtype mice 45
3.3.2.2.23 SSTTAATT55 -kplaynocs koan uti mOBsport incant rerolase e tin he OBcsap aacnd ityt hof eir HiSntCserac to tiforon mw itcoh bbHSlesCtso ne 47
colonies 48
3.2.4 Activated STAT1 and STAT3 can compensate for the lack of STAT5 in OBs 49
4 DISCUSSION 52

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Table of contents

4.1 4.1.Eff1 ectLons go-f te17rm- βt-reE2at omn entth e of HwSilCdt ynipe chme ice with 17-β-E2 leads to an increase of 52
bone mass but not to an increase of bone-adhered HSCs 52
H4.S1.C2s in Lonthge -tverasmc tulrear atnicmenthe ofof tmhice Be Mw ith 17-β-E2 leads to an increase in competent 53
4.1.3 Is the ERα or the ERβ the mediating molecule for the HSC number increase
4.und1.4er 1177--ββ--EE22 ttrreateatmmeentnt? of mice affects the environmental niche cells and not the 53
HSCs directly 55
F4.B1.M5 D1 17c-eβ-llsE 2 regulates the mRNA levels of different adhesion molecules in 57
4.me2 chGaHni selme vates HSC numbers in the endosteal niche via a STAT5-dependent 59
4.2.1 GH increases HSC numbers in the vascular and endosteal niche of wildtype
mice 59
4.4.2.2.32 SAcTtAivTat5 eisd iSmTpATort1 anta ind n SOTABsT 3 ancd tan heicor mintpeernsacattioe n fowr itth he HSlacCks of STAT5 in OBs 6062

5 MATERIALS AND METHODS 64
5.1 Materials 64
5.5.1.1.21 CMheatmeriicalsals 6464
5.5.1.1.43 MBufedifersa f aor nd celsl oclutultiuronse 6766
5.1.5 Primers for genotyping 68
5.5.1.1.76 WPriesmterersn fblor otr ealant-tiibmode PiesC R 6969
5.5.1.1.98 IFnvACestS aigatntibed odiknesoc kout mice 7070
5.2 Methods 71
5.5.2.2.12 IPsColatRsio fn or ofg DenNotA yfpinrogm mouse tail biopsy for genotyping 7171
5.5.2.2.43 DRiNgesA tisiolon atofio Dn fNroA mi n pRriNmA arys acmellspl esa nd cell lines 7676
5.2.5 Determining the quantity and quality of isolated RNA 76
5.2.6 cDNA synthesis from RNA samples using reverse transcription 77
5.2.7 cDNA check using β-actin PCR 78
5.5.2.2.98 RSelealec-ttiiomn e Pof CpriR mers for real-time PCR 7978
5.5.2.2.1110 MMicagronetarric ays ortan ofaly cselis l ofpo FpulBMatDio1 nsc wellsit h afttehe r 17aut-βo-ME2A tCSreat ment 7980
5.5.2.2.1312 SFADSC-S PdAGue E to acnd ell Wsesurftacern e mblotol aecunalyless is 8080
5.2.14 Isolation of primary OBs 81
5.5.2.2.1615 CTrulteaturme ofent stofro primaml caryell liOBsne FwBitMh DG1H 8282
5.2.17 The CAFC assay 82
5.5.2.2.1918 ILsDolatA Ðio in n ofv ivvo asaculnalyar sais nd via eBndMos ttraeal nsHplSantCsat ion into lethally-irradiated mice 8383
5.5.2.2.2120 ÔHEsotmablinisg hasmsentay Õ ofw citoh mCFproSEm-lisabed elBed M BcelM lsc fellsor in vivo LDA 8584

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5.2.22 Von Kossa - staining
5.2.23 Animal breeding and husbandry
5.2.24 Applications on mice

6 REFERENCES

7 ABBREVIATIONS

8 TABLE OF FIGURES

9 SELBSTNDIGKEITSERKLRUNG

10

11

LEBENSLAUF

POSTER, VORTRGE, VERFFENTLICHUNGEN

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Table of contents

85 86 86

87

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Abstract / Zusammenfassung

1 Abstract / Zusammenfassung
Abstract 1.1 Hematopoietic stem cells (HSCs) are the most widely studied adult stem cells in
vertebrates. Despite this, little is known about the regulation of HSC maintenance in
their specialized microenvironment.
The aim of this thesis was to study the influence of osteogenic hormones on the
regulation of HSCs. Thereby we could prove that, although estradiol increases
osteoblastic cell numbers and bone mass, it does not have any advantageous effects
on the endosteal HSC niche. Surprisingly, estradiol displayed alterations in the
microenvironment of the vascular niche, by upregulating distinct adhesion molecules
and thereby correlating with an increase of HSCs in the vascular niche. Therefore,
we suggest an enhanced retention of HSCs in the vascular niche under the influence
of estradiol, also proven by a decrease of HSCs in the peripheral blood.
Furthermore, we investigated the effects of long-term growth hormone (GH)
administration, which is known to increase bone mineral density and thereby
influence the endosteal HSC microenvironment. We clearly showed increased HSC
numbers in the vascular and the endosteal niche of wildtype mice after GH
administration. Additionally, we proposed a Janus kinases/Signal Transducers and
Activators of Transcription (Jak/STAT)-signaling-dependent mechanism of GH in the
endosteal niche. To test this hypothesis, we investigated the influences of GH in a
conditionally-mutated mouse model (STAT5OB), where Jak/STAT signaling is
disrupted in osteoblasts by the loss of STAT5. Unexpectedly, these mice showed
increased numbers of HSCs in the endosteal niche and displayed strikingly
enhanced endosteal HSC numbers after GH treatment compared to wildtype
controls. Loss of STAT5 in osteoblasts led to strong activation of STAT3 and, in
particular, STAT1, suggesting a