Mitogen-activated protein kinases function in arthritis [Elektronische Ressource] = Die Funktion von Mitogen-aktivierten Proteinkinasen in der Arthritis / vorgelegt von Christina Böhm

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Publié le : samedi 1 janvier 2011
Lecture(s) : 25
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Source : D-NB.INFO/1012700224/34
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Mitogen-activated Protein Kinases Function in Arthritis
Die Funktion von Mitogen-aktivierten Proteinkinasen in der Arthritis



Der Naturwissenschaftlichen Fakultät
der Friedrich-Alexander-Universität Erlangen-Nürnberg
zur
Erlangung des Doktorgrades Dr.rer.nat.













vorgelegt von
Christina Böhm
aus Erlangen



Als Dissertation genehmigt von der Naturwissenschaftlichen Fakultät der
Friedrich-Alexander-Universität Erlangen-Nürnberg






















Tag der mündlichen Prüfung: 19.05.2011


Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink

Erstberichterstatter: Prof. Dr. Falk Nimmerjahn

Zweitberichterstatter: PD. Dr. Jochen Zwerina


2 This work was performed at the Department of Internal Medicine 3
Rheumatology and Immunology, University Erlangen-Nuremberg


























3 Table of Contents
Table of Contents
Abstract 8
Zusammenfassung (german summary) 10

1. Introduction 13
1.1 Bone 13
1.1.1 Development of bone 13
1.1.2 Bone cells 15
1.1.3 Bone remodeling 21
1.2 Rheumatoid Arthritis 23
1.2.1 TNF signaling 25
1.2.2 Mitogen-activated protein kinases (MAPKs) 26
1.2.3 MAPKs in Rheumatoid Arthritis 27
1.2.4 p38 MAPK in Rheumatoid Arthritis 27
1.2.5 The p90 ribosomal S6 kinase (Rsk) 28
1.2.6 Rsk2 and Rheumatoid Arthritis 30
1.3 Aim of the Work 31
2. Material and Methods 32
2.1 Materials 32
2.2 Methods 37
3. Results 48
Part I 48
3.1 p38α function in TNF-induced arthritis 48
3.1.1 Generation and characterization of p38α deleted mice 48
3.1.1.1 p38α is predominantly expressed in the monocytic lineage
and is activated by TNF-α 48
3.1.1.2 Efficient inducible Mx-cre-mediated deletion of p38α 49
3.1.2 Deletion of p38α improves clinical outcome of arthritis 50
3.1.2.1 Clinical assessment of mice exhibits less signs of arthritis 50
3.1.2.2 Absence of p38α protects from systemic bone loss 52
3.1.2.3 p38α cell autonomously regulates osteoclastogenesis 54
3.1.3 Deletion of p38α does not change activation of other MAPKs
but increases activity of NF-κB 54
3.1.4 Decreased IL-1ß expression protects mice from inflammatory
bone loss 56
4 Table of Contents
Part II 58
3.2 p90 ribosomal S6 kinase 2 (Rsk2) function in TNF-induced
arthritis 58
3.2.1 Absence of Rsk2 exacerbates inflammatory cartilage and
bone destruction 58
3.2.1.1 Absence of Rsk2 in the hTNFtg mouse impairs survival and
aggravates clinical outcome of arthritis 58
3.2.1.2 Increased TNF-induced local bone loss in the absence of Rsk2 59
-/y3.2.1.3 Enhanced inflammation in hTNFtg;Rsk2 mice correlates with
higher cytokine and matrix metalloproteinase production 61
-/y3.2.1.4 Transfer of Rsk2 bone marrow does not
exacerbate TNF-mediated arthritis 64
-/y
3.2.1.5 Rsk2 mice are more susceptible to antigen-induced arthritis 65
3.3. Role for Rsk2 in the regulation of bone turnover by TNF-α 67
-/y 3.3.1 Decreased systemic bone mass in hTNFtg;Rsk2 mice 67
-/y 3.3.2 hTNFtg;Rsk2 mice have less osteoblasts and more osteoclasts 70
3.3.3 Absence of Rsk2 does not affect osteoclastogenesis in vitro 72
3.3.4 Defect in osteoblast differentiation in vitro 74
3.3.5 Osteoblast and osteocyte markers are decreased in long bones
-/y
of hTNFtg;Rsk2 mice 75
-/y3.3.6 Osteocyte death in hTNFtg;Rsk2 mice 77
-/y3.3.7 In vivo increased apoptosis of osteoblasts of hTNFtg;Rsk2 mice 79
-/y3.3.8 Rsk2 osteoblasts are more susceptible to TNF-induced apoptosis 80
4. Discussion 83
Part I 83
4.1 The p38 MAPK isoform p38α regulates inflammatory bone loss 83
4.2 p38α directly regulates osteoclastogenesis 85
4.3 Inflammatory bone loss is regulated by p38α mediated
IL-1ß expression 87
4.4 Concluding Remarks Part I 88
Part II 89
-/y4.5 hTNFtg;Rsk2 mice develop severe arthritis 90
4.6 Rsk2 controls inflammatory bone loss 93
4.7 Concluding Remarks Part II 97

5 Table of Contents
5. Abbrevations 98
6. Acknowledgements 101
7. References 102
Curriculum Vitae 121



























6 Table of Contents




























7 Abstract
Abstract
The objective of this PhD thesis was to investigate the role of two different kinases,
the mitogen-activated protein kinase (MAPK) p38 and the extracellular-signal-
regulated kinase (ERK) 1/2 activated kinase p90 ribosomal S6 kinase (Rsk2) in
Tumour necrosis factor (TNF)-mediated inflammatory bone loss.
We first used human TNF transgenic (hTNFtg) mice crossed with floxed p38α mice to
overcome the embryonic lethality of the full knock out of p38α. We used Poly (I:C)-
induced Mx-Cre-mediated conditional deletion of p38α to timely control p38α
expression. Having first confirmed that p38α is the main p38 isoenzyme expressed in
the osteoclast precursors and in the mature cells, we demonstrated that mice lacking
p38α in hematopoietic cells were protected against TNF-induced bone destruction at
the site of inflammation and against TNF-mediated systemic bone loss. The
protection was due to decreased osteoclast numbers in vivo. The phenotype was cell
autonomous since absence of p38α in monocytes resulted in a decreased RANKL-
induced osteoclast differentiation in vitro. Osteoclast differentiation was not
completely blocked in absence of p38α suggesting a partly compensation by other
important osteoclastogenic factors. Indeed, RANKL-mediated activation of the other
MAPKs was not altered and degradation of IκBα, the inhibitor of NF-κB was
increased in absence of p38α resulting in an increased activation of NF-κB. The
protection of p38α deficient mice from inflammatory bone loss was Interleukin (IL)-1ß
dependent since IL-1ß expression in vivo and in in vitro culture of monocytes was
decreased in the absence of p38α. In summary, this work indicates a key role for the
p38α isoform in RANKL- and TNF-mediated IL-1ß expression thereby playing a major
role in the regulation of inflammatory bone loss observed in arthritis.

-/y
In a similar approach, we generated hTNFtg mice lacking Rsk2 (hTNFtg;Rsk2
mice). A drastic exacerbation and acceleration of arthritis symptoms accompanied by
early mortality was observed in these mice. The increased inflammatory processes
were promoted by higher secretion of inflammatory cytokines and expression of
matrix metalloproteinases compared to hTNFtg control mice. This resulted in a very
early onset of inflammatory cartilage and bone destruction at the site of inflammation
as well as in a drastic systemic osteopenia. Osteopenia was caused by a lack of
functional osteoblasts, the bone forming cells and an enhanced recruitment of
osteoclasts, the bone resorbing cells leading to an uncoupling of normal bone
8 Abstract
remodeling. Bone formation and mineralization property of osteoblasts from
-/yhTNFtg;Rsk2 mice was strongly decreased in vivo and in vitro whereas no cell
intrinsic defect in osteoclast differentiation was observed in vitro. The decreased
numbers of osteoblasts was caused by an enhanced susceptibility of the osteoblastic
lineage to TNF-mediated apoptosis in vitro and in vivo. This resulted in an increased
in vivo apoptosis of osteocytes, the bone cells which represent the late stage of
-/y
osteoblast differentiation. Apoptosis in hTNFtg;Rsk2 mice was linked to decreased
phosphorylation of Bcl-2-associated death promoter (BAD), a known substrate of
Rsk2, which caused cell death of osteoblasts and osteocytes. Thus the enhanced
TNF-induced bone loss in mice lacking Rsk2 is likely mediated by the combined
decreased bone formation due to apoptosis of osteoblasts and by increased
osteoclast numbers in response to the elevated pro-inflammatory cytokine
environment triggering osteoclast differentiation as well as by increased osteocyte
death, which is believed to mediate osteoclast recruitment. In summary, Rsk2, a
kinase activated in osteoblasts by TNF-α, exerts a negative auto-regulatory loop
against TNF-induced inflammmatory bone loss.

















9 Zusammenfassung
Zusammenfassung
Ziel dieser Arbeit war es die Rolle zweier verschiedener Kinasen und zwar die der
Mitogen-aktivierten Protein Kinase (MAPK) p38 und der durch die extrazellulär
Signal-regulierte Kinase (ERK)1/2 aktivierten p90 ribosomalen S6 Kinase (Rsk2) in
Zusammenhang mit dem Tumor Nekrose Faktor (TNF)-vermittelten
Knochenverlustes zu untersuchen.
Für das erste Projekt wurden Mäuse, die die gefloxten p38α Allele tragen mit der
humanen Tumor Nekrose Faktor transgenen (hTNFtg) Maus gekreuzt. Die Deletion
von p38α wurde durch die Kreuzung mit Mäusen, welche die durch Poly(I:C)
induzierbare Mx-Cre Rekombinase exprimieren, bewerkstelligt. Es wurden gefloxte
p38α Mäuse verwendet, da die komplette Deletion von p38 embryonal lethal ist.
Vorhergehende Untersuchungen konnten bestätigen das p38α die am höchsten
exprimierte Isoform in Osteoklasten Vorläuferzellen und reifen Osteoklasten ist.
Mäuse, in denen p38α deletiert ist, sind vor TNF-α vermittelten Knochenerosionen an
den entzündeten Gelenken sowie vor TNF-α induzierten systemischen
Knochenverlust geschützt. Wir konnten weiterhin zeigen, dass dieser Schutz vor
Knochenabbau aus einer verringerten Präsenz an Knochen resorbierenden
Osteoklasten resultiert. Der protektive Effekt wurde nicht von äußeren Einflüssen
hervorgerufen, sondern durch ein vermindertes Differenzierungsvermögen von
Osteoklasten Vorläuferzellen zu reifen Osteoklasten. Dies konnten wir in vitro in
Abwesenheit von p38α nachweisen. Da die Osteoklastogenese nicht komplett
verhindert wurde, war davon auszugehen das andere Osteoklastogenese fördernde
Faktoren nicht von der p38α Deletion betroffen waren. Die Aktivierung anderer
MAPKs war in der Tat nicht verändert. Interessanterweise wurde aber eine erhöhte
Degradation von IκBα, welche zur Aktivierung des Transkriptionsfaktors NF-κB führt,
in Osteoklasten Vorläuferzellen festgestellt. Aufgrund dessen konnte keine komplette
Inhibierung der Osteoklasten Differenzierung gefunden werden.
Da die p38α- defizienten Mäuse vor inflammatorischen Knochenverlust geschützt
sind, haben wir untersucht ob andere Einflüsse, wie sie durch pro-inflammatorische
Zytokine in der Arthritis gegeben sind, in diesen Mäusen abgeschwächt sind. Die
Expression des Osteoklasten fördernden Zytokins Interleukin (IL)-1ß war sowohl in
vivo als auch in vitro kultivierten Monozyten signifikant gesenkt. Diese Arbeit zeigt,
dass die p38α Isoform eine wichtige Rolle in der RANKL- und TNF-vermittelten IL-1ß
10

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