role and regulation of fra-2 during skeletal muscle development nezeka alli a dissertation ...

240 Pages·2014·3.7 MB·English

ROLE AND REGULATION OF FRA-2 DURING SKELETAL MUSCLE
DEVELOPMENT

NEZEKA ALLI

A DISSERTATION SUBMITTED TO
THE FACULTY OF GRADUATE STUDIES
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

GRADUATE PROGRAM IN BIOLOGY
YORK UNIVERSITY
TORONTO, ONTARIO

JUNE 2014

© Nezeka Alli, 2014
Abstract
Regulation of skeletal muscle development and regeneration is critical to all metazoans
and also of clinical relevance as muscle wasting is manifested in a variety of disorders. The
events contributing to development and regeneration of skeletal muscle are primarily controlled
by members of the myogenic regulatory factors (MRF) and myocyte enhancer factor 2 (MEF2)
transcription factor families. Secondary factors also exert effects on myogenesis such as the
activator protein 1 (AP-1) transcription factor which has a complex role in the differentiation
process. The AP-1 subunit Fra-2 has a role in skeletal muscle development and regeneration but
it is less defined. Here, the role of Fra-2 in skeletal myogenesis was investigated thereby
extending the study of AP-1 in muscle. It was determined that Fra-2 is regulated by the ERK 1/2
MAPK pathway via phosphorylation at S320 which is important for Fra-2 protein stability. Gain
of function studies exploiting stability of Fra-2 achieved by phosphomimetic mutations impacted
differentiation negatively. Conversely, loss of function using siRNAs resulted in precocious
differentiation suggesting an overall inhibitory role for Fra-2 in myogenic cells. Intriguingly, it
was observed that AP-1 is differentially expressed in a differentiated culture of C2C12 myogenic
cells in that Fra-2 expression is restricted to monomucleated reserve cells and not in the
differentiated myotubes. Furthermore, it was determined that Fra-2 is expressed in Pax7 positive
satellite cells in a single muscle fibre culture model and that it binds to the promoter of the
mustn1 gene which, in turn, is also a novel satellite cell marker. In conclusion, Fra-2 protein
stability is regulated by phosphorylation of ERK 1/2 in myogenic cells and its expression in
quiescent reserve cells and in satellite cells suggests a possible role for Fra-2 in maintaining the
undifferentiated state in myogenic progenitor cells.
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Acknowledgements
I would like to express my appreciation and gratitude to Dr. J.C. McDermott for the
opportunity to do research in his lab. I’ve learned many valuable skills during my graduate
studies, both at and away from the bench.
I’ve enjoyed working with all members of Dr. McDermott’s laboratory. I would
especially like to thank Dr. Tetsuaki Miyake and Dr. Arif Aziz for being my mentors, when I
was an undergraduate in Dr. McDermott laboratory, and for their guidance through much of my
graduate studies.
Finally, I would like to express my gratitude to my parents and my husband for their
continued support and encouragement throughout my graduate studies.
Chapter III: Cardiotrophin-1 maintains the undifferentiated state in skeletal
muscle development

We thank Joseph Chan for technical assistance. We also thank Dr. Robert L. Perry for providing
MyHC, Myogenin, and Myc antibodies and valuable suggestions. These studies were made possible by grants
from the Natural Sciences and Engineering Research Council (NSERC) of Canada and the Canadian Institutes
of Health Research (CIHR) to J.C.M.
Chapter IV: Signal dependent Fra-2 regulation in skeletal
muscle reserve and satellite
We thank S.J. Tapscott for sharing ChIP-seq data related to AP-1 binding sites in MyoD target
genes. This work was supported by a grant from the Natural Sciences and Engineering Research Council
(NSERC) of Canada to J.C. McDermott.
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Table of Contents

Abstract ………………………………………………………………………………..ii
Acknowledgements ………………………………………………………………...…iii
Table of contents ………………………………………………………………..…….iv
List of figures ………………………………………………………………………….vi
Abbreviations …………………………………………………………………….….viii

Chapter 1: Review of literature
Embryonic skeletal muscle development
1.1 Somitogensis and the development of skeletal muscle ....................................2
1.2 Limb development in the embryo .....................................................................5

Molecular regulation of skeletal muscle development
2.1 Specification by Pax3 and Pax7 ........................................................................8
2.2 Determination and differentiation by the myogenic regulatory factors .......9
2.3 Transcriptional control of MyoD ...................................................................12
2.4 Signaling mechanisms in myogenesis .............................................................13

Skeletal muscle regeneration
3.1 Characterization in identity of satellite cells .................................................24
3.2 Molecular regulation of satellite cells ............................................................26
3.3 Signaling mechanism in satellite cells ............................................................29
3.4 Non-skeletal muscle cell types with regenerative potential ..........................31

Models used to investigate skeletal myogenesis
4.1 The C2C12 cell line ..........................................................................................33
4.2 Primary single fibre cultures ..........................................................................33

Activator Protein-1 transcription factor complex
5.1 Structural, physical and biological properties .............................................35
5.2 The Jun and Fos family of transcription factors ..........................................38
5.3 JunB ..................................................................................................................39
5.4 JunD ..................................................................................................................39
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5.5 FosB ...................................................................................................................40
5.6 Fos like antigen-1 .............................................................................................40
5.7 Fos like antigen-2 .............................................................................................41
5.8 Regulation by MAPK ......................................................................................42
5.5 Activator Protein-1 and Myogenesis ..............................................................43

Mitogen Activated Protein kinase pathways
6.1 Mechanism of signal transduction ..................................................................47

Cytokine and growth factor signaling in skeletal muscle differentiation
7.1 Insulin, Insulin like growth factors, basic fibroblast growth factor and
transforming growth factor-beta ....................................................................50
7.2 Interleukin-6 family of cytokines ....................................................................51
7.3 JAK-STAT signaling .......................................................................................52
7.4 Cardiottrophin-1 ..............................................................................................56
7.5 Cardiotrophin-1 and myogenesis ...................................................................56

Chapter II: Statement of Purpose ...........................................................................58

Chapter III: Cardiotrophin-1 maintains the undifferentiated
state in skeletal muscle development ..................................................61

Chapter IV: Signal dependent Fra-2 regulation in skeletal
muscle reserve and satellite ..............................................................109

Chapter V: Summary and Conclusions ...............................................................159

References ...................................................................................................................166

Appendices

Appendix A: Mustn1 as a novel AP-1 target gene in skeletal
muscle cells .........................................................................................201

Appendix B: Methods ...............................................................................................214

v
List of Figures
Chapter I: Review of Literature
Figure 1 Compartmentalization of somites during somitogenesis ..............................3
Figure 2 Signaling mediating myotome specification in the somites ...........................4
Figure 3 Somite segmentation and tissue derivatives ...................................................6
Figure 4 Muscle precursor cell migration and differentiation into
limb muscles ......................................................................................................7
Figure 5 Transcription factor expression during myogenesis ...................................11
Figure 6 Molecular regulation of myogenesis and transcriptional
control by MyoD .............................................................................................14
Figure 7 Signaling mechanisms in the somite that regulate muscle
specific gene expression ..................................................................................18
Figure 8 Notch signaling in somites and satellite cells ................................................19
Figure 9 Mechanism of Wnt signaling in mesodermal cells .......................................20
Figure 10 Activation of proliferation and cell cycle progression in
myoblasts by growth factors and cyclins ......................................................21
Figure 11 IGF signaling in myogenesis inhibits differentiation ..................................22
Figure 12 Mechanism of p38 signaling in skeletal muscle differentiation ..................23
Figure 13 Anatomical location of satellite cells in muscle fibre ...................................25
Figure 14 Symmetric and asymmetric satellite cell division ........................................27
Figure 15 Satellite cells activation and self-renewal .....................................................28
Figure 16 The canonical and non-canonical Wnt signaling pathways
in satellite cells ................................................................................................30
Figure 17 Culture models for skeletal muscle ...............................................................34
Figure 18 Comparison of functional domains in AP-1 family members ....................37
Figure 19 Comparison of the C terminal domain of Fos family members .................44
Figure 20 MAPK signaling cascades targeting AP-1 proteins .....................................48
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Figure 21 IL-6 family of cytokines and their receptors ................................................54
Figure 22 IL-6 cytokine induced signaling mechanism ................................................55

Chapter III: Cardiotrophin-1 maintains the undifferentiated state in skeletal myoblasts
Figure 1 CT-1 represses myogenic differentiation ......................................................79
Figure 2 CT-1 represses the expression of pro-differentiation
transcriptional regulators (MyoG and MEF2A/D) .....................................83
Figure 3 Transcriptional induction of the myoG promoter by MyoD
is repressed by CT-1 .......................................................................................83
Figure 4 Trans-activation properties of the MRF's are repressed ......................87-88
Figure 5 CT-1 inhibits the transcriptional activity of the MRF's
through activation of MEK signaling ......................................................92-93
Figure 6 STAT3 activation by CT-1 is not sufficient for inhibition
of myogenesis ..................................................................................................97
Figure 7 CT-1 delays regeneration of damaged skeletal muscle .............................101
Supplemental figure 1 and Supplemental figure 2 ..............................................................108

Chapter IV: Signal dependent Fra-2 regulation in skeletal muscle reserve and satellite
Figure 1 Fra-2 is a downstream target of the MEK 1/2-ERK 1/2
MAPK pathway ............................................................................................124
Figure 2 Identification of Fra-2 phospho-acceptor sites using
phospho-peptide mass spectrometry analysis ............................................128
Figure 3 Expression and stability of Fra-2 phospho-mutants in
myogenic cells ................................................................................................134
Figure 4 Knockdown of Fra-2 enhances differentiation ..........................................137
Figure 5 Expression and localization of Fra-2 in myogenic cells ............................140
Figure 6 Fra-2 expression in satellite cells .................................................................143
Figure 7 MEK 1/2 inhibition activates satellite cell differentiation
in primary muscle fibres ..............................................................................146
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Figure 8 Fra-2 phosphorylation may regulate muscle gene induction
in C2C12 cells ................................................................................................148
S1 CT-1 transiently activates Fra-2 in C2C12 cells ........................................156
S2 ERK 1/2 targets S320 and T322 are regulated sites on Fra-2 ..................157
S3 Neutralization of S320 and T322 on Fra-2 decreases protein
expression ......................................................................................................158

Chapter V: Summary and Conclusion
Figure 23 Summary of AP-1's role and regulation in skeletal muscle
development ..................................................................................................162

Appendix A: Chapter VI: Mustn1 as a novel AP-1 target gene in skeletal muscle
Figure 1 MyoD ChIP data analysis of potential AP-1 target genes .................208-209
Figure 2 mustn1 expression is increased in differentiating skeletal muscle ...........211
Figure 3 Fra-2 binds to the endogenous mustn1 promoter in skeletal muscle .......213

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Abbreviations
Akt Protein Kinase B
ANP Atrial natriuretic peptide
AP-1 Activator protein 1
Ash2L Ash2 (absent, small, or homeotic)-like
ATF Activating transcriptor factor
bHLH Basic helix loop helix
BMP Bone morphogenic protein
bZIP Basic leucine zipper
C/EPB CREB binding protein
CamKII Ca2+/calmodulin-dependent protein kinase II
CDK Cyclin dependent kinase
CIP/KIP Cyclin dependent kinase-interacting protein/kinases inhibitor protein
CK Casein kinase
CKI CDK inhibitors
CLC Cardiotrophin-like cytokine
CLS CBF-1, Suppressor of Hairless, Lag-2
c-met Hepatocyte growth factor receptor
CN Calcineurin

CNTF Ciliary neurotrophic factor
CRE CREB response element
CREB cAMP response element binding protein
CT-1 Cardiotrophin-1
Dsh Dishevelled
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EDL Extensor digitorum longus
EMT Epithelial to mesenchymal transition
ER Endoplasmic reticulum
ERK Extracellular signal regulated kinase
FACS Fluorescence activated cell sorting
FGF Fibroblast growth factor
FoxO1 Forkhead box protein O1
Fra Fos related antigen
Fzd Frizzled receptor
Gab2 Grb2 associated binder
gp130 Glycoprotein 130
Grb2 Growth factor bound protein 2
GSK3-β Glycogen synthases 3 β
HAT Histone acetyl transferase
HDAC Histone deacetylases
HGF Hepatocyte growth factor
IGF Insulin-like growth factor
IGFR Insulin-like growth factor receptor
IL Interleukin
INK4 Inhibitor of CDK4
IP Inositol trisphosphate
3

IRS Insulin receptor substrate
JAK Janus kinase
JLP JNK-associated leucine zipper protein
JNK c-Jun N-terminal kinases
x