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Function of the transcription factor Fra1 in adipogenesis

Die Funktion des Transkriptionsfaktors Fra1 in der Adipogenese

Der Naturwissenschaftlichen Fakultät

der Friedrich-Alexander-Universität Erlangen-Nürnberg zur

Erlangung des Doktorgrades Dr.rer.nat.

vorgelegt von Julia Iris Luther

aus Nürnberg

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

Tag der mündlichen Prüfung: 28.06.2012

Vorsitzender der Promotionskommission: Prof. Dr. Rainer Fink

Erstberichterstatter: Prof. Dr. Falk Nimmerjahn

Zweitberichterstatter: PD. Dr. Jochen Zwerina

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

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Table of Contents

Abstract ... 7

Zusammenfassung ... 8

1 Introduction ... 10

1.1 Bone ... 10

1.1.1 Bone tissue: Composition and function... 10

1.1.2 Bone development ... 10

1.1.3 Bone cells and bone remodeling ... 11

1.2 Adipose Tissue ... 12

1.2.1 Brown and white adipose tissue ... 12

1.2.2 Factors produced by adipose tissue: Adipokines ... 13

1.2.3 The co-regulation of adipose tissue and bone ... 14

1.3 Mesenchymal cell differentiation ... 15

1.3.1 Differentiation of mesenchymal stromal cells (MSC) ... 15

1.3.2 Chondrocyte differentiation ... 16

1.3.3 Myoblast and brown adipocyte differentiation ... 17

1.3.4 Transcriptional control of adipogenesis ... 17

1.3.5 Osteoblast differentiation ... 19

1.3.6 Reciprocal regulation of osteoblastogenesis and adipogenesis ... 20

1.4 AP-1 Transcription factor ... 20

1.4.1 Structure and composition of the AP-1 Transcription factor ... 21

1.4.2 Regulation of transcriptional activity ... 21

1.4.3 Functions of AP-1 ... 22

1.4.4 Functions of AP-1 in bone development ... 23

1.4.5 Functions of AP-1 in adipocyte development ... 24

1.5 Aim of the work ... 24

2. Materials and Methods ... 26

2.1 Materials ... 26

2.1.1 Buffer and staining solutions... 26

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2.1.4 Antibodies ... 32

2.1.5 Plasmids... 33

2.1.6 Molecular weight marker and loading dye... 33

2.1.7 Reagents for cell culture... 33

2.1.8 Kits and enzymes... 34

2.1.9 Chemicals ... 36

2.1.10 Consumables ... 38

2.1.11 Laboratory Instruments ... 38

2.2 Methods ... 40

2.2.1 Animal experiments ... 40

2.2.2 Cell culture ... 41

2.2.3 Methods to study RNA... 43

2.2.4 Methods to study DNA... 44

2.2.5 Methods to study protein... 45

2.2.6 Sequence analysis... 48

2.2.7 Statistical analysis ... 48

3 Results ... 50

3.1 No clear adipogenic phenotype of AP2-cre Fra1 f/f mice... 50

3.2 Lipodystrophic phenotype in mice overexpressing Fra1... 52

3.2.1 Severe lipodystrophy in Fra1 transgenic mice... 52

3.2.2 Normal brown adipose tissue (BAT)... 54

3.2.3 Immature white adipose tissue (WAT) in mice overexpressing Fra1 ... 55

3.2.4 Normal food intake and insulin levels in Fra1 transgenic mice ... 57

3.2.5 Increased insulin sensitivity, serum triglyceride and non-esterified fatty acid levels in Fra1 transgenic mice... 58

3.2.6 No ectopic lipid deposition in liver or muscle of Fra1 transgenic mice... 60

3.2.7 Decreased expression of genes involved in lipogenesis and lipolysis in the adipose tissue of Fra1tg mice... 60

3.2.8 No correction of the lipodystrophic phenotype by high fat diet... 63

3.3 Reduced differentiation to adipocytes in the WAT of Fra1 transgenic mice ... 65

3.3.1 No transdifferentiation of WAT toward BAT in Fra1 transgenic mice... 65

3.3.2 Expression of adipokines in white adipose tissue of Fra1 transgenic mice... 65

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3.3.3 No change in the expression of markers for adipocyte progenitor cells in white

adipose tissue of Fra1 transgenic mice ... 67

3.3.4 No change in AP-1 expression in the WAT of Fra1 transgenic mice ... 67

3.3.5 Decreased C/ebpα expression in white adipose tissue overexpressing Fra1 ... 68

3.4 In vitro analysis of the role of Fra1 during adipocyte differentiation ... 71

3.4.1 Cell autonomous decreased adipogenesis of Fra1 transgenic mesenchymal cells ... 71

3.4.2 Normal responses of Fra1 transgenic mesenchymal progenitors to adipogenic stimulation ... 72

3.4.3 Fra1 overexpression directly blocks adipogenesis ... 74

3.4.4 No detectable regulation of C/ebpα expression via binding of Fra1 to C/EBPβ... 75

3.4.5 Fra1 directly inhibits the transcription of C/ebpα ... 76

4. Discussion ... 79

4.1 Phenotype of the Fra1 transgenic and the Fra1 knockout mouse ... 79

4.2 Fra1 overexpression decreases adipose tissue mass ... 80

4.3 Fra1 regulates adipocyte differentiation in a cell autonomous manner... 81

4.4 Fra1 directly regulates adipocyte differentiation by reducing C/ebpαααα expression... 82

4.5 Influence of the reduced amount of mature adipocytes on metabolism... 84

4.6 Phenotype of mice with an adipocyte-specific deficiency of Fra1... 86

4.7 Conclusions ... 86

5 References ... 88

6 Appendix ... 100

6.1 Abbreviations... 100

6.2 Cloned sequence of the C/ebpαααα promoter ... 102

6.3 Acknowledgements... 104

6.4 Publications and presentations ... 105

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Abstract

A systemic neuro-endocrine regulation of bone and adipose tissue based on osteocalcin and leptin has recently been described. However, factors that locally mediate mesenchymal stem cell differentiation and commitment to osteoblasts and adipocytes, including those factors that locally integrate the systemic regulation, are poorly known. The objective of this thesis was therefore to investigate whether the AP-1 family member fos-like antigen 1 (Fra1, Fosl1), that has been described to regulate osteogenesis, could also play a role in adipogenesis.

We demonstrated that mice overexpressing Fra1 (H2-fra-1-LTR transgenic mice), which develop osteosclerosis due to a cell autonomous accelerated osteoblast differentiation also develop a severe progressive general lipodystrophy leading to the total absence of white adipose tissue (WAT). Histologically, the white adipose tissue, that is still present in young Fra1 transgenic mice, appeared immature. Markers for adipocyte maturation, that also regulate lipid storage, i.e. Glut4, Plin1, Lpl and Cd36 were down-regulated in the adipose tissue of Fra1tg mice and we could therefore show an accumulation of triglycerides and non- esterified fatty acids in the blood of these mice that are also resistant to high fat diet induced obesity.

In vitro, the adipogenic differentiation of Fra1tg primary osteoblast (POBs) isolated from the calvaria of newborn mice, that represent a common progenitor for osteoblasts and adipocytes, was strongly inhibited compared to the differentiation of POBs isolated from wild-type littermates. Similar results were obtained in experiments using an adipogenic cell line with constitutive overexpression of Fra1, indicating that this is a cell-autonomous effect dependent on Fra1. The expression of key regulators of mesenchymal cell fate decision (Runx2, Sox9 and MyoD) as well as the expression of early markers of adipogenesis (C/ebpβ and C/ebpδ) was not altered. This suggested that adipocyte differentiation rather than the commitment to the adipocyte lineage was inhibited. Accordingly, the expression of adipogenic markers essential for later stages of differentiation, namely Pparγ2 and C/ebpα was reduced. The latter was also found decreased in the white adipose tissue of Fra1tg mice. Finally, we demonstrated that the reduced expression level of C/ebpα is caused by the binding of Fra1 to the C/ebpα promoter and the direct transcriptional repression of C/ebpα by Fra1.

Thus, our data added to the known function of Fra1 in accelerating osteoblast differentiation a new cell autonomous function by inhibiting adipocyte differentiation, the latter being caused by down regulation of C/EBPα.

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Zusammenfassung

In den letzten Jahren wurde eine systemische neuroendokrine Regulation von Knochen und Fettgewebe basierend auf Osteokalzin und Leptin beschrieben. Faktoren, die lokal zur Festlegung mesenchymaler Stammzellen auf die osteogene oder adipogene Linie führen oder die Differenzierung zu Osteoblasten und Adipozyten regulieren sowie Faktoren, die lokal die systemische Regulation weiterleiten, sind jedoch weitgehend unbekannt. Ziel dieser Arbeit war es somit, die Rolle des fos-like antigen 1 (Fra1, Fosl1) eines Mitglieds der AP-1 Transkriptionsfaktor-Familie, der die Osteogenese reguliert, in der Adipogenese zu untersuchen.

Wir konnten zeigen, dass Fra1 überexprimierende Mäuse (H2-fra-1-LTR transgene Mäuse), die aufgrund einer zellautonom beschleunigten Osteoblastendifferenzierung Osteosklerose entwickeln zudem eine schwere progressive generelle Lipodystrophie entwickeln, die zu einer kompletten Degeneration des weißen Fettgewebes führt. In histologischen Schnitten erschien das weiße Fettgewebe, das in jungen Fra1 transgenen Mäusen noch vorhanden ist, unreif. Die Expression von Markergenen für reife Adipozyten, wie Glut4, Plin1, Lpl und Cd36, die auch die Einlagerung von Lipiden in die Fettzellen regulieren, war im Fettgewebe von Fra1 transgenen Mäusen erniedrigt. Dies erklärt die von uns beobachtete Akkumulation von Triglyceriden und von nicht veresterten Fettsäuren im Blut sowie die Resistenz von Fra1 transgenen Mäusen gegenüber Hoch Fett Diät-induzierter Adipositas.

In in vitro-Experimenten mit primären Osteoblasten (POBs), die aus der Calvaria neugeborener Mäuse isoliert wurden und gemeinsame Vorläuferzellen für Osteoblasten und Adipozyten darstellen, zeigten Fra1 transgene POBs im Vergleich zu POBs aus Wildtyp- Kontrolltieren eine starke Inhibierung der Differenzierung zu Adipozyten. Vergleichbare Ergebnisse wurden in Experimenten mit einer konstititiv Fra1 überexprimierenden adipogenen Zelllinie erzielt. Dies zeigte, dass der Effekt zellautonom auf Fra1 basiert. Die Expression von Schlüsselfaktoren, die für die Festlegung der mesenchymalen Zellidentität essentiell sind (Runx2, Sox9 und MyoD) sowie die Expression von Markern für die frühen Stadien der Adipogenese (C/ebpβ und C/ebpδ) war unverändert. Diese Ergebnisse deuten darauf hin, dass die Adipozytendifferenzierung, jedoch nicht die Festlegung mesenchymaler Stammzellen auf die adipogene Zelllinie inhibiert war. Dementsprechend konnten wir zeigen, dass die Expression der Marker Pparγ2 und C/ebpα, die für spätere Stadien der Differenzierung essenziell sind, erniedrigt waren. C/ebpα war zudem im weißen Fettgewebe

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Wir konnten des Weiteren demonstrieren, dass die reduzierte Expression von C/ebpα durch die Bindung von Fra1 an den C/ebpα -Promotor und die direkte transkriptionelle Repression von C/ebpα durch Fra1 verursacht wird.

Somit konnte durch die vorliegende Arbeit gezeigt werden, dass Fra1 nicht nur, wie bereits bekannt, die Differenzierung von mesenchymalen Stammzellen zu Osteoblasten beschleunigt, sondern auch zellautonom die Differenzierung zu Adipozyten inhibiert, indem es die Expression von C/ebpα verringert.

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1 Introduction

1.1 Bone

1.1.1 Bone tissue: Composition and function

Bone tissue is composed of three major cell types that regulate its development and maintenance: osteoblasts, the bone forming cells, osteoclasts, the bone resorbing cells and the latest stage of osteoblast differentiation, the osteocytes. These bone cells are embedded in extracellular matrix composed of collagen (>90% collagen I), proteoglycanes, other non- collageneous proteins and calcium phosphate as hydroxyapatite (Teitelbaum, 2000).

Bone tissue exerts several functions: it provides a structure patterning and shaping the body, as well as protecting inner organs. It allows locomotion by providing attachment sites for muscles. Furthermore, bone acts as a reservoir for calcium and phosphate. The bone marrow cavity serves as the main haematopoietic organ of the adult, providing a niche for stem cells as well as long-lived plasma cells (Clarke, 2008; Grabowski, 2009). In addition, bone tissue acts as an endocrine organ by producing factors like Osteocalcin and FGF23 (Fukumoto and Martin, 2009).

1.1.2 Bone development

There are two distinct types of bone formation: the intramembranous (or dermal) ossification and the endochondral ossification. On the one hand, the flat bones, namely the calvaria and some facial bones are formed by intramembranous ossification, whereby osteoblasts differentiate directly from mesenchymal progenitor cells (Erlebacher et al., 1995). Long bones of the axial- and appendicular skeleton and the main part of the facial bones, on the other hand, are formed via a cartilage template that is replaced by bone (Ornitz and Marie, 2002).

During this process, called endochondral ossification, mesenchymal cells aggregate, condense into compact nodules, differentiate to chondrocytes, undergo proliferation and secrete extracellular matrix, forming a cartilage scaffold (Wuelling and Vortkamp, 2010). Starting from the center, chondrocytes become hypertrophic and secrete collagen X and fibronectin, that enables the cartilage matrix that consisted mainly of collagen II to become mineralized (Gilbert et al., 2006). In addition, chondrocytes initiate angiogenesis by secreting VEGF,

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hypertrophic regions (Gilbert et al., 2006; Olsen et al., 2000). Finally, hypertrophic chondrocytes become apoptotic. Perichondreal cells differentiate to osteoblasts that produce proteins of the periosteum, a layer of connective tissue surrounding the bone and start mineralizing the matrix by accumulation of hydroxyapatite. The mineralized cartilage in the hypertrophic regions is resorbed by osteoclasts, that allows the replacement of the cartilage template by bone by osteoblasts, invading the cartilage through the newly formed blood vessels (Olsen et al., 2000) as well as the formation of the bone marrow cavity as a result of osteoclasts activity (Gilbert et al., 2006). In the epiphysis of the long bones, secondary ossification centers develop. The growth plate, found between the primary and secondary ossification center is responsible for the longitudinal growth of the bones. There, the above described processes occur in an analogous manner, resulting in the zones of resting chondrocytes, of proliferation, prehypertrophy and hypertrophy, ossification and resorption (Wuelling and Vortkamp, 2010).

1.1.3 Bone cells and bone remodeling

Bone tissue undergoes constant remodeling throughout life, resulting in longitudinal and radial bone growth and bone renewal. Bone integrity is based on the balance between bone resorption by osteoclasts and bone formation by osteoblasts.

Osteoclasts, the bone resorbing cells, originate from hematopoietic stem cell precursors of the monocyte/macrophage lineage. Progenitor recruitment and differentiation is regulated by macrophage-colony-stimulating factor (M-CSF) and Receptor activator of nuclear factor kappa-B ligand (RANKL), cytokines that are expressed by mesenchymal stromal cells and osteoblasts (Clarke, 2008). Additional factors essential for osteoclastogenesis are c-Fos, Traf6, NFATC1 and NF-κB (Teitelbaum and Ross, 2003). The first step in bone resorption is the recruitment and differentiation of precursors to multinucleated osteoclasts. Osteoclasts subsequently get attached to the bone surface via integrins and get polarised (Clarke, 2008).

They form a ruffled membrane to increase the surface active in resorption that is surrounded by a podosomal ring defining a sealed extracellular lysosomal environement (Teitelbaum, 2000). Protons are secreted to acidify this environment that enables the catabolic proteins, most importantly cathepsin K, to resorb the bone matrix (Clarke, 2008; Srivastava et al., 2001; Teitelbaum, 2000). Osteoclast number and activity is increased by parathyroid hormone, 1,25-dihydroxyvitamin D, corticosteroids as well as by pro-inflammatory cytokines, and inhibited by calcitonin, estrogen and anti-inflammatory cytokines (Srivastava et al., 2001;

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Zaidi et al., 2003). An osteoclast related imbalance of bone remodelling results in osteoporosis due to increased osteoclast numbers or activity or in osteopetrosis whereby decreased osteoclast numbers or activity results in a high bone mass (Teitelbaum, 2000;

Teitelbaum and Ross, 2003).

Bone resorption is followed by formation of new bone matrix, which is the main function of the mesenchymal stromal cell derived osteoblasts. These cells produce osteoid, uncalcified extracellular matrix, consisting mainly of type I collagen and initiate its mineralization. In addition, osteoblasts control osteoclast differentiation. Thereby, osteoclast activation is achieved by the osteoblastic secretion of RANKL and M-CSF. By secretion of osteoprotegerin (OPG), a soluble decoy receptor for RANKL, osteoclast formation can also be inhibited by osteoblastic cells. The osteoclastogenesis is therefore regulated by the ratio of RANKL to OPG (Boyce and Xing, 2007). Imbalance of bone remodeling caused by a deregulation of osteoblasts leads to osteosclerosis, whereby high bone mass is caused by an increased osteoblast number or activity or to osteopenia, a low bone mass phenotype as a result of decreased osteoblast numbers or activity.

Osteocytes represent the terminal differentiation stage of osteoblasts, embedded within lacunae in the extracellular matrix. They are linked to other cells via a network of filopodia located in canaliculi. Osteocytes are the most abundant bone cell type (Klein-Nulend et al., 2003), that account for about 90% of the total bone cell number (Bonewald, 2011). Osteocytes were described to act, based on their filopodia network, as sensors for mechanical strain, thereby regulating mechanical-induced bone remodeling (Klein-Nulend et al., 1995;

Weinbaum et al., 1994; Zhao et al., 2002). It was recently reported that osteocytes inhibit osteoclast activity and that, in turn, osteocyte apoptosis leads to osteoclast recruitment (Gu et al., 2005; Tatsumi et al., 2007). In addition, osteocyte ablation causes anti-osteoblastic effects (Tatsumi et al., 2007).

1.2 Adipose Tissue

1.2.1 Brown and white adipose tissue

Two types of adipose tissue exist: the white adipose tissue (WAT) and the brown adipose tissue (BAT). Brown adipose tissue is mainly located in the interscapular region and persists

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fat vacuoles than white adipose tissue, but a high number of mitochondria, establishing its function in thermoregulation (Hansen and Kristiansen, 2006; Tanaka et al., 1997). Important for non-shivering thermogenesis is the activity of uncoupling protein 1 (UCP1), a proton pump located in the inner mitochondrial membrane, that uses the electrochemical gradient to cause the production of heat instead of ATP (Argyropoulos and Harper, 2002).

White adipose tissue is developing after birth and acts with its large fat vacuoles as a postprandial storage for triglycerides (Tanaka et al., 1997). White adipose tissue is composed of mature adipocytes that represent about 50% of the cells, preadipocytes, fibroblasts, macrophages and endothelial cells (Trayhurn, 2007). In addition to its function in lipid storage, adipose tissue acts as an endocrine organ regulating energy metabolism and homeostasis by producing factors like the adipokines adiponectin and leptin (Confavreux et al., 2009; Harwood, 2011; Kadowaki and Yamauchi, 2005; Takeda and Karsenty, 2008).

1.2.2 Factors produced by adipose tissue: Adipokines

Adipokines are cytokines that are primarily produced and secreted by adipose tissue (Tilg and Moschen, 2006). They exert various functions, including an involvement in glucose and insulin metabolism, in immune functions (TNFα and IL-6), angiogenesis and regulation of blood pressure (Lefterova and Lazar, 2009). The function of the most important adipokines adiponectin, resistin, leptin, as well as the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin 6 (IL-6) on glucose and insulin metabolism as well as on mesenchymal cell differentiation are described in the following.

Adiponectin is the most abundant factor produced by adipocytes in the circulation whereby its level is inversely proportional to adipose tissue mass in the body (Reid, 2010). It was shown that adiponectin enhances insulin sensitivity and increases glucose uptake and fatty acid oxidation of adipose tissue and muscle. In addition, hepatic gluconeogenesis is inhibited by adiponectin (Galic et al., 2010). The functions on the metabolism were described to be exerted via the activation of AMP-activated protein kinase, an enzyme regulating glucose metabolism (Yamauchi et al., 2002a).

In contrast, resistin confers resistance to insulin. Resistin levels are increased in obesity that leads to the proposed role for resistin in the insulin resistance linked to obesity and type 2 diabetes (Steppan et al., 2001).

Leptin is the most studied adipokine that links bone and fat metabolism. Its level is directly proportional to the amount of adipose tissue in the body and leptin was shown to directly

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influence insulin resistance (Levi et al., 2011). Leptin produced by adipocytes within the bone marrow exerts a direct effect on mesenchymal stromal cells and bone cells by stimulating osteoblast differentiation and inhibiting bone resorption (Hamrick and Ferrari, 2008). In vitro studies using human MSCs showed that leptin can also inhibit osteoclast differentiation via the regulation of OPG and RANKL (Hamrick and Ferrari, 2008; Holloway et al., 2002) and, in addition, can suppress adipogenic differentiation (Thomas et al., 1999). However, leptin can regulate bone and fat by a second indirect effect involving signaling via the hypothalamus. This role in central regulation of bone and adipose tissue mass is described in more detail in the following chapter.

Adipose tissue also produces the pro-inflammatory cytokines TNFα and IL-6 providing amongst others a possible link between systemic chronic inflammation and obesity-induced insulin resistance (Galic et al., 2010; Tilg and Moschen, 2006). Macrophages are the main producers of TNFα and IL-6 in adipose tissue and expression of these cytokines is increased in obesity (Galic et al., 2010; Rasouli and Kern, 2008; Waki and Tontonoz, 2007) due to an increased infiltration of macrophages into the adipose tissue (Galic et al., 2010). TNFα and IL-6 were described to inhibit insulin signaling, increase insulin resistance (Galic et al., 2010;

Rasouli and Kern, 2008; Waki and Tontonoz, 2007) and to enhance lipolysis (Green et al., 2004; Ji et al., 2011).

1.2.3 The co-regulation of adipose tissue and bone

A systemic connection between adipose tissue and bone based on leptin and osteocalcin became a central focus of research in metabolism in the last few years (Fig. 1.1). Leptin, a hormone produced by white adipocytes, acts via the hypothalamus to inhibit appetite and to increase energy expenditure. Leptin deficient ob/ob as well as leptin receptor deficient db/db mice were therefore described to develop obesity caused by hyperphagy (Bates et al., 2003;

Cohen et al., 2001; Coleman, 1978). Bone formation is inhibited by leptin independently of its influence on body weight through the sympathetic nervous system. This pathway is activating the β2 adrenergic receptors on osteoblasts (Takeda et al., 2002), thereby increasing ATF4- dependent the expression of Esp, a gene encoding for OST-PTP, a tyrosine phosphatase that reduces osteocalcin bioactivity by γ-carboxylation (Hinoi et al., 2008; Lee et al., 2007;

Yoshizawa et al., 2009). Consequently, leptin deficiency in mice leads to an increased bone formation (Ducy et al., 2000).

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In turn, osteocalcin, an osteoblast specific protein, acts as a hormone that directly enhances pancreatic β-cell proliferation and insulin secretion and indirectly, via adiponectin increases insulin sensitivity and energy expenditure (Ferron et al., 2008; Lee et al., 2007), thereby regulating fat metabolism.

More recently, an important role for insulin signaling in osteoblasts was reported. Signaling via the insulin receptor, that is inhibited by Esp, first increases the expression of Runx2 promoting osteoblast proliferation and differentiation (Fulzele et al., 2010) and second, stimulates bone resorption due to a decreased expression of OPG. The resulting acidification in the resorption area is another mechanism leading to an increased amount of decarboxylated bioactive osteocalcin and therefore increases insulin production (Ferron et al., 2010a).

pancreas adipose tissue

brain

uncarboxylated

carboxylated

osteocalcin leptin bone

leptin leptin

insulin

Figure 1.1: Local and systemic interaction between adipose tissue and bone. Leptin, produced by adipocytes regulates fat metabolism and bone formation via the hypothalamus. In turn, osteocalcin, an osteoblast specific factor, acts on the pancreatic β-cells and the adipose tissue influencing fat metabolism. Also described in (Schett and David, 2010)

1.3 Mesenchymal cell differentiation

1.3.1 Differentiation of mesenchymal stromal cells (MSC)

A third link between osteoblasts and adipocytes exists: they both differentiate from a common mesenchymal progenitor, the mesenchymal stromal or stem cell (MSC). In fact, this MSC is the common mesenchymal progenitor for all other mesenchymal cell lineages such as

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chondrocytes, fibroblasts and myoblasts (Caplan, 2007). Mesenchymal cell fate commitment and differentiation towards the various lineages are driven by key transcription factors that confer identity to the cell. Fig. 1.2 summarizes the major transcription factors regulating MSC differentiation.

Mesenchymal stem/stromal cell

osteoblast chondrocyte fibroblast myoblast C/Ebpββββ

C/Ebpδδδδ

C/Ebpαααα Pparγγγγ2

β-catenin Runx2

Osterix

Sox9 L-Sox5, Sox6

MyoD Myogenin

Mrf4 Myf-5

brown adipocyte white

adipocyte

Sox9

Runx2/3

Prdm16

Figure 1.2: Transcription factors regulating mesenchymal cell fate decision. Mesenchymal stem/stromal cells have the capacity to differentiate into different lineages. The major transcription factors controlling lineage determination are indicated.

1.3.2 Chondrocyte differentiation

The key determinants to regulate chondrocyte differentiation are SOX9, L-SOX5, SOX6 and Runx2. In particular, SOX9 was shown to induce the condensation, proliferation and differentiation of mesenchymal progenitors to chondrocytes and to regulate the chondrogenic expression of collagen II and XI as well as aggrecan (Karsenty, 2008). Two other members of the SOX family, L-SOX5 and SOX6, are cooperating with SOX9, and have an essential function during differentiation and activation of collagen II expression (Lefebvre and Smits, 2005). Hypertrophic differentiation is inhibited by Sox genes, but activated by Runx2 and Runx3 (Hartmann, 2009).

Growth factors relevant for chondrogenesis are the members of the Transforming Growth Factor β super family (TGFβ-1, -2, -3 und BMPs), Fibroblast Growth Factors (FGFs) and

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as well as chondrocyte proliferation, differentiation and apoptosis. A central mechanism of regulation is based on the Indian-hedgehog (IHH) / parathyroid-hormone-related-Protein (PTHrP) feedback loop. IHH is expressed by the prehypertrophic chondrocytes and is positively regulating the expression of PTHrP in the cells of the periarticular zone of the bone and the perichondrium, resulting in the formation of a gradient of PTHrP. PTHrP can therefore inhibit hypertrophic differentiation of chondrocytes in the zone of proliferation and the prehypertrophic region (Vortkamp et al., 1996).

1.3.3 Myoblast and brown adipocyte differentiation

Myoblast differentiation requires activation of the regulatory marker genes Myf5 and MyoD, belonging to the myogenic bHLH protein family, myogenin and Mrf4. MyoD and Myf5 that are induced by the Wnt and Shh pathway regulate the commitment of myoblasts. Mrf4 is supposed to positively regulate the early steps of differentiation as well as promoting terminal differentiation while myogenin is necessary for terminal myoblast differentiation (Berkes and Tapscott, 2005; Gilbert et al., 2006; Kassar-Duchossoy et al., 2004; Perry and Rudnick, 2000). Recently it was shown that brown adipocytes are more related to muscle cells than to white adipocytes as they originate from Myf5 expressing precursors (Seale et al., 2008). Cell fate decision between myoblasts and brown adipocytes is proposed to be regulated by the action of PRDM16 in complex with the transcription factor C/EBPβ (Kajimura et al., 2009).

1.3.4 Transcriptional control of adipogenesis

Adipogenesis is the process by which a mesenchymal progenitor cell differentiates via a preadipocyte to a mature adipocyte. The initial step of adipogenesis, called adipocyte determination is supposed to be regulated by BMP-signaling, leading to the commitment of the progenitor cell to the adipose lineage (MacDougald and Mandrup, 2002; Otto and Lane, 2005). The second step of adipogenesis, the terminal differentiation, occurs by activation of the basic leucine zipper protein family members C/EBPβ and C/EBPδ (CCAAT/enhancer binding protein β and δ). These two transcription factors initiate the second step of terminal differentiation by directly inducing the expression of Pparγ2 (peroxisome proliferator activated receptor gamma 2) (Farmer, 2006). PPARγ2, a member of the nuclear hormone receptor superfamily, was described to be the ‘master regulator’ of adipogenesis that is necessary as well as sufficient for adipocyte differentiation as shown by the overexpression of

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Pparγ2 that can initiate adipogenic differentiation (Rosen and MacDougald, 2006). The ligand-activated transcription factor PPARγ2 subsequently dimerises with RXRα and induces, together with C/EBPβ and C/EBPδ, the expression of C/ebpα. Finally, C/EBPα and PPARγ2, that control the expression of late markers of adipocyte maturation, regulate expression of each other through a positive feedback loop (Farmer, 2006).

Figure 1.3: Adipocyte differentiation. Initiation of the terminal differentiation phase is regulated by the transcription factors C/ebpβ and C/ebpδ that activate the late markers of adipogenesis, Pparγ2 and C/ebpα.

(Green: pro-adipogenic factors, red: anti-adipogenic factors).

C/EBPα and PPARγ2 regulate the expression of various proteins establishing adipocyte function, for instance of the insulin-controlled glucose transporter Glut4, of fatty-acid-binding protein Ap2 (Fabp4), that is involved in the transport of free fatty acids, as well as of lipolytic and lipogenic genes (Lowe et al., 2011).

Numerous additional factors have been described to regulate adipocyte differentiation. For instance preadipocyte factor 1 (Pref1), a transmembrane protein that is cleaved to generate its active form (Kim et al., 2007). Pref-1 is expressed in pre-adipocytes and has to be repressed during adipocyte differentiation as it inhibits adipogenesis through regulating C/ebpβ and C/ebpδ promoter activities (Lowe et al., 2011). A known positive regulator of adipogenesis is the insulin-regulated sterol regulatory element binding protein SREBP-1c that has been described to increase PPARγ activity and to regulate the generation of Pparγ ligands in vitro as well as to increase the expression of genes regulating fatty acid synthesis (White and Stephens, 2009). Other important factors described to inhibit adipocyte differentiation are two

C/EBPααα α

C/EBPββββ C/EBPδδδδ

PPARγγγγ CHOP C/EBPγγγγ

KROX20

GATA2/3 KLF5

KLF15

SREBP1c KLF2 pCREB1

Serum mitogens

cAMP

glucocorticoids

Insulin

Pref1

RXRαααα Pparγγγγ1

adipocyte specific

genes (Glut4, Ap2)

IBMX

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transcription factors (GATA2 and GATA 3) and the Kruppel-like factor (KLF2) (Lowe et al., 2011). However, other members of the Klf family such as KLF5 and KLF15 have been described to be positive regulators of adipocyte differentiation (Lowe et al., 2011).

In vitro, differentiation to adipocytes can be induced by a cocktail of insulin, dexamethasone and 3-isobutyl-1-methylxanthine (IBMX). In several cell culture systems, the first step is a phase of mitotic clonal expansion, followed by the terminal differentiation phase. The components of the adipogenic cocktail activate specific transcription factors regulating the differentiation process. In particular, IBMX inhibits phosphodiesterases, leading to elevated cAMP levels. As a result, cAMP responsive element binding protein 1 (CREB1) is phosphorylated and is therefore able to induce the expression of C/ebpβ (Berry et al., 2010).

C/ebpβ expression is also induced by serum mitogens via KROX20 (Chen et al., 2005).

C/ebpδ expression is activated by the synthetic glucocorticoid dexamethasone via the inhibition of Pref1 expression. The third component of the adipogenic cocktail, insulin, targets Pparγ via Srebp1c (Farmer, 2006).

1.3.5 Osteoblast differentiation

The major factors regulating differentiation of mesenchymal stromal cells to pre-osteoblasts are β-catenin downstream of the wnt signaling pathway and Runx2 (Cbfa1) (Komori, 2006).

Runt related transcription factor 2, Runx2, was described to be necessary for osteoblastogenesis as mice with a homozygous deletion of Runx2 display a lack of osteoblasts. However, it was suggested that Runx2 also inhibits proliferation and terminal differentiation to osteoblasts, as overexpression of Runx2 in mice leads to a reduced number of mature osteoblasts (Nakashima and de Crombrugghe, 2003). For efficient transcriptional activity, formation of a heterodimeric transcription factor complex with Cbfβ is essential (Nakashima and de Crombrugghe, 2003; Yoshida et al., 2002). Other factors that were described to be involved in early regulation of osteoblast differentiation are ATF4, IHH, IGFs and BMP/TGF-β signalling (Karsenty and Wagner, 2002). Osterix (Osx/Sp7), one of the osteoblastic genes directly activated by Runx2, drives osteoblast maturation. As for Runx2, Osx deficiency in mice was shown to result in a complete lack of bone formation (Zhang, 2010). In vitro, osteoblast differentiation can be induced by stimulation with ascorbic acid and β-glycerophosphate.

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1.3.6 Reciprocal regulation of osteoblastogenesis and adipogenesis

Several factors have been described as regulators of cell fate decisions between osteoblasts and adipocytes by promoting commitment or differentiation into one lineage at the expense of the other (Takada et al., 2007b). PPARγ, for instance, a transcription factor that plays a crucial role in adipocyte differentiation, decreases bone formation by repressing the activity of Runx2 and Osx (Muruganandan et al., 2009). One isoform of another key transcription factor regulating adipogenesis, namely of C/EBPβ, activates factors involved in osteoblastogenesis for instance by acting as coactivator of Runx2, thereby regulating bone formation (Hata et al., 2005; Henriquez et al., 2011). Runx2 is also activated by TAZ (Wwtr1), an osteoblastogenic factor of mesenchymal stem cell differentiation, which additionally represses the function of PPARγ (Marie, 2008).

A reciprocal regulation of osteoblastogenesis and adipogenesis has also been reported for the wnt signaling pathway. Activation of the wnt signaling was described to promote osteoblastogenesis, while repressing C/ebpα and Pparγ induction, thereby inhibiting adipogenesis (Muruganandan et al., 2009). Therefore wnt signalling is inhibited during adipogenesis (Bennett et al., 2002). For the canonical wnt signaling pathway, amongst others Wnt1, 3a and 10b were described to negatively regulate adipogenesis and positively osteoblastogenesis (Kang et al., 2007), in regard to the non-canonical wnt pathway, Wnt-5a was reported inhibit PPARγ activity (Takada et al., 2007a).

A positive role in switching osteoblast versus adipocyte differentiation has also been shown for various other factors, for instance Msx2 (Ichida et al., 2004; Qadir et al., 2011), steroids like glucocorticoids (Chen et al., 2007) or the Fos family members of the AP-1 transcription factor.

1.4 AP-1 transcription factor

The AP-1 (activator protein-1) transcription factor is a sensor of changes affecting the extracellular environment. It is regulated by a great variety of stimuli, such as growth factors, cytokines, stress signals, infections and oncoproteins and is implicated in the modulation of many processes including proliferation, apoptosis, differentiation, transformation and migration (Shaulian and Karin, 2001; Shaulian and Karin, 2002).

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1.4.1 Structure and composition of the AP-1 Transcription factor

AP-1 is a dimeric transcription factor composed of one member of the Fos family of proteins (c-Fos, FosB, Fra1 or Fra2) and one member of the Jun family (c-Jun, JunB or JunD) (Zenz et al., 2008). In addition, Jun members can also form homo-dimers or associate with the structural similar members of the MAF, ATF (ATF-2, ATF-3 or B-ATF) and JDP (JDP-1 and JDP-2) subfamily (Karin et al., 1997; Shaulian and Karin, 2001). AP-1 belongs to the family of basic leucin zipper (bZIP) proteins in which the dimerisation occurs through a leucine zipper, a process required for DNA binding via a basic domain. The consensus sequence bound by AP-1, 5′-TGAG/CTCA-3′, is called TRE (TPA (12-O-tetradecanoylphorbol-13- acetate) responsive element). In addition to a bZIP domain, Jun proteins and the Fos protein members c-Fos and FosB consist of transactivation domains and binding and phosphorylation sites for different kinases (Hess et al., 2004).

Figure 1.4: Schematic representation of the AP-1 transcription factor and structure of Jun and Fos proteins. Dimerisation of a Jun and a Fos family member and DNA binding to the consensus sequence occurs through a bZIP domain (blue: bZIP domain of Jun, red: bZIP domain of Fos, yellow: DNA). In addition to the bZIP domain, Fos and Jun proteins consist of transactivation domains and binding sites for different kinases.

From (Hess et al., 2004).

1.4.2 Regulation of transcriptional activity

AP-1 activity is regulated on the transcriptional level, leading to a differing availability of AP- 1 family members depending on celltype, differentiation status and cell context. Based on the composition of the heterodimer, AP-1 complexes diverge in their transactivation potential. In

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particular, c-Fos, c-Jun and FosB have a high potential to activate transcription. Other members, like JunB, JunD, and in particular Fra1 and Fra2, the two Fos members that lack the C-terminal transactivation domain, could be weak transactivators or even exert an inhibitory function by competing for more active AP-1 complexes to be formed or to bind to the consensus sequence. However, transcription can also be regulated positively by recruiting further transcription factors (Chinenov and Kerppola, 2001; Wisdon and Verma, 1993). In addition, Jun-Fos heterodimers exhibit higher stability and stronger DNA binding affinities than Jun homodimers (Hess et al., 2004; Karin et al., 1997). Dimerisation with other structurally unrelated transcription factors and transcriptional regulators was also described including members of the C/EBP family (Wagner, 2002).

The activity of AP-1 is also regulated post-transcriptionally for instance by different kinases mainly belonging to the mitogen activated protein kinase (MAPK) pathway (Young and Colburn, 2006). The Jun N terminal Kinase (JNK) phosphorylates c-Jun, JunD and ATF-2 (Karin et al., 1997), phosphorylation by ERK is essential for Fra1 stability and activity (Doehn et al., 2009; Young and Colburn, 2006) and in combination with RSK2 for c-Fos transforming activity (Chen et al., 1996; David et al., 2005).

1.4.3 Functions of AP-1

c-Fos and c-Jun (cellular Fos and Jun, respectively) have been identified as cellular homologes of the viral oncogenes v-Fos and v-Jun, depicting their role in cell proliferation and oncogenic transformation. JunB has been described to inhibit cell proliferation, while JunD exerts pro- and anti-proliferative effects. However, both, JunB and JunD reduce cell transformation (Eferl and Wagner, 2003).

AP-1 family members also play a role in embryogenesis and organogenesis (Wagner, 2002).

In regard to the Fos family members, Fra1 was described to be essential for embryogenesis as Fra1 deficiency leads to embryonal death, due to placental defects and a lack of vascularisation (Schreiber et al., 2000). In Fra2 knockout mice, an early postnatal lethality is also observed probably as a result of heart and gastrointestinal tract defects (Karreth et al., 2004). c-Fos and Fos B, however, are not necessary during embryogenesis (Wagner, 2002). In contrast to JunD, c-Jun and JunB are required for embryonic development due to their essential role in heart and liver development and angiogenesis in the extra-embryonal tissue, respectively (Eferl et al., 1999; Schorpp-Kistner et al., 1999; Wagner, 2002).

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Furthermore, functions in the formation of fibrosis and in tumour progression (Fra1, Fra2) (Eferl et al., 2008; Kireva et al., 2011; Schroder et al., 2010; Young and Colburn, 2006), for keratinocytes (c-Jun) (Zenz and Wagner, 2006), spermatogenesis (JunD ko) (Thepot et al., 2000) and an influence on immune cells (JunB, JunD, ∆FosB) (Eferl and Wagner, 2003; Zenz et al., 2008) was described.

1.4.4 Functions of AP-1 in bone development

An important role for AP-1, especially for all the Fos protein members has been described in the control of bone homeostasis. In particular, overexpression of c-Fos leads to osteosarcoma formation due to a transformation of osteoblasts (Grigoriadis et al., 1993), and a block in osteoclast differentiation in c-Fos knockout mice was reported, resulting in osteopetrosis (Grigoriadis et al., 1994). Osteoclast size and survival however is influenced by Fra2 (Bozec et al., 2008). In addition, Fra2 knockout mice exhibit a reduction in chondrocyte (Karreth et al., 2004) and osteoblast differentiation (Bozec et al., 2010), while an increased differentiation to osteoblasts in the Fra2tg mice results in an osteosclerotic phenotype (Bozec et al., 2010).

Overexpression of Fra1 also leads to the development of osteosclerosis due to an accelerated osteoblast differentiation (Jochum et al., 2000) and a similar phenotype was described for mice overexpressing a naturally occurring splice variant of FosB (∆FosB) that lacks the C- terminal transactivation domain (Kveiborg et al., 2004; Sabatakos et al., 2000). For all three, Fra1, Fra2 and ∆FosB transgenic mice, the phenotype was reported to be due to a cell- autonomous increased osteoblast activity (Bozec et al., 2010; Jochum et al., 2000; Kveiborg et al., 2004; Sabatakos et al., 2000).

Like the Fra2 knockout mice, Fra1 deficient mice are osteopenic due to a decreased osteoblast activity (Eferl et al., 2004). No bone phenotype was described for FosB knockout mice (Zenz et al., 2008).

Deletion of c-Jun resulted in defects of the axial skeleton (Behrens et al., 2003). In addition, a role for c-Jun in promoting osteoclastogenesis was described (David et al., 2002). Mice lacking JunB develop osteopenia due to a decreased osteoblast activity (Kenner et al., 2004) and an increased bone formation occurs in JunD deficient mice (Kawamata et al., 2008).

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1.4.5 Functions of AP-1 in adipocyte development

Implications of AP-1 members in adipocyte development and function were also described. In particular, mice overexpressing ∆FosB display a reduced mass of adipose tissue (Kveiborg et al., 2004). Originally, overexpression of ∆FosB was shown to inhibit adipogenic differentiation in vitro, indicating a cell-autonomous defect within the adipocytes (Sabatakos et al., 2000). However, the decreased adipogenesis in vivo was later described to be caused by increased energy expenditure and insulin sensitivity (Rowe et al., 2009).

Recently, a reduced adipose tissue mass was described for JunB knockout mice. In these mice, no change in adipogenesis was observed as differentiation capacities of cells with a reduced level of JunB were not altered and the expression of marker genes for differentiation was not changed. Therefore, it was proposed that the reduced amount of fat is caused by a high lipolytic activity due to an increased expression of lipolytic enzymes (Pinent et al., 2011).

In addition to FosB and JunB, a possible role on adipocyte differentiation was also proposed for c-Jun, c-Fos, and Fra1, as adipogenic stimulation induced their expression in vitro (White and Stephens, 2009). However, no adipose-related phenotype has yet been described in vivo.

1.5 Aim of the work

A systemic co-control of adipose tissue and bone mass has been described. In addition, various transcription factors locally regulate the commitment between adipocytes and osteoblasts. The importance of the transcription factor AP-1 in mesenchymal stromal cell differentiation, especially for osteoblastogenesis, was reported in several mouse models. Mice with a deletion of Fra1 were described to be osteopenic, while overexpression of Fra1 resulted in progressive osteosclerosis as a result of accelerated osteoblast differentiation. A similar bone phenotype was observed in the ∆FosB transgenic mice. In addition, these mice develop a lipodystrophy and when overexpressed ∆FosB was shown to inhibit adipocyte differentiation.

The aim of this thesis was therefore to determine the in vivo potential role of Fra1 in adipogenesis. Due to the embryonic lethality of the complete Fra1 knockout, we tried to generate mice with an adipocyte specific deletion of Fra1 by crossing Fra1 floxed with Ap2- cre mice, which should express the cre recombinase in mature adipocytes. In addition, we made use of the mice ubiquitously overexpressing Fra1 under the H2k-promoter (Fra1tg

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performed in vitro differentiation experiments using Fra1 transgenic primary osteoblasts as well as an adipogenic cell line overexpressing Fra1.

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2. Materials and Methods

2.1 Materials

2.1.1 Buffer and staining solutions

Tail Preparation Buffer 50ml Tris-HCl (1M), pH 8.5 5ml EDTA (0.5M), pH 7.5-8.0

10ml SDS (10%)

20ml NaCl (5M)

dH2O ad 500ml, autoclave

add 10µl Proteinase K (20mg/ml) per 100µl buffer

Frackelton Buffer 5ml Tris-HCl, pH 7.5 (1M)

5ml NaCl (5M)

6.69g Na-Pyrophosphate

450ml dH2O

set pH to 7.05

5ml Triton X 100

Before use complete with (per ml):

1µl PMSF (200mM)

10µl Na3VO4 (10mM), pH 9.5 1µl Okadaic Acid (0.1mM)

Buffer A for cytosolic extraction 500µl HEPES (1M), pH 7.6

250µl KCl (2M)

10µl EDTA (0.5M)

50µl EGTA (0.1M)

37.5µl Spermidine (1M) 15µl Spermine (0.5M) dH2O ad 50ml

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Before use complete with (per 10 ml):

10µl DTT (1M)

50µl PMSF (0.1M)

50µl protease inhibitor cocktail 100µl Na2MoO4 (1M)

50µl Na3 VO4 (10mM), pH 9.5

Buffer B for nuclear extraction 1ml HEPES (1M), pH 7.6

4ml NaCl (5M)

100µl EDTA (0.5M)

500µl EGTA (0.1M) dH2O ad 50ml

Before use complete with (per 1ml):

1µl DTT (1M)

5µl PMSF (0.1M)

10µl protease inhibitor cocktail 10µl Na2MoO4 (1M)

5µl Na3VO4 (10mM), pH 9.5

Triton X 100 lysis buffer 150mM NaCl

(Immunoprecipitation) 1% Triton X 100 50mM Tris-HCl, pH 8

supplemented with:

1mM Na3VO4

1mM PMSF

1x protease inhibitor cocktail

SDS Sample Buffer (6x) 1.2ml Tris-HCl (0.5M), pH 7.2

4ml SDS (20%)

3.8ml Glycerol

510µl β-Mercaptoethanol

1% Bromophenol blue

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Stacking gel buffer 6.05g Tris-HCl

dH2O ad 100ml, adjust pH to 6.8, filter (0.45µm)

0.4g SDS

Running gel buffer 91g Tris-HCl

dH2O ad 500ml, adjust pH to 8.8, filter (0.45µm)

2g SDS

Stacking gel 0.75ml Stacking gel buffer

225µl Acrylamid (40%) 6.75µl SDS (10%)

7.5µl APS (10%)

4.68µl TEMED

2.19ml dH2O

Running gel 2ml Running gel buffer

2.4ml Acrylamid (40%)

80µl SDS (10%)

34.3µl APS (10%)

2.86µl TEMED

3.34ml dH2O

Running Buffer (5x) 15.1g Tris

72g Glycine

5g SDS

dH2O ad 1000ml

Blotting Buffer (10x) 11.62g Tris

5.86g Glycine

0.75g SDS

400ml Methanol dH2O ad 2000ml

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TBS (10x) 24.2g Tris

80g NaCl

dH2O ad 1000ml adjust pH to 7.6

TBS-T 1x TBS

0.1% Tween 20

Oil Red O 0.35g Oil Red O

100ml Isopropanol Before use:

dilute 3:2 in dH2O, incubate 20 min, filter (0.22µm)

2.1.2 Cell culture media and supplements

Complete medium 500ml α-MEM

10% FCS

1% Penicillin/ Streptomycin

Adipocyte differentiation medium 500ml α-MEM

10% FCS

1% Penicillin/ Streptomycin 5µg/ ml Insulin

1µM Dexamethasone

500µM IBMX

Calvaria isolation medium α-MEM

0.1% Collagenase 0.2% Dispase II

stirred for 3 hours, filtered (0.22µm)

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2.1.3 Oligonucleotides

Primers were ordered from Invitrogen.

Genotyping

H2K-fra-1-LTR fw 5´ GGG ATT AAA TGC ATG CCA AGC T 3´

rev 5´ CGA TCA CCA GAG ACC AAT CAG 3´

Fra1f/f fw 5´ GAAATGGCTCCGTGGGTAAAGGTA 3´

rev1 5´ GACAGGGTTCATCTTCATAGTTCT 3´

rev2 5´ TGTACCGGACGCTTGTCATCTCAT 3´

Ap2-cre fw 5´ GCG GTC TGG CAG TAA AAA CTA TC 3´

rev 5´ GTG AAA CAG CAT TGC TGT CAC TT 3´

qPCR

Adiponectin fw 5´ GTT GCA AGC TCT CCT GTT CC 3´

rev 5´ TCT CTC CAG GAG TGC CAT CT 3´

Ap2 fw 5´ TCA CCT GGA AGA CAG CTC CTC 3´

rev 5´ AAG CCC ACT CCC ACT TCT TTC 3´

C/ebpα fw 5´ TGG ACA AGA ACA GCA ACG AG 3´

rev 5´ CTG GTC AAC TCC AGC ACC TT 3´

C/ebpβ fw 5´ TTT CGG GAC TTG ATG CAA TC 3´

rev 5´ CCG CAG GAA CAT CTT TAA GG 3´

C/ebpδ fw 5´ GAA CAC GGG AAA GCA TGA CT 3´

rev 5´ CTT CGG CAA CCA CCT AAA AG 3´

c-Fos fw 5´ TGT GTT CCT GGC AAT AGC GTG T 3´

rev 5´ GGC AAT TCC GCC CAT AGT GA 3´

FosB fw 5´ ACC AGC TAC TCA ACC CCA G 3´

rev 5´ GGG TAA GTG TCT CTT CTC GGG 3´

Fosl1 fw 5´ GAG ACG CGA GCG GAA CAA G 3´

rev 5´ CTT CCA GCA CCA GCT CAA GG 3´

Fosl2 fw 5´ AGC CTC CCG AAG AGG ACA G 3´

rev 5´ AGG ACA TTG GGG TAG GTG AAG 3´

Gilz fw 5´ GTG GTG GCC CTA GAC AAC AAG 3´

rev 5´ TCA CAG CGT ACA TCA GGT GGT T 3´

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Glut4 fw 5´ GAA CAG CAG CCT GGG GAA CT 3´

rev 5´ GAG TCT GGG TAG GGG CAG GA 3´

Gr fw 5´ CAG CTC TGT TCC AGA CTC AGC ATG 3´

rev 5´ TAT CGC CTT TGC CCA TTT CAC TGC 3´

HPRT fw 5´ GTT AAG CAG TAC AGC CCA AA 3´

rev 5´ AGG GCA TAT CCA ACA ACA AAC TT 3´

Il-6 fw 5´ TGT GCA ATG GCA ATT CTG AT 3´

rev 5´ TCC AGT TTG GTA GCA TCC ATC 3´

Jun fw 5´ ACT CGG ACC TTC TCA CGT C 3´

rev 5´ CGG TGT AGT GGT GAT GTG CC 3´

JunB fw 5´ TCA CGA CGA CTC TTA CGC AG 3´

rev 5´ CCT TGA GAC CCC GAT AGG GA 3´

JunD fw 5´ CAT CGA CAT GGA CAC GCA AG 3´

rev 5´ CGG TGT TCT GGC TTT TGA GG 3´

Leptin fw 5´ TGA CAC CAA AAC CCT CAT CA 3´

rev 5´ TCA TTG GCT ATC TGC AGC AC 3´

MyoD fw 5´ GAT GAC CCG TGT TTC GAC TC 3´

rev 5´ AGT AGG GAA GTG TGC GTG CT 3´

Osx1 fw 5´ GGA GGC ACA AAG AAG CCA TAC GC 3´

rev 5´ TGC AGG AGA GAG GAG TCC ATT G 3´

Per1 fw 5´ CAA GGA CTT CAC CCA GGA AA 3´

rev 5´ GAT CCG GGG AGC TTC ATA AC 3´

Pgc-1α fw 5´ GTC AAC AGC AAA AGC CAC AA 3´

rev 5´ TCT GGG GTC AGA GGA AGA GA 3´

Pgc-1β fw 5´ CTT GCT TTT CCC AGA TGA GG 3´

rev 5´ CCC TGT CCG TGA GGA ACG 3´

Pparγ2 fw 5´ CTG ATG CAC TGC CTA TGA GC 3´

rev 5´ GGG TCA GCT CTT GTG AAT GG 3´

Pref1 fw 5´ CGT GAT CAA TGG TTC TCC CT 3´

rev 5´ AGG GGT ACA GCT GTT GGT TG 3´

Retn fw 5´ AGG GTG TGT GTG GGA ATT GT 3´

rev 5´ GGC CAG CCT GGA CTA TAT GA 3´

Runx2 fw 5´ TGT TCT CTG ATC GCC TCA GTG 3´

rev 5´ CCT GGG ATC TGT AAT CTG ACT CT 3´

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Sox9 fw 5´ CTG AAG GGC TAC GAC TGG AC 3´

rev 5´ TAC TGG TCT GCC AGC TTC CT 3´

Srebf1 fw 5´ GTG AGC CTG ACA AGC AAT CA 3´

rev 5´ GGT GCC TAC AGA GCA AGA GG 3´

Tnfα fw 5´ GCT GAG CTC AAA CCC TGG TA 3´

rev 5´ CGG ACT CCG CAA AGT CTA AG 3´

Ucp1 fw 5´ CTG CCT CTC TCG GAA ACA AG 3´

rev 5´ TGC ATT CTG ACC TTC ACG AC 3´

Sequencing

GLprimer2 5´ TGG AAG ACG CCA AAA ACA TAA AG 3´

RVprimer3 5´ GGG ACA GCC TAT TTT GCT AG 3´

Chromatin Immunoprecipitation

CHIP fw 5´ TTG CAG CGC AGG AGT CAG T 3´

rev 5´ TGA CTT TCC AAG GCG GTG AGT 3´

2.1.4 Antibodies

α-Tubulin mol. Probes A-11126

β-Actin (mouse), AC-15 Sigma A5441-2ml

Biotinylated anti-mouse IgG (H+L) Vector BA-9200

Biotinylated anti-rabbit IgG (H+L) Vector BA-1000

C/EBPα Santa Cruz Biotechnology sc-61

C/EBPβ (mouse) Santa Cruz Biotechnology sc-7962

C/EBPβ (rabbit) Santa Cruz Biotechnology sc-150

c-Jun Cell Signaling 9165

DYKDDDDK Tag = Anti-Flag M2 Cell Signaling 2368

Fra1 Santa Cruz Biotechnology sc-605

Insulin Santa Cruz Biotechnology sc-9168

Ki67 BD 550609

Mouse IgG HRP (goat) Promega W402B

p-p44/42 MAPK Cell Signaling 9101

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Rabbit IgG Vector I-1000

Rabbit IgG HRP (goat) Promega W401B

2.1.5 Plasmids pbabe, pbabe-Fra1

pMSCV, pMSCV-Fra1ER

pBluescript II KS +/- containing C/ebpα promoter and coding sequence (C. Calkhoven)

2.1.6 Molecular weight marker and loading dye

peqGOLD Prestained Protein Marker IV PeqLab 27-2110

Quick-Load 1 kb DNA Ladder NEB N0468S

GeneLadder 100bp plus 1.5kb Genaxxom M3094

Loading buffer DNA II Applichem A2571,0025

2.1.7 Reagents for cell culture Cell culture

α-MEM PAN Biotech GmbH P04-21500

Gibco 32561-029

DMEM Gibco 41965-039

Foetal Bovine Serum Gibco 10499-044

Penicillin/Streptomycin PAN Biotech GmbH P06-07100

Trypsin/EDTA 10x PAN Biotech GmbH P10-024100

Dulbecco’s PBS 1x Gibco 14190-094

Puromycin Invivogen ant-pr-1

Insulin Sigma 16634

IBMX Sigma I5879

Dexamethasone Sigma D2915

Estradiol Sigma E8875

Dispase II Sigma D4693-1G

Collagenase from Clostridium hisolyticum Type IA

Sigma C9891-1G

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Transfection

Opti-MEM I Reduced Serum Medium Gibco 31985

Lipofectamin 2000 Invitrogen 11668-027

Metafectene Pro Biontex T040

2.1.8 Kits and enzymes Plasmid-Preparation

QIAGEN Plasmid Midi Kit (100) Qiagen 12145

QIAGEN Plasmid Mini Kit (25) Qiagen 12123

QIAquick Gel Extraction Kit (50) Qiagen 28704

Cloning

Restriction enzymes:

PmlI NEB R0532

NruI NEB R0192

PstI NEB R0140

SacI NEB R0156

SmaI NEB R0141

T4 DNA Ligase, LC Fermentas EL0015

DNA Pol. Lg. Fragm. (Klenow) NEB M0210

Antarctic Phosphatase NEB M0289

Luciferase assay

Dual-Luciferase® Reporter Assay System Promega E1910

Passive Lysis Buffer, 5X Promega E1941

Immunoprecipitation and Western Blot

µMACS Protein A MicroBeads Miltenyi 130-071-001

Quick Start Bradford 1x Dye Reagent Bio-Rad 500-0205 Pierce ECL Western Blotting Substrate Thermo Fisher 32106 ReBlot Plus Stripping Solution, 10x Millipore 2504

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RNA-Isolation

Water, nuclease-free (30ml) Fermentas R0582

peqGold Trifast PeqLab 30-2020

peqGOLD RNAPure PeqLab 30-1020

RNAlater® Solution Ambion AM7021

TRIzol® Reagent Invitrogen 15596018

cDNA-Synthesis and RT-PCR

DNAse I Fermentas EN0521

GeneAmp® 10X PCR Buffer II & MgCl2 Applied Biosystems N8080130

GeneAmp® dNTP’s Applied Biosystems N8080007

MuLV Reverse Transcriptase Applied Biosystems N8080018 Oligo d(T)16 (50µM) Applied Biosystems N8080128

SYBR Green I-dTTP Eurogentec RT-SN2X-

03+WOUN

RNAse Inhibitor Applied Biosystems N8080119

ELISA and colorimetric assays for serum

Osteocalcin Tecomedical BT-470

Leptin Crystal Chem Inc. 90030

Insulin Crystal Chem Inc. 90080

NEFA-HR(2) R1 Wako 434-91795

NEFA-HR(2) R2 Wako 436-91995

NEFA Standard Wako 270-77000

Triglyceride Reagent Sigma T2449-10ml

Free Glycerol Reagent Sigma F6428-40ml

Glycerol Standard Solution Sigma G7793-5ml

Immunohistology

StreptABComplex/HRP Dako K0377

Liquid DAB + Substrate Dako K3468

MOM Vector BMK-2202

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Genotyping

AmpliTaq® DNA Polymerase Applied Biosystems N8080161

Taq DNA Polymerase Peqlab 01-1030

dNTPs Mix PCR 1 Roth L541.2

Proteinkinase K Invitrogen 25530031

additional kits

In Situ Cell Death Detection Kit Roche 11 684 795 001

Chip-IT Express active motif 53008

2.1.9 Chemicals

Acrylamide 40 % Roth T802.1

Agarose AppliChem A2114

Ampicillin Sigma A-9518

Ammonium peroxydisulphate Roth 9592

Bovine Serum Albumin Sigma A-9647

Bromophenol blue Roth T116

Chloroform Roth 7331.2

DEPC Roth K028

Dithiothreitol Roth 6908

DMSO Merck 802912

EDTA Roth 8040

EGTA Roth 3054

Eosin Sigma 318906

Ethanol pure Roth 5054

Ethanol denatured Roth K928

D(+)-Glucose Merck K4318837

Glycerol Roth 3783

Glycine Roth 3908.2

Hemalum Mayer Merck 1.09249.0500

HCl 37% Roth 4625

Hepes Roth 19105.4

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Hydrogen peroxide 30% Merck 1.07210.0250

Isopropanol Roth 9866.6

K(HPO4)2 Roth 3904

KCl Roth 6781

LB Agar Roth X965.2

LB Broth Roth X964.2

Lipofectamine Invitrogen 18324-012

Lumigen PS-3 Detection Reagent Amersham RPN 2132V2

Magnesium chloride hexahydrate Roth 2189.2

Magnesium sulphate heptahydrate Roth P027.1

Mercaptoethanol Roth 4227

Methanol Roth 4627

2-Methyl-2-butanol Sigma 15.246-3

Hematoxylin Solution, Mayer’s Sigma MHS1

Milk powder Blotting Grade Roth T145

Na2HPO4 Roth P030

NaH2PO4 2H20 Roth T879

Sodium bicarbonate Roth 6885.2

Na4P2O7(H2O)10 Roth T883

NaCl Roth 3957

Na-Cl solution 154 Berlin-Chemie 038115/01

Sodium hydroxide solution Roth K021

Nonidet P40 Roche 1754599

Okadaic acid Calbiochem 459618

Oil Red O Sigma 0-0625

PMSF Roth 6367

Ponceau S Applichem A2935

Protease Inhibitor Cocktail Sigma P8340

Roti-Histofix 4% Roth P087.3

Rotiphorese 50x TAE Buffer Roth CL86.2

Rotiphorese Gel 30 Roth 3029

SDS Pellets Roth CN30.2

Sodium orthovanadate Sigma S-6508

Spermine Fluka 85590

(38)

TEMED Roth 2367

TEMED Promega V 3161

TRIS Roth 4855

Triton X 100 Roth 3051

Tween 20 Roth 9127

Vectashield Mounting Medium Vector H 1200

Xylene Roth CN80.2

2.1.10 Consumables

Folded Filters Schleicher & Schuell 311647

Cell lifter Corning 3008

CL-XPosure Film (18 x 24 cm) Thermo Fisher 34089

µColumns (IP) Miltenyi 130-042-701

Contour Test Strips Bayer 06707326

MicroAmp Optical 96-Well Reaction Plate Applied Biosystems N8010560 MicroAmp Optical Adhesive Film Applied Biosystems 4311971

Nitrocellulose membrane Bio-Rad 162-0112

Precellys Steel Kit 2.8 mm PeqLab 91-PCS-MK28

Precellys Ceramic Kit 1.4 mm PeqLab 91-PCS-CK14

Specimen Molds Cryomold Sakura 4566/4557

O.C.T. Compound Sakura 4583

Microtainer SST Tubes BD 365951

Filter Tip 10-1000µl, Flasks, dishes, plates Greiner Bio One

2.1.11 Laboratory Instruments

Biological Safety Cabinet Nu-440-600E Nuair

Blotting machine Multiphor II GE Healthcare

Centrifuge Heraeus Fresco 17 Thermo Electron Corporation Centrifuge Rotina 420R Hettich Zentrifugen

Colour digital camera DP72 Olympus Colour digital camera DS-5Mc Nikon

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Electrophoresis System BioRad Mini-PROTEAN Tetra Cell

Electrophoresis System C.B.S. Scientific Co.

Glucometer Contour Bayer

Homogeniser Precellys 24 Peqlab

Incubator Heraeus Instruments

Sirius Luminometer Berthold Detection Systems MACS MultiStand, µMACS Separator Miltenyi

Mausinjektionskäfig Typ A G&P Kunststofftechnik

Metabolic cages Tecniplast

Microscope Nikon Eclipse 80i Nikon

Microscope DMIL Leica

Microtome SLIDE 2003 mpö pfm

pH meter HI 9321 HANNA Instruments

Photometer BioPhotometer Eppendorf

Real-time PCR Cycler Applied Biosystems 7300 Real-time PCR System

Thermocycler Biometra T Professional Biometra

Thermocycler GeneAmp®PCRSystem9700 Applied Biosystems Thermocycler Mastercycler Eppendorf

Thermomixer comfort Eppendorf

(40)

2.2 Methods

2.2.1 Animal experiments Animals

The Fra1 (H2-fra-1-LTR) transgenic mice (Jochum et al., 2000) had been back-crossed into C57BL/6 background by 9 successive crossings. To generate Ap2-cre Fra1f/f mice, Fra1f/f mice of a mixed background (C57BL/6 crossed with SV129) (Eferl et al., 2004) were crossed with Ap2-cre mice (The Jackson laboratory, B6.Cg-Tg(Fabp4-cre)1Rev/J, 005069).

For high fat diet-experiments, mice were fed with a diet from Research Diets/Broogarden (D12331) ad libitum for 6 weeks.

Genotyping

Tail biopsies were digested in lysis buffer at 55°C, 700rpm over night. After inactivation (95°C, 15 minutes), 1:10 diluted supernatants were used for PCR reactions. For genotyping of Fra1tg, Fra1f/f and Fabp4-cre the following PCR mix using Taq-Polymerase (Peqlab) was used:

10x PCR Buffer 2.5µl

dNTPs (10 mM) 2.5µl

forward primer (10 µM) 2.5µl reverse primer (10 µM) 2.5µl Taq Polymerase 0.125µl

H2O 13.875µl

DNA 1 or 2µl

PCR programs:

Fra1tg Fraf/f Ap2-cre

initial denaturation 94 °C 5 min

94°C 2 min

94°C 3 min denaturation 94 °C

30 sec

94°C 40 sec

94°C 30 sec

annealing 58 °C

40 sec

58°C 40 sec

52°C 1 min

extension 72 °C

1 min

72°C 1 min

72°C 1 min

No. of cycles 40 41 40

final extension 72°C

10 min

72°C 2 min

product size 1200 bp 308 bp (wt)

354 bp (floxed)

100 bp

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