Umeå University Odontological Dissertations, New Series No: 132

CD47–SIRPα: an Interaction of Importance for Bone Cell Differentiation

Cecilia Koskinen

Department of Odontology Umeå University Umeå 2014

Responsible publisher under swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7601-000-6 ISSN: 0345-7532 Cover picture: Osteoclasts and stromal cells combined in a co-culture Electronical version availble at http://umu.diva-portal.org/ Printed by: Print & Media Umeå, Sweden 2014

To my family

“Perplexity is the beginning of knowledge”

Khalil Gibran

Table of Contents

Abstract iii Populärvetenskaplig sammanfattning på svenska v List of Publications vii Abbreviations viii 1 Introduction 1 1.1 Bone tissue 1 1.2 CD47 11 1.3 SIRPα 12 1.4 The interaction between CD47 and SIRPα 14 1.5 PI3K–Akt signaling pathway 16 2 Aims 18 3 Methods 19 3.1 Animals 19 3.2 Isolation and culture of bone marrow stromal (BMS) cells 19 3.3 Isolation and culture of osteoclasts 20 3.4 Co-cultures of BMM and BMS cells 20 3.5 Gene expression analysis 21 3.6 Protein expression analysis 22 3.7 Flow cytometry 23 3.8 Histomorphometry analysis 23 3.9 Peripheral quantitative computed tomography (pQCT) 23 3.10 Statistical analysis 23 4 Results and Discussion 25 4.1 Osteoclast formation in vitro in the absence of CD47–SIRPα signaling 25 4.2 Histomorphometry analyses of femurs from CD47-deficient mice 26 4.3 Osteoclast differentiation in vitro in the absence of CD47–SIRPα signaling 27 4.4 Stromal cell/osteoblast differentiation in the absence of CD47 30 4.5 Signaling downstream of SIRPα in stromal cells upon activation by CD47 31 4.6 Co-cultures and osteoclastogenesis 33 5 Conclusions 36 6 Acknowledgements 37 7 References 40

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Abstract Bone tissue is continuously remodeled by bone-forming osteoblasts and bone-resorbing osteoclasts, in processes tightly regulated by hormones, cytokines and growth factors. CD47, a ubiquitously expressed protein, and one of its ligands, signal-regulatory protein alpha (SIRPα), are two cell- surface proteins belonging to the immunoglobulin (Ig)-superfamily. The interaction between CD47 and SIRPα is important for, amongst other processes, the fusion of macrophages into giant cells, which are closely related to osteoclasts.

The aim of the present study was to gain knowledge about the role of CD47– SIRPα interaction and resultant downstream signaling pathways in bone cell differentiation, formation and function.

The addition of antibodies against CD47 or SIRPα inhibited the formation of multinucleated osteoclasts from bone marrow monocytes (BMMs) in culture. Moreover, a significant decrease in the number of osteoclasts was detected in CD47-/- BMM cultures compared to CD47+/+ cultures. In line with these in vitro results, we found fewer osteoclasts in vivo in the trabecular bone of CD47-/- mice, as compared to CD47+/+ bone. Interestingly, an extended analysis of the trabecular bone of CD47-/- mice revealed that the bone volume, mineralizing surface, mineral apposition rate, bone formation rate and osteoblast number were also significantly reduced compared with CD47+/+ mice, indicating the importance of CD47 in osteoblast differentiation. In vitro studies of bone marrow stromal (BMS) cells from CD47-/- mice or SIRPα-mutant mice (mice lacking the signaling domain of SIRPα) showed a blunted expression of osteoblast-associated genes. Moreover, these altered genotypes were associated with reduced activity of the bone mineralization-associated enzyme alkaline phosphatase as well as a reduced ability to form mineral. To reveal the molecular mechanisms by which CD47 activation of SIRPα is important for BMS cell differentiation, we studied signaling downstream of SIRPα in the absence of CD47. In BMS cells lacking CD47, a considerable reduction in the levels of tyrosine phosphorylated SIRPα was detected, and the subsequent recruitment of the Src-homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP-2)–phosphoinositide 3-kinase (PI3K)–Akt2 signaling module was nearly abolished.

In conclusion, the interaction between CD47 and SIRPα results in the activation of the SHP-2–PI3K–Akt2 pathway, which is necessary for normal osteoblast differentiation. In CD47-/- mice and SIRPα-mutant mice, this

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interaction is perturbed, which prevents normal osteoblast differentiation and subsequent mineral formation. In addition, the altered BMS cell phenotype results in an impaired ability to stimulate osteoclast differentiation.

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Populärvetenskaplig sammanfattning på svenska Vårt skelett har flera viktiga funktioner såsom att skydda inre organ och möjliggöra kroppens rörelser genom att muskler fäster i skelettet. Dessutom lagrar skelettet kalicum som kan utvinnas när det behövs i kroppen och i skelettet återfinns också benmärgen. Benvävnaden är en del av skelettet och den byggs om och förnyas under hela vår livstid. Ombyggnaden av benvävnaden utförs av bennedbrytande celler kallade osteoklaster och benbildande celler kallade osteoblaster. Cellernas utmognad och aktivitet styrs av olika signaler så som hormoner, tillväxtfaktorer och vid belastning av skelettet. Under friska förhållanden bygger osteoblasterna upp den benvävnad som osteoklasterna brutit ner så att jämnvikt råder. Vid olika sjukdomstillstånd som påverkar skelettet t ex parodontit (tandlossning), osteoporos (benskörhet) och reumatoid artrit (ledgångsreumatism) är samspelet mellan osteoklaster och osteoblaster satt ur spel, oftast med en minskad benmängd som resultat. I strävan efter att förstå vilka cellsignaler som negativt påverkar cellernas bildning och aktivitet vid sjukdom är det av yttersta vikt att förstå vilken cellsignaleringen som råder under friska förhållanden.

Signal regulatory protein α (SIRPα) och CD47 är signalerande proteiner som finns på cellens yta. De har förmågan att binda till och samverka med varandra. Samverkan mellan SIRPα och CD47 har bland annat visat sig vara viktig för fusion av makrofager (storätar-celler), en typ av celler som är närbesläktade med osteoklaster.

Avhandlingens syfte har varit att undersöka betydelsen av CD47 och SIRPα för skelettcellers utveckling och funktion.

För att studera CD47 och SIRPαs betydelse för osteoklastbildning odlades osteoklastförstadieceller från benmärg både från normala möss (CD47+/+) och från möss som saknar genen som kodar för CD47-proteinet (CD47-/-), vilket gör att dessa celler inte kan bilda CD47-protein. I CD47-/--odlingar bildades det färre osteoklaster jämfört med i CD47+/+-odlingar. Dessutom fann vi ett lägre uttryck av gener som kodar för proteiner kopplade till osteoklastutmognad i CD47-/--odlingar jämfört med i CD47+/+-odlingar. Undersökning av skelettet i lårbenen från CD47-/- möss avslöjades att även där var antalet osteoklaster färre jämfört med i lårbenen från CD47+/+-möss. Ytterligare analyser av lårbensskelett hos CD47-/--möss visade dessutom att CD47-/--möss hade lägre benvolym, lägre benbildnings förmåga och färre osteoblaster än CD47+/+-möss. För att studera CD47 och SIRPα’s betydelse för osteoblaster odlades osteoblastförstadieceller från benmärg från SIRPα- mutant möss (saknar den signalerande delen av proteinet) och CD47-/--möss v

följt av analyser av gener som kodar för proteiner kopplade till osteoblastutveckling. Dessutom utvärderades osteoblasternas förmåga att bilda mineral. Både i SIRPα-mutant och CD47-/- osteoblastodlingar återfanns ett lägre uttryck av viktiga osteoblastgener och proteiner samt en försämrad förmåga att bilda mineral i jämförelse med normala osteoblastodlingar. Utöver detta visade det sig CD47–SIRPα-samverkan vara viktig för osteoblasternas förmåga att frisätta osteoklast-stimulerande proteiner.

För att utreda hur CD47–SIRPα-samverkan kan påverka skelettcellernas utmognad undersöktes hur signalsystemet från SIRPα-proteinet in i cellen fungerar. I normala osteoblaster aktiverade CD47 SIRPα och den intra- cellulära delen fosforylerades som i sin tur rekryterade src homology-2 domain containing protein tyrosine phosphatase (SHP-2). SHP-2 aktiverade enzymet phosphoinositide 3-kinase (P3IK) och efterföljande Akt2. I CD47-/-- osteoblaster där aktiveringen av SIRPα fallerade skedde en avsevärt lägre aktivering av SHP-2 och PI3K–Akt2.

Sammanfattningsvis, under normala förhållanden aktiverar CD47 SIRPα som leder till SHP-2–PI3K–Akt2- signalering, resulterande i normal utmognad av osteoblaster. Då interaktionen mellan CD47 och SIRPα saknas blir osteoblastdifferentieringen störd och till följd av detta hämmas mineralbildningen. Den förändrade osteoblastdifferentieringen leder också till försämrad kapacitet att understödja osteoklastbildning.

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List of Publications

The thesis is based on the following papers, which will be referred to by their roman numbers (I–III):

I. Pernilla Lundberg, Cecilia Koskinen, Paul A. Baldock, Hanna Löthgren, Åsa Stenberg, Ulf H. Lerner, Per-Arne Oldenborg. Osteoclast formation is strongly reduced both in vivo and in vitro in the absence of CD47/SIRPα-interaction. Biochemical and Biophysical Reserach Communication 352 (2007) 444-448

II. Cecilia Koskinen, Emelie Persson, Paul Baldock, Åsa Stenberg, Ingrid Boström, Takashi Matozaki, Per-Arne Oldenborg, Pernilla Lundberg. Lack of CD47 Impairs Bone Cell Differentiation and Results in an Osteopenic Phenotype in vivo due to Impaired Signal Regulatory Protein α (SIRPα) Signaling. The Journal of Biological Chemistry 2013, 288:29333-29344

III. Cecilia Koskinen, Sara Engman, Rima Sulniute, Takashi Matozaki, Per-Arne Oldenborg, Pernilla Lundberg. Reduced SIRPα phosphorylation and concommitant SHP-2–PI3K–Akt2 signaling decrease osteoblast differentiation. Manuscript.

Reprints were made with kind permission from the publishers.

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Abbreviations α-MEM Alpha minimal essential medium ALP Alkaline phosphatase BMC Bone marrow cell BMM Bone marrow monocyte BMP Bone morphogenetic protein BMS Bone marrow stromal BMU Basic multicellular unit/bone metabolic unit c-Fms Colony Stimulating factor 1 receptor CTX C-terminal telopeptide of collagen type I

D3 1α,25(OH)2-vitamin D3 DAP12 DNAX activation protein 12 DC Dendritic cell DC-STAMP Dendritic cell-specific transmembrane protein ECM Extracellular matrix IAP Integrin-associated protein IGF Insulin-like growth factor IL Interleukins ITAM Immunoreceptor tyrosine-based activation motif ITIM Immunoreceptor tyrosine-based inhibitory motif JAK Janus kinase M-CSF Macrophage-colony stimulating factor MAPK Mitogen-activated protein kinase micro-CT Micro computed tomography MSC Mesenchymal stem cell NFATc1 Nuclear factor of activated T-cells, cytoplasmic 1 NFκB Nuclear factor kappa B NO Nitric oxide OPG Osteoprotegrin PBS Phosphate buffered saline pQCT Peripheral quantitative computed tomography PTH Parathyroid hormone PI3K Phosphoinositide 3-kinase RANK Receptor activator of nuclear factor κB RANKL Receptor activator of nuclear factor κB ligand RTqPCR Quantitative real-time reverse transcription polymerase chain reaction Runx2 Runt-related transcription factor 2 SH2 Src-homology-2 SHP-2 SH2 domain-containing protein tyrosine phosphatase-2 SIRP Signal-regulatory protein SP Surfactant TGFβ Transforming growth factor beta TRAP Tartrate acid phosphatase TSP Thrombospondin

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INTRODUCTION

1 Introduction 1.1 Bone tissue 1.1.1 Function The human skeleton is a rigid and strong organ composed of cartilage and bone, a highly specialized connective tissue. The bones of the skeleton provide structural support for the rest of the body and enable the attachment of skeletal muscles, joints, tendons and ligaments to thereby facilitate movement of the body. Furthermore, bones protect the inner organs, contain bone marrow and act as a mineral deposit, which is necessary for maintaining mineral homeostasis in the body (reviewed in Seeman and Delmas 2006; Clarke 2008).

1.1.2 Structure and composition 1.1.2.1 Structure The skeleton is composed of approximately 80% cortical bone and 20% trabecular bone. Cortical bone, also called compact bone, is a dense and solid structure, covered by the periosteum, and is mainly found in long bones that surround the bone marrow. Trabecular bone (also known as spongy or cancellous bone) has a honeycomb-like morphology. It is usually found at the ends of long bones, although islands of trabecular bone can be found interspersed in the bone marrow cavity. All the mineralized bone (both cortical and trabecular) is covered by osteoid, a non-mineralized bone tissue. Trabecular bone has a higher bone surface area per unit than cortical bone and therefore shows a higher rate of surface-dependent events, including remodeling and metabolic activity (reviewed in Clarke 2008).

1.1.2.2 Composition Both cortical and trabecular bone tissue comprise a mixture of organic (bone proteins) and inorganic (mineral) components. Between 50% and 70% of bone tissue is constituted by mineral, which provides mechanical rigidity and strength. The major inorganic component is hydroxyapatite

[Ca10(PO4)6(OH2)], but small amounts of carbonate, magnesium and acid phosphate are also detected in the inorganic matrix. The organic matrix, which provides elasticity and flexibility, is composed of about 85–90% of collagenous proteins, mostly type I collagen. The remaining part of the organic matrix is made up of non-collagenous proteins comprising proteoglycans (e.g. aggrecan, versican, decorin), glycoproteins (e.g. alkaline phosphatase, osteonectin, bone sialoprotein), glycoproteins with cell- attachment activities (e.g. thrombospondins, vitronectin, fibronectin) and γ- carboxylated proteins (e.g. osteocalcin). Moreover, growth factors such as transforming growth factor beta (TGFβ), bone morphogenetic proteins 1

INTRODUCTION

(BMPs) and insulin-like growth factors (IGFs) have been found to be deposited in the bone matrix (reviewed in Clarke 2008).

Non5collagenous( (protein(( Cor$cal(bone( Organic(( ( matrix( Collagen( type(I( ((( (

Trabecular( bone( In5organic(( matrix( (mineral)(

Figure 1. Structure and composition of cortical and trabecular bone.

1.1.3 Bone cells, bone modeling and remodeling Bone tissue is continuously reshaped, repaired and reconstructed to maintain its structural composition and ensure mineral homeostasis. Hormones (e.g. estrogen and glucocorticoids), the central nervous system (Lerner and Persson 2008), mechanical forces (Lee, O'Brien et al. 2006) and local factors (Clarke 2008) stimulates osteoblasts and osteoclasts, the cells responsible for bone modeling and remodeling processes.

Osteoblasts synthesize bone matrix and regulate its mineralization, but are also capable of differentiating into osteocytes or bone-lining cells (Hill 1998). Osteoclasts, by contrast, degrade bone tissue, although they are needed both for the formation of the skeleton and the regulation of bone mass. Bone modeling is the process of shaping or modulating the bone tissue, and is performed by osteoblasts; unlike remodeling, it does not necessarily involve osteoclast activity and bone resorption. Bone remodeling requires both osteoclasts and osteoblasts, in a complex, balanced and well- regulated process. Both bone modeling and remodeling occur throughout life (Seeman and Delmas 2006). Bone degrading diseases such as osteoporosis, osteopetrosis, rheumatoid arthritis or periodontitis cause disturbance or imbalance in the remodeling process and promote either osteoclast or osteoblast differentiation and/or activity, resulting in altered bone volume.

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INTRODUCTION

Mesenchymal+stem+cell+ Hematopoe0c+stem+cell+

Preosteoblast+ Preosteoclast+

Osteoblast+ Osteoclast+ Bone+lining+cells+ Osteoid+ Mineralized+0ssue+ Osteocytes+

Figure 2. Schematic illustration of bone cells and their origin.

1.1.3.1 Osteoblasts Osteoblasts, the cells responsible for synthesizing bone tissue, derive from multipotent mesenchymal stem cells (MSCs) (Pittenger, Mackay et al. 1999). In response to the activation of cell-lineage specific transcription factors, MSCs differentiate into stromal cells, including osteoblasts, and other cells of mesenchymal origin — for example, fibroblasts, chondrocytes or adipocytes. Runt-related transcription factor 2 (Runx2) and Osterix have been identified as master regulatory transcription factors of osteoblastogenesis, since neither Runx2-deficient nor osterix -deficient mice form mature osteoblasts (Otto, Thornell et al. 1997; Nakashima, Zhou et al. 2002).

Mature osteoblasts are preceded by preosteoblasts — cells that resemble the osteoblasts but lack the ability to form mineral. Active osteoblasts are characterized by their cuboidal morphology, mineral formation capacity and the strong expression of alkaline phosphatase (ALP), an enzyme essential for mineral deposition (Stein and Lian 1993). Osteoblasts also have the ability to express and release bone matrix proteins such as type I collagen and osteocalcin, a calcium-binding protein. In addition, osteoblasts express receptors for the calcium-homeostasis-regulating hormones parathyroid hormone (PTH) and 1α,25(OH)2-vitamin D3 (D3) (Horwood, Elliott et al. 1998). Moreover, osteoblasts express and release macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL), two cytokines that are essential for osteoclast proliferation, differentiation and survival. Osteoblasts also release osteoprotegrin (OPG), a soluble decoy receptor that controls the amount of

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INTRODUCTION

RANKL available to bind to receptor activator of nuclear factor κB (RANK) (Teitelbaum and Ross 2003).

1.1.3.1.1 Intracellular signaling in osteoblasts Osteoblast differentiation from MSCs, requires cell activation via systemic factors including osteotropic hormones (e.g. intermittent PTH, D3, sex steroids) and neuropeptides (Lundberg, Bostrom et al. 1999), and local factors including Wnt proteins (Day, Guo et al. 2005), growth factors (e.g. BMPs (Li and Cao 2006)), cell–extracellular matrix (ECM) interactions and cell–cell interactions (Marie 2009; Marie 2012).

Several different intracellular signaling pathways are involved in osteoblast differentiation and activation, stimulating or inhibiting further cellular events through the activation of different transcription factors. For example, PTH binding to its receptor induces G-protein signaling, leading to increased osteoblast differentiation via enhanced Runx2 expression. BMP-2 and BMP- 7 have been shown to be very potent in stimulating osteoblastogenesis (Li and Cao 2006). They exert their effect by binding to BMP receptors coupled to Smads. C-Smads and R-Smads form transcription factor complexes that are able to co-operate with Runx2 to induce osteoblast differentiation (Lee, Kim et al. 2000; Zhang, Yasui et al. 2000). Activation of BMP-2 can also induce PI3K–Akt2 signaling, resulting in increased Runx2 expression (Mukherjee, Wilson et al. 2010). Binding of extracellular Wnt3a and Wnt10b to the Frizzled receptor and lipoprotein related protein 5 and 6 (LRP5/6) activates the canonical pathway, enabling the protein β-catenin to enter the cell nucleus and mediate gene transcription (Day, Guo et al. 2005). The Wnt pathway in combination with heparin may also engage PI3K–Akt, resulting in altered Runx2 expression (Ling, Dombrowski et al. 2010). Moreover, PI3K–Akt signaling and extracellular signal-regulated kinase (ERK) 1/2, a mitogen-activated protein kinase (MAPK), can be activated when integrins (α5β1, α2β1, α4β1) physically connect osteoblasts to the ECM (reviewed in Marie 2009).

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INTRODUCTION

BMP$ Growth$$ factors$ BMPR$ $ Wnt Smad$ PTH$ Frizzled$$ LRP5/6$ +Heparin$ G2$ protein$ cAMP$

PI3K$ Akt$ β2catenin$ MAPK$ Runx2$

ECM$ Integrin$ ECM$ Integrin$

Osteoblast$survival,$ proliferaMon$and$differenMaMon$

Figure 3. Schematic illustration of cellular signaling in osteoblasts. There are several signaling pathways of importance for osteoblast differentiation and activation. Lipoprotein related protein 5 and 6 (LRP5/6), bone morphogenetic protein (BMP), mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K) and integrins–extra cellular matrix (ECM) exert their action through regulation of the transcriptions factor runt-related transcription factor 2 (Runx2).

1.1.3.2 Bone-lining cells and osteocytes About 50–70% of mature osteoblasts undergo apoptosis after involvement in bone formation (Lynch, Capparelli et al. 1998). The remaining cells become inactivate bone-lining cells or differentiate into osteocytes. As osteoblasts become inactive, their morphology changes and they exhibit a thin and elongated shape. These cells are known as bone-lining cells and they cover the majority of the bone surface that is not undergoing remodeling. It is proposed that the bone-lining cells participate in the remodeling process by uncovering the mineralized bone and by forming a canopy over the remodeling area (Chambers and Fuller 1985; Hauge, Qvesel et al. 2001; Andersen, Sondergaard et al. 2009). Osteoblasts that are incorporated in the newly formed bone matrix within lacunas differentiatie into osteocytes. They have long filopodial processes that enable contact with other osteocytes as well as with bone-lining cells and osteoblasts. Osteocytes have been proposed to function as mechanosensors, converting mechanical skeletal force into biological signals (Cowin, Moss-Salentijn et al. 1991). Osteocytes are also suggested to be involved in osteoclastogenesis via the release of RANKL (Nakashima, Hayashi et al. 2011). Furthermore, osteocytes release sclerostin, a bone-formation inhibitor that impedes canonical Wnt pathway 5

INTRODUCTION signaling in osteoblasts, resulting in impaired differentiation and mineralization (Li, Zhang et al. 2005). Sclerostin has also been shown to promote osteoclast formation and activity (Wijenayaka, Kogawa et al. 2011).

1.1.3.3 Osteoclasts Osteoclasts are multinucleated cells derived from the monocyte/macrophage cell line of hematopoetic stem cells (Udagawa, Takahashi et al. 1990) that have the ability to decalcify and degrade the bone matrix. The differentiation of osteoclasts is referred to as osteoclastogenesis (Boyle, Simonet et al. 2003).

1.1.3.3.1 Intracellular signaling pathways in osteoclast differentiation Several different factors regulate the process of osteoclast differentiation and subsequent fusion. Osteoblasts, bone marrow stromal (BMS) cells and T- lymphocytes all release or express cytokines that are necessary for promoting osteoclastogenesis. M-CSF, which is produced by osteoblasts and stromal cells, is required for the survival and proliferation of osteoclast precursors. The critical role of M-CSF in osteoclastogenesis was demonstrated in M-CSF-deficient osteopetrotic mice (op/op) (Suda, Tanaka et al. 1993), which lack functional osteoclasts. This defect was corrected by M-CSF administration in vivo (Hattersley, Owens et al. 1991; Suda, Tanaka et al. 1993). Expression of the tyrosine kinase transmembrane receptor for M-CSF, c-Fms, is induced by the early transcription factors PU.1 and microphthalmia-associated transcription factor (MITF). Upon M-CSF binding, c-Fms, autophosphorylates and elicits downstream signaling through ERK1/2 and PI3K–Akt pathways, regulating the proliferation, differentiation and survival of osteoclasts and their precursors (Ross and Teitelbaum 2005).

Osteoblasts, T-cells and endothelial cells are capable of expressing and releasing RANKL, the key cytokine that mediates osteoclastogenesis (Lacey, Timms et al. 1998; Yasuda, Shima et al. 1998). Osteoblastic RANKL expression is stimulated by PTH, D3, tumor necrosis factor alpha and various interleukins (e.g. IL-1, IL-6, IL-11, IL-17, IL-20) (reviewed in Souza and Lerner 2013). The osteoclast precursors that have proliferated in respons to M-CSF express RANK, and subsequent binding by RANKL induces commitment to the osteoclastic cell phenotype, which correlates with the expression of tartrate acid phosphatase (TRAP), a key osteoclast marker. Upon further stimulation by both M-CSF and RANKL, the preosteoclasts fuse and form multinucleated giant cells (Boyle, Simonet et al. 2003).

Activation of RANK by RANKL initiates an intracellular signaling cascade mediated by TRAF6, an adapter molecule essential for osteoclastogenesis 6

INTRODUCTION

(Naito, Azuma et al. 1999). TRAF6 activates nuclear factor kappa B (NFκB), a transcription factor that plays an important role in osteoclast differentiation (Franzoso, Carlson et al. 1997), either through a non- canonical or canonical pathway (Souza and Lerner 2013). RANK also activates another important transcription factor complex through TRAF6; AP-1 (Asagiri and Takayanagi 2007), which partly consists of c-Fos (Grigoriadis, Wang et al. 1994). Moreover, it has been shown that RANKL, via NFκB and AP-1, induces the expression of nuclear factor of activated T- cells, cytoplasmic 1 (NFATc1), a transcription factor crucial for osteoclast differentiation (Takayanagi, Kim et al. 2002).

Although RANKL is the master cytokine of osteoclastogenesis, additional co-stimulatory pathways ensure osteoclast differentiation. Several small transmembrane adapter proteins (e.g. DAP12 (DNAX activation protein 12) and FcRγ) and their associated receptors (e.g. triggering receptor expressed on myeloid cells 2 (TREM2) and osteoclast-associated immunoglobulin-like receptor (OSCAR)) are engaged in co-stimulation, which initiates signaling via immunoreceptor tyrosine-based activation motifs (ITAMs) (reviewed in Long and Humphrey 2012; Souza and Lerner 2013). One known signaling pathway initiated via co-stimulatory proteins is the calcium–calmodulin– calcineurin pathway that mediates the activation of NFATc1 (Takayanagi 2007).

During the final step of osteoclastogenesis, preosteoclasts fuse to form multinucleated cells. Several proteins, such as dendritic-cell-specific transmembrane protein (DC-STAMP) and osteoclast stimulatory transmembrane protein (OC-STAMP) are involved in the fusion event (Oursler 2010). Besides the ability to resorb bone tissue, mature osteoclasts are characterized by several osteoclast-associated markers such as TRAP, calcitonin receptor, cathepsin K and β3-integrin (Boyle, Simonet et al. 2003). The genes encoding these proteins are directly regulated by NFATc1 or by NFATc1 in complex with other transcription factors (reviewed in Takayanagi 2007).

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INTRODUCTION

Osteoblast/stromal&cell&

RANKL& RANK& M+CSF& ?" c+Fms& TRAF6& P& OSCAR/TREM2&

FCRγ/" PI3K& Grb2& MITF& NFκB& AP+1& DAP12" Akt& ERK& 2+ " (Ca ) i" Calmodulin"

PU.1& Calcineurin"

NFATc1&

auto+& amplificaDon&

Cell&survival,&proliferaDon,&& inhibiDon&of&apoptosis& Osteoclast&differenDaDon&

Figure 4. Schematic illustration of signaling pathways in osteoclasts. Macrophage-colony stimulating factor (M-CSF), receptor activator of nuclear factor κB (RANK), RANK ligand (RANKL), phosphoinositide 3-kinase (PI3K), growth factor receptor-bound protein 2 (GrB2), extracellular signal- regulated kinase (ERK) microphthalmia-associated transcription factor (MITF), nuclear factor kappa B (NFκB), osteoclast-associated immunoglobulin-like receptor (OSCAR), triggering receptor expressed on myeolid cells 2 (TREM2), DNAX activation protein 12 (DAP12), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) are all involved in intracellular osteoclastic signaling.

1.1.3.4 The remodeling cycle Bone remodeling occurs in restricted areas, known as resorption loci, through the concerted action of bone cells aided by a capillary blood supply in a basic multicellular unit/bone metabolic unit (BMU). Briefly, a temporary BMU is formed, resorption of old bone occurs, and new bone is formed. The BMU subsequently dissolves, leaving inactive osteoblasts positioned on the newly formed bone. The signaling events preceding determination of the BMU and the resorption loci are complex and not yet fully understood (reviewed in Raggatt and Partridge 2010). The average length of the remodeling cycle in cancellous bone is about 200 days, with approximately 150 days devoted to bone formation (Eriksen, Gundersen et al. 1984; Eriksen, Melsen et al. 1984).

1.1.3.4.1 Initiation The process of remodeling begins with the degradation of the osteoid. During the initial step, bone-lining cells become activated and regain a rounded shape due to stimulation by various factors such as hormones (PTH,

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INTRODUCTION

D3), growth factors, cytokines as well as osteocytic signaling (Raggatt and Partridge 2010). It is proposed that the bone-lining cells are stimulated by the same signals necessary for osteoclast formation (Chambers and Fuller 1985). Activated bone-lining cells and osteoblasts secrete proteolytic enzymes (e.g. matrix metalloproteinases) responsible for osteoid degradation. The uncovering of mineralized bone is followed by the formation of a canopy over the remodeling area/BMU, which creates a good microenvironment for further bone resorption (Hauge, Qvesel et al. 2001; Andersen, Sondergaard et al. 2009)

The initial step is followed by osteoblastic recruitment of mononuclear osteoclast progenitors from the endosteum and periosteum by various chemoattractants such as osteocalcin, type I collagen (Malone, Teitelbaum et al. 1982) and the more recently discovered monocyte chemoattractant protein-1 (MCP-1) (Li, Qin et al. 2007). The precursors proliferate, differentiate and finally become active osteoclasts with bone-resorbing capacity due to M-CSF and RANKL stimulation.

1.1.3.4.2 Resorption When mature osteoclasts reach the site of resorption, their cytoskeletons are re-organized and they attach to the extracellular bone matrix via αvβ3- integrins. Attachment to the bone matrix induces the formation of actin rings that face the resorption site and create a sealing zone. The plasma membrane becomes polarized, enabling the formation of ruffled borders as well as a functional secretory domain. The ruffled borders are considered to be the actual resorbing organ and they secrete large amounts of acid and proteolytic enzymes into the resorption lacunae (known as Howship’s lacunae). Acidification of the resorption lacunae results in dissolution of the bone tissue mineral, and is performed either via v-type ATPase pumps (proton pumps) delivering hydrogen ions to the sealed zones, or via the secretion of acidic vesicles that fuse with the cell membrane in the ruffled border. Alongside, or shortly after, the secretion of acidic vesicles, the major matrix- degrading protein cathepsin K is released into the resorption lacunae. Together with metalloproteinases and TRAP, cathepsin K degrades the organic bone matrix. The degradation products are endocytosed into the osteoclast, transported through the cell and secreted via the functional secretory domain into the extracellular fluid (reviewed in Vaananen 2005).

1.1.3.4.3 Formation/Mineralization The resorption phase is followed by the apoptosis of osteoclasts or their emigration from the area enabling the formation of new bone tissue. The bone formation process is guided by local signaling processes, so called coupling processes, which are still not fully elucidated. It is proposed that 9

INTRODUCTION coupling factors, such as IGFI, IGFII and TGFβ, are stored in the bone matrix and released during bone resorption, and that TGFβ appears to be a key signal for recruiting mesenchymal cells/pre-osteoblasts to the resorption site (Tang, Wu et al. 2009). However, a lack of bone-resorbing osteoclasts does not result in altered osteoblast-mediated bone formation, indicating that coupling factors can be released from sites other than degraded bone matrix.

It has also been hypothesized that osteoclasts themselves are able to produce coupling factors, and several coupling mechanisms have been proposed (Martin and Sims 2005). The soluble molecule sphingosine 1-phosphate is secreted by osteoclasts and induces the recruitment of osteoblast precursors as well as promoting osteoblast survival (Pederson, Ruan et al. 2008). Moreover, bidirectional signaling between the cell-anchored receptor EphB4 (on osteoblasts) and the tethered ephrin-B2 ligand (on osteoclasts) enhances osteoblastogenesis and suppresses osteoclast differentiation (Zhao, Irie et al. 2006).

As the osteoblasts return to the resorption area they synthesize type I collagen and non-collagenous protein to form the osteoid. Finally, the osteoblasts ensure mineralization of the newly produced osteoid, through a process that is still not fully understood. However, it is proposed that the osteoblasts transport matrix vesicles containing ALP to the ECM, and that these vesicles function as initial sites of mineralization as hydroxyapatite crystals are formed within them. The crystals are transferred into the ECM through the vesicle membrane, and fill the spaces between the collagen proteins. The function of ALP is to degrade inorganic pyrophosphate in the ECM to provide free organic phosphate that reacts with calcium to form hydroxyapatite (reviewed in Orimo 2010).

10

INTRODUCTION

1.2 CD47 CD47, or integrin-associated protein (IAP), is a ubiquitously expressed cell- surface glycoprotein belonging to the immunoglobulin (Ig)-superfamily. The amino (N)-terminus comprises a highly glycosylated IgV-like domain, and is followed by five transmembrane regions and a cytoplasmic tail with four different splice variants, where isoforms 2 and 4 are the predominant forms (Brown and Frazier 2001). Due to the lack of a cytoplasmic signaling domain, CD47 facilitates intracellular signaling partly by associating with heterotrimeric G-proteins (Frazier, Gao et al. 1999), via PLICs (protein linking IAP to cytoskeleton) (Wu, Wang et al. 1999) or via the cytoplasmic tail of integrins (Blystone, Lindberg et al. 1995). CD47 was first discovered in leukocytes and placenta as a plasma membrane protein associated with the integrin αvβ3 (Brown, Hooper et al. 1990). It has since been shown that CD47 interacts with several integrins, thrombospondin-1 (TSP-1) and signal-regulatory proteins (SIRPs), mediating intracellular signaling in different cell types.

1.2.1 CD47, thrombospondin and integrins TSP-1 is an ECM glycoprotein that is known to be involved in cell proliferation, differentiation and motility (Adams and Lawler 2004). The interaction between CD47 and TSP-1 has been shown to stimulate αvβ3- mediated cell spreading on vitronectin (Gao, Lindberg et al. 1996), amongst other things. CD47 together with TSP-1 also interacts with integrin αIIbβ3 and α2β1, stimulating the activation, spreading and aggregation of platelets (Chung, Gao et al. 1997; Chung, Wang et al. 1999; Isenberg, Romeo et al. 2008). Moreover, CD47 associates with integrin α2β1 and regulates the chemotaxis of smooth muscle cells (Wang and Frazier 1998). CD47 induces the activation and adhesion of integrin α4β1 on sickle red blood cells via TSP-1(Brittain, Han et al. 2004), and synergize with α4β1 to regulates the migration of B-lymphocytes (Yoshida, Tomiyama et al. 2000). Furthermore, one of the major functions of the interaction between CD47 and TSP-1 is to inhibit nitric oxide (NO) signaling in vascular cells (Isenberg, Hyodo et al. 2007).

1.2.2 CD47 and SIRPs CD47 is also known to bind to the SIRP-family members SIRPα and SIRPγ. CD47 binding to SIRPγ is suggested to be important for supporting T-cell proliferation (Piccio, Vermi et al. 2005), whereas the interaction with SIRPα facilitates a number of cellular events, which will be discussed in more detail below.

11

INTRODUCTION

1.3 SIRPα SIRPα (also known as SHPS-1, MFR, BIT, P84 and CD172A) was first discovered in 1996 in rat fibroblasts as a binding protein for Src-homology-2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP-2), and was proposed to function in cell adhesion and growth factor-induced cell signaling (Fujioka, Matozaki et al. 1996). SIRPα is mainly expressed by myeloid cells and neurons (Adams, van der Laan et al. 1998) but is also detected in fibroblasts and endothelial cells (Barclay and van den Berg 2013).

Similar to CD47, the SIRP family (comprising SIRPα, SIRPβ and SIRPγ) belongs to the Ig-superfamily (Barclay and Brown 2006). SIRPα, a 110–120 kDa cell-surface glycoprotein, contains an extracellular domain with three Ig-like domains (IgV, IgC, IgC), a single transmembrane domain and an intracellular domain. The intracellular domain contains four tyrosine phosphorylation sites that form two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which contribute to further downstream signaling (Cant and Ullrich 2001). Moreover, SIRPα contains two intracellular proline-rich regions, which also function as binding domains (van Beek, Cochrane et al. 2005). Both SIRPβ and SIRPγ have similar extracellular domains to SIRPα but lack the cytoplasmic ITIM-containing domain. Instead, SIRPβ has a 6-amino acid short cytoplasmic tail that binds to DAP12, which regulates positive cell signaling through an ITAM (Barclay and Brown 2006), whereas SIRPγ, with a 4-amino acid short cytoplasmic tail, does not have any known intracellular signaling motifs (van Beek, Cochrane et al. 2005).

1.3.1 SIRPα, surfactant and growth factors SIRPα not only interacts with CD47, but it also interacts with lung surfactant A and surfactant D (SP-A, SP-D) (Wright 2005). It has been proposed that SP-A and SP-D, by binding to SIRPα, inhibit alveolar macrophage phagocytosis and thereby regulate inflammatory responses in the lung (Janssen, McPhillips et al. 2008). SP-D, unlike CD47, binds to the membrane-proximal domain of SIRPα in human neutrophils and differentiated neutrophil-like cells (Fournier, Andargachew et al. 2012). Moreover, SIRPα signaling can be regulated via epidermal growth factor, platelet-derived growth factor, IGF, neutrophins, lysophosphatidic acid, growth hormone and CSF (Cant and Ullrich 2001).

12

INTRODUCTION

SP*A,&SP*D&

Integrin&

PLICs ITIM& ITIM& & CD47& & & & SIRPα&

TSP*1& G*protein&

cAMP&

Growth&&

factors&

&

& & SIRPγ&

Figure 5. Schematic illustration of CD47 and SIRPα interactions. Surfactant A, D (SP-A, SP-D), Signal regulatory protein α, γ (SIRPα, γ), inhibitory motif (ITIM), Thrombospondin-1 (TSP-1), protein linking IAP to cytoskleton (PLIC).

1.3.2 Signaling downstream of SIRPα Downstream signaling induced by activated SIRPα involves tyrosine phosphorylation of ITIMs and the subsequent recruitment and activation of SHP-1 and/or SHP-2. Both SHP-1 and SHP-2 have the ability to dephosphorylate proteins and thereby mediate specific biological functions coupled to SIRPα (reviewed by Cant and Ullrich 2001; van Beek, Cochrane et al. 2005; Matozaki, Murata et al. 2009). It has been proposed that SHP-1, which is mainly expressed in epithelial and hematopoietic cells, has a negative regulatory function, whereas SHP-2, expressed in neuronal and myeloid cells, contributes to the positive regulation of cell signaling (Neel, Gu et al. 2003; Oldenborg 2004). SIRPα has also been proposed to bind to other signaling molecules, and might thereby function as a scaffold to facilitate downstream signaling (Timms, Swanson et al. 1999). One of these molecules is Src kinase-associated phosphoprotein 2 (Skap2), which, through SIRPα, mediates integrin-stimulated cytoskeletal rearrangement in macrophages (Alenghat, Baca et al. 2012). SIRPα also modulates the action of growth hormones via the Janus kinase 2 (JAK2) pathway (Carter-Su, Rui et al. 2000) and induces macrophage NO production through JAK–signal

13

INTRODUCTION transducer and activator of transcription (STAT) signaling and PI3K–Rac1 signaling (Alblas, Honing et al. 2005).

1.4 The interaction between CD47 and SIRPα In 1999, CD47 was discovered to be a ligand for SIRPα in neural tissue (Jiang, Lagenaur et al. 1999) and in hematopoietic cells (Seiffert, Cant et al. 1999). The Ig domain of CD47 binds to the N-terminal IgV domain on SIRPα via hydrogen bonds (Hatherley, Graham et al. 2008); the interaction can induce bidirectional signaling (Jiang, Lagenaur et al. 1999).

The interaction between CD47 and SIRPα is probably best known for its role as a ‘marker of self’ first discovered in erythrocytes, which enables the immune system to distinguish self from foreign; erythrocytes lacking CD47, but not wild-type erythrocytes, are phagocytosed rapidly after being injected into wild-type mice (Oldenborg, Zheleznyak et al. 2000). These findings have been further consolidated and SIRPα’s importance for the survival of erythrocytes has been demonstrated (Oldenborg, Gresham et al. 2001; Ishikawa-Sekigami, Kaneko et al. 2006; Uluçkan, Becker et al. 2009). In addition, SIRPα negatively regulates platelet clearance (Yamao, Noguchi et al. 2002), and the interaction between CD47 and SIRPα also facilitates the transendothelial and transepithelial migration of granulocytes and monocytes (Liu, Merlin et al. 2001; de Vries, Hendriks et al. 2002; Liu, Buhring et al. 2002; Liu, O'Connor et al. 2004). Furthermore, it has been shown that SIRPα expressed on dendritic cells (DCs) interacts with CD47 expressed on T-cells, leading to bidirectional signaling that causes suppression of normal DC maturation and inhibition of cytokine production by mature DCs (Latour, Tanaka et al. 2001). In vitro, the CD47–SIRPα interaction promotes T-cell activation and induction of antigen-specific cytotoxic T-lymphocytes response by DCs (Seiffert, Brossart et al. 2001). In addition, it has been suggested that there is a role for the CD47–SIRPα interaction in neuronal network formation in the hippocampus (Murata, Ohnishi et al. 2006), and the interaction between these two proteins in cultured neurons supports the formation of spines and filopodia (Miyashita, Ohnishi et al. 2004).

In 1995, Saginario et al. discovered a protein that was highly upregulated in fusing macrophages (Saginario, Qian et al. 1995). The protein was purified and named macrophage fusion receptor (MFR), and was shown to be identical to SIRPα (Saginario, Sterling et al. 1998). By using a soluble extracellular domain of MFR/SIRPα, Saginario et al. were able to prevent macrophage fusion, indicating that MFR is involved in the fusion event of macrophages (Saginario, Sterling et al. 1998). Consistent with the findings regarding SIRPα and macrophage fusion, it has been shown that CD47 is

14

INTRODUCTION also expressed and upregulated during macrophage fusion (Han, Sterling et al. 2000). By blocking CD47 with monoclonal antibodies or by adding a GST–CD47 fusion protein, Han et al. further showed that the formation of macrophages depended on functional CD47 (Han, Sterling et al. 2000). Additionally, it has been demonstrated that CD47 is involved in the multinucleation of rat alveolar macrophages through its transitory interaction with SIRPα (Han, Sterling et al. 2000). Vignery has suggested that reported data regarding the CD47–SIRPα interaction and macrophage fusion are applicable to the fusion event during osteoclastogenesis, due to the shared origin, morphology and similar functions between macrophages and osteoclasts (Vignery 2000).

15

INTRODUCTION

1.5 PI3K–Akt signaling pathway PI3K is an enzyme that, among other functions, promotes the binding and phosphorylation of the Akt family of serine/threonine protein kinases. Many growth factors and cytokines induce the phosphorylation of SHP-2, which, in turn, activates PI3K (Wu, O'Rourke et al. 2001). Upon activation, PI3K converts phosphatidylinositol-4,5-bisphosphate (PIP2) to phosphatidyl- inositol-3,4,5-bisphosphate (PIP3), which recruits Akt and phosphoinositide - dependent protein kinase 1 (PDK1). PDK1 has the ability to phosphorylate Akt, which enables further downstream signaling to regulate cellular events such as cell survival, proliferation, migration and differentiation (reviewed in Chen, Crawford et al. 2013). Three isoforms of Akt (Akt1–3) are found in mammalian cells: Akt1 and Akt2 are widely expressed, whereas Akt3 is mostly expressed in brain tissue.

Growth'factors' PIP2' PIP Cytokines' 3' PDK1' P' PI3K' Akt' SHP92' P'

Prolifera>on'Differen>a>on'Survival'Migra>on' Figure 6. Schematic illustration of PI3K signaling. Phosphoinositide 3-kinase (PI3K),

Phosphatidylinositol-4,5-bisphosphate (PIP2), phosphatidylinositol-3,4,5-bisphosphate (PIP3), phosphoinositide-dependent protein kinase 1 (PDK1), Src-homology-2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP-2), phosphate (P).

CD47 and SIRPα have both been shown to be involved in PI3K signaling. As described earlier, macrophage NO production is partly mediated via SIRPα–SHP-2 signaling to the PI3K–Rac1 pathway (Alblas, Honing et al. 2005). Furthermore, the migration of polymorphonuclear neutrophils across endothelial and epithelial monolayers is known to depend on CD47 expression on the cell surface, and signaling downstream of CD47 via PI3K partly regulates the rate of migration (Liu, Merlin et al. 2001).

It has also been demonstrated that PI3K and Akt1/2 have specific roles in bone metabolism. Specifically, the PI3K–Akt2 pathway is important for normal preosteoblast–osteoblast differentiation and survival (Ling, Dombrowski et al. 2010; Guntur, Rosen et al. 2012) and PI3K can inhibit osteoblast apoptosis via activation of Akt. PI3K–Akt signaling was also

16

INTRODUCTION needed in a murine osteoblast cell line in order for the cells to undergo calcification (Chen, Huang et al. 2013). Furthermore, inhibiting PI3K–Akt signaling in osteoblastic cells and immature mesenchymal cells blocked the osteoblastic differentiation induced by the forced expression of Runx2 (Fujita, Azuma et al. 2004), and the absence of BMP-2-mediated activation of Akt2 in BMS cells led to decreased expression of Runx2 and osterix (Mukherjee, Wilson et al. 2010). Moreover, mice with an osteoblast specific deletion of Pten, an endogenous PI3K inhibitor, exhibited an increasing bone mineral density throughout life. In vitro, osteoblasts lacking Pten differentiated faster in comparison to controls and demonstrated significant increased levels of phosphorylated Akt (Liu, Bruxvoort et al. 2007).

PI3K–Akt signaling is also important for osteoclasts and has been shown to be induced downstream of c-Fms, thereby mediating the survival of osteoclast precursors (Kikuta and Ishii 2013). Furthermore, PI3K–Akt signaling is reported to induce osteoclast differentiation by regulating the NFATc1 signaling pathway (Moon, Kim et al. 2012).

17

AIM

2 Aims The overall aim of the present thesis was to gain knowledge about the interaction of CD47 and SIRPα in relation to bone-cell differentiation and bone remodeling both in vitro and in vivo.

The specific purposes of the present thesis were to investigate:

• The role of the CD47–SIRPα interaction in osteoclastogenesis (Papers I and II).

• The role of the CD47–SIRPα interaction and signaling in osteoblast differentiation and function (Papers II and III)

• The role of CD47 in bone remodeling in vivo (Paper II)

18

METHODS

3 Methods 3.1 Animals Male mice were used in all experiments. CD47-/- mice and their wild-type counterparts were on a Balb/c background, backcrossed to Balb/c. The SIRPα-mutant mice (lacking the intracellular signaling domain of SIRPα) and their wild-type counterparts were on a C57BL/6 background, backcrossed to C57BL/6J. CsA mice were from our own inbred colony. Animals were kept in accordance with local guidelines and maintained in a specific pathogen-free barrier facility. The Local Animal-Ethics Committee approved all animal procedures.

3.2 Isolation and culture of bone marrow stromal (BMS) cells Femurs and tibiae from 5–9-week-old mice were dissected in phosphate buffered saline (PBS). Cartilage ends were removed before bone marrow cells were flushed from the long bones. Subsequently, the cells were seeded in alpha minimal essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (benzylpenicillin, gentamycin sulphate, streptomycin), hereafter referred to as culture medium, in 60 cm2 culture dishes or in 24-well plates (at 106 cells/cm2) and incubated for 24 h (37°C, 5% CO2). Thereafter, the culture medium was changed every 2–3 days. To promote the formation of mineralized nodules, ascorbic acid and β- glycerophosphate were added to the cultures after 7 days. The cultures were harvested after 7–15 days, after which mRNA was extracted, osteoblast differentiation and mineralization capacity were assessed using histochemical stainings, and ALP activity was measured.

Osteoblast differentiation and mineralization capacity were visualized by staining for the presence of ALP, as well as for the presence of phosphate and calcium in the ECM, using von Kossa and Alizarin Red stains, respectively, according to standard histochemical procedure. To demonstrate the content of ALP, cells were washed with PBS, fixed with acetone and subsequently stained by using Naphthol AS-MX phosphate, disodium salt and Fast Blue BB. To visualize mineralized ECM, cells were washed with PBS and fixed using 4% paraformaldehyde prior to being stained with either 5% silver nitrate under daylight conditions for 5–15 min (von Kossa stain) to detect phosphate or with Alizarin Red according to the manufacturer’s protocol to detect calcium. The BMS cell cultures were photomicrographed and the mineral content was quantified using Cell^B software.

19

METHODS

3.3 Isolation and culture of osteoclasts 3.3.1 Spleen cell cultures Spleen cells from 5–9-week-old mice were obtained by dissecting the spleen, free of adhering tissue, followed by gently rubbing it against a grooved Petri dish to dissociate the cells. Erythrocytes were lysed in red blood cell lysis buffer (0.16 M NH4Cl, 0.17 M Tris, pH7.65) before the remaining cells were seeded in culture medium at a density of 106 cells/cm2. The cells were allowed to settle for 24 h before the culture medium was changed and M-CSF and RANKL were added. The culture medium was then changed every second day and the culture was harvested after 5 or 6 days. The cells were fixed and stained for TRAP.

3.3.2 Non-adherent bone marrow monocyte (BMM) culture (Referred to as bone marrow cell (BMC) culture in paper I, but as BMM culture in paper II and throughout the Results and Discussion). BMCs were collected as described for BMS cells above. Subsequently, the cells were washed with EDTA, the erythrocytes were lysed using ice-cold water, and the remaining cells were incubated in 37°C, 5% CO2. After 2 h the non- adherent cells (i.e., the bone marrow monocytes/macrophages) were collected, counted and seeded in culture wells (at 2 x 105 cells/cm2) in culture medium supplemented with M-CSF ± RANKL in order to stimulate osteoclast formation. The culture medium was changed every second day and the culture was harvested after 4–6 days. The cells were fixed and stained for TRAP.

3.3.3 Bone marrow cell (BMC) culture BMCs were collected as described for BMS cells above and seeded at a density of 106 cells/cm2 in culture wells or on bone slices with culture medium. After 24 h, the medium was changed and either PTH or D3 was added in order to stimulate osteoclast differentiation. After 6–7 days, the culture was harvested; mRNA was extracted, and cells were fixed and stained for TRAP. In addition, TRAP activity was measured and the levels of M-CSF and RANKL protein in the culture medium were analyzed.

3.4 Co-cultures of BMM and BMS cells Cells used for co-culture experiments were harvested from mice at 5–9 weeks of age. BMS cells were isolated as described previously. The cells were plated in 60 cm2 culture dishes in culture medium that was changed every third day. After 14 days of incubation (37°C, 5% CO2) adherent cells were detached, counted and seeded at 105 cells/cm2 in 96-well plates before being incubated for 24 h. Meanwhile, non-adherent BMM cells were isolated and treated as described for BMMs. After 2 h incubation, these non-adherent

20

METHODS cells were collected, counted and seeded onto the stromal cells at 6 x 104 cells/cm2. The co-cultures were stimulated with D3 in order to promote osteoclast differentiation. After 5–7 days, the cultures were harvested, fixed and stained for TRAP.

In spleen cell culture, BMM cultures, BMC cultures and co-cultures, the number of osteoclasts was visualized by TRAP staining and light microscopy. TRAP staining was performed using a commercially available kit according to the manufacturer’s protocol. Cells that were TRAP positive and that contained 3 or more nuclei were counted as osteoclasts. Levels of the C-terminal telopeptide of collagen type I (CTX) in BMC culture medium were measured as a marker of osteoclast resorption activity, and the formation of resorption pits on bone slices was also used to visualize osteoclast activity in BMCs.

3.5 Gene expression analysis Gene expression analyses were performed on BMCs and BMS cells.

3.5.1 RNA extraction and cDNA synthesis Total RNA from the cells was extracted using lysis solution from a commercially available kit according to the manufacturer’s protocol, treated with DNase I in order to eliminate genomic DNA, and quantified using a NanoDrop spectrophotometer. Equal amounts of RNA (0.05–1.0 µg) from samples belonging to the same experiments were reverse transcribed into single-stranded cDNA using a standardized kit with avian myeloblastosis virus (AMV) and oligo(dT) primers. Reactions without AMV reverse transcriptase were included as a negative control to ensure that no genomic DNA was present in the samples. mRNA expression was then analyzed using quantitative real-time reverse transcription (RT)- polymerase chain reaction (PCR).

3.5.2 Quantitative real-time RT-PCR RT-qPCR analysis was performed using TaqMan Universal PCR Master Mix and TaqMan gene expression assays containing fluorescently labeled probes, oligonucleotides and primers, which were ordered as Assay-on- demand or Inventoried kit. β-actin and beta-2-microglobin were used as endogenous controls to adjust for any variability in amplification due to differences in the starting concentrations of mRNA. Using the standard curve method, the relative expression of the gene of interest was calculated based on the threshold cycle (Ct) values of the investigated gene and the Ct values of the endogenous controls.

21

METHODS

3.6 Protein expression analysis 3.6.1 Enzyme-linked immunosorbent assay (ELISA) ELISAs were performed to identify the amount of CTX, M-CSF, RANKL and OPG within cells or released into the culture medium from BMCs, using commercially available kits. Briefly, samples were immobilized on a plastic surface prior to the addition of an enzyme-conjugated antibody against the protein of interest. Subsequent addition of the enzyme substrate resulted in a color change, measured using a spectrophotometer, which indicated the amount of the investigated protein.

3.6.2 ALP activity assay Cells from BMS cell cultures were washed with PBS prior to lysis in deionized water. Alkaline buffer was used to establish a basic milieu supporting optimal ALP enzymatic function. Cleavage of the substrate p- nitrophenyl by ALP released p-nitrophenol, which becomes yellow in basic milieu. The reaction was stopped using 0.15 M NaOH and the intensity of the color/absorbance was measured at 405 nm.

3.6.3 TRAP activity assay Cells from BMC cultures were washed with PBS prior to lysis in Triton-X. Tartaric acid buffer was used to establish an acidic milieu, which supported optimal TRAP enzymatic function. Cleavage of the substrate p-nitrophenyl by TRAP released p-nitrophenol. 0.15 M NaOH was used to stop the reaction and promote the color change. The intensity of the color/absorbance was measured at 405 nm.

3.6.4 Western blot BMS cells were cultured for 14 days, then lysed with ice-cold lysis buffer for preparation of whole cell lysates or immunoprecipitates. Immunoprecipitation was performed using protein G-Sepharose coated with anti-SIRPα mAbs overnight at 4°C. Washed immunoprecipitates and whole cell lysates were boiled in reduced SDS-PAGE sample buffer with β- mercaptoethanol and separated by 10% SDS-PAGE, followed by transfer to nitrocellulose membrane under standard conditions. Non-specific binding was blocked by using 5% non-fat dried milk powder in TBST (pH 7.6) followed by incubation of the nitrocellulose membrane in TBST (pH 7.6) containing the appropriate primary antibody (mAb Akt2, SHP-2, phosphoAkt2, phosphoSHP-2). Primary antibodies were detected with peroxidase-conjugated goat anti-rat or anti-mouse mouse IgG.

22

METHODS

3.7 Flow cytometry BMS cells were cultured for 14 days, then washed and resuspended in cold PBS before being incubated on ice for 30 min with anti-SIRPα mAb P84 or an IgG isotype control. Cells were then washed with PBS and analyzed by flow cytometry.

3.8 Histomorphometry analysis 18-week-old and 28-week-old male wild-type or CD47-/- mice were used for histomorphometry analysis. The mice were injected with the fluorescent compound calcein 10 days and 3 days prior to being euthanized. Right femurs were dissected free of soft tissue, and bisected at the midpoint of the shaft before being embedded, undecalcified, in methyl-methacrylate. 5 µm sagittal sections were prepared for analysis. For the assessment of mineralized tissue, the sections were stained using the von Kossa stain, and trabecular bone volume was measured in a sample region extending 4.5 mm proximal to the distal growth plate including all trabecular bone within the cortical boundaries. Osteoblast numbers were estimated from sections stained with von Kossa and toluidine blue. Only osteoblasts adjacent to the osteoid surface were included in the analysis. The sections were also stained for TRAP as a measure of the osteoclast surface and osteoclast number; only osteoclasts adjacent to trabecular bone were included.

Dynamic histomorphometry parameters were analyzed using fluorescence microscopy to visualize single and double calcein labels. The mineralizing surface (MS) was estimated using the amount of bone surface (BS) covered with single and double labels (sLS and dLS) using the equation MS = ((0,5 x sLS) + dLS) x 100/BS, expressed as percentage of the BS. The mineral apposition rate (MAR) was estimated by the distance between the calcein labels divided by the time interval between the calcein injections: MAR = interlabel distance/7, expressed in µm/d. The bone formation rate (BFR) was calculated as BFR = MS/BS x MAR, expressed in µm2/µm3/day.

3.9 Peripheral quantitative computed tomography (pQCT) pQCT was used to measure cortical parameters in tibiae from 18-week-old and 28-week-old male CD47-/- or CD47+/+ mice. Scans were performed using a voxel size of 70 µm, a scan speed of 5 mm/sec and a slice width of 1 mm. Bones were scanned in a single slice 4 mm distal from the proximal margin of the tibiae.

3.10 Statistical analysis Statistical analyses were performed using one-way analysis of variation (ANOVA) with Levene’s homogenicity test, and post-hoc Bonferroni or Dunnett’s T-test. A Student’s t-test was used when appropriate. All 23

METHODS experiments were performed at least twice with comparable results, to ensure reproducibility, and all data are represented as the means ± SEM. Significance levels were set to p < 0.05 (*), 0.01 (**) or 0.001 (***).

24

RESULTS AND DISCUSSION

4 Results and Discussion Throughout life, each bone in a healthy skeleton continuously undergoes balanced modeling and remodeling processes. These processes occur in order to adapt to changing biomechanical forces, to replace old and microdamaged bone and to maintain normal mineral homeostasis. In diseases affecting the bone tissue such as periodontitis, rheumatoid arthritis and osteoporosis, the balanced remodeling processes are disturbed, resulting in bone loss. To understand the disturbed and uncoordinated bone remodeling processes in disease, it is of great interest to investigate the balanced molecular control of bone cells in health. Over recent decades, significant advances have been accomplished in understanding important signaling pathways and mechanisms regulating bone homeostasis. However, the mechanisms and signaling systems involved are still far from being fully understood. The interaction between CD47 and SIRPα may be a small, yet important, piece of the giant bone-remodeling puzzle.

4.1 Osteoclast formation in vitro in the absence of CD47–SIRPα signaling (Paper I) The two papers published on CD47 and SIRPα’s involvement in macrophage fusion were the starting point for the thesis and of our investigation (Saginario, Sterling et al. 1998; Han, Sterling et al. 2000). Vignery (Vignery 2000) reviewed the results regarding CD47 and SIRPα and their importance for macrophage fusion and presented these results as applicable to osteoclast fusion, but it had not been shown that cells from the osteoclast cell lineage were affected by the interaction between CD47 and SIRPα. Therefore, our objective was to elucidate the role of CD47 and SIRPα in osteoclastogenesis using hematopoietic cells derived from either spleen cells or bone marrow monocyte/macrophages (referred to as BMCs in paper I, but as BMMs in paper II and throughout the Results and Discussion sections), in contrast to previous studies that had used mature rat alveolar or peritoneal macrophages (Saginario, Sterling et al. 1998; Han, Sterling et al. 2000).

The spleen cell cultures and BMM cultures were treated with M-CSF and RANKL in order to promote the formation of multinucleated bone-resorbing TRAP-positive osteoclasts. Function-blocking monoclonal antibodies against either CD47 (miap 301) or SIRPα (P84) were added to some of the cultures. When the function of SIRPα or CD47 was blocked in either spleen cell cultures or BMM cultures, the number of osteoclasts was significantly reduced in both cell culture types compared to groups treated only with M- CSF and RANKL. The morphology of the cells and the number of nuclei per

25

RESULTS AND DISCUSSION osteoclast were unaffected by mAb P84 in either spleen cell cultures or in BMM cultures. BMM cultures prepared from either CD47+/+ or CD47-/- mice yielded similar results to those obtained using the function-blocking antibodies — that is, there was a significantly reduced number of osteoclasts in the absence of CD47. As far as we know, we were the first to publish results that strongly suggest that the physical interaction between CD47 and SIRPα is important in order to form osteoclasts. In our study, inhibiting the interaction did not seem to affect the size or the number of nuclei in the formed osteoclasts, which indicated that it is not only the fusion event that is controlled by the CD47–SIRPα interaction.

A subsequent study by Uluçkan et al. (Uluçkan, Becker et al. 2009) also demonstrated that osteoclast formation from CD47-/- cell cultures was impaired in comparison to CD47+/+ cultures. Interestingly, the Uluçkan study showed that pre-incubating the cells with M-CSF and increasing the RANKL concentration restored osteoclast formation in CD47-/- cultures. To confirm these findings of restored osteoclast formation, we pre-incubated CD47-/- BMM cultures with M-CSF and either increased the RANKL concentration or used a more potent truncated form of RANKL, which rescued osteoclast formation (data not published). In summary, BMM cultures from CD47-deficient mice, or cultures in which either CD47 or SIRPα were blocked by function-blocking antibodies, exhibited a reduced number of osteoclasts, although an increased concentration of the important cytokines M-CSF and RANKL rescued osteoclast formation.

4.2 Histomorphometry analyses of femurs from CD47-deficient mice (Papers I and II) The interesting in vitro findings showing an osteoclast defect in the absence of CD47–SIRPα signaling prompted us to further investigate the osteoclast phenotype in vivo in CD47-deficient mice. Femurs from two different ages, 18 weeks and 28 weeks, were analyzed and we were the first to show that, at both ages, the number of osteoclasts was significantly reduced. Likewise, the trabecular bone surface covered by osteoclasts was significantly lower in CD47-/- mice compared to CD47+/+ mice at both ages (papers I and II). Maile et al. also investigated the CD47-/- bone phenotype but were unable to find any significant differences in the number of osteoclasts; however, they did demonstrate a 40% reduction (non-significant) in the amount of eroded surface, a marker of osteoclast function, indicating a defect in CD47-/- osteoclasts (Maile, DeMambro et al. 2011). Uluçkan et al. investigated osteoclast perimeter in vivo, and were unable to detect a difference in the perimeter between CD47+/+ and CD47-/- mice (Uluçkan, Becker et al. 2009). However, these findings were demonstrated in C57/BL6 mice and our

26

RESULTS AND DISCUSSION findings were demonstrated in Balb/c mice, and the measurement of perimeter does not necessarily reflect the true number of osteoclasts.

Our extended analyses of the bone phenotype of CD47-/- mice (paper II) unexpectedly revealed significantly lower trabecular bone volume in CD47-/- mice compared to CD47+/+ at both 18 weeks and 28 weeks of age. These findings were confirmed by micro computed tomography (micro-CT) measurements of 16-week-old CD47-/- mice on a C57BL/6 background performed by Maile et al. (Maile, DeMambro et al. 2011). However, in contrast to our findings, Uluçkan et al. demonstrated a small, but significant, increase in trabecular bone volume in CD47-/- mice compared to CD47+/+ mice analyzed by micro-CT and histomorphometry (Uluçkan, Becker et al. 2009). A possible reason for the discrepancy in the results could be that Uluçkan et al.’s study used tibiae or lumbar vertebral bodies from 5-week- old mice with a C57BL/6 background, whereas we studied femurs from 18- week-old and 28-week-old Balb/c mice. Given that we observed fewer osteoclasts and yet lower trabecular bone volume, our results indicated that it was not only osteoclasts that were affected by the lack of CD47. Indeed, further analysis demonstrated that the number of osteoblasts and the osteoid surface in both 18-week-old and 28-week- old mice were significantly lower in CD47-/- mice compared to CD47+/+ mice. These findings were accompanied by a lower mineralizing surface, a lower mineral apposition rate as well as a lower bone formation rate in 18-week-old CD47-/- mice compared to CD47+/+ mice. In contrast, at 28 weeks of age, the dynamic bone formation parameters were not significantly different between the genotypes. Interestingly, the dynamic bone formation parameters decreased in CD47+/+ mice but remained at the same levels in CD47-/- mice between 18 weeks and 28 weeks, indicating that CD47-/- mice age prematurely. The results from our histomorphometry analyses of osteoblasts and dynamic bone formation in 18-week-old mice were in agreement with the findings of Maile et al., which demonstrated fewer osteoblasts, a lower mineralizing surface, a lower mineral apposition rate and a lower bone formation rate in the trabecular bone of 16-week-old CD47-/- mice compared to CD47+/+ mice (Maile, DeMambro et al. 2011).

4.3 Osteoclast differentiation in vitro in the absence of CD47– SIRPα signaling (Paper II) To further unravel CD47–SIRPα’s importance in osteoclast differentiation and formation, a crude culture system was used to imitate in vivo osteoclastogenesis (referred to as BMC culture in paper II as well as in the Results and Discussion sections). The crude BMC cultures contain both mesenchymal progenitors and hematopoietic progenitors flushed from the bone marrow. The BMCs were stimulated with either PTH or D3 in order to 27

RESULTS AND DISCUSSION initiate stromal cells to release M-CSF and RANKL, which, in turn, promoted osteoclast differentiation. Notably, crude BMC culture is sometimes directly stimulated with M-CSF and RANKL in order to promote osteoclastogenesis, an approach that bypasses the importance of stromal cells.

The results from our experiments clearly demonstrate that both PTH and D3 were able to actively promote osteoclast formation in CD47+/+ BMC cultures, but that in cultures from CD47-/- mice, neither PTH nor D3 supported osteoclast formation. In addition to decreased osteoclastogenesis, we detected fewer resorption pits and a decrease in the amount of CTX released into the medium from D3-stimulated CD47-/- BMCs cultured on bone slices, compared to CD47+/+ BMC cultures. These data primarily point to a defect in CD47-/- stromal cells/osteoblasts that reduces the ability to promote osteoclast differentiation, resulting in a reduced osteoclast activity, and not to a defect in the osteoclasts per se.

Interestingly, Maile et al. showed that stimulating BMC cultures with M- CSF and RANKL resulted in the formation of fewer multinucleated TRAP- positive cells in CD47-/- cultures compared to CD47+/+ cultures; however, equal numbers of TRAP-positive cells (independent of the number of nuclei) were detected. Therefore, Maile et al. suggested a fusion defect rather than a differentiation problem in CD47-/- cells, which was supported by the finding of equal levels of expression of calcitonin and integrin β3 in both genotypes. In contrast to the fusion aspect, Maile et al. showed that fewer osteoclasts were formed in D3-stimulated BMC cultures from CD47-/- mice (Maile, DeMambro et al. 2011), indicating a defect in the ability of the stromal cells to release M-CSF and/or RANKL, or an altered expression of the cytokine receptors c-Fms and RANK. Moreover, when osteoclast formation was driven by the addition of exogenous M-CSF and RANKL to the BMC culture, this could have compensated for the lack of CD47, in correlation with previous findings (Uluçkan, Becker et al. 2009). Therefore, it is possible that Maile et al. only detected a late fusion defect, since normal proliferation and differentiation of osteoclast precursors occurred in the presence of sufficient levels of M-CSF and RANKL. van Beek et al. strengthened the hypothesis that increased M-CSF and RANKL concentrations can overcome defective osteoclastogenesis as they showed that BMC cultures from wild-type mice and SIRPα-mutants (lacking the signaling SIRPα domain) produced equivalent numbers of osteoclasts when stimulated with RANKL and M-CSF (van Beek, de Vries et al. 2009).

In addition to the involvement of CD47–SIRPα signaling in osteoclastogenesis, it has been suggested that CD47 is important for 28

RESULTS AND DISCUSSION myeloma-induced differentiation of DCs to osteoclasts (Kukreja, Radfar et al. 2009). Upon cell-to-cell contact with myeloma cells, DCs form multinucleated giant cells, which are considered to be osteoclasts due to TRAP-positivity and the ability to resorb bone. DCs co-cultured with myeloma cells showed a significant increase in TSP-1 expression, and when anti-TSP-1 antibodies were added to the co-cultures to inhibit TSP-1 binding to CD47, the formation of osteoclasts decreased (Kukreja, Radfar et al. 2009). Moreover, Kukreja et al. demonstrated that the CD47–TSP-1 interaction is important for osteoclast formation from human monocytes and that blocking TSP-1 in vivo had a significant effect on osteolysis and hypercalcemi (Kukreja, Radfar et al. 2009).

We further analyzed the expression of osteoclast-associated genes in the absence of CD47 (Paper II). As described earlier, NFATc1 is one of the most important transcription factors during osteoclast differentiation and is responsible for initiating the transcription of several osteoclast-associated genes. NFATc1 is activated downstream of RANK–TRAF6 signaling as well as via co-stimulatory pathways such as OSCAR–FcRγ (Takayanagi 2007). In CD47+/+ BMC cultures, but not in CD47-/- BMC cultures, D3 upregulated the mRNA expression of nfatc1 compared to a non-treated control group. The inability of D3 to induce nfatc1 expression in CD47-/- BMC cultures might be linked to results showing a decreased expression of the genes encoding OSCAR and RANK in D3-stimulated CD47-/- BMCs compared to CD47+/+ BMCs.

As a probable consequence of decreased nfatc1 expression in CD47-/- D3- stimulated BMCs, the expression of the osteoclast-associated genes Acp5 (encoding TRAP) and calcr (which encodes the calcitonin receptor) were not increased. The result showing that the mRNA expression of Acp5 was not upregulated by D3 in CD47-/- BMC cultures was further confirmed by the observation that neither PTH nor D3 was able to promote TRAP protein activity in CD47-deficient BMC cultures. The mRNA levels of the osteoclast fusion protein DC-STAMP were substantially increased in response to D3 in CD47+/+ cultures, but only a minor increase was seen in CD47-/- cultures.

To our knowledge, no other groups have investigated osteoclastic gene expression in the absence of CD47. However, van Beek et al. have shown that the expression of the genes encoding DC-STAMP, the calcitonin receptor, cathepsin K and TRAP are unaltered in BMC cultures from SIRPα-mutants compared to wild-type cultures, although the cultures were stimulated with M-CSF and RANKL, and hence the function of the stromal cells was bypassed as detailed above (van Beek, de Vries et al. 2009). 29

RESULTS AND DISCUSSION

Taken together, the results indicate that the gene expression of several important markers of osteoclast differentiation was significantly decreased in D3-stimulated CD47-/- cultures compared to CD47+/+ cultures, which probably explains the reduction in osteoclast numbers in CD47-/- mice. But the knowledge that osteoclast formation could be rescued by additional M- CSF and RANKL and the results demonstrating lower trabecular bone volume and fewer osteoblasts in vivo strongly indicate a key defect in stromal cell differentiation in the absence of CD47.

4.4 Stromal cell/osteoblast differentiation in the absence of CD47 (Paper II) Knowing that CD47-/- mice most likely have an altered stromal cell phenotype, BMC cultures from CD47-deficient mice were further analyzed. Significantly reduced gene and protein expression of M-CSF and RANKL was observed in CD47-/- cultures compared to CD47+/+ cultures. Moreover, we were able to show that the levels of OPG protein and mRNA were lower in CD47-/- BMC cultures compared to CD47+/+ cultures (data not published). Maile et al. also investigated the relative expression of tnfsf11 and tnfrsf11b (the genes encoding RANKL and OPG) and, in accordance with us, showed a decrease in tnfrsf11b expression but, unlike us, showed an increase in tnfsf11 expression in CD47-/- cultures compared to CD47+/+ cultures. The consequent altered gene expression of tnfsf11 and tnfrsf11b resulted in that a higher tnfsf11:tnfrsf11b ratio was obtained, which might result in the increased differentiation of osteoclasts (Maile, DeMambro et al. 2011). However, it is hard to interpret their data, as it is not clear how the BMC cultures were stimulated and what type of control was used.

Further examination of stromal cell/osteoblast in BMC cultures was performed in order to explore the phenotype of CD47-/- stromal cells. The expression of the osteoblast-associated genes encoding Runx2, osterix, ALP and α-1-collagen was comparable between CD47-/- and CD47+/+ cultures after 3 days, but lower in CD47-/- cultures compared to CD47+/+ cultures after 6 days of culture. These results indicate an impaired osteoblast differentiation ability in CD47-/- cultures.

Interestingly, the adipocyte-specific gene PPARG, which encodes peroxisome proliferator-activated receptor gamma, was slightly, but yet significantly, upregulated in CD47-/- cultures compared to CD47+/+ cultures after 3 days, indicating that CD47-/- stromal cells differentiate towards an adipocytic lineage instead of an osteoblastic cell lineage.

The findings of lower levels of Akp1 mRNA expression in CD47-/- BMC cultures were confirmed by assays showing that ALP activity was 30

RESULTS AND DISCUSSION significantly lower in CD47-/- cultures compared to CD47+/+ cultures. As a result of the altered stromal cell/osteoblast phenotype in CD47-/- mice, fewer mineral deposits were formed. In CD47+/+ BMS cell cultures stimulated with osteogenic medium, mineral deposits (indicated by positive von Kossa staining) were observed after 14 days and 21 days. In the CD47-/- BMS cell cultures, on the other hand, no mineral was formed by 14 days. After 21 days, von Kossa-positive deposits were detected in CD47-/- cultures, although the extent of these was not the same as those seen in CD47+/+ cultures. Likewise, Maile et al. proposed that CD47 is important for osteoblastogenesis on the basis that less ALP-positive and von Kossa- positive staining was seen in vitro in CD47-/- cultures compared to CD47+/+ cultures (Maile, DeMambro et al. 2011).

The lack of osteoblasts in CD47-/- cultures was further explored by Maile et al. in calvaria osteoblast cell lines, and was suggested to be the result of a reduced interaction of CD47 with SIRPα. The consequent reduced phosphorylation of SIRPα resulted in the impaired activation of MAPK, which has previously been shown to be of importance for osteoblast proliferation (Maile, DeMambro et al. 2011).

In conclusion, CD47-/- stromal cells/osteoblasts have an altered phenotype compared to CD47+/+ stromal cells, with reduced expression of osteoblast- associated genes and impaired M-CSF and RANKL protein expression. Likewise, ALP activity is lower in the absence of CD47, and CD47-/- stromal cell/osteoblast cultures have a reduced ability to form mineral deposits compared to CD47+/+ cultures.

4.5 Signaling downstream of SIRPα in stromal cells upon activation by CD47 (Paper II and III) To further understand the mechanism behind the changed bone marrow stromal cell/osteoblast phenotype, we hypothesized that the defect was due to the absence of CD47-mediated SIRPα signaling. Therefore, we investigated whether tyrosine phosphorylation of SIRPα could be involved in mediating osteoblast differentiation, as it is known that SIRPα becomes phosphorylated following binding by CD47 and that this tyrosine phosphorylation is important for mediating adhesion-dependent cellular functions (Johansen and Brown 2007).

First, we demonstrated, by flow cytometry, that SIRPα was expressed at equal levels in both CD47+/+ and CD47-/- stromal cells. SIRPα was then immunoprecipitated from stromal cells of both genotypes and subsequently western blotted to assess the extent of tyrosine phosphorylation. Tyrosine-

31

RESULTS AND DISCUSSION phosphorylated SIRPα was barely detectable in CD47-/- stromal cells compared to CD47+/+ cells, indicating a defect in SIRPα signaling in the absence of CD47.

Since the western blot results demonstrated altered SIRPα phosphorylation in stromal cells due to a lack of interaction with CD47, it was of great interest to further investigate signaling downstream of SIRPα in stromal cells. Phosphorylated SHP-2 facilitates positive regulation of intracellular signaling downstream of phosphorylated SIRPα (Oldenborg 2004), and one possible signaling pathway activated by SHP-2 is PI3K and, subsequently, Akt2 (Wu, O'Rourke et al. 2001). The PI3K–Akt2 pathway is known to regulate osteoblast differentiation (Fujita, Azuma et al. 2004; Ling, Dombrowski et al. 2010; Mukherjee, Wilson et al. 2010; Guntur, Rosen et al. 2012; Chen, Huang et al. 2013) and therefore we hypothesized that the SIRPα–SHP-2 signaling pathway was perturbed in CD47-/- stromal cells. Our western blot analyses showed that SHP-2 was co-immunoprecipitated with SIRPα from CD47+/+ BMS cell cultures but not from CD47-/- cultures, despite the presence of equal amounts of SIRPα protein in the lysates. We also detected much higher levels of phosphorylated SHP-2 and phosphorylated Akt2 in CD47+/+ stromal cell lysates than in CD47-/- stromal cell lysates. These findings strengthened our hypothesis that a lack of interaction between SIRPα and CD47 affects the SHP-2–PI3K–Akt2 pathway and could explain the defect seen in stromal cell/osteoblast differentiation in the absence of CD47.

During recent years, the positive effects of PI3K signaling in osteoblasts have been demonstrated both as increased differentiation and higher survival rate, consistent with our findings. However, Kaneshiro et al. showed that SHP-2–Akt2 signaling negatively regulates osteoblast differentiation, since inhibition of PI3K increased osteoblast differentiation (Kaneshiro, Ebina et al. 2013). However, these results involved interleukin-6-induced stimulation of an osteoblastic cell line, which implicates signaling through the gp130 subunit of the type I cytokine receptor in the activation of SHP-2 and Akt2.

After demonstrating that signaling downstream of SIRPα (activated by CD47) is important for normal stromal cell–osteoblast differentiation, it was of interest to discover whether the results regarding stromal cells from CD47-/- mice were due to a lack of SIRPα signaling. In order to find out, we investigated the stromal cell/osteoblast phenotype of SIRPα-mutant mice. As described earlier, SIRPα-mutant mice express normal levels of the extracellular SIRPα-domain, but the intracellular signaling domain is deleted and thus cannot be phosphorylated upon ligand binding (Okazawa, Motegi et

32

RESULTS AND DISCUSSION al. 2005). Our results from BMS cell cultures demonstrated that mRNA levels of Runx2 were significantly decreased in SIRPα-mutant stromal cells compared to wild-type stromal cells, similar to the decreased expression of Runx2 seen in stromal cell/osteoblast cultures from CD47-/- mice. This decreased Runx2 expression in SIRPα-mutant stromal cells could possibly be explained by decreased signaling through SHP-2–PI3K–Akt2 in the absence of the SIRPα signaling domain. Moreover, consistent with our Runx2 findings, the levels of mRNAs encoding osterix, osteocalcin and ALP were significantly decreased in the absence of the SIRPα signaling domain, pointing to a stromal cell/osteoblast defect in SIRPα-mutants, in agreement with the defect seen in CD47-/- stromal cells/osteoblasts.

In addition, the ALP activity and the ability to form mineral were significantly impaired in SIRPα-mutant stromal cells compared to wild-type cells. In accordance with the in vitro mineral formation defect seen due to impaired SIRPα–SHP-2–Akt2 signaling, Chen et al. have shown that the levels of osteocalcin and the ability to form mineral are decreased in an osteoblastic cell line treated with a PI3K inhibitor or depleted of Akt2 by short interfering RNA (Chen, Huang et al. 2013). Additionally, van Beek et al. have shown that SIRPα-mutant mice exhibit a decreased cortical bone volume (van Beek, de Vries et al. 2009), although this in vivo finding was explained by an increased bone-resorbing activity of osteoclasts lacking SIRPα rather than a defect in osteoblasts.

Taken together, the results from SIRPα-mutant BMS cultures demonstrate that SIRPα-mediated downstream signaling is necessary for normal osteoblastogenesis. Moreover, in combination with the results from CD47-/- stromal cells, signaling downstream of SIRPα is dependent on its activation by CD47.

4.6 Co-cultures and osteoclastogenesis (Paper II) To confirm the hypothesis that the defect in stromal cells caused by impaired signaling downstream of SIRPα prevents the induction of osteoclastogenesis, we performed co-cultures combining wild-type, CD47-/- or SIRPα-mutant BMM progenitors with wild-type, CD47-/- or SIRPα- mutant BMS cells. As expected, wild-type BMS cells in combination with wild-type BMM progenitors yielded multinucleated osteoclasts, whereas co- culturing SIRPα-mutant BMS cells or CD47-/- BMS cells with wild-type BMMs produced significantly fewer osteoclasts. However, when wild-type BMS cells were combined with BMMs from either SIRPα-mutant mice or CD47-/- mice, the number of osteoclasts was equivalent to those in co- cultures containing only wild-type cells. This implies that the impaired

33

RESULTS AND DISCUSSION osteoclast formation seen in CD47-/- BMC cultures is likely to be caused by the lack of a CD47–SIRPα interaction and subsequent downstream signaling in stromal cells, as we have been able to show that normal osteoclast formation occurs when BMMs from SIRPα-mutants or CD47-deficient mice are cultured with wild-type stromal cells.

The CD47–SIRPα interaction is involved in many different cellular events, by affecting different intracellular signaling pathways. As mentioned earlier our findings are just a small piece of a giant puzzle and our finding does not preclude the importance of other possible mechanisms controlling bone cell differentiation, governed by the CD47–SIRPα interaction. However, taken together, our presented results indicate that, during normal conditions, SIRPα and CD47 have the ability to interact and thereby promote downstream signaling through SHP-2 and the PI3K–Akt2-pathway in stromal cells. This pathway increases the expression of genes encoding the crucial osteoblastic transcription factors Runx2 and osterix, which enter the cell nucleus and induce the transcription of osteoblast-associated genes. This increased gene expression is coupled to osteoblast differentiation, leading to the formation of mature bone-forming osteoblasts. Moreover, the CD47– SIRPα-interaction partly controls the release and expression of the master osteoclast cytokines M-CSF and RANKL, in a yet unknown way, and thereby indirectly controls osteoclastogenesis.

RANKL!

Stromal!cell! M%CSF! Osteoclast!differen

SIRPα! osteocalcin*

! ! ! α,1,coll* P! ALP* CD47! SHP%2! P! PI3K! Akt2! Runx2! Osteoblast!differen

Bone!forma

Figure 7. Proposed involvement of the CD47–SIRPα-interaction in mediating bone-cell differentiation. Signal Regulatory Protein α (SIRPα), Src-homology-2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP-2), Phosphoinositide 3-kinase (PI3K), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), macrophage-colony stimulating factor (M-CSF), receptor activator of nuclear factor κB (RANKL)

34

RESULTS AND DISCUSSION

A lack of interaction between CD47 and SIRPα perturbs downstream signaling, thereby affecting both osteoblast and osteoclast differentiation, resulting in decreased bone formation as well as decreased bone resorption.

RANKL!

Stromal!cell! M%CSF! Osteoclast!differen

SIRPα! osteocalcin*

! ! ! α,1,coll* X* P! ALP* CD47! SHP%2! P! PI3K! Akt2! Runx2! Osteoblast!differen

Bone!forma

Figure 8. Lack of CD47–SIRPα-interaction resulting in decreased bone-cell differentiation. Signal Regulatory Protein α (SIRPα), Src-homology-2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP-2), Phosphoinositide 3-kinase (PI3K), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), macrophage-colony stimulating factor (M-CSF), receptor activator of nuclear factor κB (RANKL)

35

CONCLUSION

5 Conclusions The observations presented in this thesis show that the interaction between CD47 and SIRPα is of importance for bone-cell differentiation. In summary, the findings supporting our conclusion are:

• Signaling downstream of SIRPα via the SHP-2–PI3K–Akt2 pathway is diminished in the absence of CD47 binding in stromal cells/osteoblasts.

• Impaired SIRPα signaling results in an altered stromal cell/osteoblast phenotype with a reduced ability to form mineral and to express M-CSF and RANKL.

• The reduced stromal cell/osteoblast capacity to express M-CSF and RANKL affects osteoclastogenesis, resulting in the formation of fewer osteoclasts in CD47-deficient BMC cultures.

• The in vitro findings are confirmed in vivo, as CD47-deficient mice exhibit fewer osteoblasts, lower dynamic bone formation parameters, and fewer osteoclasts.

36

ACKNOWLEDGMENTS

6 Acknowledgements I would like to express my deepest thanks to all of you who in different ways have supported me during my PhD student years. In particular, I would like to express my warm appreciation and sincere gratitude to the following people:

First of all, associate professor Pernilla Lundberg, my friend, colleague and supervisor. I admire your never-ending energy, your ability to focus and your genuine passion for science. Thank you for giving me the opportunity to be a part of the most exciting research project, for always supporting me and for always being honest and trustworthy. Your excellent guidance has helped me to accomplish this work!

Professor Per-Arne Oldenborg, my co-supervisor, thank you for sharing your great knowledge with me, and for your enthusiasm, fruitful discussions and guidance through these years!

Professor Ulf Lerner, many years ago you awoke my curiosity for research, for which I am grateful. Thank you for always willingly answering my questions and for showing an interest in my work!

Paul Baldock, co-author, thank you for inviting me to the Garvan Institute of Medical Research and for introducing me to the art of histomorphometry.

My co-workers and the lab-experts: Rima Sulniute, thank you for good friendship and for producing invaluable results in the past few months. Ingrid Boström thank you for sharing your osteoblast culture-skills and your deep laborative knowledge with me. Anita Lie thank you for always finding solutions to my lab-related problems and for sharing my fascination for beautiful osteoclasts. Inger Lundgren, Chrissie Roth and Birgit Andertun thank you for untiring answering lab-questions and helping me in the lab! I also appreciate that all of you have shown interest in my life outside the lab, and that you have brought delicious ‘fika’ to the lab throughout the years.

Barbro Borgström, thank you for helping me with the mice whenever I needed.

Anders Johansson, thank you for contributing to a good working environment and for sharing your knowledge about both laboratory and scientific issues.

37

ACKNOWLEDGMENTS

Lillemor Hägglund, thank you for always being friendly, positive and helping me with administrative issues, I really appreciate it.

Py Palmqvist, thank you for showing sincere interest in my work and for your encouragement during the years.

Former PhD students Anna Brechter, Susanne Granholm, Peyman Kelk, Zivko Mladenovic and Nelly Romani Vestman as well as present PhD students Fredrik Strålberg, Ali Kassem and Åsa Stenberg, thank you for interesting talks about research but primarily for talks about everything other than research.

‘Pernillas girls’, my co-authors, Emelie Persson, Sara Engman and Elin Kindstedt, thank you for nice companionship in the lab. I hope you will continue to do great research!

Maria Ransjö, Emma Persson and Pedro Souza, researchers who have been in the lab for various lengths of time, thank you for broadening my research horizons.

Professor Ingegerd Johansson, thank you for being my research education examiner and an inspiring scientist.

I would also like to acknowledge former and present colleagues in the county council of Västerbotten, thank you for supporting me and showing an interest in my research. A special thanks to my friends at the periodontology section, Ulf Folkesson, Carola Höglund Åberg, Tara Hedayati and Christopher Appelqvist for good laughs and interesting odontological discussions. To all the nurses at the periodontology section, thank you for being so professional and so warm-hearted! Eva Utterberg, thank you for trying to satisfy my requests regarding research-time.

Linus Andersson, thank you for helping me with the cover of the thesis!

A special thanks to my fantastic friends in Umeå and scattered around Sweden — no one mentioned, no one forgotten! You are invaluable, and each of you brightens even the darkest day.

‘Matlaget’, thank you for everyday dinners, fabulous parties and great friendship.

38

ACKNOWLEDGMENTS

Gunvor and Einar, heads of the Larsson-Holm ‘clan’, I appreciate your hospitality, your ability to make everyone feel welcome and, most of all, thank you for including me in the clan. The Larsson-Holm clan, thank you for making every birthday party and family dinner a memorable experience!

To my grandparents Lea and Lars, thank you for always caring and being proud of me.

To my mother Lilian and father Lars-Owe, thank you for believing in me and loving me unconditionally. Doris and Hasse, you have extended and enriched my family. Mom & Hasse, Dad & Doris, the four of you are invaluable and I sincerely thank you for being there for my family and me.

To my brother, Robert, and sister, Susanne, and their families. Thank you for good times, for encouraging me through the years, and for your never- ending love.

To my best friend and the love of my life, Hampus, words cannot express how grateful I am to you for standing by me no matter what, and for sharing your life with me. My girls, Milla and Maja, you are the best things that ever happened to me. Thank you for being you!

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