Transduction pathways implicated in osteoblastic cells activation induced by parathyroid hormone (PTH)

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Thesis

Reference

Transduction pathways implicated in osteoblastic cells activation induced by parathyroid hormone (PTH)

REY, Alexandre François

Abstract

Le récepteur à l'hormone parathyroïdienne (PTH1R) est couplé aux protéines G et sa stimulation induit les cascades de signalisation Gαs/AMPc/PKA et Gαq/PLCβ/PKC. La liaison de la β-arrestine au récepteur engendre sa désensibilisation et son internalisation. Le PTH1R active également les voies des MAPKs. Nous avons étudié les déterminants structurels du récepteur et les molécules impliquées dans l'activation d'ERK1/2, ainsi que le rôle des MAPKs dans la différenciation ostéoblastique. Nous montrons que l'activation d'ERK1/2 implique deux voies de signalisation. La première impliquant la liaison de Gαq à la 2ème boucle intracellulaire du récepteur. La seconde impliquant la liaison de la β-arrestine au C-proximale du récepteur, ainsi que de c-Src aux motifs polyprolines de la partie C-distale du récepteur. La deuxième étude montre que la PTH active ERK1/2 et p38 dans les ostéoblastes. L'activation de p38 dépend de l'AMPc/PKA et p38 est impliqué dans la différenciation ostéoblastique et la minéralisation.

REY, Alexandre François. Transduction pathways implicated in osteoblastic cells activation induced by parathyroid hormone (PTH). Thèse de doctorat : Univ. Genève, 2008, no. Sc. 3982

URN : urn:nbn:ch:unige-5480

DOI : 10.13097/archive-ouverte/unige:548

Available at:

http://archive-ouverte.unige.ch/unige:548

Disclaimer: layout of this document may differ from the published version.

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UNIVERSITE DE GENEVE

Département de biologie cellulaire FACULTE DES SCIENCES

Professeur Jean-Claude Martinou

Département de Réhabilitation et Gériatrie FACULTE DE MEDECINE Service des Maladies Osseuses Professeur Joseph Caverzasio

Professeur Serge Ferrari

_____________________________________________________________________

Transduction Pathways

implicated in Osteoblastic Cells Activation induced by Parathyroid Hormone (PTH)

THESE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologique

par

Alexandre François Rey de

Chermignon (VS)

Thèse n° 3982

GENEVE

Atelier d’impression ReproMail 2008

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Remerciements/Acknowledgments

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Premièrement, j’aimerais remercier le Professeur René Rizzoli sans qui ce projet n’aurait pas vu le jour et dont les supports intellectuel et financier furent essentiels.

First, I would like to thank Prof. René Rizzoli for being the initiator of all this project and his intellectual and financial supports all along.

Ensuite j’aimerais remercier Serge et Joseph pour leurs conseils scientifiques et leur patience.

Second, I would like to thank Serge and Joseph for their scientific guidance and their patience.

De plus, j’aimerais remercier tous mes collègues, spécialement Danielle Manen, Madeleine Lachize, Pierre Apostolides pour leurs conseils techniques et Estelle Bianchi pour son précieux soutien.

Third, I would like to thank all my colleagues, specially Danielle Manen, Madeleine Lachize, Pierre Apostolides for their technical assistance and Estelle Bianchi for her supporting presence.

Finalement, j’aimerais remercier toutes les personnes qui sont dans mon coeur et qui savent combien elles sont importantes à mes yeux.

And finally I would like to thank all the people who know they are in my heart and also know, how they are important to me.

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Summary

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Parathyroid hormone (PTH), a peptide of 84 amino acids, is the major hormone responsible of calcium and phosphate homeostasis and bone remodeling. The actions of PTH and PTH-related peptides (PTHrP) are mediated by a G protein coupled receptor (GPCR), referred to as PTH receptor 1 (PTH1R) and is expressed in bone, cartilage and kidney. Ligand binding to PTH1R not only stimulates Gs-mediated activation of adenylyl cyclase, which in turn stimulates cAMP production and subsequent activation of protein kinase A (PKA), but also stimulates phospholipase C (PLC) by Gq leading to the activation of protein kinase C (PKC). PTH1R desensitization implies -arrestin recruitment to the receptor, which thus induces G proteins uncoupling and receptor internalization. In addition, PTH1R may activate the ERK1/2 mitogen-activated protein kinase (MAPK) pathway through a multitude of transduction cascades including second messengers of both classical G proteins pathways, as well as -arrestin and transactivation of the epidermal growth factor (EGF) receptor. Moreover, PTH1R structural determinant such as the juxta- membrane region of the receptor that binds the G subunit is implicated in ERK1/2 activation. Other GPCRs, such as the 2-adrenergic receptor, activates ERK1/2 through c-Src interaction with -arrestin, whereas the 3-adrenergic receptor through c-Src interaction with the receptor polyproline (PxxP) motifs. Concerning PTH1R, little is known on PTH1R structural determinants implicated in ERK1/2 activation as well as the role of c-Src.

There are mainly three families of MAPKs: ERK1/2, JNK and p38. ERK1/2 activation cascade is important for proliferation, development and differentiation, whereas activation of JNK and p38 are important for development, inflammation and apoptosis. MAPK signaling in osteoblasts may play an important role on osteoblastic proliferation and differentiation and thus on bone formation. PTH1R-mediated ERK1/2 activation is correlated with osteoblast proliferation, whereas nothing has been described on PTH-induced JNK and p38 activation. Moreover, cAMP pathway seems to be important for PTH anabolic effects in vivo.

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In order to clarify the molecular mechanisms underlying PTH activity, we first studied the PTH1R structural determinants and their interacting molecules implicated in ERK1/2 activation. Then we studied the role of MAPKs and their molecular mechanisms in the PTH anabolic effects on osteoblastic cell functions. For the purpose of molecular pharmacology studies we used Human Embryonic kidneys (HEK293) cells transiently expressing PTH1R or mutated receptors and cytoplasmic molecules. For functional pharmacology experiments we used murine osteoblastic (MC4) cells naturally expressing PTH1R.

Our structure-function studies indicate that PTH1R-mediated ERK1/2 activation engages both the 2^nd intracellular¨loop (IC2) and C-terminus of the receptor. IC2 indeed is necessary for Gq-mediated PKC activation, which is directly involved in ERKs phosphorylation (“classical pathway”). In addition PKC phosphorylates distinct serine residues in PTH1R proximal C-terminus, thereby recruiting beta- arrestins. In turn beta-arrestins interact with activated c-Src bound to polyproline (PxxP) motifs in the PTH1R distal C-terminus. The stable molecular complex constituted by PTH1R, beta-arrestins and c-Src is internalized into the cytoplasm and provides a second, “terminal pathway” for ERKs activation.

Our functional studies in osteoblasts in turn indicate that PTH activates both ERK1/2 and p38 MAP kinases, the latter through a camp-PKA-dependent pathway. We also show that PTH-stimulated p38 activation leads to enhanced alkaline phosphatase activity and matrix mineralization, whereas ERKs activation mediates osteocalcin production, which are indices of osteoblastic cell differentiation.

In conclusion, we have delineated PTH1R structure function relationships implicated in ERK1/2 activation, and osteoblastic cell differentiation, in addition to cAMP signaling.

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Résumé

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L’hormone parathyroïdienne, un peptide de 84 acides aminés, est la principale hormone responsable de l’homéostasie du calcium et du phosphate ainsi que du remodelage osseux. Les actions de la PTH et de ses peptides apparentés (PTHrP) sont médiées par un récepteur couplé aux protéines G appelé, le récepteur à la PTH de type 1 (PTH1R) qui est principalement exprimé dans l’os, le cartilage et le rein.

L’activation du récepteur par ses agonistes induit non seulement l’activation de l’adényle cyclase par la protéine Gs, conduisant à la production d’AMPc et par conséquent à l’activation de la protéine kinase A (PKA), mais stimule également la phospholipase C  (PLC) par la protéine Gq, qui à son tour active iCa/IP3 et ainsi la protéine kinase C (PKC). La désensibilisation du récepteur est engendrée par le recrutement de la -arrestine au récepteur, ce qui a comme effets de découpler les protéines G et d’internaliser le récepteur. De plus, le PTH1R est capable d’activer des mitogen-activated protein kinases (MAPKs) ERK1/2 à travers différentes voies de signalisation, incluant les seconds messagers des deux voies classiques des protéines Gs et Gq ainsi que via une transactivation du récepteur à l'EGF (epidermal growth facteur) médiée par les -arrestins. Il existe peu d’information quant aux déterminants structurels du PTH1R impliqués dans l’activation d’ERK1/2. Seule la région juxta-membranaire du C terminus, qui lie la sous-unité G, a été impliquée dans l’activation d’ERK1/2. De plus, certains GPCR activent ERKs via c-Src, tel le récepteur 2-adrénergique, grâce à la liaison de c-Src à la -arrestine, ou récepteur

3-adrénergique à-travers la liaison de c-Src aux motifs polyprolines (PxxP) du récepteur lui-même. Le rôle de c-Src dans l’activation d’ERKs par la PTH est

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inconnu, mais il doit être évoqué, étant donné la présence de plusieurs motifs PxxP dans la partie distale de PTH1R c-terminus.

Outre ERK, il existe deux autres groupes principaux de MAPKs : JNK et p38. En général, l'activation de la voie ERK1/2 est importante pour la prolifération et la différenciation cellulaires ainsi que pour le développement, alors que les voies JNK et p38 sont importantes pour le développement, l’inflammation et l’apoptose. Dans les ostéoblastes, l'activation des MAPKs joue un rôle important dans la prolifération et la différenciation ostéoblastiques et par conséquent sur la formation osseuse, du moins in vitro. En particulier, l'activation de ERK1/2 médiée par le PTH1R est impliquée dans la prolifération ostéoblastique, alors que concernant l’activation de JNK et p38, aucune information n’a été décrite. Il faut toutefois noter qu'in vivo, la voie de l’AMPc est essentielle aux effets de la PTH, alors que dans certaines cellules ostéoblastiques, cAMP-PKA peut activer MAPK.

Afin de clarifier les mécanismes moléculaires de l’activité de la PTH, nous avons tout d’abord étudié les déterminants structurels du récepteur ainsi que les molécules impliquées dans l’activation d’ERK1/2. Ensuite nous avons étudié le rôle des MAPKs et leurs mécanismes moléculaires sur les effets anaboliques de la PTH dans la différenciation ostéoblastique. Pour étudier les mécanismes moléculaires de la signalisation nous avons utilisé des cellules rénales humaines embryonnaires (HEK293 cells) exprimant transitoirement le PTH1R ou des récepteurs mutés, en association à des molécules cytoplasmiques, tandis que pour l’étude fonctionnelle de

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la signalisation, nous avons utilisé des cellules ostéoblastiques de souris (MC4 cells) exprimant naturellement le PTH1R.

La première étude montre que l’activation d’ERK1/2 médiée par le PTH1R requiert deux voies de signalisation. En termes de ‘structure-fonction’, la première que l’on nomme ‘classique’, implique la liaison de Gq à la seconde boucle intracellulaire du récepteur, et la seconde voie, que l’on nomme ‘terminale’, implique la liaison de la - arrestine à la partie proximale du C terminus du récepteur aussi bien que la liaison de c-Src aux motifs polyproline (PxxP) de la partie distale du C terminus du récepteur. De plus, il existe une coopération entre la voie classique et terminale qui se situe en amont de l’activation d’ERK1/2, qui se traduit par la phosphorylation des résidus sérine par la PKC afin de permettre la liaison de la -arrestine au récepteur.

Nous montrons également que le complexe moléculaire formé du PTH1R, de la - arrestine et de c-Src est important dans la stabilisation de l’internalisation du récepteur.

La deuxième étude montre que la PTH active les voies des MAPKs ERK1/2 et p38 dans les ostéoblastes. Nous démontrons que l’activation de p38 médiée par le PTH1R est clairement dépendante de la cascade de signalisation AMPc/PKA et que p38 est également impliqué dans l’augmentation de l’activité de l’ALP ainsi que de la minéralisation de la matrice. De plus, nous montrons que l’activation de ERK1/2 induit par la PTH est responsable de la production d’ostéocalcine.

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En conclusion, nous avons déterminé le rôle essentiel des régions IC2 et C-terminus du PTH1R, spécifiquement des motifs PxxP, et de la liaison à c-Src, dans l'activation de ERK en réponse à la PTH. De plus, nous montrons qu’outre l’AMPc, l’activation des MAPKs est impliquée dans la différenciation ostéoblastique.

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Abbreviations

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AC: adenylyl cyclase

ALP : alkaline phosphatase

ATDC5 : murine chondrocyte cell line AT1R: angiotensinogen 1A receptor

2(3)-AR: 2(3)-adrenergic receptor BGP: bone gla protein (osteocalcin) BMD : bone mineral density

BMU: basic multicellular unit Bone morphogenic protein: BMP Ca²⁺: calcium

cAMP : cyclic adenosine 3’,5’-monophosphate CATK: cathepsin K

Cbfa1 : core-binding protein a-1 (Runx2)

CPTHR: carboxyl (C)-terminal parathyroid hormone receptor CREB: cAMP responsive element-binding

CSF-1: colony-stimulating factor-1 c-Src: c-Src tyrosine kinase

1,25(OH)2D3 : 1,25-dihydroxyvitamin D Dkk1(2): dickkoff 1 (2)

Calvariae cells : primary murine calvariae cell cultures COL1 : type 1 collagen

DAG : diacyl glycerol ECM : extracellular matrix

EGF(R): epidermal growth factor (receptor)

ERK1/2: extracellular signal-regulated kinases1/2 FGF: fibroblast growth factor

FSH: follicule-stimulating hormone GDP : guanosine di-phosphate GPCR : G-protein coupled receptor GRK: G-protein-coupled receptor kinase GTP : guanosine tri-phosphate

HEK293 : human embryonic kidney cell line IC2: second intracellular loop

IFN: interferon Ig: immunoglobulin IGF: insulin growth factor IL: interleukin

IP3 : inositol tri-phosphate

JNK: c-Jun amino-terminal kinase KO : knocked out

Lrp5(6): lipoprotein receptor-related protein 5 (6) MG63 : human osteosarcoma cell line

MAPK : mitogen-activated protein kinase MKKK : MAPK kinase kinase

MKK : MAPK kinase

MC3T3 : murine osteoblastic cell line

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MC4 : subclone of MC3T3 NMU: neuromedin U NPY : neuropeptide Y OB: osteoblast

OC: osteoclast

OPG : osteoprotegrin Osteopontin: OPN

PDE4: phosphodiesterase 4

PDGF : plateled derived growth factor PGF-2 : prostaglandin factor-2

PKA : protein kinase A PKC : protein kinase C PLC : phospholipase C

POB: primary osteoblastic cells PTH: parathyroid hormone iPTH: intermittent PTH

PTHrP : parathyroid hormone related protein PTH1R : parathyroid hormone type 1 receptor PTH2R: parathyroid hormone type 2 receptor PxxP: polyproline

RANK: Receptor activator for Nuclear Factor B RANKL : RANK ligand

ROS17/2.8 : rat osteoblast-like osteosarcoma cell line BSP: bone sialoprotein

SaOS2 : human osteoblast-like osteosarcoma cell line SERM : selective estrogen receptor modulator

SRE: Serum Responsive Element SRF: Serum response factor TCF: ternary complex factors

TGF- : transforming growth factor  TKR : tyrosine kinase receptor

TNF: tumour necrosis factor

TRAP: tartrate-resistant acid phosphatase

TIP39: tuberoinfundibular peptide of 39 residues UMR106 : rat osteoblast-like osteosarcoma cell line V2R: vasopressin receptor

WT: wild type

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General introduction

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1. Bone tissue

Bone is a mineralized connective tissue that serves three functions: 1) mechanical, as support and site of muscle attachment for locomotion; 2) protective, for vital organs and bone marrow; and 3) metabolic, as a reserve of ions, especially calcium and phosphate, for maintenance of serum homeostasis which is essential to life.

Anatomically, two types of bone are present in the skeleton: long bones (femur, tibia, humerus, etc.) and flat bones (skull bones, ileum, etc.). External examination of a long bone shows two wider extremities (the epiphyses), a cylindrical tube in the middle (the diaphysis), and a zone between them (the metaphysis). The external part of bones is formed by a thick layer of calcified tissue, the cortex (Figure 1), which, in the diaphysis encloses the medullar cavity where the haematopoietic bone marrow is housed. The internal space of the metaphysis and the epiphysis is filled with a network of thin calcified trabeculae; this is the cancellous bone or trabecular bone (Figure 1). The spaces enclosed by these thin trabeculae are also filled with hematopoietic bone marrow. Consequently there are two bone surfaces at which bone is in contact with the soft tissues: an external surface (the periosteal surface) and an internal surface (the endosteal surface) (Figure 1). These surfaces are lined with osteogenic cells organized in layers, the periosteum and the endosteum. Cortical and trabecular bone are made of the same cells and the same matrix proteins, but there are functional differences. Cortical bone has mainly a mechanical and protective function whereas trabecular bone has in general a metabolic function. Nonetheless, in vertebral bodies trabecular bone may also have a role in bone strength.

The fundamental constituents of bone are cells (bone-lining cells, osteoblasts, osteocytes and osteoclasts) and the mineralized extracellular matrix. Osteoblasts (OB) (bone-forming cells) synthesize and secrete the matrix constituents (collagen and ground substance). Osteocytes are mature bone cells made from osteoblasts that have made bone tissue around themselves. Osteoclasts (OC) are large cells that break down bone tissue. The last type of cells are bone-lining cells. These are made from osteoblasts along the surface of most bones in the skeleton. Bone-lining cells are thought to regulate the movement of calcium and phosphate into and out of the 18

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bone. The extracellular matrix is extremely abundant, composed of collagen fibers (type I, 90% of the total protein) usually oriented in a preferential direction, and noncollagenous proteins (ground substance) such as glycoproteins, proteoglycans, osteopontin (OPN), bone sialoprotein (BSP) and bone gla-protein (BGP) also known as osteocalcin. Osteoblasts are responsible of the extracellular matrix synthesis and mineralization. The mineral phase is constituted of crystals of hydroxyapatite [3Ca3(PO4)2(OH)2] and are found on collagen fibers, within them, and in the ground substance. The preferential orientation of collagen fibers alternates in adult bone from layer to layer, giving to this bone a typical lamellar structure. The lamellae can be parallel to each other if deposited along a flat surface, or concentric if deposited on a surface surrounding a channel centered on a blood vessel (haversian system) (1).

A B C

Cortical Trabecular Bone Bone

Figure 1. Microquantitative computed tomography (micro-CT) image of a sagittal section of a human radius. On the enlarged picture, the cortex (cortical bone) is subdivided in three compartments: periosteal (A), intracortical (B) and endocortical (C). Trabecular or cancellous bone is made of interconnected trabeculae.

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1.1 Osteoblast differentiation and bone formation

The progression of osteoblast maturation requires a sequential activation and suppression of genes that encode the phenotypic and functional proteins of the osteoblast populations. Signaling proteins, transcription factors and regulatory proteins support the temporal expression of genes that characterize stages of osteoblast differentiation and extracellular matrix synthesis and mineralization (bone formation) (Figure 2).

Osteoprogenitor cells arise from multipotential mesenchymal stem cells that give rise to a number of cell lineages including those for osteoblasts (OB) (2). The differentiation towards the osteoblastic lineage is controlled by genes related to the Hedgehog family among which Indian hedgehog (Ihh) and Sonic hedgehog (Shh) are implicated (3, 4). The transcription factors such as Cbfa1 (core-binding protein a-1, also known as Runx2) and osterix (5) are crucial for osteoblastic differentiation.

Signaling molecules such as -catenin activated by Wnt proteins (6-8) (see below) and Smad proteins activated by the bone morphogenic proteins (BMPs) are also powerful regulators of osteoblastic differentiation from the mesenchymal stem cells (Figure 2) (4).

Differentiation towards the osteoblastic versus adipocytic lineage strongly depends on specific transcription factors such a Cbfa1 for the osteoblastic lineage (9-11) and PPAR (peroxisome proliferator-activated receptor gamma) for the adipocytic lineage (12). In fact, when PPAR is more expressed than Cbfa1, differentiation towards the adipocytic lineage is more important and thus induces a decreased number of osteoblasts (‘Fat gain is bone loss’). This correlates with the fact that with ageing bone marrow adipogenesis is increased (13) and moreover, PPAR activators such as glitazones decrease bone mass and are associated with an increased risk of fractures (14-17). Another transcription factor implicated in bone formation is Sox4.

In fact, Sox4 knockout mice are lethal but hererozygote mice have an intrinsic osteoblast defect that produces cortical and trabecular osteopenia. Proliferation,

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differentiation and mineralization of cultured Sox4^+/- osteoblasts is dramatically impaired.

Among the local signaling factors implicated in the remodeling process are Wnt ligands. Their role was inferred after the identification of mutations in the Wnt co- receptor low-density lipoprotein receptor-related protein 5 (Lrp5) in patients with heritable skeletal diseases. ‘Loss-of-function’ mutations in Lrp5 were found to cause the Osteopetrosis-Pseudoglioma syndrome (OPPG), an autosomal recessive disorder characterized by extremely low BMD and skeletal fragility (18). Missense mutation in Lrp5 that are thought to create a ‘gain of function’ cause autosomal dominant high bone mass phenotypes in which BMDs are well above the population mean (19, 20).

Lrp5, a single-transmembrane-domain receptor, interacts with the frizzled receptor complex to inhibit the phosphorylation of -catenin by glycogen synthetase kinase-3 (GSK-3). As phosphorylated -catenin is more susceptible to ubiquitin-mediated degradation, by inhibiting GSK-3 activity, Lrp5 allows accumulation of -catenin and its nuclear ingress. Activated -catenin then interacts cooperatively with Tcf/Lef transcription factors to stimulate osteoblast differentiation. Mutations in Wnt members, ablation of the Wnt co-receptor Lrp5 or Lrp6, or use of Wnt inhibitors such as sclerostin, soluble frizzled-related proteins, Wnt inhibitors factors and the Dickkoff family members Dkk1 and Dkk2, reduce osteoblastogenesis and bone formation (21-24).

Once the osteoblastogenic cells are committed towards the osteoblastic lineage, they will temporally express proteins involved in extracellular matrix biosynthesis and mineralization. The extracellular matrix is extremely abundant, composed of collagen fibers (type I, 90% of the total protein) usually oriented in a preferential direction, and noncollagenous proteins (ground substance) such as glycoproteins, proteoglycans, osteopontin (OPN), bone sialoprotein (BSP) and bone gla-protein (BGP) also known as osteocalcin. The expression of these proteins provide a panel of osteoblast phenotypic markers that reflect stages of osteoblastic differentiation (Figure 1). Based on bone nodule formation in vitro, the process has been subdivided into three stages: 1) proliferation, 2) extracellular matrix development and

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maturation, and 3) mineralization, with characteristic changes in gene expression at each stage. Thus, the proliferation phase is associated with the expression of early genes such as c-fos and histone H4 (25, 26). Then, expression of the osteoblast- associated genes collagen type I (COLL I), alkaline phosphatase (ALP), osteopontin (OPN), osteocalcin (BGP), bone sialoprotein (BSN), and parathyroid hormone receptor (PTH1R) is asynchronously upregulated, acquired, and/or lost as the osteoprogenitor cell differentiate and extracellular matrix matures and mineralizes (Figure 2) (27). In general, ALP increases then decreases when mineralization is well progressed. ALP catalyses the hydrolysis of phosphate esters at the osteoblast cell surface to provide a high phosphate concentration for bone mineralization process.

OPN peaks twice, during proliferation and then again later but before certain other matrix proteins including BSP and BGP. OPN as well as BSP are proteins that link bone cells and the hydroxyapatite of mineralized matrix. BSP is transiently expressed very early and then upregulated again in differentiated osteoblasts. Osteocalcin (BGP) appears approximately concomitantly with mineralization (27). BGP is thought to play a role in mineralization and calcium homeostasis. It has been postulated that it may also function as a negative regulator of bone formation (28).

Moreover, very recently osteoblast-secreted osteocalcin has been found to exert an endocrine regulation of sugar homeostasis (29). In fact, mice deficient in the osteoblastic-specific protein kinase OST-PTP have hyperinsulinemia, hypoglycemia and increased sensitivity to insulin, demonstrating that bone exerts humoral control over both pancreatic -cells and insulin target tissues. Osteocalcin knockout mice have an opposite phenotype, and removing one osteocalcin allele corrects the OST- PTP phenotype. Moreover, administraion of uncarboxylated osteocalcin improves glucose tolerance in vivo.

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Cbfa1 Osterix

-catenin Sox4

Histone c-Fos COLL I OPN

ALP BSP COLL I

BGP OPN Lineage

commitment

Proliferation

Differentiation Growth

Mineralization Matrix

maturation A.

B.

Osteocyte Mesenchymal

stem cell

Osteoprogenitor Preosteoblast Mature osteoblast

Figure 2. Growth and differentiation of osteoblasts. A. Temporal expression of osteoblast genes during the stages of proliferation, matrix maturation and mineralization. B. The reciprocal and functionally coupled relationship between cell growth and differentiation-related gene expression is illustrated by arrows. Broken vertical lines between the three principal stages indicate transition points requiring downregulation of cell growth and extracellular matrix signaling events for the progression of differentiation. (COLL I: collagen type I; OPN: osteopontin; ALP:

alkaline phosphatase; BSP: bone sialoprotein; BGP: osteoclacin; OPN: osteopontin).

In summary, osteoblasts are key cells for bone formation during skeletal growth, renewal of bone matrix and bone repair. Their functions are 1) to synthesize collagen I and non collagenous proteins (ALP, BSP, OPN, OC, OPN) of the bone extracellular matrix; 2) direct the arrangement of the extracellular matrix fibrils; 3) contribute to the mineralization of bone matrix due to ALP that increases levels of phosphate (necessary for mineralization) by degrading inorganic pyrophosphates; 4) synthesize growth factors for the autocrine/paracrine regulation of bone formation, such as insulin growth factor (IGF) I and II, transforming growth factor (TGF-) and bone morphogenetic proteins (BMPs) (30); and 5) synthesize cytokines and other

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molecules for the coupling of osteoblasts to osteoclasts in order to regulate bone remodelling.

1.2 Osteoclast differentiation and bone resorption

The osteoclast (OC) is a tissue-specific macrophage polykaryon created by the fusion and differentiation of monocyte/macrophage precursors cells (Figure 3). The initial stage of differentiation is under the control of PU.1, a transcription factor responsible for the development of both osteoclasts and macrophages (31). Expression of CD200 is potently induced de novo in macrophages at the onset of fusion and by RANKL (Receptor activator for Nuclear Factor B Ligand). OCs deficient in CD200 had a defect in multinucleation and in signaling domnstream of receptor of NF-B (RANK) (possible cross-talk between CD200 receptor and RANK), which are essentiel for osteoclastogenesis. Moreover, CD200-deficient mice have a lower number of OCs and a higher bone density (32). The close contact between osteoblasts/stromal cells and osteoclastic precursors is also essential for osteoclast differentiation. In fact, two osteoblasts/stromal-derived factors are necessary and sufficient to induce osteoclast differentiation, the tumour necrosis factor (TNF)-related cytokine RANKL, and the polypeptide growth factor CSF-1 (colony-stimulating factor-1) (33, 34). Both factors are required to induce proteins-specific expression of the osteoclast lineage, including tartrate-resistant acid phosphatase (TRAP), cathepsin K (CATK) and the

3-integrin (33), leading to the development of mature OCs. Osteoclast differentiation also involves cell-fusion. This will increase the area in contact with bone relative to the circumference of the cell bone-attachment, which must be sealed to produce acid environment.

The mature, multinucleated OC is then activated by RANKL binding to its receptor RANK. In response to activation of RANK (35), the osteoclast undergoes structural changes, such as the formation of a tight junction with the bone surface and the rearrangements of the cytoskeleton to form a ‘ruffled membrane’ (capacity of polarization) (Figure 3). This infolding of the plasma membrane appears only when

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the cell is attached to the mineralized bone surface. Once this external ‘vacuole’ is formed between the plasma membrane and the bone surface, it is acidified by the production of hydrogen ions generated by a vacuolar-proton pump encoded by the gene Atp6i (36). This electrogenic pump drives chloride cosecretion through Cl^- channel. This HCl secretion is accompanied with the export of the proteolytic enzymes TRAP and pro-CATK (Figure 3). Through this process bone is dissolved and degradation products, such as collagen fragments, calcium and phosphate are removed by vesicular transcytosis and released into the circulation. Moreover, osteoblasts/stromal cells may produce a protein named osteoprotegerin (OPG) that may act as a decoy receptor by blocking RANKL binding to RANK, and thus negatively regulating osteoclastic differentiation and bone resorption (37).

Interestingly, bone marrow immune cells also have an important role on osteoclastogenesis (38-40). B cells also produce OPG and B-cell knockout mice were found to be osteoporotic and deficient in bone marrow OPG. Moreover, through CD40 ligand to CD40 receptor stimulation, T cells have been shown to promote OPG production by B cells in vivo (41). However, T-cells are a primary source of RANKL for osteoclast activation.

Preosteoclast Fused polykaryon

Activated osteoclast Mature

osteoclast

M-CSF

RANKL RANKL

Cl^- Ruffled membrane

Bone

Figure 3. Osteoclastogenesis and osteoclast activation. Preosteoclast differentiates into mature osteoclast, a fused polykaryon arising from 10 to 20 individual cells. M- CSF (CSF-1) and RANKL are essentiel for osteoclastogenesis. Under the action of M- CSF and RANKL the fused polykaryon is recruited, adhere to bone and differentiate into a mature osteoclast. Then, RANKL activates the osteoclast to secrete HCl and proteolytic enzymes responsible for the degradation of bone matrix. v3 integrins attach the osteoclast to the bone surface in order to form a sealed compartment. OPG can neutralize RANKL and negatively regulate osteoclastogenesis and osteoblast activation. Adapted from Boyle, Nature (2003).

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1.3 Bone remodeling

In vertebrate skeleton, bone is in a dynamic state being continuously destroyed and reformed in order to prevent degradation of function as bone becomes older, to preserve constant bone mass, to repair microdomage and to maintain calcium homeostasis (42, 43). This physiological process named ‘bone remodeling’ is possible through the coordinated actions of osteoclasts and osteoblasts which are responsible for bone resorption and formation, respectively. This turnover or remodeling of bone mostly occurs at the bone surfaces.

Bone replacement begins by osteoclastic resorption followed by osteoblastic formation. These processes are not independent but closely linked and take place within small anatomical structures which are called ‘basic multicellular units’ (BMU) (44). In the skeleton, some 10⁶BMUs are activated but are geographically and chronologically separated from each others. The different stages of the bone remodeling sequence are shown in Figure 4. The sequence begins with the activation and retraction of the bone-lining cells. This activation may be induced by the osteocytes within the matrix. In fact, osteocytes may sense microdomage or send apoptotic signals in order to initiate bone remodeling (45, 46). Then, pre-osteoclasts derived from hematopoietic cells of the monocyte/macrophage lineage in the bone marrow or from circulating precursors, migrate from a blood vessel to the active site, where they merge to form multinucleated osteoclasts. They will assemble to create the hemicone or cutting cone in order to begin to excavate a cavity or a tunnel. The front line of this cavity becomes sclerotic and is called ‘the cement line’. This stage is short in time (about 2-4 weeks) and is characterized by continued arrival of new osteoclasts to replace the ones that underwent apoptosis. The resorption phase is terminated when all osteoclasts disappear by apoptosis. Then, after a reversal phase, the resorption cavity becomes lined by osteoblasts. Osteoblasts synthesize the extracellular matrix or osteoid material (formation phase) that, after a lag period, mineralizes to form new bone. The formation phase is much longer than the

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resorption phase and lasts about 4-6 months. It ends when the cavity is refilled to form a completed osteon (47-50).

BONE MARROW

Osteoclast (OC) precursors

OBs Lining cells

OCs Lining

cells

Osteoblast (OB) precursors

Figure 4. Illustration of a BMU showing the different phases and cellular activity of bone remodeling through time. It is a two dimensions illustration, but in vivo, the new BMU progresses in three dimensions with osteoclasts continuing to resorb bone (resorption phase), leaving behind them the eroded bone (reversal phase) where it becomes lined by osteoblasts. Adapted from Riggs et al. JBMR (2005).

The components of the bone remodeling sequence can be assessed quantitatively only by histomorphometry of bone biopsies. Therefore, Riggs and Parfitt have proposed an equation that describes the remodeling sequence at each BMU (50):

 BS (m/y) = Ac.f (/y) x (W.Th – E.De) (m)

where  BS is the rate at which bone is added or removed from bone surfaces, Ac.f is the activation frequency, W.Th is the wall thickness of the completed osteon, E.De is the erosion depth of the cavity, (W.Th – E.De) is the remodeling balance and represents the net gain or loss of bone at the BMU level.

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This equation has two major components: activation frequency and remodeling balance.

Activation frequency is the probability that bone remodeling will occur anywhere at any time and is the summation of several processes, most specifically the recruitment and differentiation of osteoclast precursors. Activation frequency is also proportional to the number of new BMUs being formed. Because activation frequency is not the same in all individuals and varies among disease states by up to 10 fold, it is therefore the main target of bone remodeling by pharmacological agents. The other component, the Remodeling balance represents the algebraic difference of the formation phase minus the resorption phase at the BMU level. If the cavity is not completely filled by osteoblasts, the balance will be negative, whereas if it is overfilled the balance will be positive. Remodeling balance does not vary as much as activation frequency does. It represents only 1/100 of the variability of the activation frequency. However, it is this small change in remodeling balance that allows bone to be gained or lost, because steady-state changes in activation frequency will not cause changes in bone mass if the remodeling balance is zero, hence the concept of coupling (see section 1.3.1) (51). However, changes in activation frequency may have short-term effects on bone mass because of expansion or contraction of the remodeling space, but these do not continue after steady state is achieved. Therefore, steady-state changes in bone mass can be estimated by the product of activation frequency (Ac.f) x remodeling balance (W.th – E.De).

1.3.1. The Concept of Coupling

The remodeling of bone also involves a strict coupling of bone resorption and formation. This coupling process is made possible through local and systemic humoral mechanisms. The notion that coupling is mediated locally by an autocrine/paracrine process, comes from a significant amount of experimental evidence. Many cytokines present in the extracellular matrix or synthesized by bone cells have been shown to be involved in bone remodeling. For instance, osteoblast- stimulating factors such as insulin growth factor (IGF) I and II, transforming growth factor (TGF-) and bone morphogenetic proteins (BMPs) are released from the matrix 28

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during bone resorption and may be important positive regulators of bone formation in increasing osteoblastic differentiation (52-56). Other growth factors, such as platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) may also play an important role in physiological remodeling in enhancing migration and proliferation of osteoblastic precursors (57, 58). As already described, the notion of local coupling has culminated with the cloning of two molecules that are responsible for the interaction between cells of the osteoblastic and osteoclastic lineage: OPG which is an inhibitor of osteoclast differentiation, and RANKL which is an osteoclast differentiation factor (33, 34, 37).

Moreover, recent findings on ephrins have been shown to be implicated in a new and possibly major coupling system between osteoblasts and osteoclasts in bone remodeling. In fact, Zhao et al. have used mouse models to demonstrate bidirectional signaling between osteoclasts and osteoblasts. This bidirectional signaling is mediated by the transmembrane ephrinB2 ligand in osteoclasts and EphB4, a tyrosine kinase receptor, in osteoblasts. According to the scenario suggested by the authors, reverse signaling from osteoblasts to osteoclasts attenuates osteoclasts differentiation and forward signaling from osteoclasts to osteoblasts favors bone formation at sites where bone resorption had recently occurred (reversal phase) (59).

The control of coupling is not only local but also systemic. Hormones such as parathyroid hormone (PTH) and 1,25-dihydroxy vitamin D increase bone resorption and bone formation, whereas calcitonin decreases bone resorption (60-64). Growth hormone, acting through both systemic and local IGF production, can stimulate bone formation and resorption (52). Sex steroid hormones also act on bone remodeling. In fact, androgen increases bone formation, whereas oestrogen decreases bone resorption by decreasing remodeling, but formation is decreased less than resorption and bone mass increases (65). Other hormones such as thyroid hormones can also stimulate bone resorption and formation and are critical for maintenance of normal bone remodeling (66). Moreover, a new theory that implies Follicule-stimulating hormone (FSH), which rises in post-menopausal women, has been demonstrated to be responsible for hypogonadal bone loss. Accordingly, bone loss would not only be 29

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due to oestrogen deficiency (responsible for the decrease in osteoblast activity), but also due to FSH increase which would trigger bone resorption directly (67).

Lastly, a novel systemic control of bone remodeling has been described: the central control of bone formation, which is mediated by a neuroendocrine mechanism. This central regulation involves leptin, an adipocyte-secreted anorexigenic hormone that controls body weight, reproduction and bone remodeling, and which binds to and exerts its effects through the hypothalamus (68-70). It has been demonstrated that leptin acts on the hypothalamus to stimulate the sympathetic nervous system and that the latter leads to 2-adrenergic receptors stimulation of osteoblasts to inhibit bone formation. 2-adrenergic receptors on osteoblasts regulate their proliferation, and a -adrenergic agonist decreases bone mass in leptin-deficient and wild-type mice while a -adrenergic antagonist increases bone mass in wild type and ovariectomized mice (71). Moreover, it has been shown that the sympathetic nervous system also favours bone resorption by increasing expression in osteoblast progenitor cells of the RANKL (72). Thus, leptin-regulated neural pathways control both aspects of bone remodeling.

Neuropeptide Y (NPY) is a downstream modulator of leptin action, possibly at the level of the arcuate nucleus (in the hyopthalamus) where NPY neurons are known to express both leptin receptors and Y2 receptors. Central administration of NPY causes bone loss and it has been shown that Y2 receptor-deficient mice and selective deletion of hypothalamic Y2 receptors in mature conditional Y2 knockout mice have an increased bone mass. This hypothalamus specific Y2 receptor deletion stimulates osteoblast activity and bone formation (73). It has been shown more precisely that the Y2-mediated anabolic pathway stimulates cortical and cancellous bone formation, whereas the leptin-mediated pathway has opposing effects in cortical and cancellous bone (74). Moreover, an vitro study suggested that the specific mechanism by which deletion of Y2 receptors increases bone mass implies a greater number of mesenchymal progenitors and an altered expression of Y1 receptors within the bone cells (75).

Another central control of bone remodeling has recently been described implicating neuromedin U (NMU), another anorexigenic neuropeptide. NMU knockout mice

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have high bone mass owing to increase in bone formation. Central administration of NMU decreased fat mass and bone mass in NMU-deficient mice and in leptin- deficient mice indicating that NMU regulates bone mass centrally and acts independently of leptin. Paradoxically, leptin increased bone volume in NMU knockout mice suggesting that NMU acts downstream of leptin. Interestingly, central administration of leptin or injection of isoproterenol (a sympathomimetic) did not reduce bone mass in NMU knockout mice, indicating that NMU knockout mice were resistant to the anti-osteogenic effects of both leptin and sympathetic nervous system activity. Therefore it has been suggested that NMU affects only the negative regulator of bone remodeling by leptin, that is, the molecular clock genes (mediators of the inhibition of bone formation by leptin) (76).

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1.3.2 Pathophysiology of osteoporosis and treatment effects on bone remodelling

Abnormalities of bone remodeling can produce a variety of skeletal disorders.

Osteoporosis is by far the most common metabolic disorder of the skeleton (77). This disorder has been divided into type 1, or postmenopausal osteoporosis, and type 2, or age-related osteoporosis, on the basis of possible differences in pathophysiological mechanisms involved. Studies have suggested that oestrogen deficiency is important for the pathogenesis of both types of osteoporosis and in both men and women (78, 79). Nevertheless in type 2 osteoporosis there is a relative deficiency of calcium and vitamin D (insufficient sun exposition, reduced intestinal absorption of vitamin D, decreased food intake, decreased renal 1-alpha-hydroxylase activity), which induces hyperparathyroidism and hence increases bone resorption.

Osteoporosis is defined as a decrease in bone mass and (strength) alterations in microstructure, such as trabecular thinning and loss of connectivity, cortical thinning and porosity leading to increased propensity to fracture. The loss of bone mass and strength can be contributed to by 1) failure to reach an optimal peak bone mass as a young adult, 2) excessive resorption of bone after peak bone mass has been achieved, or 3) an impaired bone formation response during bone remodeling. Studies using bone markers show that there is accelerated bone remodeling at menopause (80-82) and that bone formation may increase overall, but that the rate is inadequate to replace the bone lost by resorption. This is because oestrogen deficiency augments erosion depth by prolonging the resorption phase through increased osteoclastic lifespan due to reduced apoptosis (83). However in spite of stimulated osteoblast differentiation, the net increase in bone formation is inadequate to compensate for enhanced bone resorption because of an augmentation in osteoblastic apoptosis, a phenomenon also induced by oestrogen deficiency (84). Moreover, Follicule- stimulating hormone (FSH), which rises in post-menopausal women, has also been demonstrated to trigger bone resorption (67). In addition, oestrogen deficiency also leads to an increased expression of cytokines such as IL-1, IL-6, IFN and TNF,

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whose action is to stimulate bone resorption. Hence, T cells and dentritic cells have been implicated in oestrogen dependent bone loss (85-89).

The physiopathological mechanism of postmenopausal and age-related osteoporosis is an overall increase in bone remodeling in which bone resorption exceeds bone formation and, as a consequence bone mass decreases and fracture risk rises. At present, treatment of postmenopausal osteoporosis to prevent fractures include mostly inhibitors of bone resorption such as selective oestrogen receptor modulators (SERMs) and bisphosphonates (90, 91). Potent bisphophonates currently available decrease bone turnover, initially allow osteoblasts to refill the remodeling space, and prevent osteoclasts to further degrade cancellous bone structure, i.e. thinning and perforation of trabeculae; they later increase the degree of mineralization; and perhaps diminish cortical porosity (92). Due to their high affinity for the bone matrix, their effect may be lasting for months to years after treatment withdrawal.

However, bisphosphonates do not actively build new bone. In contrast, daily intermittent administration of parathyroid hormone stimulates bone remodeling, which directly increases trabecular thickness and bridging, and may prompt some de novo bone modeling and subsequent thickening of the cortex (93). However, as a result of its increased bone turnover, PTH causes cortical porosity and decreases the degree of mineralization, at least initially. A large randomized prospective clinical trial in postmenopausal women treated with daily sub-cutaneous injections of recombinant PTH (1-34) for 22 months reported a 17% increase in both spine and femur bone mineral density (BMD) and a 53-65% reduction in fractures (94). Of note, discontinuation of PTH leads to a rapid decline in bone mineral density (BMD), because PTH increased the remodelling space without further stimulation of osteoblast activity once PTH is stopped. Therefore, it is advisable to administer an antiresorptive agent such as a bisphosphonate after treatment with PTH in order to maintain the densitometric gains achieved with PTH (95, 96).

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2. Parathyroid hormone (PTH)

Parathyroid hormone (PTH), a peptide of 84 amino acids, is a major regulator of calcium and phosphate homeostasis and bone remodeling (97). The parathyroid glands are the principle site for PTH synthesis and secretion; these processes are primarily regulated by blood levels of calcium and 1,25-dihydroxyvitamin D (1,25(OH)2D3), which are, in turn, under the control of PTH. Calcium negatively regulates PTH secretion via activation of the Calcium Sensor Receptor (98, 99).

1,25(OH)2D3 regulates PTH secretion both indirectly (via calcium) and directly (100).

The two major target organs of PTH action are bone and kidney (97). In the kidney, PTH acts at two sites: in the proximal tubule, PTH inhibits phosphate reabsorption and activates 1,25(OH)2D3 formation; in the distal tubule, PTH stimulates calcium absorption. In bone PTH targets its complex actions to cells of the osteoblast lineage, and stimulate osteoclastogenesis indirectly through activation of osteoblastic cell with consequent resorption of bone matrix and release of calcium. The primary physiological consequences of increasing serum PTH are an increase in serum calcium, a decrease in serum phosphate, an increase in circulating 1,25(OH)2D3, and an increase in bone remodeling.

2.1 PTH effects in vivo

Although constant, high levels of PTH cause increased bone resorption, low and intermittent doses of PTH, promote bone formation and increase bone mineral density (101). Concerning PTH effects in vivo, a classical clinical example where bone is exposed to a constant PTH concentration, is hyperparathyroidism (can be mimicked by continuous PTH infusion). This pathophysiological situation leads to an increased cortical porosity, a decreased cortical thickness by endocortical resorption, but trabecular microstructure may be improved (61, 102-106). In the case of daily intermittent PTH (iPTH) exposure, cortical porosity is also increased but

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cortical thickness and trabecular microstructure are drastically increased (61, 94, 107- 111).

Hence, increased levels of circulating PTH increase bone remodeling, and this has dramatically different effects in bone depending on PTH dosage regimen.

Intermittent exposure to PTH as a once- or twice-daily subcutaneous injection increases bone formation over resorption, resulting in a net gain in bone mass in humans and rodents (94, 112-116). Thus, daily intermittent PTH (iPTH) is associated with increased cancellous bone volume, density and strength. iPTH has complex effects on cortical bone, including increases in cortical porosity and cortical thickness that result in neutral to positive effects on cortical strength (61, 94, 107-111). In contrast, continuous infusion of PTH (or hyperparathyroidism) increased serum calcium in humans (117) and rats (61, 118) and preferentially stimulated bone resorption with smaller effects on formation, causing a net loss of bone mass (61, 62, 101). Nevertheless, PTH-derived molecules such as PTH-Fc (PTH fused to the Fc fragment of human IgG1) that has a more prolonged half-life, has been shown to be more powerful than PTH. Mice or rats only treated once or twice a week with PTH- Fc significantly improved cortical bone volume and density compared to animals treated with daily PTH (119).

There are two general mechanisms proposed for the PTH-related anabolic effect, which require its direct action upon the osteoblast lineage: 1) promoting the differentiation of committed osteoblast precursors (120) and 2) inhibiting osteoblast apoptosis (121, 122). Because PTH affects both bone formation and resorption, and because the activities of osteoclasts and osteoblasts are linked through the normal process of bone remodeling, it is likely that the anabolic effect of PTH relates either directly or indirectly to bone remodeling. This could mean that PTH treatment results in the activation of new BMUs, that the balance of formation against resorption is improved within BMUs or a combination of the two. This view is consistent with the fact that the anabolic effect of PTH is greater on trabecular bone and on endocortical bone (94, 110). Indeed, bone remodeling takes place where osteoclasts can attach, i.e. on the trabecular and endocortical bone surfaces, whereas bone modeling takes place where only osteoblasts are present, i.e. on the periosteal 35

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and ‘quiescent’ bone surfaces in general. Therefore, PTH effect is particularly marked on the endocortical surface, which is actively remodeling in old age (123, 124). On the contrary, the periosteal response to intermittent PTH is only observed at the highest PTH concentrations and therefore appears to be less prominent compared to the response in the endosteal and trabecular compartments (125). This observation underscores that PTH may also have different effects on osteoblasts, depending on their localization in bone. In summary, PTH effects in vivo are dependent on the dose and mode of administration, as well as on bone compartments.

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2.2 PTH effects on osteoblasts in vitro

2.2.1 On osteoblast proliferation

In vitro, PTH induces various contradicting responses. In osteoblast-like cells such as UMR 106 and ROS 17/2.8 osteosarcoma cells, incubated with PTH at 10^-8M for 24 hours, DNA synthesis was decreased (126, 127). On the contrary, at a dose of 10^-11 M, PTH was able to induce cell proliferation of the same cell line, i.e. UMR 106 cells (128). Moreover, in mouse non cancerous osteoblasts such as MC3T3 and in primary osteoblasts, PTH induced cell proliferation (129). Thus, PTH-induced cell proliferation in vitro depends on the dose and the cell type.

In vivo, intermittent injection of PTH in rats increase the number of osteoprogenitor cells from bone marrow (130, 131). However, the in vivo assessment of [³H]- thymidine incorporation into osteoblastic cells in rats showed no difference in the percentage of labeled osteoblasts between vehicle- and PTH-treated animals, whereas incorporation of [³H]-proline in the distal epiphysis was increased (120).

These observations suggest that PTH does not stimulate osteoblastic proliferation but rather stimulates differentiation of osteoprogenitor cells in vivo. It is commonly accepted that differentiation requires exit from the cell cycle, and numerous in vitro studies have shown that proliferation decreases as osteoblast differentiation proceeds. Consistent with the possibility that PTH increases osteoblast differentiation by suppressing proliferation, in vivo and in vitro studies have shown that short-term exposure to PTH causes exit of osteoblast progenitors from the cell cycle. Thus, PTH reduced the expression of histone H4, a marker of the cell cycle (132), as well as expression of the cell cycle inhibitors p27KIP1 and p21Cip1 (133), in metaphyseal bone of young rats, a site rich in replicating osteoblast progenitors (134).

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2.2.2 On osteoblast differentiation

In vitro, expression levels of specific genes associated with the various stages of osteoblastic differentiation, such as alkaline phosphatase (ALP), type I collagen (COLL I) and osteocalcin (BGP), can also be either stimulated or inhibited by PTH depending on the maturation stage of cell culture and on the duration of treatment (135-137). Indeed, PTH may preferentially stimulate osteoblast differentiation in immature osteoblasts and rather inhibit it in mature osteoblasts. Moreover, the longer osteoblasts are exposed to PTH, the less they may differentiate. However, under well-defined experimental conditions, intermittent PTH treatment increases mineralized nodule formation in cultures of mouse (MC3T3) osteoblastic cells as well as in primary osteoblastic cell cultures from mice, rats and humans (135, 137). This mineralized nodule formation is associated with high levels of expression of ALP, COLL I and BGP.

It has been shown in vitro, that some of the anabolic effects of PTH may also be mediated by an autocrine production of Insulin-like growth factor 1 (IGF-1) (138).

Thus in osteoblastic cells from newborn rat calvariae and exposed to PTH, osteoblast differentiation was inhibited by anti-IGF-1 neutralizing antibodies (137). In another model, using cultured marrow cells treated with PTH, osteoblast differentiation was also inhibited by anti-IGF-1 antibodies (139). In vivo, IGF-1 knocked mice showed an impaired bone formation induced by PTH (140, 141).

Among the genes implicated in osteoblast differentiation, the transcription factor Cbfa1 (core-binding protein a-1, also known as Runx2) is essential (9-11). Cbfa1- deficient mice die at birth and exhibit a complete lack of bone formation (10), and intermittent PTH administration in vivo stimulates Cbfa1 protein in the rat proximal tibiae metaphysis (142). However, in vitro, PTH regulates expression of Cbfa1 in a time- and dose-dependent manner in UMR-106. Regulation occurred both at the Cbfa1 mRNA level, and at the posttranslational level to increase the activity of Cbfa1 protein. The dose-dependent activity on mRNA and protein expression suggests an anabolic function for PTH at lower doses. However, higher doses of PTH decreased Cbfa1 mRNA and protein level. Moreover, it was found that PTH also activates a 38

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PKA-dependent increase in Runx2 activity (142). Increased Runx2 levels may be secondary to cAMP-dependent down-regulation of cyclin D1, which besides controlling the cell cycle, also targets Runx2 for proteasomal degradation (143).

2.2.3 On osteoblast apoptosis

In vitro and in vivo, PTH is anti-apoptotic and increases life-span of mature osteoblasts, without affecting the generation of new osteoblasts. This antiapoptotic effect was sufficient to account for the increase in bone mass measured by BMD on distals femurs of mice. The anti-apoptotic effect of PTH was confirmed (in the same study) in vitro in cultures of calvarial osteoblasts, MC3T3 murine osteoblastic cells and MG-63 human osteoblastic cells. In fact, osteoblasts incubated with dexamethasone which induces a pro-apoptotic effect, was inhibited with the addition of PTH (122). Another in vitro study showed that PTH effects on apoptosis were dramatically dependent on cell status. In preconfluent MC3T3 murine osteoblasts and multipotential mesenchymal cells (C3H10), PTH protected against dexamethasone-induced reduction cell viability, whereas in postconfluent cells PTH resulted in reduced cell viability (144).

2.2.4 On osteoblast-mediated activation of osteoclasts

The discovery of the osteoprotegerin (OPG)/RANK ligand (RANKL)/RANK system and of its actions on osteoclastogenesis represents an important step in the understanding of bone physiology. Whereas RANKL promotes osteoclast formation by binding to RANK on the osteoclast surface, OPG inhibits RANKL activity by binding it, thereby inhibiting osteoclat differentiation and activation (33, 34, 37).

In vitro and in vivo studies have clearly demonstrated that PTH increases the number and activity of osteoclasts by upregulation of RANK/RANKL system (145, 146). PTH has been found to stimulate RANKL mRNA levels, to decrease OPG production, and 39

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to increase the RANKL/OPG ratio in osteoblasts/stromal cells (147-149). These findings have been confirmed also by in vivo demonstration that PTH inhibits the expression of OPG in bone with the kinetics of an immediate early gene (150).

Furthermore, OPG has been shown to inhibit bone resorption induced by administration of PTH (151). In summary, PTH dose- and time-dependently increases RANKL and decreases OPG expression in vivo and in vitro by osteoblastic cells, while low-dose intermittent PTH increases OPG and reduces RANKL/OPG ratio (139, 147).

In summary, all these findings clearly show that the actions of PTH depend on multiple mechanisms and various factors such as the dose, method of administration, and the osteoblastic cell model used. Such conflicting results have plagued the scientific community and hinder our understanding of PTH action in bone.

Nevertheless all these studies tend to indicate that transient activation of PTH1R activates multiple interconnected pathways leading to decreased proliferation of osteoblastic progenitors, increased osteoblastic survival, increased osteoblastic differentiation and the production and/or activation of osteogenic growth factors.

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3. PTH receptor and its transduction signaling pathways

The PTH/PTH-related peptide (PTHrP) receptor (PTH1R), which is expressed in bone, cartilage and kidney, is a member of the family of seven transmembrane receptors that are coupled to G proteins, also named G-protein-coupled receptors (GPCRs). The G proteins are heterotrimeric and comprising ,  and  subunits. The

 subunit is responsible for GTP and GDP binding for GTP hydrolysis, whereas the  and  subunits are associated in a tightly linked  complex. Activation of PTH1R by its agonists stimulates formation of cyclic 3’,5’-adenosine monophosphate (cAMP) by activating adenylyl cyclase (AC) through the action of stimulatory G-alpha proteins (Gs) coupled to the receptor (Figure 5). In turn, cAMP binds to the regulatory subunit of the enzyme PKA (protein kinase A) that releases the active catalytic subunit of the enzyme. PTH has also been demonstrated to activate phospholipase C (PLC) by Gq leading to the formation of diacylglycerol (DAG) which activates protein kinase C (PKC) and 1,4,5-inositol triphosphate (IP3), resulting in increased intracellular free Ca²⁺ (152-154). Moreover, PTH1R may also transduce its signaling through Gi which suppresses cAMP accumulation (155, 156).

Figure 5. The Gs and Gq transduction signaling pathways mediated by PTH1R.

See text for details. Adapted from Swarthout et al., Gene (2002).

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Thus, PTH1R is capable to coupling to several subclasses of G proteins, including Gs, G^q and G^i/o. Structure-function studies of the PTH1R revealed that the second and third intracellular loops (IC2 and IC3 loops) contain determinants for G protein coupling. Replacing the EKKY amino acid sequence located in the IC2 loop of PTH1R with the amino acid sequence DSEL blocks PTH-mediated signaling via PLC

without affecting signaling via adenylyl cyclase (157). Regions of the PTH1R’s IC3 loop contain elements required for coupling to both AC and PLC (158). On the contrary, serial truncations of PTH1R cytoplasmic C-terminus up to amino acid 480 does not affect PTH signaling via AC or PLC but had a modest reduction in receptor expression (159). Moreover, it has been shown that the juxta-membrane region of PTH1R C-terminus between amino acids 468 and 491 is capable of binding G protein heterotrimeric complexes via direct G interactions (160). Indeed, mutations that disrupt G interaction block PTH signaling via PLC and mitogen- activated protein kinase (MAPK) and markedly reduce signaling via AC (160).

Others molecules such as -arrestin, phosphodiesterases 4 (PDE4), and c-Src are implicated in PTH1R-mediated signaling and will be further described (see section 4).

There are other members of the PTH/PTHrP receptor family. The second type of PTH receptor is the ‘PTH2 receptor’ (PTH2R). In rats, PTH2R is expressed in the central nervous system and many other tissues (peptide-secreting cells of gastrointestinal tract; placenta; testis; cardiac and vascular endothelium) but, unlike the PTH1R, not in renal tubules and bone (161-163). PTH2R (but not PTH1R) is activated by its endogenous ligand, the tuberoinfundibular peptide of 39 residues (TIP39) (164). PTH2R may also be activated by PTH but not by PTHrP (162, 165-167).

PTH2R exhibits dual signaling in response to PTH, with generation of cAMP, cytoplasmic Ca²⁺ transients and protein kinase C mobilization (167-169).

A third type PTH receptor, termed the ‘type-3 zPTH receptor’ or ‘zPTH3R’. was cloned from a zebrafish cDNA library (170). When expressed in mammalian (COS-7) cells, the zPTH3R activates adenylyl cyclase but not PLC with human PTH(1–34)

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(171). The importance of the PTH3R to human physiology is uncertain, however, because no evidence has been produced to date for the existence of a mammalian homolog of this receptor.

Carboxyl (C) fragments of intact PTH-(1–84), such as PTH-(39–84) or PTH-(53–84), do not bind or activate the PTH1R (172). Nevertheless, recent evidence points to the existence of a putative type PTH receptor that recognize the carboxyl (C)-terminal region of intact PTH-(1–84) (CPTHRs) and that are possibly expressed by osteocytes (173). It has been shown that COOH-terminal fragments of PTH elicit increases of [Ca²⁺]i in osteocytes (174). Moreover, in ROS 17/2.8 cells, fragments from within the sequence PTH-(35– 84), which cannot activate the PTH1R, regulate expression of alkaline phosphatase, osteocalcin, collagen 1, and IGF-binding protein-5 (175, 176).

All these data strongly suggest the existence of a fourth PTH receptor (CPTHR), but its nature and structure have remained elusive so far.

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3.1 PTH effects mediated by the cAMP pathway

Studies in vitro and in vivo have shown that the N-terminal 1-34 synthetic fragment of the 84 amino acids of PTH mediates full PTH activity (177), eliciting a cAMP response and stimulating bone formation (178). There are functional sub-domains within this 34 amino acid peptide (PTH(1-34)) that mediate unique responses and enable the dissection of PTH action. The first two amino acids are generally necessary to mediate cAMP induction (179), amino acids from 3 to 7 appear to be required for calcium mobilization and amino-acids 29-32 are required for PKC activation (180). Further investigations with N-terminally truncated PTH peptides revealed that PTH(3-34) which fails to stimulate adenylyl cyclase but apparently retain the capacity to stimulate PLC pathway lacks anabolic activity in vivo (181). In another study using PTH(3-38) which also failed to stimulate cAMP production in vitro, Hilliker et al. did not either find any anabolic activity on rat bones (182). C- terminally substituted analog of PTHrP(1-34), RS-66271, which is less potent that PTH(1-34) in stimulating inositol phosphate production but exhibits equivalent activity in stimulating cAMP in vitro, induced the same increase of trabecular and cortical bone mass in rats as PTH(1-34) did (183).Therefore all these data show that cAMP signaling pathway seems necessary for PTH anabolic activity.

At the cellular level, the actions of PTH appear to be predominantly mediated by the cAMP dependent-PKA pathway, leading to the phosphorylation of transcription factors such as Cbfa1/Runx2 (142, 184) and cAMP-response element-binding protein (CREB) (185), which in turn, will regulate the transcriptional activation of multiple osteoblastic genes. Cbfa1/Runx2, which plays a central role in osteoblast differentiation, has been described to be able to induce promoter activity of the osteoclacin, osteopontin and collagenase-3 genes (9, 186-191). CREB induces the transcription of c-fos, an immediate early gene, which is important for osteoblast proliferation and differentiation (185, 192). Moreover, the activator protein-1 (AP-1) family of genes encoding for transcription factors such as c-fos, junB, c-jun and fra-2, has been shown to be implicated in osteoblast proliferation and differentiation and is 44

Transduction pathways implicated in osteoblastic cells activation induced by parathyroid hormone (PTH)

Thesis

Reference

Transduction pathways implicated in osteoblastic cells activation induced by parathyroid hormone (PTH)

Transduction Pathways

implicated in Osteoblastic Cells Activation induced by Parathyroid Hormone (PTH)

Table of contents

Remerciements/Acknowledgments

Summary

Résumé

Abbreviations

General introduction

1. Bone tissue

1.1 Osteoblast differentiation and bone formation

1.2 Osteoclast differentiation and bone resorption

1.3 Bone remodeling

2. Parathyroid hormone (PTH)

2.1 PTH effects in vivo

2.2 PTH effects on osteoblasts in vitro

3. PTH receptor and its transduction signaling pathways

3.1 PTH effects mediated by the cAMP pathway