ß-arrestin2 regulates age-related and parathyroid hormone-induced changes in the expression of genes involved in bone remodeling/turnover

Thesis

Reference

ß-arrestin2 regulates age-related and parathyroid hormone-induced changes in the expression of genes involved in bone

remodeling/turnover

BIANCHI, Estelle

Abstract

La ß-arrestin2 est une molécule cytoplasmique impliquée dans la régulation de la signalisation intracellulaire, principalement par les récepteurs couplés aux protéines G, tel le PTH/PTHrP récepteur. L'administration quotidienne de PTH (i.PTH), un traitement approuvé contre l'ostéoporose, augmente la masse osseuse et réduit le risque de fracture, alors que l'hyper-parathyroïdisme ou l'infusion de PTH (c.PTH) mène à des effets cataboliques osseux, spécialement sur l'os cortical. Une analyse par microarray nous a permis d'identifier 2 réseaux de gènes centrés sur p38 MAPK - NFkB et TGFB1 spécifiquement régulée en réponse à i.PTH et par la ß-arrestin2. Nous avons également identifié des nouveaux gènes cibles exprimés dans l'os et les ostéoblastes, régulés par la ß-arrestin2 et la PTH. Nos résultats finalement suggèrent un rôle pour la ß-arrestin2 dans la régulation des changements physiologiques sous-tendant les modifications de la masse/structure osseuse se produisant avec l'âge.

BIANCHI, Estelle. ß-arrestin2 regulates age-related and parathyroid hormone-induced changes in the expression of genes involved in bone remodeling/turnover. Thèse de doctorat : Univ. Genève, 2009, no. Sc. 4136

URN : urn:nbn:ch:unige-39261

DOI : 10.13097/archive-ouverte/unige:3926

Available at:

http://archive-ouverte.unige.ch/unige:3926

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

UNIVERSITÉ DE GENÈVE

Département de Zoologie et Biologie Animale FACULTÉ DES SCIENCES Professeur François Karch

Département de Réhabilitation et Gériatrie FACULTÉ DE MÉDECINE Service des Maladies Osseuses Professeur Serge Ferrari

ß-arrestin2 regulates age-related and parathyroid hormone-induced changes in the expression of genes

involved in bone remodeling/turnover

THÈSE

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

par

Estelle BIANCHI de

Genève

Thèse N°4136

Genève

Centre d’Edition des HUG 2009

Table of contents

Acknowledgments p.5

Summary p.6

Résumé p.8

Abbreviations p.10

I. General introduction p.13

I.1. Bone composition p.14

I.2. Osteoblast differentiation p.18

I.3. Osteoclastogenesis and bone resorption p.23

I.4. Bone remodeling p.25

I.5. Osteoblast-osteoclast coupling p.28

I.5.1 OPG/RANKL/RANK p.28

I.5.2. Bidirectional ephrin signaling p.29

I.6. Age-related bone loss p.31

I.7. Parathyroid hormone p.34

I.7.1 Parathyroid hormone (PTH) p.34

I.7.2. PTH paradoxical effects on bone p.36

I.7.3. PTH receptors p.39

I.7.4. PTH1R signaling p.41

I.8. ß-arrestins p.45

II. Materials and methods p.49

II.1. Isolation of primary osteoblasts from mouse calvariae p.50

II.2. Cell splitting p.50

II.3. Cell culture and PTH treatments p.51

II.4. Newborn mice sexing p.51

II.5. Alizarin red staining p.52

II.6. RNA isolation p.53

II.6.1. RNA isolation from cells p.53

II.6.2. RNA isolation from bone p.53

II.7. Microarray analysis p.54

II.8 Comparison of PTH-regulated genes between WT and Arrb2^-/-

osteoblasts p.55

II.9. Additional selection criteria p.55

II.10. Quantitative real-time PCR p.56

II.11. Mice genotyping p.58

II.12. Animals and PTH treatments p.59

II.13. Serum biochemistry p.59

II.14. In vivo bone mass and body composition measurements p.60

II.15. Ex vivo microstructure measurements p.60

III. Results p.62

III.1. Results, study 1: p.63

III.1.1. Hypothesis, study 1 p.64

III.1.2. Study 1, part 1, in vitro (published paper) p.65

III.1.3. Study 1, part 2, in vivo p.76

III.1.3.1. Design, study 1, part 2 p.77

III.1.3.2. Results, study 1, part 2 p.78

III.2. Results, study 2 p.95

III.2.1. Hypothesis, study 2 p.96

III.2.2. Results, study 2 p.97

III.2.3. Summary of the results, study 2 p.114

IV. Discussion p.117

IV.1. Discussion study 1 p.118

IV.1.1. Discussion, study 1, part 1 p.119

IV.1.2. Discussion, study 1, part 2 p.120

IV.1.3. Conclusions, study 1 p.125

IV.2. Discussion and conclusions, study 2 p.126

IV.3. Conclusions and perspectives, study 1 & 2 p.131

V. References p.135

VI. Annexes p.152

VI.1. Annexes, study 1 p.153

VI.2. Annexes, study 2 p.182

Acknowledgments

First I would like to thank Professor Serge Ferrari for sharing his knowledge with me, for his patience and his encouragements which accompanied me along these years of work. I am also very grateful to him for giving me the opportunity to share my work at international meetings.

Second, I would like to thank Professor René Rizzoli for welcoming me in the Service of bone diseases.

Third, I would like to thank all colleagues from the Service of bone diseases and more particularly the ones that contributed to my work in any manner, i.e. Fanny Cavat, Madeleine Lachize, Oliver Perron, Danielle Manen, Dominique Pierroz, Nicolas Bonnet, Tara Brennan and Alexandre Rey.

Fourth, I would like to thank people from the Genomics Platform NCCR Frontiers in Genetics, more particularly Patrick Descombes, Olivier Schaad and Christelle Barraclough for their expertise and technical support for microarrray and real-time analyses.

Last but not least, I would like to thank my boyfriend, family and friends for their encouragement and support during this thesis.

Summary

ß-arrestin2 is a cytoplasmic molecule involved in the regulation of intracellular signaling by a variety of transmembrane receptors, primarily G protein-coupled receptors.

Thus, binding of parathyroid hormone (PTH) to its receptor, the PTH/PTH related peptide (PTHrP) receptor leads to ß-arrestins recruitment and receptor uncoupling from G proteins.

PTH is critical for calcium homeostasis and bone formation and mineralization. Daily administration of PTH (intermittent PTH, i.PTH) an approved treatment for osteoporosis, increases bone mass and reduces fracture risk, while hyperparathyroidism or PTH infusion (continuous PTH, c.PTH) results in predominant bone catabolic effects especially on cortical bone. Hence, PTH induces bone formation by directly acting on osteoblasts, but also through osteoblast-osteoclast coupling, resulting in increased bone remodeling, i.e. bone formation and resorption. Thus, the receptor activator of NFκB ligand (RANKL) / osteoprotegerin (OPG) ratio is indicative of osteoclastogenic activity in bone, i.e. a high ratio favors bone resorption and a low ratio favors bone formation. In ß-arrestin2-deficient (Arrb2^-/-) mice, the response to i.PTH is complex, suggesting an increase of periosteal bone formation, but a predominant resorption at trabecular and endocortical surfaces. Moreover in bone catabolic circumstances, the absence of ß-arrestin2 results in enhanced bone turnover, due to increased RANKL/OPG ratio. The process of bone (re)modeling ensures adaptation of size, shape, microarchitecture and mineral content, as well as repair of bone damages, in response to growth, aging and mechanical constraints. Alterations of the bone remodeling balance with age results in osteoporosis.

To unravel the role of ß-arrestin2 in bone remodeling, we first used microarray analysis of primary osteoblastic cultures from WT and Arrb2^-/- mice calvariae exposed to i.PTH or c.PTH, to identify i.PTH-induced bone genes targeted for regulation by ß-arrestin2.

Then we studied the expression of these targeted genes in addition to known bone-related genes in femoral trabecular and cortical bone from WT and Arrb2^-/- mice treated with i.PTH or c.PTH. Finally we examined the age-related changes in the expression of some genes related to bone turnover in trabecular and cortical bone from WT and Arrb2^-/- mice, parallel to changes of ß-arrestins expression and bone microarchitecture modifications with age.

Thus, microarray analysis identified a p38 MAPK - NFκB and a TGFB1 gene networks regulated by i.PTH and ß-arrestin2. We also identified novel gene targets expressed in osteoblasts and bone, regulated by ß-arrestin2 and PTH. We finally showed that age-related changes in bone structure were paralleled by down-regulation of ß-arrestin genes and other bone genes.

In conclusion, these studies delineated a role for ß-arrestin2 in the modulation of gene expression in response to PTH-induced changes of bone turnover, identified specific PTH- induced genes expressed in osteoblasts and bone targeted for regulation by ß-arrestin2, and finally suggest a role for ß-arrestin2 in the physiological changes underlying modifications of bone mass/structure occurring with age.

Résumé

La ß-arrestine2 est une molécule cytoplasmique impliquée dans la régulation de la signalisation intracellulaire par une variété de récepteurs transmembranaires, principalement les récepteurs couplés aux protéines G. Ainsi, la liaison de l’hormone parathyroidienne (PTH) à son récepteur, le PTH/PTH related peptide (PTHrP) récepteur, conduit au recrutement de la ß-arrestine2, et au découplage du récepteur des protéines G. La PTH est critique pour l’homéostasie du calcium, la formation et la minéralisation osseuse. L’administration quotidienne de PTH (PTH intermittente, i.PTH), un traitement approuvé contre l’ostéoporose, augmente la masse osseuse et réduit le risque de fracture, alors que l’hyper-parathyroïdisme ou l’infusion de PTH (PTH continue, c.PTH) mène à des effets cataboliques osseux, spécialement sur l’os cortical. La PTH induit ainsi la formation osseuse en agissant directement sur les ostéoblastes, mais aussi en agissant via le couplage des ostéoblastes aux ostéoclastes qui résulte en un remodelage osseux accru (i.e. formation et résorption osseuse augmentées). Ainsi, le ratio receptor activator of NFκB ligand (RANKL) / ostéoprotégérine (OPG) est indicatif de l’activité ostéoclastogénique dans l’os, i.e. un ratio élevé est en faveur de la résorption osseuse et un ratio bas de la formation osseuse. Chez la souris déficiente en ß- arrestine2 (Arrb2^-/-), la réponse à la PTH intermittente est complexe, suggérant une augmentation de la formation osseuse au niveau du périoste, mais une résorption osseuse plus marquée au niveau des surfaces trabéculaires et endocorticales. De plus dans des circonstances cataboliques pour l’os, l’absence de ß-arrestine2 résulte en un remodelage osseux accentué, à cause d’une augmentation du ratio RANKL/OPG. Le processus de remodelage osseux assure l’adaptation de la taille, de la forme, de la microarchitecture et du contenu en minéral, ainsi que la réparation des dommages induits, en réponse à la croissance,

le vieillissement et la contrainte mécanique. Les altérations du remodelage osseux observées avec l’âge peuvent mener à l’ostéoporose.

Pour définir le rôle de la ß-arrestine2 dans le remodelage osseux, nous avons tout d’abord utilisé une analyse par microarray de cultures primaires d’ostéoblastes isolés à partir de calvariae de souris WT et Arrb2^-/-, exposées à la PTH intermittente ou continue, afin d’identifier des nouveaux gènes induits par i.PTH et régulés par la ß-arrestine2. Nous avons ensuite étudié l’expression de ces gènes cibles, en plus de gènes connus pour être impliqués dans la biologie osseuse, dans le compartiment trabéculaire et cortical du fémur de souris WT et Arrb2^-/-, préalablement traitées avec i.PTH ou c.PTH. Finalement nous avons examiné le changement d’expression de gènes liés au remodelage osseux avec l’âge dans les compartiments osseux trabéculaire et cortical de souris WT et Arrb2^-/-, parallèlement aux modifications d’expression des ß-arrestines et des changements de la masse/structure osseuse avec l’âge.

Ainsi, l’analyse par microarray a identifié 2 réseaux de gènes centrés sur p38 MAPK - NFκB et TGFB1 spécifiquement régulés en réponse à i.PTH et par la ß-arrestine2. Nous avons également identifié des nouveaux gènes cibles exprimés dans l’os et les ostéoblastes, régulés par la ß-arrestine2 et la PTH. Nous avons finalement montré que parallèlement aux changements de masse/structure osseuse liés à l’âge, l’expression génique des ß-arrestines est diminuée ainsi que celle d’autres gènes osseux.

En conclusion, ces études montrent un rôle pour la ß-arrestine2 dans la modulation de l’expression des gènes en réponse à des changements du remodelage osseux induits par la PTH, ont permis l’identification de gènes spécifiquement induits par la PTH et régulés par la ß-arrestine2 dans l’os et les ostéoblastes, et finalement suggèrent un rôle pour la ß-arrestine2 dans la régulation des changements physiologiques sous-tendant les modifications de la masse/structure osseuse se produisant avec l’âge.

Abbreviations

ALP: alkaline phosphatase

AMP: adenosine mono-phosphate Arrb2^-/-: ß-arrestin2 deficient ATP: adenosine tri-phosphate BGLAP: bone gla-protein BMC: bone mineral content BMD: bone mineral density BMPs: bone morphogenic proteins BMU: bone multicellular unit

BRC: bone remodeling compartment BV/TV: bone volume / total volume

cAMP: cyclic adenosine 3’,5’-monophosphate CaR: calcium-sensing receptor

CSF1: colony stimulating factor 1

CSF1R: colony stimulating factor 1 receptor DAG: diacylglycerol

EFN: ephrin ligand

EGFR: eptihelium growth factor receptor ELISA: enzyme-linked immunosorbent assay EPH: ephrin receptor

ERK: extracellular signal-regulated kinase FGFs: fibroblast growth factors

GDP: guanosine di-phosphate GH: growth hormone

GO: Gene Ontology

GPCRs: G protein-coupled receptors GPI: glycosyl-phosphatidyl inositol GRKs: G protein-coupled receptor kinases GTP: guanosine tri-phosphate

HPT: hyperparathyroidism

IBSP: integrin-binding sialoprotein (bone sialoprotein)

IC: intracellular

IGF1: insulin-like growth factor 1

IGF1R: insulin-like growth factor 1 receptor IHH: indian hedgehog

ILs: interleukins

IP3: inositol tri-phosphate

IPA: Ingenuity Pathway Analysis IRS1: insulin receptor substrate 1 IRS2: insulin receptor substrate 2 JUNKs: c-Jun amino-terminal kinases MAPKs: mitogen-activated protein kinases M-CSF: macrophage colony stimulating factor MSC: mesenchymal stem cells

NFκB: nuclear factor kappa b

NHERFs: sodium/hydrogen exchanger regulator factors OPG: osteoprotegerin

OPPG: osteoporosis pseudoglyoma PCR: polymerase chain reaction PDEs: phosphodiesterases

PHPT: primary hyperparathyroidism PKA: protein kinase A

PKC: protein kinase C PLC: phospholipase C PTH: parathyroid hormone

i.PTH: intermittent parathyroid hormone c.PTH: continuous parathyroid hormone PTH1R: parathyroid hormone 1 receptor PTH2R: parathyroid hormone 2 receptor PTHrP: parathyroid hormone-related protein RANK: receptor activator of nuclear factor kappa b

RANKL: receptor activator of nuclear factor kappa b ligand SLRPs: small leucine-rich proteoglycans

SOST: sclerostin

TGFB: transforming growth factor beta Trab: trabecular

TRACP5b: tartrate-resistant acid phosphatase form 5b TRAP: tartrate-resistant acid phosphatase

Veh: vehicle WT: wild-type

I. GENERAL INTRODUCTION

I.1. Bone composition

The adult skeleton is a single organ comprising the cartilage and bone tissues. It is composed of more than 200 bones spread throughout the body. Depending on their locations, the bones serve as structural support for vital organs, movement, and bone marrow protection and/or maintenance of mineral homeostasis. Bone is also the primary site of hematopoiesis and therefore the site of a complex interplay between bone tissue and the immune system.

Bone consists of two parts: the axial skeleton that includes the bones of the head and trunk, and the appendicular skeleton that comprises the bones of the limbs and pelvic girdle.

Moreover, anatomically two main types of bone are distinguished: the flat bones, such as the mandible or the skull and the long bones, such as the femur or the radius. Long bones consist of a hollow tube (called midshaft or diaphysis) that ends with cone-shaped extremities each composed of the metaphysis region (directly adjacent to the diaphysis), the epiphysis region (at bone extremities) and the growth plate located in between (Figure 1).

Figure 1. Representation of a longitudinal cross section of a long bone ¹.

Two types of bone coexist: the trabecular bone, also called cancellous or spongy bone, and the cortical bone, also called compact or dense bone (Figure 2). Trabecular bone is found principally in the axial skeleton and in the metaphyses and epiphyses of long bones. Its structure is highly porous and represents a network of rod- and plate-shaped trabeculae surrounded by an interconnected pore space filled in with bone marrow (Figure 2). Cortical bone is always found on the outside of bones (bone cortex) and surrounds the trabecular bone.

Thus, in long bones the diaphysis is almost entirely composed of cortical bone enclosing the medullar cavity that contains bone marrow, whereas the metaphysis and epiphysis are filled in with a network of mineralized trabeculae surrounded by cortical bone. The bone cortex is formed by a thick layer of mineralized bone subdivided in two regions, the outer fibrous sheath called the periosteum and the inner surface directly in contact with bone marrow,

cancellous bone. The proportion between these two types of bone (cortical versus trabecular bone) varies tremendously with skeletal sites. Thus, the ratio of cortical:trabecular bone in the radius diaphysis is 95:5, in the femoral head 50:50 and in the human vertebrae 25:75.

Figure 2. Photograph of a thick ground section of the proximal part of the tibia showing the cortical (compact) bone and the trabecular (cancellous) bone, plate- or rods-like shaped ².

Bone is constituted from cells and the mineralized extracellular matrix. The bone forming cells or osteoblasts are bone marrow-derived stromal cells that are responsible for the extracellular matrix deposition and for its mineralization. Contrarily to osteoblasts, osteocytes do not produce the extracellular matrix, but are embedded in the mineralized matrix and are characterized by long processes extending in the lacunocanalicular system creating an interconnected network essential for intercellular communication between neighboring osteocytes and the bone-lining osteoblasts. Thus, chemical and mechanical signals are transmitted through this network, allowing bone adaptation and adequate response to external

stimuli. Therefore osteocytes are predominantly associated with calcium homeostasis and mechanosensory functions.

The bone resorbing cells or osteoclasts are derived from hematopoietic stem cells and are formed by the fusion of mononuclear precursor cells, resulting in large and multinucleated cells (for details, see chapter I.3.). The bone matrix is essentially constituted of collagen type I alpha 1 or 2 (COL1A1 or COL1A2). Randomly-oriented collagen fibrils formed the woven bone (also called, immature or primitive bone) normally found in embryonic or newborn skeleton. The lamellar bone is a more mature form of bone tissue characterized by collagen fibrils aligned in thin sheets called lamellae. In addition to collagen, bone matrix contains non-collagenous proteins and mineral deposits that are found in spaces between the collagen fibrils. Bone gla-protein (BGLAP) also known as osteocalcin, is one of the most abundant non-collagenous proteins in bone and one of the more extensively studied. This bone-specific protein presents calcium and mineral binding properties and may also regulate osteoclast and osteoblast precursor activities. A lot of other non-collagenous proteins are important in mineral binding. Indeed, such as secreted phosphor protein 1 (SPP1) also known as osteopontin, integrin-binding sialoprotein (IBSP) also known as bone sialoprotein, and thrombospondin are involved in cell-binding through arginine-glycine-aspratic acid (RGD) sequence, that is recognized by integrin transmembrane proteins providing a link between cell cytoskeleton and the extracellular matrix. Growth factors, such as transforming growth factor beta (TGFBs) and insulin-like growth factor (IGFs) and cytokines, such as osteoprotegerin (OPG), interleukins (ILs) and bone morphogenic proteins (BMPs) are present in small quantities in bone matrix. Such proteins are important for the regulation of cell differentiation, activation, growth and turnover ^{1; 3}.

I.2. Osteoblast differentiation

Osteoblasts are derived from mesenchymal stem cells (MSC). These pluripotent cells can differentiate into a variety of cells, including myoblasts, adipocytes, chondrocytes and osteoblasts ⁴. Progression along the osteoblastic lineage requires the sequential activation and suppression of the expression of gene encoding signaling proteins, transcription factors and regulatory proteins (Figure 3).

Figure 3. Transcriptional control of myoblastic, adipocytic, chondrocytic and osteoblastic differentiation ⁵.

Commitment of MSC towards the chondrocyte lineage requires Sox9 induction, towards adipogenesis, Pparγ activation and towards myoblastic lineage, MyoD stimulation ⁶. Two transcription factors have been shown to be essential for osteoblast commitment and differentiation: the runt-related transcription factor 2 (Runx2, also known as Cbfa1) and the zinc finger motif containing factor, osterix (Sp7, also known as Osx) ⁴. The essential role of Runx2 in osteoblast differentiation was revealed with Runx2-deficient mice that present a

cartilaginous skeleton with a total absence of osteoblasts ^{7; 8; 9}. These mice also exhibit a delayed chondrocyte maturation indicating that Runx2 is involved in both osteoblast and chondrocyte differentiation ¹⁰. Osterix-deficient mice, that have no defect in cartilage but lack osteoblasts and mineralized bone matrix, revealed its specific role in osteoblast differentiation

11. Osterix is not expressed in absence of Runx2, whereas Runx2 expression is normal in osterix-deficient mice, indicating that osterix functions downstream Runx2 activity. These two transcription factors regulate positively or negatively osteoblast-specific genes including Col1, Spp1, Sparc (osteonectin) and Bglap 11; 12; 13; 14

. MSC differentiation in osteoblasts is also regulated by other factors, such as distaless (Dlx), Msx and Hox homeodomain gene families, and by the local microenvironment where morphogens are produced.

In addition to these transcription factors, signaling pathways, including Indian Hedgehog (IHH), TGFB, BMP and WNT signaling, were shown to play important role in regulating osteoblast differentiation, by regulating Runx2 and/or osterix expression. Thus, IHH was found to be indispensable for osteoblast development, because Ihh-deficient mice completely lack osteoblasts in bones formed by endochondral ossification ¹⁵. IHH also promotes osteoblast differentiation through regulation of Runx2 expression ¹⁶. BMP signaling was first shown to be able to induce bone formation at ectopic sites ¹⁷ and subsequently to activate Runx2 and osterix expression ^{18; 19}. TGFB signaling effect on bone formation can be positive or negative depending on the context, inducing osteoblastic differentiation in the early stages and inhibiting late stage of osteoblasts maturation ²⁰. As for BMP signaling, TGFB was shown to regulate Runx2 expression ^{19; 21}. Canonical Wnt signaling is a key pathway for regulation bone formation (Figure 4) ²².

WNTs are secreted glycoproteins that transduce their signal through receptors of the frizzled family and LRP5/6 coreceptors to intracellular ß-catenin. In the absence of WNT ligand, ß-catenin is part of a complex that facilitates its phosphorylation and its subsequent

proteasomal degradation. In presence of WNT ligand, the complex is dissociated, leading to ß-catenin cytoplasmic accumulation and translocation to the nucleus, where in association with TCF/LEF transcription factors it induces transcription of target genes. Mutations in LRP5 are responsible not only for the autosomal recessive disorder osteoporosis pseudoglioma (OPPG), characterized by low bone mass, deformations and frequent fractures

23, but also for a high bone mass phenotype, when mutations result in a constitutively active LRP5 protein ²⁴. Moreover, Lrp6 has been shown to be essential for normal osteogenesis, as Lrp6-deficient mice die during the perinatal period with embryonic phenotypes comparable to a combination of various Wnt loss-of-function mutations, including truncated axial skeleton and limb patterning defects ²⁵. Consistently, a spontaneous point mutation in the mouse Lrp6 gene results in delayed ossification and reduced bone mass at adult age ²⁶. In addition, mutations of the sclerostin gene (Sost) lead to sclerosteosis ²⁷ and Van Buchem disease ²⁸, respectively. The effect of the canonical WNT signaling in osteoblasts is less clear, acting positively or negatively depending on the osteoblast marker and stage of differentiation considered ²⁹. Until recently it was assumed that the effect of Wnt signaling in bone were due to direct action in osteoblasts precursors, osteoblasts and osteocytes. However new findings suggest that, at least in mice, WNT signaling transduced by LRP5 in the duodenal enterochromaffin cells regulates serotonin synthesis, which acts in an endocrine manner to regulate bone cell metabolism ³⁰. Of note, non-canonical WNT signaling has also been shown to be involved in different stages of bone cell metabolism, but this will not be developed in this work ²².

Figure 4. The canonical Wnt/ß-catenin signaling pathway and its extracellular regulation. (A) Activation by Wnt of the canonical Wnt signaling. (B) In the presence of Dickkopf (Dkk) and Krement (Krm) a tertiary protein complex is made with Lrp5/6 for internalization, thus inhibiting Wnt signaling. (C) Prevention of Wnt signaling by Sclerostin (Sost) binding to Lrp5/6 ²².

Some systemic factors, such as parathyroid hormone (PTH), sexual hormones, glucocorticoids and vitamin D also influence osteoblast differentiation and bone formation (PTH effects on bone are discussed chapter I.7.2.).

Once the mesenchymal stem cells are committed towards the osteoblastic lineage, they go through the osteoblastogenesis process that can be divided in three major stages:

proliferation, extracellular matrix maturation and mineralization, which are characterized by sequentially expressed distinctive osteoblast markers (Figure 5). The first proliferating phase supports expansion of osteoprogenitors, and is characterized by the expression of genes known to activate the proliferation (e.g., c-myc, c-fos, c-jun), genes typical of cell cycle progression (e.g., histones and cyclins) as well as growth factors such as IGFs, FGFs, TGFB, cell adhesion proteins (e.g., fibronectin) and the major component of the bone matrix, COL1.

During the second differentiation period the bone matrix is matured and organized through continuous COL1 synthesis and cross-link maturation. In addition, genes necessary for

subsequent matrix mineralization are up-regulated (e.g., ALP). The onset of the third stage is characterized by the expression of genes involved in hydroxyapatite deposition in the bone matrix. Thus, several mineral-binding proteins, such as SPP1, BGLAP are maximally expressed in this bone matrix mineralization phase.

Figure 5. Stages of osteoblast differentiation in vitro. Gene expression levels of gene markers reaching peak of expression at characteristic stage of differentiation. Adapted from ³¹.

I.3. Osteoclastogenesis and bone resorption

The osteoclasts, the only cell type able to resorb bone, are multinucleated giant cells issued from hematopoietic precursors of the monocyte and macrophage lineage, the principal physiological precursors being bone marrow macrophages. Recruitment of osteoclasts from their mononuclear precursors requires the presence of non hematopoietic marrow stromal cells, that produce two cytokines essential and sufficient for basal osteoclastogenesis the membrane-bound and soluble receptor activator of NFκB ligand, RANKL and macrophage- colony stimulating factor, CSF1 (or M-CSF) ³². Thus, proximity between osteoblastic lineage and hematopoietic cells is required to permit binding of RANKL and CSF1 to their respective receptors, RANK (receptor activator of NFκB) and CSF1R (or c-Fms) expressed on cells of the macrophage and monocyte lineage. The first step in osteoclast development (Figure 6) is undistinguishable from immune cell differentiation, and PU.1 that is a B cell transcription factor is essential for early osteoclast development ³³. Then osteoclast precursors acquire CSF1R, the receptor for CSF1 that is necessary for their survival, proliferation and differentiation. With the activation of RANK by osteoblast-expressed RANKL, osteoclast precursors become committed, and cell fusion results in mature osteoclasts. Both, RANKL and CSF1 are required to produce proteins that typify the osteoclast lineage, such as the tartrate-resistant acid phosphatase (TRAP), the cathepsin K, the calcitonin receptor and the ß3-integrins. Osteoclasts are also secondary regulated by steroid hormones, attachment proteins and cytokines.

Upon RANKL stimulation mature osteoclasts become polarized with the formation of a ruffled membrane ³⁵, adhere to bone through integrins, creating a sealed compartment where acidification necessary for bone resorption will occur. This compartment that is isolated from the general extracellular space is acidified by an electrogenic proton pump and Cl^- channel to pH about 4.5, and the exposed organic matrix essentially made of COL1 is subsequently degraded by the cathepsin K lysosomal enzyme (Figure 7) ³⁴. The degraded fragments are endocytosed and transported in vesicles to the basolateral membrane where they are discharged ³⁶.

Figure 7. Osteoclast action ³⁷.

I.4. Bone remodeling

Bone homeostasis is tightly controlled and largely dependent upon the coupling of bone resorption to bone formation and the cellular communication between osteoblasts and osteoclasts. Thus, bone remodeling is a necessary continuous process for adaptation and repair, mending microscopic skeletal damages and replacing aged bone ³⁸. This process is a surface phenomenon that takes place in bone multicellular units (BMUs), asynchronously at various places in the skeleton in both, cortical and trabecular bone. The loss of this coupling and consequently the disruption of bone homeostasis is the result of a wide range of pathologic diseases, including osteoporosis, osteopetrosis and cancer-induced bone diseases.

The normal remodeling sequence in bone follows a scheme of quiescence, activation, resorption, reversal, formation and return to quiescence ³⁹. However, in term of osteoclast- osteoblast communication, it is more convenient to define bone remodeling as three phases:

initiation, transition and termination (Figure 8). The initiation phase comprises the osteoclast precursor recruitment, differentiation and activation of osteoclasts, as well as maintenance of bone resorption. The transition phase is the period that includes arrest of bone resorption, osteoclast apoptosis, osteoblast recruitment and differentiation. The bone surface is therefore prepared for the following bone formation. The termination phase includes new bone formation (osteoid), mineralization and return to quiescence.

Figure 8. Three phase model of bone remodeling ⁴⁰.

The osteoclastic bone resorption lasts about 2-3 weeks in human bone, whereas transition period is of 1-2 weeks and the osteoblastic bone formation is a much slower process lasting about three months ^{1; 40}. The precise mechanisms responsible for osteoclast recruitment is still not completely understood, but are thought to result of microdamages (microcracks) sensed by the osteocytes ³⁹. It is now likely that osteoclast precursors arrive to bone remodeling sites through the circulation, while it is less clear for preosteoblasts. Interestingly, Hauge et al. demonstrated that bone remodeling takes place in specialized vascular structure covered with a dome of flattened cells displaying osteoblastic phenotype, called bone remodeling compartments (BRCs) (Figure 9), forming a barrier between the marrow space and individual BMU ^{41; 42}. This compartmentation might occur among other reasons, to keep the factors liberated from bone localized and to increase their concentration, but also to isolate them from the direct influence of the hematopoietic niche.

Figure 9. Connections between osteocyte network, lining cells, and the bone remodeling compartment (BRC) ⁴¹.

I.5. Osteoblast – osteoclast coupling

I.5.1 OPG/RANKL/RANK

The concept of coupling is based on the idea that osteoblats and osteoclasts influence respectively their differentiation and activity. Osteoblasts express a large number of cytokines that regulates osteoclast precursor differentiation, such as ILs, RANKL, CSF1 and OPG ⁴³. Inversely, activated osteoclasts through resorbing activity may liberate growth factors such as TGFBs and BMPs from bone matrix and/or directly produced by osteoclasts that subsequently act on osteoblasts, but it is still controversial if osteoclasts are necessary for osteoblastic bone formation ⁴⁴

The discovery of the OPG/RANKL/RANK system highlights the precise mechanism of how preosteoblastic or stromal cells controlled osteoclast development (Figure 10).

Membrane-bound or soluble RANKL expressed by preosteoblastic/stromal cells bind to the RANK receptor on the surface of osteoclastic precursor cells. Similarly CSF1 (M-CSF) secreted by preosteoblastic cells bind to its receptor CSF1R (c-Fms) on preosteoclasts. These two cytokines, CSF1 and RANKL, are necessary and sufficient to induce osteoclastogenesis

32. CSF1 essentially contribute to proliferation, survival and differentiation of osteoclast precursors, while RANKL is crucial for differentiation, fusion into multinucleated cells, activation, and survival of osteoclastic cells. The osteoprotegerin (OPG), a decoy receptor of RANKL, blocks the entire system by inhibiting RANKL-RANK interaction ⁴⁵. Thus, the RANKL/OPG ratio is indicative of osteoclastogenic activity in altered bone remodeling diseases. A large number of factors influence osteoclastogenesis by regulating this system, including the parathyroid hormone (PTH) that acts on both OPG and RANKL ⁴⁶ and TGFB that acts on OPG ⁴⁷. Estrogen was also shown to influence this system; it stimulates OPG production in human osteoblastic cells ⁴⁸ and its deficiency leads to increased RANKL

expression in preosteoblastic, T and B cells isolated from bone marrow of early postmenopausal women compared to premenopausal and estrogen-treated postmenopausal women ⁴⁹.

Figure 10. OPG/RANKL/RANK sytem ⁵⁰.

I.5.2. Bidirectional ephrin signaling

More recently, a new concept for coupling of bone resorption to bone formation has emerged, involving bidirectional signaling between the ephrin receptor EphB4 on osteoblasts, and the ephrin ligand EfnB2 on osteoclasts ⁵¹. Currently, 14 Eph receptors and 8 ephrin ligands exist in human genome. Eph receptors belong to the receptor tyrosine kinase family and are divided in two classes: type A (EphA1-A8) and type B (EphB1-B6), depending on their affinity for ephrin ligands type A (EfnA1-A6) or type B (EfnB1-B3) ⁵². Normally, EfnA ligands bind to EphA receptors and EfnB ligands to EphB receptors, respectively, but there are two exceptions: the receptor EphA4 can bind to ephrin A and B ligands and the ligand EfnA5 can bind to EphB2 in addition to EphA receptors ^{52; 53}. Type B ephrins are transmembrane proteins, while type A ephrins are glycosyl-phosphatidyl inositol (GPI)- anchored. Interaction between ephrins and Eph receptors results in bidirectional signaling. Of note, the EphB4 receptor can stimulate reverse signaling only through EfnB2 ligand.

Thus, reverse signaling through EfnB2 into osteoclasts inhibits osteoclatogenesis, via inhibition of c-Fos expression, while forward signaling through EphB4 on osteoblasts stimulates differentiation of the osteoblastic cells, probably via RhoA repression ⁵¹ (Figure 11).

Figure 11. Ephrin – Eph signaling and bone homeostasis ⁵⁴.

Consistently with this signaling, mice overexpressing EphB4 specifically in osteoblasts exhibited an increase in bone mass, bone mineral density and bone formation rate with a decreased osteoclasts number ⁵¹. In addition, ephrins have been shown to be regulated by PTH, which might influence its effects on osteoclast-osteoblast coupling ^{55; 56}. Although it has long been accepted that resorption of bone is needed to promote bone formation during the transition phase of bone remodeling, this ephrin signaling brought evidences that the mature osteoclast itself may be the inducer of bone-forming activity by acting directly on osteoblast precursors or bone lining cells. This concept implies that the distance between osteoblasts and osteoclasts is close enough for a direct physical contact, an idea that remains controversial. Moreover, the system is more complex, because osteoblasts have been shown to express not only ephrin receptors, but also ephrin ligands, at least Efnb1 and Efnb2 ⁵¹.

I.6. Age-related bone loss

Bone acquisition increases during childhood and adolescence and reach a peak during the second decade of life. Sex steroids are major determinants in the achievement of peak bone mass and are responsible for the differences observed between sexes, but at the peak, the volumetric bone mineral density (gram of hydroxyapathite/cm³) is comparable in both sexes, i.e. degree of mineralization ⁵⁷. Age-related bone loss is described as the reduction in bone mass related to aging that affects both, men and women, and starts after the third decade of life and continues thereafter ⁵⁸; it is the primary underlying cause of fractures in elderly people ⁵⁹.

In women, three phases of bone loss can be distinguished: first, an early phase of trabecular bone loss in premenopausal women, second a transient phase beginning at menopause of rapid loss of trabecular bone associated with less dramatic cortical bone loss and third around 8 to 10 years following menopause a slower age-related phase of bone loss involving a similar amount of cortical and trabecular bone that continues indefinitely. The rapid phase of bone loss occurring at menopause affects mostly trabecular bone and is therefore mainly associated with vertebral and wrist fractures ⁵⁹. This postmenopausal osteoporosis clearly results from loss of ovarian function, as it can be prevented by estrogen replacement ⁶⁰. Estrogen deficiency results in an increased intensity of bone remodeling, i.e.

activation frequency of BMUs, increased osteoclast formation and activity and decreased osteoclast apoptosis ^{61; 62}. This high bone turnover results in microarchitectural damages, including loss of trabecular cross-struts that weaken bone, resorption cavities on trabecular surfaces (remodeling sites) that act as stress-risers, i.e. points of focus for strain where microfractures become more likely and increased cortical porosity due to enhanced Haversian bone remodeling occurs mainly in postmenopausal women ^{63; 64}.

As men lack the equivalent of menopause, they do not exhibit the rapid phase of bone loss, but however present a similar pattern of age-related bone loss ⁶⁵. Indeed, lack of estrogen has long been considered as the major cause of bone loss, but other factors, such as declining levels of androgen (in men) and vitamin D and poor nutrition also contribute to age-related bone loss ⁶⁶. Elderly men and women exhibit a decline of bone turnover ^{65; 67}, parallel to decreased trabecular bone, cortical thinning and expansion of the marrow cavity, i.e.

endocortical resorption ^{65; 68}, that can also be accompanied by an increase of the cortical shell diameter, due to periosteal apposition ⁶⁹. These age-related changes in bone mass result from periosteal apposition (which takes place on the outside of the bone) and endosteal bone resorption (which takes place on the inside of the bone). Although men and women have similar endosteal bone resorption, periosteal apposition is less affected in men, resulting in less dramatic bone loss in men (Figure 12) ⁶⁵.

Figure 12. Position and extent of bone loss in men and women ⁶⁵.

In addition to increased bone resorption, age-related bone loss is associated with a progressive decline in bone formation, as illustrated by decreased osteoblast number, function and survival ⁶⁸. Moreover, the predominant feature of senile osteoporosis is the infiltration of fat in the bone marrow at the expense of osteoblastogenesis, indicating that the decline in

bone formation observed with age might be due to the preferential differentiation of mesenchymal stem cells (MSC) into adipocytes instead of osteoblasts ⁷⁰. Thus, aging bones exhibit decreasing levels of osteoblastogenesis and increasing osteoclastogenesis and bone marrow adiposity. Increased osteoclastogenesis due to increased RANKL is associated to decline in estrogen ⁴⁹, whereas the concomitant reduction in osteoblastogenesis and increase in adipogenesis results from the age-related increase in expression of the adipogenic peroxisome proliferating-activated receptor gamma (PPARG) and decline in expression of the osteogenic RUNX2, inducing a shift in bone marrow precursor differentiation into adipocytes more than osteoblasts. In addition, infiltrated adipocytes secrete adipokines and toxic fatty acids that in turn would affect osteoblast function and survival ⁷¹.

Although during the early postmenopausal phase, serum ionized calcium is maintained at a constant level by a decrease in serum PTH levels, compensating for the increased bone resorption induced by estrogen deficiency, during the late, slow phase of bone loss, serum PTH progressively increases simultaneously with biochemical markers of bone turnover, i.e. serum osteocalcin and urine N-telopeptide of type 1 collagen (NTx) ⁷². Thus, the increase in serum PTH levels with age in both sexes represents secondary hyperparathyroidism, resulting from intestinal calcium malabsorption, but also from vitamin D deficiency that is very common in elderly people ⁶⁷.

Aging mice recapitulate spontaneously most of the changes in bone mineral density (BMD) and microstructure observed in humans. However, mice present neither the equivalent of menopause ⁷³ nor the intra-cortical harversian bone remodeling occurring in humans, implying that cortical porosity and thinning observed in rodents result only from endocortical bone (re)modeling. C57BL/6J mice exhibit in both sexes, an age-related decline in trabecular bone volume, despite increasing total BMD due to continuous expansion of cortical bone 74; 75; 76; 77; 78

.

I.7. Parathyroid hormone

I.7.1. Parathyroid hormone (PTH)

Mammalian parathyroid hormone (PTH) is a single chain polypeptide of 84 amino acids expressed almost exclusively in the parathyroid gland, with lesser expression in rodent hypothalamus and thymus ⁷⁹. The PTH gene is first translated into a precursor, a prepro-PTH peptide, including a 25 amino acid pre sequence important for the passage through the membrane of the endoplastic reticulum, a 6 amino acid pro sequence removed before hormone is transported to secretory vesicles and a 84 amino acid mature PTH sequence ⁸⁰, that is then processed producing the mature PTH (1-84). PTH is distributed through the circulation to target sites, especially to kidney and bone. PTH is secreted by the parathyroid glands in response to small decline in blood ionized calcium, to maintain the normocalcemic state (Figure 13).

Figure 13. Metabolism and clearance of the parathyroid hormone (PTH) ⁸¹.

To accomplish this task PTH promotes bone resorption and release of calcium from the skeleton reservoir; by inducing renal tubular reabsorption of calcium and excretion of phosphate; and by indirectly enhancing intestinal calcium absorption by increasing the renal production of the active vitamin D metabolite, 1,25(OH)₂ vitamin D. Inversely, 1,25(OH)₂ vitamin D and calcium act coordinately to suppress PTH gene expression and to inhibits parathyroid cells proliferation ⁸². The parathyroid cells sense the changes in calcium levels through a G protein-coupled receptor (GPCR), the calcium-sensing receptors (CaR), localized in its plama membrane ⁸³. Calcium binding to extracellular specific sites of the CaR activates the G-dependent cAMP/PKA and PLC/PKC signaling pathways, inhibiting thereafter by unknown mechanisms the synthesis and secretion of PTH. Inversely, decline in blood ionized calcium results in decreased signaling through the CaR, and consequently in increased PTH secretion ⁸⁴. Therefore calcimimetic drugs are used as CaR agonists to suppress PTH secretion, especially in the management of hyperparathyroidism ⁸⁵. Another mechanism used for PTH inactivation is its cleavage by calcium-sensitive cathepsins within the parathyroid glands. Under condition of hypercalcemia, PTH(1-84) is cleaved at higher rate between residues 34-35 or 36-37, and amino terminal fragments required for binding to the PTH/PTHrP receptors are rapidly degraded within the parathyroid glands, resulting in reduction of biologically active PTH ⁸⁶. However, cleavage of circulating PTH(1-84) to carboxyl-terminal fragments can also occur in the peripheral tissues, such as liver and kidney

87. PTH mid- or carboxy-terminal fragments have been shown to have some biological effects, but their biological roles remain unclear ^{88; 89}.

I.7.2. PTH paradoxical effects on bone

PTH is the only approved anabolic treatment for osteoporosis, stimulating both, bone formation and resorption, the balance remaining positive for formation. PTH has to be administrated intermittently to promote bone formation in a way that yields a transitory peak blood level 90; 91; 92

. Indeed, to be effective this treatment consists of daily subcutaneous injections of PTH(1-34) also named teriparatide or of the intact human recombinant PTH(1- 84) ⁹³. Intermittent PTH (i.PTH) is associated with increased cancellous bone volume, density and strength. Its effects on cortical bone are more complex, but include increased cortical thickness and porosity resulting in positive bone strength 92; 94; 95; 96

. Inversely, continuous elevation of PTH levels, associated with PTH infusion or hyperparathyroidism (HPT) characterized by excessive PTH secretion due to impairment of its regulation, leads to bone loss ⁹⁷. More specifically, continuous PTH (c.PTH) has usually neutral effect on cancellous bone, but net catabolic effect on the cortical bone 98; 99; 100; 101

.

Both i.PTH and c.PTH increase bone turnover, but to a different extent due to different RANKL/OPG ratio. Thus i.PTH by reducing the RANKL/OPG ratio induces an imbalance of the system in favor of bone formation, whereas c.PTH stimulates less bone formation than i.PTH, due to an imbalance of the system in favor of bone resorption, resulting from an increased RANKL/OPG ratio. At the cellular level, single exposition of osteoblastic cells to PTH induces an increase in the RANKL/OPG ratio, due to increased RANKL and decreased OPG mRNA expression 102; 103; 104; 105; 106

. Similar effects were observed in vivo after a single injection of PTH ^{107; 108}. The effects of long-term PTH treatments are less clear. As expected, exposure of osteoblasts to i.PTH is associated with an increase of mineralization and of the expression osteoblastic differentiation markers, such as ALP and osteocalcin mRNA, whereas c.PTH inhibits osteoblast differentiation and mineralization, enhances the number of TRAP

positive cells and increases the RANKL/OPG ratio at mRNA level 107; 109; 110; 111; 112

. However, patients treated for 12 months to i.PTH also exhibited an increase in serum RANKL/OPG ratio ^{113; 114}.

PTH promotes bone resorption by inducing new osteoclast recruitment and differentiation. The effect of PTH on bone resorption could be due to direct PTH action on osteoclasts through functional PTH receptors, but it is still controversial 115; 116; 117

. More likely, these PTH effects are mediated through the PTH/PTHrP receptor on cells of the osteoblast lineage, then acting on osteoblast-osteoclast coupling via the OPG/RANKL/RANK system. Direct cell to cell contact might be required for PTH-induced osteoclast activation, but uncertain ¹¹⁸. It has also been shown that PTH stimulates osteoblasts to secrete proteins such as collagenase and plasmingen activators, which may favor osteoclastic bone resorption

119; 120

.

PTH acts directly on cells of the osteoblastic lineage, enhancing their activity and stimulating their differentiation possibly through the inhibition of Dkk1 and Sost, two inhibitors of the canonical WNT signaling, but also by inhibiting their apoptosis 121; 122; 123

. Indeed, O’Brien et al. demonstrated using a PTH receptor constitutively active in osteocytes that PTH acts through the PTH/PTHrP receptor to inhibit Sost and activate LRP5 signaling, two factors required for bone formation ¹²⁴.

PTH related peptide (PTHrP) has also been shown to play a role in PTH effects on bone, as the increased trabecular volume observed in PTH-deficient mice is due to reduced PTH-induced osteoclastic and persistent PTHrP-stimulated osteoblastic bone formation ¹²⁵. IGF1 is another factor required for PTH-induced bone anabolic effects, as Igf1-deficient mice fail to respond to intermittent PTH treatment ¹²⁶. PTH-induced elevation of osteoblast number and bone formation may also occur indirectly through stimulation of bone resorption which

releases growth factors embedded in the bone matrix, that in turn promote bone formation ¹²⁷. A summary of the potential anabolic effects of PTH on target cells is shown Figure 14.

Figure 14. Potential cellular targets for the anabolic effects of PTH ¹²⁸.

All these findings underscore that the molecular mechanisms mediating the various PTH effects are complex and depend on PTH doses, on the mode of administration or time of exposure to the hormone and on bone compartment considered. Thus for instance, we still do not understand why and how anabolic PTH treatment increases trabecular thickness simultaneously to Haversian intracortical remodeling, and why induction of periosteal effects need much higher PTH concentrations than intermittent PTH-induced effect on trabecular or intracortical bone.

Bone marrow compartment Bone marrow compartment

I.7.3. PTH receptors

The PTH/PTHrP receptor, also called parathyroid hormone 1 receptor (PTH1R), is a G protein-coupled receptor (GPCR), with seven membrane-spanning domains. It belongs to the class B of GPCRs family, that exhibit a large amino-terminal extracellular domain containing six conserved cysteine residues. PTH related peptide (PTHrP) signals also through this receptor as amino-terminal (1-34) region of PTH and PTHrP are sufficient to activate this receptor ¹²⁹. Due to high homology in amino-terminal (1-34) region, particularly within their first 13 residues, PTH and PTHrP effects are indistinguishable at least on the mineral ion homeostasis ¹³⁰. PTHrP is an autocrine and paracrine factor with among others, an essential role in normal skeletal development ¹³¹.

The PTH1R, as expected, is highly expressed in bone and kidney, but also in other tissues ¹³². Mice with a deletion of the PTH1R gene exhibit neonatal lethality with severe defects in endochondral bone formation ¹³³. Interestingly, the ablation of the PTHrP gene results in a similar phenotype, indicating that dysfunctional endochondral bone formation is due to the disruption of the PTHrP paracrine effects through PTH1R in the growth plate ¹³¹. PTH-deficient mice exhibit reduced trabecular bone volume during fetal life ¹³⁴, but increased trabecular bone mass in postnatal life ¹³⁵. In PTHrP-haploinsufficient mice, trabecular bone volume is normal at birth, but reduced by 3 months of age ¹³⁶. By examining the postnatal double mutant PTH-deficient PTHrP-haploinsufficient mice, Miao et al. showed that PTHrP is required for the maintenance of normal trabecular bone mass, but also for the high trabecular bone volume observed in PTH-deficient mice ¹²⁵. PTH seems to be the major regulator of cortical bone turnover, as cortical thickness is increased in PTH-deficient mice and not affected in PTHrP-haploinsufficient mice ¹²⁵. However in the PTH-deficient PTHrP-

haploinsufficient double mutant mice, cortical thickness is not affected, suggesting a role of PTHrP on cortical bone in absence of PTH ¹²⁵.

Deletion of the PTH gene leads to hypocalcemia and hyperphosphatemia, whereas mutations activating constitutively the PTH1R result in opposite effects, hypercalcemia and hypophosphatemia, underlying the crucial role of PTH in mineral ion homeostasis ^{137; 138}.

PTH is also able to bind to another receptor presenting 51% of amino acids sequence homology to PTH1R, the PTH2R. In human PTH2R is responsive to PTH but not PTHrP, whereas in rodents PTH as well as PTHrP fail to activate this receptor. PTH2R is expressed only in a few tissues with a predominant expression in the hypothalamus, but unlike PTH1R not in bone and kidney. Moreover, TIP39 seems to be the natural ligand for PTH2R ^{139; 140}. These data altogether suggest a distinct physiological role for the PTH2R receptor.

A third type PTH receptor, named type-3 zPTH receptor or zPTH3R was cloned from zebrafish, but to date its importance is uncertain, as there is no evidence for a mammalian homolog of this receptor ¹⁴¹.

Carboxyl (C) terminal PTH fragments, such as PTH(39-84), or PTH(53-84) were for a long time thought to be inactive, because they presented no agonist activity at the PTH1R ⁹⁰. Nevertheless, some evidences point at the biological activity of these fragments and at the existence of a putative PTH receptor (CPTHR) binding specifically to COOH-terminal portion of PTH. Thus, PTH(7-84) as other N-terminal truncated peptides, was shown to counteract the classical biologic actions of PTH(1-34) and PTH(1-84) on calcium homeostasis and to influence osteoclast differentiation in vitro ^{142; 143}. In addition, these C-terminal PTH fragments were found to be present at high concentration in the circulation, dependently on serum calcium levels and to exert unique biological effects in appropriately selected assays, both in vivo and in vitro ^{144; 145}. Divieti et al. also demonstrated that the activation of this receptor in PTH1R-null osteocytes might be responsible for disturbed survival ¹⁴⁶. Hence,

these biologically active N-terminal truncated peptides might mediate their effects through the CPTHR, but more work is required for its identification ^{88; 145}.

The facts that synthetic PTH(1-34) could reproduce the principal actions of the full- length PTH, that synthetic C-terminal PTH fragments were not capable to signal through the PTH1R and that pure PTH(1-84) is difficult to synthesize, led to the recognized widespread use of synthetic PTH(1-34) as a surrogate for the intact hormone in current research ¹⁴⁵.

I.7.4. PTH1R signaling

As already mentioned the PTH1R is a GPCR, and as indicated by his name it is coupled to the G proteins. G proteins are heterotrimeric and comprise 3 subunits: the α subunit and the ß and γ subunits associated in a tightly linked ßγ dimer. Binding of a ligand to a GPCR induces conformational changes of the receptor allowing him to function as a guanine nucleotide exchange factor (GEF), that permit the hydrolysis of GTP to GDP on the α subunit. This exchange leads to the G protein dissociation into α subunits bound to GTP and the ßγ dimers, which both activate several effectors. To start a new cycle, the α subunit then hydrolyzes the GTP to GDP and allows re-association to the heterotrimeric G protein ¹⁴⁷. PTH binding to the PTH1R can activate the adenylyl cyclase through the Gαs, leading to cyclic 3’,5’-adenosine monophosphate (cAMP) formation, that in turn binds to the regulatory subunit of the protein kinase A (PKA), resulting in the release of the active catalytic subunit of the enzyme. PTH can also activate the phospholipase C (PLC) through Gαq, leading to formation of diacylglycerol (DAG), which then activates protein kinase C (PKC) and 1,4,5- inositol trisphosphate (IP3), resulting in increased intracellular free Ca²⁺ (Figure 15) ¹⁴⁸.

Figure 15. PTH mediated signaling ¹⁴⁸.

The receptor sites involved in coupling to G proteins are currently investigated by mutagenesis. Thus, replacing the EEKY by DSEL amino acids in the second intracellular (IC) loop was shown to selectively impair the Gαq- without affecting the Gαs -linked signaling ¹⁴⁹, whereas mutations in the third IC loop blocks both the Gαq and the Gαs pathways ¹⁵⁰. Serial truncations up to amino acid 480 of the PTH1R cytoplasmic C-terminal tail does not affect PTH signaling via Gαq or Gαs, but seems to reduce mutant receptors expression ¹⁵¹. Disruption of the C-terminal tail domain between amino acids 268 and 491 blocks signaling via PLCß/Ca²⁺ and MAPK and markedly reduces signaling via cAMP/PKA, due to impaired binding of the G protein ßγ subunit to the PTH1R ¹⁵². In addition, the intracellular tail of PTH1R was suggested to be important for coupling to a pertussis toxin-sensitive Gαi protein that inhibits adenylyl cylcase ^{149; 153}. This receptor tail was also shown to contain a highly conserved domain required to bind to family of sodium/hydrogen exchanger regulator factors (NHERFs) which might be key determinants for PLC versus adenylyl cyclase signaling ^154;

155.

Although it is clear that an intact PTH or PTHrP N-terminal region is needed for effective cAMP/PKA signaling, it is still not evident for PKC activation. Thus, various N-

145

intact N-terminal is needed to signal properly through Gαq/PLC/DAG/IP3/PKC signaling ¹⁵⁶. These discrepancies might be explained by the fact that PTH1R can activate PKC by at least two mechanism: one, PLC-dependent requiring an intact N-terminal PTH/PTHrP, and one another, PLC-independent triggered by the N-truncated ligands comprising at least the PTH(29-32) region or PTHrP(25-34) respectively ¹⁴⁵.

The PTH1R is required to mediate the PTH-induced bone resorption ¹⁵⁷. Agents increasing cAMP levels such as forskolin, as well as Ca²⁺ ionophores and phorbol esters, were all reported to promote bone resorption in organ culture, suggesting that both cAMP/PKA and PKC/IP3 might be implicated PTH-induced bone resorption. However, experiments with Ca²⁺

ionophores also showed inhibition of bone resorption. Similarly, both pathways are probably involved in hematopoietic precursor differentiation to osteoclast-like cells ⁸¹. PTH effects on the RANKL/OPG system, i.e. stimulation of RANKL and inhibition of OPG, are mimicked by agents increasing cAMP levels and disrupted by agents impairing cAMP signaling ^{102; 105;}

106; 158; 159. Mice with a disruption of the subunit Gαs specifically in osteoblasts exhibit a marked reduction in endosteal osteoclast number and bone resorption, further underlying that cAMP is the primary second messenger in PTH-induced bone resorption ¹⁶⁰. The signaling mechanisms responsible for anabolic responses of the skeleton to i.PTH administration are still confused. Normally, PTH is able to increase the number and/or the functional activity of mature osteoblasts and to stimulate ostoblast differentiation in vitro ^{109; 112}. The activation of transcription factors essential for osteoblast differentiation and/or proliferation, such as Creb, Runx2 and osterix, is mediated through cAMP/PKA signaling ^{161; 162}. PTH N-terminal fragments activating predominantly the cAMP/PKA pathway, such as PTH(1-30) et PTH(1- 31), are anabolic agents for bone ^{163; 164}. Mice with a deletion of the subunit Gαs specifically in osteoblasts present a marked impairment of trabecular bone formation ¹⁶⁰. Altogether these findings suggest that the cAMP/PKA signaling pathway plays a major role in the mediation of