Induction de l'apoptose des ostéoclastes humains par la Prostaglandine D[indice inférieur 2] : récepteurs et mécanismes de transduction impliqués

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Universite de Sherbrooke

Induction de l'apoptose des osteoclastes humains par la Prostaglandine D2: recepteurs et mecanismes de transduction impliques

Par Li Yue

Programme de Pharmacologie

These presentee a la Faculte de medecine et des sciences de la sante en vue de 1’obtention du grade de Philosophiae Doctor (Ph.D.) en Pharmacologie

Sherbrooke, Quebec, Canada Decembre, 2013

Membres du ju ry devalu atio n

Dr. Artur J. de Brum-Femandes, Departement de Pharmacologie, FMSS, UdeS Prof. Jean-Bemard Denault, Departement de Pharmacologie, FMSS, UdeS

Prof. Patrick McDonald, Departement de Pediatrie, FMSS, UdeS

Prof. Martin G. Sirois, Departement de Pharmacologie, Faculte de Medecine, Universite de Montreal

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I dedicate this thesis to

son and to m y husband

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recepteurs et m ecanism es de tra n sd u ctio n im pliques Par Li Yue

Programme de Pharmacologie

These presentee a la Faculte de medecine et des sciences de la sante en vue de I’obtention du diplome de Philosophiae Doctor (Ph.D.) en Pharmacologie, Faculte de medecine et des

sciences de la sante, Universite de Sherbrooke, Sherbrooke, Quebec, Canada, J 1H 5N4 La prostaglandine D2 (PGD2) est un mediateur lipidique qui active directement deux recepteurs specifiques, DP et CRTH2, regulant ainsi des processus inflammatoires, immunitaires et apoptotiques. Les osteoclastes (OC) sont de larges cellules multinucleees participant au metabolisme et remodelage de l’os, ainsi qu’a la reparation de fracture osseuse. Nos travaux ont mis en evidence l’expression des recepteurs DP et CRTH2 chez des OCs humains. Cependant, les effets de la PGD2sur l’apoptose des OCs sont inconnus. L’objectif de la presente etude a ete de determiner si la PGD2 induit l’apoptose et les mecanismes qui en decoulent dans les OC humains. Les OCs humains differencies ont ete traites avec la PGD2, les agonistes et antagonistes de ses recepteurs. Le traitem ent des OCs avec la PGD2, en presence de naproxene, qui permet d ’inhiber la production endogene de prostaglandines, augmente de fa?on dependante de la dose et en fonction du temps le pourcentage d ’OCs apoptotiques. Ceci a egalement ete observe lors du traitement des OCs avec l’agoniste specifique DK-PGD2 du recepteur CRTH2, mais pas avec le traitement du compose BW 245C, antagoniste du recepteur DP. En absence de naproxene, l’antagoniste CAY 10471 du recepteur CRTH2 reduit le taux d ’apoptose des OCs tandis que le compose BW A868C, antagoniste du recepteur DP, n ’a aucun effet. L ’apoptose des OCs par la PGD2 via CRTH2 est associee a l’activation de la caspase-9, et non pas la caspase-8, ce qui entraine le clivage de la caspase-3. Afin de determiner plus precisement les mecanismes menant a ces resultats, les OCs ont ete traites avec les inhibiteurs de M EK-1/2, PI3K et IKK2/N F-kB. Le traitement des OCs avec la PGD2 et l’agoniste de CRTH2 diminue la phosphorylation des proteines ERK1/2 et Akt, tandis que la phosphorylation de P-arrestine-1 est augmentee. Par ailleurs, les niveaux de phosphorylation d ’E R K l/ 2 et Akt ont ete augmentes alors que le taux de proteines P-arrestine-1 phosphorylees a ete diminue par l’antagoniste de CRTH2. En outre, le traitement des OCs avec I’inhibiteur de MEK-1/2 augmente l’apoptose des OCs induite par PGD2 et l’agoniste de CRTH2. Cependant, l’antagoniste de CRTH2 diminue I’activite de la caspase-3 induite par l’inhibiteur de MEK1/2. Le traitement des OCs avec I’inhibiteur de la PI3K diminue la phosphorylation d ’ER K l/2, tandis que la phosphorylation d ’E R K l/2 augmentee par l’antagoniste de CRTH2 a ete attenuee par l’inhibiteur de PI3K. Les agonistes et antagonistes du recepteurs DP n ’ont pas d ’effet sur la phosphorylation d ’E R K l/2, Akt, P-arrestine-1 ni sur l’activite de la caspase-3 chez les OCs. Le traitement des OCs avec PGD2 et les ligands de ses recepteurs ne modifie pas la phosphorylation de RelA/p65. De plus, l’activite de la caspase-3 n ’est pas alteree dans les OCs traites avec l’inhibiteur d ’IKK2. En conclusion, PGD2, en se liant a CRTH2, induit l’apoptose des OCs via la voie apoptotique intrinseque qui est associee a la regulation des voies de signalisation des proteines P-arrestine-1, ERK1/2, et Akt, mais pas celle du IKK2/N F-kB.

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SU M M A RY

P rostaglan d in D2 induces hum an osteoclast apoptosis an d its underlying m echanism s By

Li Yue

Program o f Pharmacology

Thesis presented at the Faculty o f Medicine and Health Sciences for the obtention o f Doctor o f Philosophy (Ph.D.) degree in Pharmacology, Faculty o f Medicine and Health

Sciences, Universite de Sherbrooke, Sherbrooke, Quebec, Canada, J1H 5N4

Prostaglandin D2 (PGD2) is a lipid m ediator that directly activates two specific receptors, DP and CRTH2, thereby regulating inflammation, immune response and apoptosis. Osteoclasts (OCs) are large multinucleated cells that participate in bone metabolism, remodeling, and fracture repair. Our previous data show the expression o f DP and CRTH2 in human OCs. However, it is unknown w hether PGD2 affects OC apoptosis. The objective o f the thesis was to determine whether PG D 2 induces human OC apoptosis and the underlying mechanisms implicated in this effect. The differentiated human OCs were treated with PGD2, and its receptors agonists/antagonists. Treatment with PGD2 in the presence o f naproxen to inhibit endogenous prostaglandins production increased OC apoptosis in a dose- and time-dependent manner, as did the specific CRTH2 agonist compound DK-PGD2 but not the DP agonist compound BW 245C. In the absence o f naproxen, the CRTH2 antagonist compound CAY 10471 reduced OC apoptosis whereas the DP antagonist BW A868C had no such effect. PGD2/CRTH2-induced OC apoptosis was associated with the activation o f caspase-9 (an intrinsic apoptosis pathway-initiator caspase), but not caspase-8 (an extrinsic apoptosis pathway-initiator caspase), leading to caspase-3 cleavage. To further determine the mechanisms underlying these findings, human OCs were treated with the inhibitors o f MEK-1/2, PI3K and IKK2/NF-kB. Treatments with PGD2 and a CRTH2 agonist decreased ERK1/2 and Akt phosphorylation, whereas both treatments increased p-arrestin-1 phosphorylation. Both ERK1/2 and A kt phosphorylation were augmented, whereas the phosphorylated P-arrestin-1 was reduced by a CRTH2 antagonist. Furthermore, treatm ent o f OCs with a MEK-1/2 inhibitor increased OC apoptosis induced by PGD2 and by a CRTH2 agonist. However, a CRTH2 antagonist diminished the MEK-1/2 inhibitor-induced increase in caspase-3 activity. In addition, treatment o f OCs with a PI3K inhibitor decreased ERK1/2 phosphorylation, whereas increased ERK1/2 phosphorylation by CRTH2 antagonist w as attenuated by a PI3K inhibitor. Both DP receptor agonist and antagonist did not affect either Akt, ERK1/2, P-arrestin-1 phosphorylation or a specific MEK-1/2 inhibitor-induced increase in caspase-3 activity in OCs. Treatment o f OCs with PGD2 and its receptor ligands did not alter RelA/p65 phosphorylation (ser536). M oreover, the caspase-3 activity was not altered in OCs treated with an IKK2/NF-kB inhibitor. In summary, PGD2 induces human OC apoptosis through a CRTH2-dependent intrinsic apoptosis pathway, which is associated with regulation o f the P-arrestin-1, ERK1/2, and Akt, but not with IKK2/NF-kB, signaling pathways.

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O B JE C T IV E S ... 42 R E S U L T S ...43 A R T IC L E 1 ... 43 R esu m e ... 46 A b stra c t... 48 In tro d u c tio n ...48 M aterials and m e th o d s... 49 R e su lts...53 D iscu ssio n ... 62

A cknow ledgm ents...65

F u n d in g ... 65 C onflict o f in terest s ta te m e n t... 65 R eferences... 65 A R T IC L E 2 ... 71 R esu m e...73 A b stra c t...76 In tro d u c tio n ...77 M aterials a n d m e th o d s... 78 R e su lts...81 D iscu ssio n... 92 C onclusions...95

A cknow ledgm ents...96

F u n d in g ...96

C onflict o f in terest s ta te m e n t... 96

R eferences... 97

D ISC U SSIO N ...104

1. PG D2 an d its recep to rs in O C a p o p to sis ... 105

2. E xtrinsic/intrinsic apoptosis p ath w ay s in PG D 2-induced osteoclast ap o p to sis... 106

3. Signaling pathw ays involved in PG D 2/C R T H 2-induced osteoclast ap o p to sis 107 3.1 (3-arrestin-1 signaling in PGD2/CRTH2-induced osteoclast apoptosis... 107

3.2 PI3K/Akt pathway during PGD2-induced osteoclast apoptosis... 108

3.3 MAPK/ERK signaling during PGD2-induced osteoclast ap o p to sis...109

3.4 NF-kB signaling pathway in PGD2-induced osteoclast apoptosis...111

4. C o n trib u tio ns o f this study to know ledge a d v a n c e m e n t...113

C O N C L U S IO N ... 114

P E R S P E C T IV E S ... 115

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L IST O F R E F E R E N C E S ... 119

ANNEXES...143

1. Supplementary figures...143

1.1 Experimental design for treatment with kinase inhibitors... 143

1.2 Morphology o f human differentiated o steoclasts... 144

1.3 The percentage o f TRAP staining c e lls ... 145

1.4 PGD2/CRTH2 induced osteoclast apoptosis in a time-dependent m an n er...146

1.5 Effect o f PGE2 on osteoclast apoptosis... 147

1.6 Dose-dependent effect o f PI3K inhibitor on osteoclast apoptosis... 148

2. Conference abstracts and presentations...149

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LIST OF FIGURES

IN T R D U C TIO N

F ig u re 1. Representative diagram o f a long bone structure... 3

F ig u re 2. Bone remodeling cycle...6

F ig u re 3. Three important bone cells: OBs, osteocytes and OCs... 7

F ig u re 4. Differentiation o f OBs and osteocytes from mesenchymal stem cells... 9

F ig u re 5. Lacunae containing osteocytes within the calcified m atrix...10

F ig u re 6. OC differentiation from hematopoietic stem cells...11

F ig u re 7. Mechanism o f osteoclastic bone resorption...13

F ig u re 8. Regulation o f OC differentiation and activation by M -CSF/RANKL/OPG 15 F ig u re 9. Schematic representation o f necrosis and apoptosis... 16

F ig u re 10. Overview o f extrinsic and intrinsic apoptosis pathways... 19

F ig u re 11. Pro-apoptosis and anti-apoptosis members o f Bcl-2 family... 20

F ig u re 12. Pro-apoptotic and anti-apoptotic pathways involved in OC apoptosis... 21

F ig u re 13. Overview o f PI3K/Akt, MEK/ERK and N F-kB signaling pathway in OC apoptosis...22

F ig u re 14. Overview o f PI3K/Akt signaling pathw ay... 24

F ig u re 15. Four MAPK cascades... 25

F ig u re 16. Role o f MEK1/2-ERK1/2 in OC apoptosis... 26

F ig u re 17. Canonical and non-canonical pathways o f N F-kB activation... 28

F ig u re 18. Role o f N F-kB pathway in OC apoptosis...30

Fig u re 19. Metabolism o f arachidonic acid to prostaglandins...32

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Figure 22. Regulation o f cellular apoptosis by PGD2, its metabolites and its

receptors... 41

A R T IC L E 1

F igure 1. TRAP staining and TACS Blue Labeling analysis o f human differentiated OCs... 54 F igure 2. Concentration-response curves o f PGD2 and its agonist or antagonist on

apoptosis in human OCs, and PGD2 production by human O C s... 57 F igure 3. Caspases activities after stimulation with PGD2 and agonists o f its receptors in OCs... 58 Figure 4. Caspases activities after stimulation with PGD2 receptors antagonists in

OCs... 59 Figure 5. Levels o f caspase-8 and their cleaved forms in OCs in response to PGD2, an agonist or antagonist o f its receptors treatments using western blot analysis...60 F ig u re 6. Levels o f caspase-9 and their cleaved forms in OCs in response to different treatments using western blot analysis... 61

A R T IC L E 2

F ig u re 1. Representative light micrographs o f identifications for human differentiated OCs... 83 F ig u re 2. Participation o f MEK-ERK1/2 pathway in apoptosis o f OCs...85 F ig u re 3. Modulation o f (3-arrestin-l phosphorylation at Ser412 during human OC

apoptosis... 87 F igure 4. Involvement o f Akt phosphorylation in OC apoptosis...88 F igure 5. Association o f Akt and ERK1/2 signaling pathways in PGD2-induced OC apoptosis... 90 F ig u re 6. No effect on OC apoptosis through IKK2/N F-kB signaling pathw ay...91

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X

DISCUSSION

Figure 23. A schematic model showing the regulation o f PGD2 in OC apoptosis... 113 ANNEX

S upplem entary Figure 1. The experimental design for treatment o f kinase inhibitors with or without PGD2 and its receptors agonists/antagonists... 143 S upplem entary Figure 2. Representative light micrographs o f morphology in human differentiated OCs... 144 S upplem entary Figure 3. The percentage o f TRAP-positive cells with no less than three nuclei, TRAP-positive cells and TRAP-negative cells...145 S upplem entary Figure 4. Time-response curves o f PGD2 and its receptors

agonists/antagonists on apoptosis in human O Cs... 146 S upplem entary Figure 5. Effect o f PGE2 and PGD2 on apoptosis in in vitro-

differentiated OCs using the TACS Blue Label Kit...147 S upplem en tary Figure 6. Concentration-response curve o f PI3K inhibitor in inducing OC apoptosis in the presence o f 10 nM o f PGD2... 148

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LIST OF TABLES

T ab le 1. Characteristics o f DP and CRTH2 receptors...35 T ab le 2. PGD2 receptors agonists and antagonists... 36

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LIST OF ABBREVIATIONS

AA Arachidonic acid

AC Adenylate cyclase

BH Bcl-2 homology

BMP Bone morphogenetic protein

cAMP Cyclic adenosine monophosphate

CFU-GM Colony-forming unit-granulocyte/macrophage

CRTH2 Chemoattractant receptor homologous molecule expressed on T-helper type 2 cells

COX Cyclooxygenase

DAG Diacylglycerol

DK-PGD2 13,14-dihydro-15-keto-PGD2

DMSO Dimethyl sulfoxide

DP D-type prostanoid

15dPGJ2 15-deoxy-A12,14-PGJ2

ECL Enhanced chemiluminescence

EIA Enzyme immunoassay

ERK Extracellular signal-regulated kinase

FasL Fas ligand

FBS Fetal bovine serum

FOXO Forkhead box O

GPCR G protein-coupled receptor

Gsk3p Glycogen synthase kinase-30

GRK GPCR kinase

H-PGDS Hematopoietic PGD synthase

IkB Inhibitor o f kB

IKK IkB kinase

IL Interleukin

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MAPK Mitogen-activated protein kinase

MAPKK MAPK kinase

MAP3K MAPK kinase kinase

M-CSF Macrophage-colony stimulating factor

MEK MAPK-ERK kinase

MEKK MAPK kinase kinase

mTOR Mammalian target o f rapamycin

NEMO NF-kB essential modulator

N F-kB Nuclear factor kB

NIK _NF-kB inducing kinase

OB _Osteoblast

OC _Osteoclast

OPG _{Osteoprotegerin}

PARP _{Poly (ADP ribose) polymerase}

PBMCs _{Peripheral blood m ononuclear cells}

PDK _{Phosphoinositide-dependent protein kinase}

PDTC _{Pyrrolidine dithiocarbamate}

PG _{Prostaglandin}

p g d2 _{Prostaglandin D2}

p g e2 _{Prostaglandin E2}

p g h2 _{Prostaglandin H 2}

PDK _{Phosphatidyl inositol 3-kinase}

PIP2 _{Phosphatidylinositol 4,5-bisphosphate} PIP3 _{Phosphatidylinositol 3, 4, 5-trisphosphate}

PKA _{Protein kinase A}

PKC _{Protein kinase C}

PLC _{Phospholipase C}

PTH _{Parathyroid hormone}

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RANKL Receptor activator for nuclear factor kB ligand

RB Ruffled border

RSK Ribosomal protein S6 kinase

RTK Receptor tyrosine kinase

TGF Transforming growth factor

Th2 _{T-helper type 2}

TMRM Tetramethyl rhodamine methyl ester

TNF Tumor necrosis factor

TRAF TNF receptor associated factor

TRAIL TNF-related apoptosis-inducing ligand

TRAIL-R TRAIL-receptor

TRAP Tartrate-resistant acid phosphatase

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Bone is a specialized connective tissue that undergoes continuous processes o f remodeling and turnover. Both processes rely on a balance between bone resorption by osteoclasts (OCs) and bone formation by osteoblasts (OBs). M any bone diseases are characterized by an imbalance o f bone turnover: when the amount o f bone resorption by OCs exceeds that laid down by OBs, bone diseases happen, such as rheumatoid arthritis (RA) and osteoporosis (Singh et al. 2012). The OCs alternate between migration and resorption phases during their life span, until they die by apoptosis. Hence, the induction o f OC apoptosis can decrease bone resorption, which redresses the imbalance o f bone remodeling and turnover in metabolic bone diseases.

Prostaglandins (PGs) are lipid mediators synthesized from arachidonic acid (AA) through the catalysis by cyclooxygenases (COXs) and the action o f different synthases. Among these PGs, prostaglandin D2 (PGD2) is a key mediator in various pathophysiological processes and diseases including vasodilatation (Cheng et al. 2006), pain (Eguchi et al. 1999), sleep (Hayaishi 2002), bronchoconstriction (Brannan et al. 2006) and asthma (Oguma et al. 2008). PGD2 and its metabolites are also reported to be involved in the apoptosis o f different cells (Ward et al. 2002; Chen et al. 2005; Wang and M ak 2011). Previous study from our laboratory has shown that OCs in culture express PGD2 receptors: D-type prostanoid (DP) receptor and chemoattractant receptor homologous molecule expressed on T-helper type 2 cells (CRTH2) (Durand et al. 2008). The activation o f the DP receptor by PGD2 on OCs reduces actin ring formation leading to inhibition o f bone resorption, whereas the activation o f the CRTH2 receptor increases lamellipodia resulting in migration o f OCs thereby controlling bone resorption and osteoclastogenesis (Durand et

al. 2008). However, to our knowledge there are no studies regarding the effects o f PGD2 on

OC apoptosis. The aim o f the present study is to investigate whether PGD2 can induce human OC apoptosis and the potential mechanisms implicated in this effect.

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In this dissertation, a brief review on bone physiology, PG metabolism, and cellular apoptosis is provided. Furthermore, the results regarding the effect o f PGD2 on human differentiated OC apoptosis and underlying mechanisms are presented in the following two articles and in the Supplementary Figures. Finally, the interpretation o f the findings is provided in the discussion section.

1. Bone anatom y an d physiology 1.1 Bone anatomy

The adult human skeleton system usually consists o f 206 bones. Bone is a specialized connective tissue that provides mechanical support for tendons, ligaments and joints, and that protects vital organs against damage. It also produces red and white blood cells, as well as stores calcium and phosphate to maintain mineral homeostasis. Bones have complicated shapes, which is roughly divided into four categories: long bones (arms and legs), short bones (tarsals o f ankle and carpals o f wrist), flat bones (ribs and cranium), and irregular bones (facial bones and vertebrae). The most familiar shape is the long bone (Downey and Siegel 2006). Figure 1 shows the structure o f a long bone.

The long bone has two irregular ends, a proximal and a distal epiphysis. At the joint, the epiphysis is covered with cartilage that is made o f type II collagen. The cartilage protects epiphyses from friction and shock at freely moveable joints. The diaphysis is the long narrow shaft (main section) o f the bone. A t the center o f the diaphysis is a medullary (marrow) cavity, which contains bone marrow. The function o f bone m arrow is to generate red and white blood cells, as well as to store fats. A visible line called epiphyseal line forms at the junction o f the epiphysis and diaphysis, once the adult bone has reached maximum length and the whole plate has calcified (Clarke 2008).

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cavity is the central cavity within a bone where bone marrow is stored. The bone marrow in the medullary cavity only has yellow bone marrow for fat storage. However, the red bone marrow is required for the formation o f red blood cells, which can be found in the flat bones o f adult and long bones o f children. Numerous arteries and veins pass through the long bone to build up a rich network o f blood vessels in the bone marrow. Endosteum is a thin layer o f connective tissue lining the medullary cavity o f a bone (Del Fattore et al. 2010).

1.2 Bone organic and inorganic components

Bone is composed o f both organic and inorganic components. The 22% o f whole cortical bone is organic component, 69% o f which is inorganic component, and 9% o f which is water (Stenner et al. 1984).

1.2.1 Organic components

The main organic component (-90% ) o f bone is collagen, and the rest includes proteoglycans, matrix proteins, cytokines and growth factors. Collagen is the most abundant matrix protein in the body, which forms cartilage, bone and tendons. The main form o f collagen is collagen type I (Viguet-Carrin et al. 2006). The proteoglycans are composed o f glycosaminoglycan-protein complexes, which are responsible for compressive strength and mineralization inhibition. M atrix proteins enhance mineralization and bone formation, and they also contain non-collagenous proteins, such as osteocalcin, osteonectin and osteopontin. Osteocalcin produced by OBs is the most abundant non-collagenous protein in the matrix. It is directly involved in regulation o f bone mineral density, and has chemotactic activity for a number o f cells including OCs and OBs. Moreover, osteonectin secreted by platelets and OBs has an effect on mineral organization in matrix or calcium modulation (Young 2003). In addition, the cell-binding protein osteopontin, expressed by both OCs and OBs, influences bone equilibrium by inhibiting mineral deposition and promoting OC differentiation and activity (Standal et al. 2004). Sclerostin is a secreted glycoprotein expressed in osteocytes and chondrocytes, which inhibits bone formation by OBs. The mechanisms underlying the inhibitory effect o f sclerostin on bone formation by OBs are associated with the inhibition o f Wnt signaling pathway (Hoeppner et al. 2009;

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van Amerongen and Nusse 2009; Silverman 2010); but not due to the antagonism o f bone morphogenetic protein (BMP) signals (van Bezooijen et al. 2007; Krause et al. 2010). Sclerostin also regulates bone mineralization and mineral metabolism through affecting hormone secretion (Ryan et al. 2013). Sclerostin is negatively regulated by parathyroid hormone (PTH), but is induced by calcitonin (Gooi et al. 2010; Bellido et al. 2013). Clinical trials have shown that sclerostin antibody (AM G 785) exhibits potential benefit in the treatment o f osteoporosis and other skeletal disorders (Lewiecki 2011; Padhi et al.

2011).

1.2.2 Inorganic components

Inorganic components include calcium hydroxyapatite and calcium phosphate. The inorganic matrix gives the hardness and rigidity o f bone, and it is composed o f a crystalline complex o f calcium and phosphate. The 99% o f inorganic component is hydroxyapatite, which is also known as bone mineral and provides compressive strength. Calcified bone includes approximately 5% water, 25% organic matrix, and 70% inorganic mineral-hydroxyapatite (Sommerfeldt and Rubin 2001).

1.3 Bone cells

There are five types o f bone cells, including osteoprogenitor cells, OBs, osteocytes, OCs, and bone-lining cells. Osteoprogenitor cells localize in the deeper layer o f periosteum and the bone marrow, which originate from stem cells. Osteoprogenitor cells can be differentiated into OBs and are also called preosteoblasts (Clines 2010). The OBs are bone-forming cells derived from pluripotent mesenchymal cells o f the marrow stroma, whereas OCs originate from cells o f the hematopoietic lineage. Once trapped in bone matrix, OBs become the star-shaped osteocytes. Bone-lining cells are quiescent OBs covering the bone as a barrier for certain ions (Ng al. 1997; Boyle et al. 2003). The

differentiation and function o f the bone cells are modulated by a variety o f osteotropic hormones and cytokines. These cells are also regulated through cell-cell contact by cytokines. The details regarding the formation, differentiation and function o f OBs, osteocytes, and OCs are described in the section o f Bone Cell Physiology.

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differentiate into preosteoblasts and ultimately mature OBs. Some OBs are embedded in bone matrix, and become osteocytes.

The OBs are mononuclear cells that participate in bone formation. Usually OB shape varies from flat to plump. These cells reside on bone-form ing surfaces, and are responsible for bone matrix production and its subsequent mineralization. In practice, OBs are specialized fibroblasts that can produce bone matrix in addition to fibroblastic products. For example, OBs can produce osteocalcin and bone sialoprotein, which are non-collagenous bone matrix proteins. Especially, osteocalcin is the predominant non-collagenous protein expressed by OBs, which forms about 1% o f extracellular matrix protein (Huang et al. 2005). Bone sialoprotein increases OB differentiation and matrix mineralization in vitro. OBs can secrete unmineralized organic matrix o f bone (called osteoid) during differentiation. Osteoid is made up o f 90% type I collagen and 10% ground substance (Kalfas 2001). Ground substance is an amorphous gel-like substance, and its main components are proteoglycans. Ground substance is observed in cartilage, W harton’s jelly o f umbilical cord and vitreous humor o f eye (Prolo 1990; Kalfas 2001; Ryu et al. 2013). It occupies the cavities and clefts between the cells and fibers o f connective tissues, which acts as a support for the cells and fibers.

OBs express various genetic markers, such as macrophage-colony stimulating factor (M-CSF), alkaline phosphatase, osteocalcin, osteopontin, and osteonectin (Szulc et al. 2005; Ringe et al. 2008). OBs also produce proteoglycans, such as decorin and biglycan, which can store calcium ion for calcification and regulate growth o f hydroxyapatite by obstructing excess calcification. OBs can synthesize cytokines, such as insulin like growth factor I, insulin like growth factor II, BMP and transforming growth factor (TGF)-(3. These cytokines are embedded in calcified bone matrix, and have critical roles in OB differentiation and function. Actually, both OBs and osteoprogenitor cells are the primary sources for many bone resorption regulating factors, including PGs, TGF-P, interleukins (ILs), and leptin (Teitelbaum 2000; Compston 2001; Lee et al. 2002), which are important mediators in regulating OC differentiation and function. Thus, OBs can regulate the differentiation o f OCs.

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maintenance o f the sealing zone, which are required for effective osteoclastic bone resorption (Nakamura et al. 1999). Calcitonin receptor is a marker o f OC differentiation (Quinn et al. 1999). The resorption pits generated by OCs are often found within the bone matrix, which can be used as a functional assay o f OCs (Rumpler et al. 2013).

2.3.3 Bone resorption by osteoclasts

The OCs are detected in pits o f the bone surface where they dissolve bone tissue by removing its mineralized matrix and degrading the organic bone. This process is named bone resorption.

Bone resorption is a multi-stage process that has at least four steps. The process is initiated by the attachment o f the OCs to bone matrix, where they becom e highly polarized. There are four distinct membrane domains formed: sealing zone, ruffled border (RB), functional secretory domain and basolateral domain. The sealing zone separates the resorptive space from the surrounding bone (Teitelbaum 2000). The sealing zone is the attachment o f the O C’s plasma membrane to the underlying bone, and it is bound by belts o f specialized adhesion structures known as podosomes. The second step is the formation o f a specialized membrane (RB) that acts as cell’s resorptive organelle, after plasma membrane polarization. The RB touching the surface o f the bone tissue promotes removal o f the mineral component o f bone. In the third step hydrogen (H+) and chloride ions (CF) are secreted into the resorption cavity by this highly permeable membrane RB. H+ is released through the action o f carbonic anhydrase (H20 + C 0 2 —* H CCV + H+), acidifying and aiding dissociation o f the mineralized bone matrix into ionic forms o f phosphoric acid and carbonic acid, Ca2+, H20 and other materials. The vacuolar-type H+-ATPase proton pump is a macromolecular complex located on the RB plasma membrane o f OCs, and utilizes the energy from ATP hydrolysis to expel H+, thereby regulating extracellular acidification for bone demineralization in bone resorption. This continuous release o f H+ dissolves mineralized bone matrix and concomitantly elevates the proteolytic enzymes activity to degrade the organic matrix. However, the passive C F conductance by chloride channel is required for the acidification o f resorption lacunae (Schlesinger et al. 1997; Rousselle and Heymann 2002). HCO3 7C F exchange at the cell’s non-resorptive surface keeps the

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2.3.4 Regulation o f osteoclast form ation, differentiation and activation

The OCs alternate between migration and resorption phases during their life span until they die by apoptosis. Apoptosis shortens the life span o f these cells, and limits the amount o f bone resorption. The OC formation, differentiation and activation are regulated by numerous cytokines, growth factors and hormones, such as M -CSF (Nakanishi et al. 2013), RANKL (Ikeda et al. 2008) and tum or necrosis factor (T N F)-a (Kudo et al. 2002). TN F-a binds to two OC surface receptors, TNF receptor 1 and 2 (Kobayashi et al. 2000), while M-CSF and RANKL bind to the corresponding receptors (c-fms for M-CSF; and RANK for RANKL) on OC precursors. Finally, OC precursors migrate to a resorption site, differentiate and fuse, thereby forming multinucleated giant cells. Among the factors essential for OC differentiation and activation, both IL-1 and IL- 6 directly promote OC generation (Kudo et al. 2002; Kudo et al. 2003), whereas estrogen, TGF-P, interferon-y, IL-4, 1L-12 alone or in synergy with IL-18, inhibit OC survival (Bendixen et al. 2001; Horwood et al. 2001; Huang et al. 2003; Krum et al. 2008; Houde et al. 2009). Specific integrin receptors avP3 are responsible for the identification o f bone by OCs, thereby facilitating the attachment o f OC to the mineralized bone surface. Moreover, both PTH and

1,25-(OH)2D3 can induce OC formation.

It has been shown that OB lineage cells regulate OC differentiation and activation (Suda et

al. 1999; Mackie 2003). This is because OBs in the bone marrow express M-CSF and

RANKL, which can trigger the differentiation o f OC precursors into OCs. The mechanisms o f M-CSF and RANKL regulation in OCs are associated with several signaling pathways including extracellular-signal-regulated kinases (ERKs), mitogen-activated protein kinases (MAPKs) and NF-kB (Takayanagi 2007). OB lineage cells can produce osteoprotegerin (OPG) which prevents OC differentiation and activation. This is due to the binding o f OPG to RANKL, which interferes with binding o f RANKL to RANK. These findings suggest the dual effects o f OB lineage cells in OC differentiation and activation. Figure 8 presents the regulation o f OC differentiation and activation by M-CSF/RANKL/OPG.

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3.1.2 Assays available fo r measurement o f apoptosis

Many different assays are available for apoptosis detection. The commonly used approaches for apoptosis detection are listed as follows (also available in http://www.aDODtosisworld.com/ADQDtosisAssavs.htmn:

• Caspase Assays: caspases include a group o f specific cysteine proteases that are activated during apoptosis. There are various commercial kits and antibodies for determination o f caspases for apoptosis initiation (caspases-2, -8, -9 and -10), apoptosis execution (caspases-3, - 6 and -7) and cytokine activation (caspases-1, -4, -5 and -13). The protein (total and cleaved) levels and activities o f these caspases can be determined by Western blotting and commercially available fluorescent kits, respectively. It should be noted that the commercially available fluorescent kits to detect caspase activity are not highly specific, which is due to the overlap o f cleavage motifs in substrates (M cStay

et al. 2008).

• DNA Fragmentation Assays: The enzyme responsible for apoptotic DNA fragmentation is the caspase-activated DNAse that cleaves DNA to generate small fragments (180-200 base pair) during apoptosis. Both apoptotic DNA ladder and terminal transferase mediated DNA nick end labeling (TUNEL) apoptosis detection kits are available to determine DNA fragmentation. An agarose-gel electrophoresis is used to detect DNA fragmentation, while TUNEL staining relies on the presence o f nicks in the DNA, which can be recognized by terminal deoxynucleotidyl transferase.

• TACS Blue Label Staining: this assay is designed for the in situ detection o f apoptosis in tissue and cultured cells. It is based on incorporation o f bromodeoxyuridine at the 3 ’ OH ends o f DNA fragments that are generated during apoptosis. An insoluble blue precipitate occurs in nuclei where DNA undergoes double-stranded breaks. Blue stained cells are referred to apoptotic cells.

• Protein Cleavage Assays: poly (ADP ribose) polymerase (PARP) that is one of the cleavage targets of caspase-3, although caspase-7 is better at cleaving PARP-1 than caspase-3 (Boucher et al. 2012). Cleaved PARP can be measured by Western blotting using a specific antibody.

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• Mitochondrial Assays: tetramethyl rhodamine methyl ester (TMRM) is suitable for cytofluorometric measurements o f mitochondrial membrane potential in cells. TMRM Assay Kit is commercially available to determine mitochondrial membrane potential. • Annexin V Assays: The Annexin V staining via fluorescence microscopy is a simple

and effective method to detect one o f the earliest events in apoptosis-the extem alization o f phosphatidylserine-in living cells. This assay employs annexin V, which has a strong and specific affinity for phosphatidylserine, to monitor the phosphatidylserine translocation during apoptosis.

• Other Assays such as Cell Permeability Assays, Cell Proliferation and Senescence Assays are also available to study apoptosis.

3.2 Extrinsic and intrinsic apoptosis pathways

Many mammalian cells undergo apoptosis during normal development or in response to various stimuli, such as growth factor withdrawal, DNA damage, chemical treatment and oxidative stress. Apoptosis involves cysteine-proteases activated by dimerization and cleavage (Fuentes-Prior and Salvesen 2004), which includes initiator caspases (e.g., caspases-2, -8, -9 and -10) and their targets, the effector caspases (e.g., caspases-3, - 6 and -7).

Two most common pathways have been shown to initiate cellular apoptosis: the extrinsic and the intrinsic pathways. The extrinsic pathway, a death receptor pathway, uses caspase- 8 and - 1 0 activation leading to the propagation o f the apoptosis signal after stimulation o f upstream death receptors o f the TNF receptor superfamily. The death receptors include CD95 (Fas), which binds to Fas ligand (FasL), and the TNF-related apoptosis-inducing ligand (TRAIL) receptors, which binds to TRAIL. The intrinsic pathway is mitochondrion-activated and involves the Bcl-2 family members, which can be induced through the release o f apoptogenic factors such as cytochrome c from the mitochondrial intermembrane space (Saelens et al. 2004; Jin and El-Deiry 2005). Bcl-2 family proteins contain Bcl-2 homology (BH) domains, which controls the release o f caspase-activating proteins from the mitochondria. This family consists o f about 20 pro- and anti-apoptotic proteins. Pro-apoptotic members are sub-divided into two groups including pro-apoptotic

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3.4.1 PI3K/Akt signaling pathway

PI3K/Akt pathway is one o f the key pathways in regulating cell motility and invasion, growth, metabolism, survival and proliferation. In addition, this pathway mediates the anti-apoptotic function in many cell types (M oon et al. 2012). PI3K/Akt pathway can be activated by the signals from GPCRs, cytokine receptors, integrins, and receptor tyrosine kinases (RTKs). PI3K can activate the small GTPase Rac, which is capable to mediate cell motility and invasion. Activated PI3K also translocates to the membrane and phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2), then PIP2 is converted to produce phosphatidylinositol-3,4,5-trisphosphate (PIP3) after activation o f growth factors and cytokine receptors. PIP3 recruits signaling proteins with pleckstrin-homology-domains to the cell membrane, including serine/threonine kinase Akt and its activator phosphoinositide-dependent protein kinase (PD K )-l (Figure 14). Activation o f PI3K pathway exhibits an inhibitory effect on T-helper type 2 (Th2) cells apoptosis (Xue et al. 2009). The PI3K/Akt pathway mediates the regulation o f several hormones and autacoids in cellular apoptosis. For example, neuregulin-1, a new autacoid, protected against apoptosis in myocytes by activating PI3K/Akt pathway (Fang et al. 2010). Treatment with 17(3-estradiol reduced light-induced retinal neuronal apoptosis, which is associated with PI3K activation and RelA/p65 nuclear translocation (M o et al. 2013).

The phosphorylation o f Akt on Thr308 and Ser473 results in full activation to regulate mammalian target o f rapamycin (mTOR), glycogen synthase kinase-3p (Gsk3P), Bad, forkhead box O (FOXO) proteins, p21, p27 and other factors (Liu et al. 2009; Chen et al. 2011). Thus, Akt is involved in multiple cellular processes, including cell motility and invasion, growth, metabolism, survival and proliferation. Akt has three closely related isoforms: A ktl, Akt2 and Akt3. Previous studies demonstrate that the individual Akt isoforms have different distributions and dissimilar effects (Dummler and Hemmings 2007). A ktl is expressed in most tissues and is implicated in growth and development. A ktl is also able to induce cell survival by blocking apoptosis (Chen et al. 2010). Akt2 is expressed mostly in insulin-responsive organs such as skeletal muscle, adipose tissue and liver. Akt2 is necessary to promote glucose transport. In vitro experiments show that Akt2 is a special insulin-signaling molecule. Akt2-null mice are insulin resistant, and exhibit mild

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3.4.3 N F -kB signaling pathw ay

NF-kB is a well-known transcription factor. In an inactivated state, NF-kB is sequestered in the cytoplasm by an inhibitor o f kB (IkB), which prevents the N F-kB:IkB complex from translocating to the nucleus. In mammalian cells, the N F-kB family consists o f five proteins: pl05/p50 (NF-kB 1), pl00/p52 (NF-kB2), RelA/p65, RelB and c-Rel. Only RelA/p65, c-Rel and RelB contain a transactivation domain at their C-termini. N F -k B l/p l0 5 and NF-i<B2/pl00 are synthesized as large inactive precursors o f the p50 and p52 proteins, respectively (Hayden and Ghosh 2012). These NF-kB subunits can form various homodimers or heterodimers, such as p50/p50, p52/p52, RelA/RelA homodimers, as well as RelA/p50, RelB/p52, and RelA/c-Rel heterodimers. Previous studies have revealed that p50 and RelA/p65 are expressed in various types o f cells, while RelB expression is confined in specific sites, such as lymph nodes and thymus. However, c-Rel expression is limited to hematopoietic cells (Li and Verma 2002). NF-kB plays an important role in regulation o f inflammation, immunity, differentiation, proliferation, and apoptosis (Hayden and Ghosh 2011; Novack 2011; Wullaert et al. 2011), thus implicating NF-kB in the pathogenesis o f numerous diseases, including inflammatory and autoimmune diseases, cardiovascular disease, cancer, diabetes, and metabolic bone diseases.

Two NF-kB signaling pathways have been identified: the canonical and non-canonical NF-kB pathways. The canonical NF-kB pathway (also known as classical N F-kB pathway) is regulated by IkB kinase (IKK) complex in response to a variety o f stimuli including reactive oxygen species, inflammatory stress, and ionizing radiation. These stimuli cause the phosphorylation o f IKK complex, which contains two catalytic subunits IK K a (IKK1) and IKKp (IKK2), and a regulatory subunit IKKy (NF-kB essential modulator, NEM O). Activated IKK complex phosphorylates two serine residues located in an IkB regulatory domain (e.g., serines 32 and 36 in human IkBoi), which leads to the ubiquitination and proteasomal-dependent degradation o f IkB. This causes the disassociation o f RelA from ficBa, and permits RelA/p50 NF-kB heterodimers to translocate into the nucleus. In the nucleus, NF-kB binds to a consensus sequence o f target genes, thereby activating their transcription. The expression o f IkB is also modulated by N F-kB, and newly synthesized

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In addition, the posttranslational modifications {e.g., phosphorylation and acetylation) o f NF-kB subunits are considered as a second level o f NF-kB activation (Schmitz et al. 2001; Anrather et al. 2005; Neumann and Naumann 2007; Yang et al. 2010). Several sites o f RelA/p65 phosphorylation have been identified, but the m ost extensively studied are Ser276, Ser468 and Ser536 (Zhong et al. 1998; Schmitz et al. 2004; Neumann and Naumann 2007). For example, RelA/p65 phosphorylation at Ser536 can be induced by multiple kinases including IKK2 (Sakurai et al. 1999; Buss et al. 2004).

Several studies demonstrate the regulation o f N F-kB in OC apoptosis. N F-kB activation leads to the differentiation and survival o f OCs, while inhibition o f NF-kB induces OC apoptosis (Ozaki et al. 1997; Abbas and Abu-Amer 2003; Penolazzi et al. 2003; Vaira et al. 2008). There are two approaches employed to inhibit N F-kB activity: 1) transfect cells with a dominant-negative IkB mutant, which blocks RelA/p65 nuclear translocation; and 2) treat cells with decoy oligodeoxynucleotides mimicking the nonsymmetric N F-kB binding site, thereby preventing NF-kB from binding to its target binding sites. It has been shown that both dominant-negative IkB mutant and decoy oligodeoxynucleotides targeting NF-kB induce OC apoptosis (Abbas and Abu-Amer 2003; Penolazzi et al. 2003). This is in line with the observations that the agents pyrrolidine dithiocarbamate (PDTC), N-tosyl-l-phenylalanine chloromethyl keton and gliotoxin, which can inhibit NF-kB activation, stimulate OC apoptosis (Ozaki et al. 1997). Interestingly, both a dominant-negative IkB mutant (blocking RelA/p65 nuclear translocation) transfection and RelA/p65 deletion increase caspase-9 activity, whereas caspase- 8 activity is not altered by RelA/p65 deletion. These findings suggest that intrinsic apoptotic pathway is involved in the regulation o f NF-kB in OC apoptosis (Abbas and Abu-Amer 2003; Vaira et al. 2008). These observations also indicate that canonical N F-kB pathway regulates OC apoptosis. However, a study has shown that NF-kB p i 00 limits osteoclastogenesis implicating the involvement o f non-canonical NF-kB pathway in OC survival and apoptosis (Yao et al. 2009). Figure 18 shows the role o f N F-kB pathway in OC apoptosis. Thus, understanding

the function o f N F-kB in OCs would be pivotal in developing novel strategies for treatments o f bone diseases. There are conflicting reports on the role o f the N F-kB pathway

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exert at least part o f their beneficial effects by regulating OC apoptosis (W einstein and Manolagas 2000; Benford et al. 2001; Wu et al. 2005).

As described, induction o f OC apoptosis can decrease bone resorption, thereby affecting the rate o f bone remodeling and turnover, which might be a novel therapeutic approach in intervening the diseases with bone loss (Piva et al. 2009). However, the underlying mechanism in regulation o f OC apoptosis is yet unknown. Understanding the mechanisms regulating OC apoptosis may not only improve the efficacy o f existing therapies, but also discover novel strategies for drug developm ent in pathologies characterized by excessive bone loss. The Canadian Orthopaedic Foundation reports that over 20% o f Canadians (> 6 million people) present bone and jo in t health problems: arthritis, rheumatism and osteoporosis. This organization also shows that bone and joint disorders cost the economy about $ 17-billion every year in health resources and lost productive force. The Public Health Agency o f Canada reports that osteoporosis affects more than 200 million people worldwide, while 1.5 million Canadians 40 years o f age or older have been diagnosed with osteoporosis (http://www.osteoporosis.ca/index.php/ci id/8867/la id/l.htm ) (Goeree et al. 2006). Hence, the potential benefits o f novel targeted treatm ents for OC apoptosis in these metabolic diseases are enormous.

4. Prostaglandins

4.1 Prostaglandin synthesis

Prostaglandins are lipid mediators synthesized from AA through catalysis by COXs and the action o f different synthases (Figure 19). Briefly, COXs transform AA released from the plasma membrane to prostaglandin H2 (PGH2), which is an intermediate substrate further metabolized by specific synthases to produce PGs. These PGs elicit their biological effects through activation o f cell surface GPCRs, which play important roles in regulating a variety o f pathophysiological processes including bone formation (Li et al. 2007) and resorption (Krieger et al. 2000; Miyaura et al. 2003) by influencing the cross-talk between OBs and OCs (Khosla 2001; Li et al. 2002). Therefore, the modulation o f PGs signaling pathway will affect bone loss and fracture repair (Simon et al. 2002; Gerstenfeld et al. 2003).

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plasma, with a half-life o f approximately 30 minutes (Fukushima 1990; Schuligoi et al. 2007). Furthermore, 75% o f the radioactive labeled PG D2 was excreted into the urine within 5 hours (Liston and Roberts 1985). Therefore, the rapid degradation o f PGD2 should be beared in mind when analyzing the functions o f endogenous and exogenous PGD2 in

vivo.

PGD2 is rapidly degraded into a series o f metabolites: AI2-PGD2; 13,14-dihydro-15-keto-PGD2(DK-PGD2); PGJ2; A12-PGJ2; 15-deoxy-A1214-PGJ2 (15dPGJ2); 9 a , lip -P G F2 and 15-deoxy-A1214-PGD2 (Figure 20). DK-PGD2 is a metabolite o f PGD2 through 15-hydroxy PG dehydrogenase pathway (Rangachari and Betti 1993). Both AI2-PGD2 and DK-PGD2are the selective agonists o f CRTH2 receptor (Hirai et al. 2001; Gazi et al. 2005). DK-PGD2 cannot exist in human urine because it experiences p-oxidation, which is further metabolized into lip -h y d ro x y compounds (Liston and Roberts 1985; Morrow et al. 1991). Moreover, the PGD2-derived 9 a , 1IP-PGF2 is formed by the action o f PGD2 11-ketoreductase in the liver, lung and kidneys. 9 a , llp -P G F2 is also a CRTH2 agonist (Sandig et al. 2006), and plasm a 9 a , 11P-PGF2 is a sensitive m arker for mast cell activation in bronchial asthma (Bochenek et al. 2004). The level o f 9 a , 11P-PGF2 is also highly increased in systemic mastocytosis patients (Awad et al. 1994). PGD2 can be converted to PGJ2, which can further isomerize with albumin to A12-PGJ2 or then is quickly transformed to 15dPGJ2. All PGD2, PGJ2 and A12-PGJ2 have high affinity binding to liver fatty acid binding protein and intracellular protein, which participate in the metabolism o f free fatty acids (Khan and Sorof 1990). The J series o f PG D2 derivatives (PGJ2, A12-PGJ2 and 15dPGJ2) exhibit a function on inflammation (Shibata et al. 2002; Peeraully et al. 2006; Pierre et al. 2009). However, 15-deoxy-A12,14-PGD2 is a composite analog o f PGD2, and it is a potential precursor to 15dPGJ2 which is identified as a ligand for peroxisome proliferator-activated receptor y (Forman et al. 1995). Thus, the study o f PGD2 metabolites would enhance the understanding o f PGD2 in regulating cell function.

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Table 1. Characteristics of DP and CRTH2 receptors

DP receptor Gas (Boie et al. 1995)

al nervous system, bone, retina, lungs, intestine,

lature and nasal mucosa (Boie et al. 1995; Gerashchenko et al. Wright et al. 2000; Gervais et al. 2001; Nantel et al. 2004; d et al. 2008)

ells, OBs, OCs, dendritic cells, basophils, leukocytes, al killer cells, epithelial cells (Nantel et al. 2004; Gallant et al.

Durand et al. 2008)

;ases OPG production, bone resorption, osteoclastogenesis, •cular pressure and venous vasodilatation; stimulates dyl cyclase; increase Ca2+, cAMP, mucin secretion, arterial ;ension(Okuda-Ashitakaet al. 1993; Woodwards/al. 1993;

't al. 1995; Walch et al. 1999; Wright et al. 2000; Moreland et al.

Gallant et al. 2005; Van Hecken et al. 2007; Durand et al. 2008)

CRTH2 receptor

Gai/o and Gaq (Sawyer et al. 2002; Nagata and Hirai 2003)

Thym us, bone, brain, spleen, heart, and digestive system (Sawyer et al. 2002; Durand et al. 2008)

Eosinophils, O Bs, OCs, basophils, macrophages, m onocytes and Th2 cells (Sawyer et al. 2002; Gosset et

al. 2003; Gallant et al. 2005; Durand et al. 2008; Tajima et al. 2008)

D ecreases RANKL expression, osteoclastogenesis and cA M P formation; increases O B s chem otaxis, OC migration, eosinophil and Th2 cell m otility and Ca2+ concentration; modulate eosinophil m orphology and degranulation (Gervais et al. 2001; Hirai et al. 2001; Monneret et al. 2001; Gallant et al. 2005; Durand et al. 2008)

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T able 2. PG D 2 recep to rs agonists a n d an tag o n ists

C om pound A ffinity D P (nM ) A ffinity C R T H 2 (nM ) D esignation p g d2 1.7 ± 0 .3 2.4 ± 0.2 DP and CRTH2 agonists BW 245C 0.4 ± 0.1 > 80000 DP agonist 13,14-dihydro-15-keto-PGD2 (DK-PGD2) > 6 0 0 0 2.9 ± 0 .3 CRTH2 agonist

BW A868C (Giles et al. 1989) 1.7 ND DP antagonist

CAY 10471 (Ulven and

Kostenis 2005) 1200 0.6 CRTH2 antagonist

ND= not determined. Taken from (Sawyer et al. 2002).

4.4 PGD2 signal transduction

4.4.1 G protein coupling-dependent signal transduction

The two known DP and CRTH2 receptors o f PGD2 are both GPCRs. PGD2 binding to DP results in receptor coupling to Gas-type G proteins, leading to activation o f adenylate cyclase (AC) and production o f intracellular cyclic adenosine monophosphate (cAMP), thereby activating protein kinase A (PKA). In contrast to DP, CRTH2 couples to Gaj-type G proteins, leading to inhibition o f AC activity and cAMP generation. CRTH2 can also couple to Gaq-type G proteins, which activates phospholipase C (PLC), and subsequently increases the level o f diacylglycerol (DAG), inositol triphosphate (IP3), and intracellular calcium, leading to activation o f protein kinase C (PKC). Figure 21 shows PGD2-induced G protein coupling-dependent signal transduction via DP and CRTH2 receptors.

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known as P-arrestin-1) and arrestin-3 (also known as P-arrestin-2) are ubiquitous. Arrestin-mediated pathway can synergize or oppose G-protein dependent signals, which may be due to the differences in agonist concentration, phosphorylation state o f the receptor, alteration o f receptor conformation, and availability o f downstream effectors (Azzi et al. 2003; Wang and DeFea 2006; Yee et al. 2006; Sun et al. 2007). There are two steps for arrestins to inhibit GPCR binding to G proteins and downstream signals. First, GPCR is phosphorylated on serine/threonine residues by a member o f GPCR kinases (GRKs). The second step is that arrestin binds to the receptor, which inhibits further G protein-dependent signals and downstream targets receptors for internalization (Gurevich and Gurevich 2004). It has been shown that P-arrestins can scaffold a number o f kinases, such as Src, PI3K, Akt, PKC and PKA, MAPK/ERK, leading to their activation or inactivation (Beaulieu et al. 2005; Wang and DeFea 2006; DeWire et al. 2007; Wang et al. 2007; Defea 2008; Cheung

et al. 2009; Coffa et al. 2011). For example, increased phosphorylation o f p-arrestin-1

(Ser412) impairs its activity, leading to disruption o f G protein-mediated M APK/ERK signals by insulin (Hupfeld et al. 2005).

Previous reports have shown that CRTH2 can elicit signals via regulation o f P-arrestin in an alternative G protein-independent pathway (Azzi et al. 2003; Baker et al. 2003; Wei et al. 2003; Mathiesen et al. 2005). It was reported that PGD2-mediated CRTH2 activation in G protein-independent pathway led to P-arrestin translocation to the receptor (M athiesen et al. 2005), where P-arrestin further promotes the internalization o f CRTH2 and DP receptors (Gallant et al. 2007). Interestingly, PGD2 induces the production o f human P-defensin-3 in human keratinocytes by the CRTH2/Gi/Src/M EK/ERK pathway (Kanda et al. 2010), suggesting an intertwined and reciprocal regulation between G-protein coupling dependent and independent signals. There is no report regarding the role o f P-arrestin in PGD2-induced signal transduction in OCs.

4.5 PGD2 and diseases

PGD2 is an eicosanoid product that is synthesized in the central nervous system and peripheral tissue (Jowsey et al. 2001; Ricciotti and FitzGerald 2011). It is mainly produced by mast cells and other immune cells, such as dendritic cells and Th2 cells. PGD2 has a

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wide variety o f functions involved in physiological processes and in pathogenesis o f diseases. For example, in the brain, PGD2 is implicated in neurophysiological functions, including sleep-wake regulation (Hayaishi 2002), body temperature regulation (Onoe et al. 1988), hormone release (Koh et al. 1988) and pain perception (Eguchi et al. 1999). It is well known that PGD2 is involved in asthma (Oguma et al. 2008) and inflammation (Ricciotti and FitzGerald 2011; Joo and Sadikot 2012). It should be noted that the study o f the function o f PGD2 in inflammation is complicated because it exerts both pro-inflammatory and anti-inflammatory effects depending on the inflammatory milieu.

Accumulating evidence shows that the inflammatory response is involved in the pathogenesis o f various diseases including cancer, stroke, cardiovascular diseases and arthritis (Ricciotti and FitzGerald 2011). Asthm a is a chronic inflammatory disease o f the airways, which is characterized by bronchospasm, reversible airflow obstruction, and variable and relapsing symptoms (Fireman 2003). PGD2 is involved in pathogenesis o f asthma by inducing augmentation o f capillary permeability (Flower et al. 1976), bronchoconstriction (Brannan et al. 2006), mucous generation (Marom et al. 1981) and vasodilatation (Cheng et al. 2006). Previous studies reveal that PGD2 inhibits inflammatory eosinophil apoptosis and increases eosinophil survival (Gervais et al. 2001). PGD2 also increases Th2-induced immune response (Chen et al. 2007) and inhibits IL-12 production in dendritic cells (Gosset et al. 2003). There are tw o distinct types o f PGD2 synthases: hematopoietic PGD synthase (H-PGDS) and lipocalin-type synthase (L-PGDS), which catalyze PGH2 to generate PGD2. The PGD2 synthases and metabolites, and its two receptors also play critical roles in asthma and inflammation (Oguma et al. 2008; Ricciotti and FitzGerald 2011; Joo and Sadikot 2012).

A recent study has shown that synovial fluids from patients with inflammatory arthritis contain significantly increased levels o f PGD2 as compared to PGE2 levels (M oghaddami et

al. 2013). Furthermore, an animal study reveals that PGD2 level is increased during the development o f collagen-induced arthritis, and that treatment with PGD2 and DP agonist BW245C significantly lowers the inflammatory response and jo in t damage (Maicas et al. 2012). Both PGD2 and its metabolite 15dPGJ2 reduced the generation o f matrix metal loproteinases in cytokine-activated chondrocytes, suggesting its chondroprotective

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effects (Fattahi and Mirshafiey 2012). These findings indicate the protective role o f PGD2 in inflammatory arthritis.

4.6 PGD2 and cellular apoptosis

PGD2 and its metabolites o f the J series have been shown to regulate cell apoptosis (Kim et

al. 2003; Chen et al. 2005; Chambers et al. 2007; Shin et al. 2009). For example, PGD2 or its metabolites (e.g., A12-PGJ2 and 15dPGJ2) cause apoptosis in granulocytes (Ward et al. 2002), neuroblastoma cells (Kondo et al. 2002), non-small cell lung carcinoma cells (W ang and M ak 2011) and human leukemia cells (Chen et al. 2005). Interestingly, PGD2 can also exhibit anti-apoptotic function in eosinophils (Gervais et al. 2001) and in human Th2 cells (Xue et al. 2009). Both DP and CRTH2 receptors are involved in the regulation o f PGD2 in cell apoptosis. For instance, the onset o f apoptosis in eosinophils is delayed by DP receptor agonist (Gervais et al. 2001). Furthermore, PGD2 prevents the apoptosis o f human Th2 cells by CRTH2-dependent pathway (Xue et al. 2009). It is also reported that PGD2 induces apoptosis o f non-small cell lung carcinoma cells through its metabolite 15dPGJ2, which is not associated with either DP or CRTH2 (W ang and M ak 2011). These findings demonstrate that PGD2 regulates cellular apoptosis via its DP and CRTH2 receptors as well as its metabolite-mediated signals (Figure 22).

Several studies have shown that both intrinsic and extrinsic apoptotic pathways mediate the regulation o f PGD2 in apoptosis. For example, PGD2 induces apoptosis o f cardiac myocytes via Fas/FasL-dependent extrinsic pathway, whereas cytochrome od ep en d en t pathway associates with the induction o f apoptosis o f non-small cell lung carcinoma cells by PGD2’s metabolite 15dPGJ2(Wang and Mak 2011; Qiu et al. 2012). Numerous signal pathways are associated with the regulation o f PGD2 in cellular apoptosis. For instance, PGD2 inhibits the apoptosis o f human Th2 cells through activation o f the PI3K pathway (Xue et al. 2009), while PGD2 metabolites induce caspase-dependent neutrophil apoptosis via inhibition o f NF-kB activation (Ward et al. 2002). Therefore, the effects o f PGD2 are dependent on both the specific cell population and signaling pathway that is activated. However, the effects o f PGD2 on OC apoptosis are unknown.

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HYPOTHESIS

The previous findings from my laboratory have shown that human OCs express both DP and CRTH2 receptors o f PGD2 (Durand et al. 2008). It has been shown that PG D2 exhibits function in regulating cellular apoptosis (Ward et al. 2002; Chen et al. 2005). However, it remains elusive whether PGD2 has any effect on OC survival and apoptosis. We hypothesized that PGD2 has a regulatory role in OC apoptosis.

OBJECTIVES

The specific objectives o f the present study are to:

1) Characterize the effects o f PGD2 and its receptors on human in v/Vro-differentiated OC apoptosis;

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ARTICLE 1

Contribution

This article was written entirely by Li Yue under the supervision o f Dr. Artur J. de Brum-Femandes. Li Yue made substantial contributions to study conception and design, experimental execution, acquisition o f all data, and data analysis and interpretation.

Article published in the journal Bone, September 2012 51(3): 338-46. Epub 12 June 2012

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