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Universite de Sherbrooke
Induction de l'apoptose des osteoclastes humains par la Prostaglandine D 2 : 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 jury devaluation 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 © L i Yue, 2013
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R ESU M E Induction de l'apoptose des osteoclastes hum ains p a r la pro stag lan d in e D2: recepteurs et m ecanism es de tran 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 traitement 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 MEK-1/2, PI3K et IKK2/NF- 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 ’ER 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 ’ER 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/NF- kB. Mots-cles: Osteoclastes, Apoptose, Prostaglandine D2, ERK1/2, Akt, P-arrestine-1
SUM M ARY P rostaglandin D 2 induces hum an osteoclast apoptosis and 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 D 2 (PGD 2 ) is a lipid mediator 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 whether PGD2 affects OC apoptosis. The objective o f the thesis was to determine whether PGD2 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 Akt phosphorylation were augmented, whereas the phosphorylated P-arrestin-1 was reduced by a CRTH2 antagonist. Furthermore, treatment 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). Moreover, 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. Keywords: Osteoclasts, Apoptosis, Prostaglandin D2, ERK1/2, Akt, P-arrestin-1
V
TABLE OF CONTENTS L IST O F F IG U R E S ......................................................................................................................viii L IST O F T A B L E S.......................................................................................................................... xi LIST O F A B B R EV IA T IO N S......................................................................................................xii IN T R O D U C T IO N ............................................................................................................................1 1. Bone anatom y and physiology............................................................................................2 1.1 Bone anatom y...................................................................................................................... 2 1.2 Bone organic and inorganic com ponents......................................................................... 4 1.2.1 Organic com ponents....................................................................................................4 1.2.2 Inorganic components................................................................................................. 5 1.3 Bone cells..............................................................................................................................5 1.4 Bone remodeling..................................................................................................................6 2 Bone cell physiology..................................................................................................................7 2.1 Osteoblasts............................................................................................................................7 2.2 Osteocytes.............................................................................................................................9 2. 3 Osteoclasts.......................................................................................................................11 2.3.1 Osteoclast form ation..................................................................................................11 2.3.2 Osteoclast identification........................................................................................... 11 2.3.3 Bone resorption by osteoclasts................................................................................ 12 2.3.4 Regulation o f osteoclast formation, differentiation and activation.................... 14 3. O steoclast apo p to sis............................................................................................................... 15 3.1 Cell apoptosis......................................................................................................................15 3.1.1 Difference between apoptosis and necrosis........................................................... 15 3.1.2 Assays available for measurement o f apoptosis................................................... 17 3.2 Extrinsic and intrinsic apoptosis pathw ays.................................................................... 18 3.3 Osteoclast apoptosis andextrinsic/intrinsic apoptosis pathways................................ 20 3.4. Signaling pathways involved in osteoclast apoptosis................................................ 22 3.4.1 PI3K/Akt signaling pathway.................................................................................... 23 3.4.2 ERK1/2 signaling pathw ay...................................................................................... 24 3.4.3 NF- kB signaling pathw ay.........................................................................................27 3.5 Bone diseases and osteoclast apoptosis......................................................................... 30 4. P ro stag lan d in s.........................................................................................................................31 4.1 Prostaglandin synthesis.....................................................................................................31 4.2 PGD 2 metabolism...............................................................................................................32 4.3 PGD 2 and its receptors...................................................................................................... 34 4.4 PGD2 signal transduction..................................................................................................36 4.4.1 G protein coupling-dependent signal transduction.............................................. 36 4.4.2 G protein coupling-independent signal transduction...........................................37 4.5 PGD 2 and diseases.............................................................................................................38 4.6 PGD 2 and cellular apoptosis............................................................................................ 40 4.7 PGD2 and osteoclast function..........................................................................................41
H Y PO TH ESIS
42
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 esum e..................................................................................................................................... 46 A b stract.................................................................................................................................... 48 In tro d u ctio n .............................................................................................................................48 M aterials and m eth o d s.........................................................................................................49 R esu lts.......................................................................................................................................53 D iscussion................................................................................................................................ 62 A cknow ledgm ents..................................................................................................................65 F u n d in g .................................................................................................................................... 65 Conflict o f interest statem e n t.............................................................................................. 65 R eferences................................................................................................................................ 65 A R T IC L E 2 ................................................................................................................................. 71 R esum e......................................................................................................................................73 A b stract.....................................................................................................................................76 In tro d u ctio n .............................................................................................................................77 M aterials and m e th o d s......................................................................................................... 78 R esu lts.......................................................................................................................................81 D iscussion................................................................................................................................ 92 C onclusions..............................................................................................................................95 A cknow ledgm ents..................................................................................................................96 F u n d in g .....................................................................................................................................96 C onflict o f interest statem en t.............................................................................................. 96 R eferences................................................................................................................................ 97 D ISC U SSIO N .................................................................................................................................104 1. PG D 2 and its receptors in O C a p o p to sis........................................................................ 105 2. E xtrinsic/intrinsic apoptosis pathw ays in PG D 2-induced osteoclast apoptosis... 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 apoptosis........................109 3.4 NF-k B signaling pathway in PGD2-induced osteoclast apoptosis...........................111 4. C ontributions 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 A C K N O W L E D G E M E N T S....................................................................................................... 118
vii LIST 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 osteoclasts...................................................... 144 1.3 The percentage o f TRAP staining c e lls ....................................................................... 145 1.4 PGD 2 /CRTH 2 induced osteoclast apoptosis in a time-dependent m anner..............146 1.5 Effect o f PGE 2 on osteoclast apoptosis........................................................................ 147 1.6 Dose-dependent effect o f PI3K inhibitor on osteoclast apoptosis........................... 148 2. Conference abstracts and presentations......................................................................149 3. Publications.......................................................................................................................150
LIST OF FIGURES
IN TRD U C TIO N F igure 1. Representative diagram o f a long bone structure....................................................... 3 F igure 2. Bone remodeling cycle....................................................................................................6 F igure 3. Three important bone cells: OBs, osteocytes and OCs............................................. 7 F igure 4. Differentiation o f OBs and osteocytes from mesenchymal stem cells................... 9 F igure 5. Lacunae containing osteocytes within the calcified matrix.....................................10 F igure 6. OC differentiation from hematopoietic stem cells....................................................11 F igure 7. Mechanism o f osteoclastic bone resorption...............................................................13 F igure 8. Regulation o f OC differentiation and activation by M -CSF/RANKL/OPG
15
F igure 9. Schematic representation o f necrosis and apoptosis................................................ 16 Figure 10. Overview o f extrinsic and intrinsic apoptosis pathways....................................... 19 Figure 11. Pro-apoptosis and anti-apoptosis members o f Bcl-2 family................................. 20 F igure 12. Pro-apoptotic and anti-apoptotic pathways involved in OC apoptosis.............. 21 F igure 13. Overview of PI3K/Akt, MEK/ERK and N F- k B signaling pathway in OC apoptosis............................................................................................................................................22 F igure 14. Overview of PI3K/Akt signaling pathway.............................................................. 24 F igure 15. Four MAPK cascades.................................................................................................. 25 F igure 16. Role o f MEK1/2-ERK1/2 in OC apoptosis............................................................. 26 Figure 17. Canonical and non-canonical pathways o f NF- k B activation.............................. 28 Figure 18. Role o f NF- k B pathway in OC apoptosis.................................................................30 Figure 19. Metabolism o f arachidonic acid to prostaglandins..................................................32 Figure 20. PGD 2 metabolites......................................................................................................... 34
Figure 21. PGD2-induced G protein coupling-dependent signaling via its receptors..........37 Figure 22. Regulation o f cellular apoptosis by PGD2, its metabolites and its receptors............................................................................................................................................ 41
A R TIC LE 1 Figure 1. TRAP staining and TACS Blue Labeling analysis o f human differentiated OCs.................................................................................................................................................... 54 Figure 2. Concentration-response curves o f PGD2 and its agonist or antagonist on apoptosis in human OCs, and PGD2 production by human O Cs............................................. 57 Figure 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 Figure 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 Figure 1. Representative light micrographs o f identifications for human differentiated OCs.................................................................................................................................................... 83 Figure 2. Participation o f MEK-ERK1/2 pathway in apoptosis o f OCs................................85 Figure 3. Modulation o f (3-arrestin-l phosphorylation at Ser412 during human OC apoptosis........................................................................................................................................... 87 Figure 4. Involvement o f Akt phosphorylation in OC apoptosis............................................88 Figure 5. Association o f Akt and ERK1/2 signaling pathways in PGD2-induced OC apoptosis........................................................................................................................................... 90 Figure 6. No effect on OC apoptosis through IKK2/NF- k B signaling pathway..................91
X
DISCUSSION Figure 23. A schematic model showing the regulation o f PGD 2 in OC apoptosis............. 113 ANNEX Supplem entary Figure 1. The experimental design for treatment o f kinase inhibitors with or without PGD 2 and its receptors agonists/antagonists........................................................... 143 Supplem entary Figure 2. Representative light micrographs o f morphology in human differentiated OCs.......................................................................................................................... 144 Supplem 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 PGD 2 and its receptors agonists/antagonists on apoptosis in human OCs...................................................................... 146 S upplem entary Figure 5. Effect o f PGE 2 and PGD 2 on apoptosis in in vitrodifferentiated OCs using the TACS Blue Label Kit..................................................................147 Supplem entary Figure 6. Concentration-response curve o f PI3K inhibitor in inducing OC apoptosis in the presence o f 10 nM o f PGD2............................................................................. 148
xi
LIST OF TABLES
T able 1. Characteristics o f DP and CRTH2 receptors................................................................35 T able 2. PGD 2 receptors agonists and antagonists..................................................................... 36
xii
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 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
Ik B
Inhibitor o f k B
IKK
Ik B kinase
IL
Interleukin
IP3
Inositol triphosphate
molecule expressed on
JNK
C-Jun N-terminal kinase
L-PGDS
Lipocalin-type PGD synthase
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
NF- kB
Nuclear factor k B
NIK
NF- kB inducing kinase
OB
Osteoblast
OC
Osteoclast
OPG
Osteoprotegerin
PARP
Poly (ADP ribose) polymerase
PBMCs
Peripheral blood mononuclear cells
PDK
Phosphoinositide-dependent protein kinase
PDTC
Pyrrolidine dithiocarbamate
PG
Prostaglandin
pgd2
Prostaglandin D2
pge2
Prostaglandin E2
pgh2
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
RA
Rheumatoid arthritis
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
TUNEL
Terminal transferase mediated DNA nick end labeling
INTRODUCTION
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). Many 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 D 2 (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 of different cells (Ward et al. 2002; Chen et al. 2005; Wang and Mak 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.
2 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 and 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 marrow 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).
3
Cartilage
Proximal epiphysis
Epiphyseal line (growth line) Spongy (cancellous) bone (containing red marrow) Endosteum Compact bone Medullary (marrow) cavity Artery and vein
Diaphysis •<
Yellow marrow Periosteum
Distal epiphysis }
Figure 1. R epresentative d iag ram o f a long http://encvclopedia.lubopitko-bg.com/Bone.htmn
bone
stru c tu re .
(Taken
from
On the outer layer o f the long bone, there is a membrane called the periosteum containing blood vessels, nerve fibers, OBs, and OCs. The periosteum is a fibrous connective tissue sheath, which plays a vital role in fracture repair, bone formation and appositional growth. The bones have two components: trabecular bone (also known as spongious bone or cancellous bone) and compact bone (also called cortical bone). The layer below periosteum includes compact bone, which is steady, firm, smooth and densely packed as an intricate calcium matrix (Clarke 2008). The layer beneath the compact bone is the trabecular bone, which is composed o f the sturdy collagen matrix. Moreover, it has structural rigidity and elasticity to resist mechanical stress. Trabecular bone is also highly vascularized, and mainly contains red marrow in its central cavity (Kozielski et a l 2011). The medullary
4 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 of 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. Matrix 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;
5 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 (AMG 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 of 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.
6 1.4 Bone remodeling Under normal conditions, bone is subjected to continuous turnover and remodeling via bone formation and resorption. Bone remodeling cycle continues throughout life, and it is typically “in balance” to keep healthy bone. The remodeling cycle has the successive phases as follows: 1) Resorptive phase: activated OCs digest a discrete area o f mineralized bone matrix, 2) Reversal phase: OB precursors proliferate and differentiate into OBs, which are recruited from the adjacent bone marrow stromal cell population into the resorption lacuna after the completion o f osteoclastic resorption, 3) Formation phase: OBs fill the resorption lacunae with deposition o f new bone matrix, which is initially unmineralized and called osteoid. OBs mature into osteocytes once embedded in osteoid. When formation phase is complete, OBs become quiescent lining cells on the newly bone-forming surface until activated (Hadjidakis and Androulakis 2006). Figure 2 shows bone remodeling cycle.
Hematopoietic Stem Cells
©\
©
Mesenchymal Stem Cells
N
Pre-osteoclasts
00
I
% Lining Cells
Resting Bone
\
Mononuclear Pre-osteoblasts O^ ^ b la s ts Cells Osteoclasts
ft# Resorption
Reversal
Matrix Deposition
Osteocytes
Mineralization
Figure 2. Bone rem odeling cycle. The first step is the resorption o f bone mineral and matrix by activated OCs in the bone remodeling cycle. The second step is formation o f the cement line in resorption lacunae, and OBs produce newly synthesized matrix. Finally, matrix mineralization and the differentiation o f some OBs into osteocytes accomplishes the remodeling cycle. Taken from (Kapinas and Delany 2011).
7
2 Bone cell physiology As mentioned above, there are five types o f bone cells. The OBs, osteocytes and OCs will be discussed in the following paragraphs, because these cells are mainly involved in bone formation, resorption, and remodeling. Figure 3 shows OBs, osteocytes and OCs in a section o f bone.
Figure 3. Three important bone cells: OBs, osteocytes and OCs. http://www.siumed.edu/~dking2/ssb/remodel.htm)
(Taken from
2.1 Osteoblasts The OBs differentiate from multipotent mesenchymal stem cells (osteoprogenitor cells), which reside in the deeper layer o f periosteum and the bone marrow. The osteoprogenitors express the master regulatory transcription factor C bfal/R unx2 that is necessary for OB differentiation in response to growth factors, such as BMPs and fibroblast growth factors (Shui et al. 2003; Agata et al. 2007). Finally, these precursor cells proliferate and
8 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-forming 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
of
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 of 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.
9 2.2 Osteocytes Mesenchymal stem cells in the bone marrow differentiate slowly into preosteoblasts until they have the location and phenotype o f OBs. Then OBs mature into osteocytes once they are trapped in osteoid (Senba et al. 2012). Approximately 10-20% of OBs can differentiate into osteocytes. Figure 4 shows the differentiation o f OBs and osteocytes from mesenchymal stem cells.
A Mesenchymal Stem cell
Pre-OB
Osteocyte
Figure 4. Differentiation of OBs and osteocytes from mesenchymal stem cells.
An osteocyte is a star shaped cell, which contains a nucleus and a thin ring piece o f cytoplasm. Osteocytes account for 90-95% o f adult bone cells, and these cells have an average half-life o f 25 years. Osteocytes are considered terminally differentiated OBs, which are incapable o f mitotic division. These cells can communicate with each other or with other cells including endothelial cells and hematopoietic cells via canaliculi, which are used for exchange o f nutrients and waste through gap junctions (Link 2013). In mature bone, osteocytes reside inside spaces called lacunae. Figure 5 shows osteocytes sitting in lacunae within the calcified matrix. The osteocytes play an important role in bone formation and resorption. There are controversial reports regarding the role o f osteocytes in bone formation. Osteocytes have been reported to activate bone formation, due to release o f anabolic factors (e.g., prostaglandin E 2 (PGE 2 ), nitric oxide, and COX-2) after mechanical stimuli (Burger and Klein-Nulend 1999). This is corroborated by the study that bone formation was severely inhibited after osteocyte ablation (Tatsumi et al. 2007). However, osteocyte density is negatively associated with bone formation, and osteocytes inhibit OB function and bone
10 formation via sclerostin (Qiu et al. 2002; W inkler et al. 2003; van Bezooijen et al. 2004). In general, osteocytes suppress bone resorption because osteocyte death is followed by bone resorption (Verborgt et al. 2000; Jilka et al. 2007; Tatsumi et al. 2007; Moriishi et al. 2012). Under normal conditions, osteocytes secrete high levels o f TGF-p, thereby suppressing bone resorption. In aged bone, the expression o f TGF-P is reduced whereas the expression o f OC stimulatory factors, such as receptor activator for nuclear factor kB (NF-kB) ligand (RANKL) and M-CSF, is increased in osteocytes, which augments bone resorption and net bone loss (Heino et al. 2002; Heino et al. 2009). Both inflammatory factors and glucocorticoids can induce osteocyte apoptosis. Apoptotic osteocytes are reported to release apoptotic bodies that express RANKL to recruit OCs (Bonewald 2011). It is possible that osteocyte apoptosis is associated with reduced mechanotransduction, which may result in the development o f osteoporosis via regulation o f OC function (Heino et al. 2009).
(A)
(B)
calcified m atrix
Osteocyte in lacuna Canaliculus Osteoblast
Newly calcified bone matrix Canaliculi
L acuna
Figure 5. L acunae containing osteocytes w ithin th e calcified m atrix. Osteocytes occupy small spaces named lacunae, which are found in the calcified matrix o f bone. Cytoplasmic processes o f the osteocytes extend away from one to other osteocytes in small channels called canaliculi. (A) is taken from http://classconnection.s3.amazonaws.com/981/flashcards/598981/ipg/ossification21310523 544517.jpg): and (B) is taken from http://www.histology.leeds.ac.uk/bone/assets/osteon.gif
11 2. 3 Osteoclasts 2.3.1 Osteoclast form ation OCs are large multinucleated cells that are 30-100 pm in diameter. Normally, they contain 5-20 nuclei but may have as many as 200 nuclei. These cells are generated by the fusion o f mononuclear progenitors o f the monocyte-macrophage family. Myeloid stem cells derived from hematopoietic cells further differentiate to monocytes, macrophages and other cells. The colony-forming unit-granulocyte/macrophage (CFU-GM) is the earliest identifiable hematopoietic precursor o f OCs (Menaa et al. 2000). CFU-GM-derived cells differentiate to mononuclear pre-OC, which can fuse together to form multinucleated quiescent OCs under proper stimuli. Upon activation, OCs becomes polarized, and forms distinct membrane domains (Roodman 2006). Figure 6 presents the differentiation o f OCs from hematopoietic stem cells.
o - 0 ' O -%-(8k Sten/cell
OC Precursor
Pre-OC
Quiescent
Activated OC
Figure 6. OC differentiation from hematopoietic stem cells.
2.3.2 Osteoclast identification In general, OCs differ from other bone cells in their morphological characteristics including giant size and multiple nuclei, as above described. These cells also express specific markers, such as tartrate resistant acid phosphatase (TRAP), cathepsin K, integrin a v p 3 , and calcitonin receptor. TRAP is an important cytochemical marker and enzyme o f OCs, which is able to generate reactive oxygen species and to further degrade collagen matrix (Halleen et al. 2003), while cathepsin K is a collagenolytic and papain-like cysteine protease involved in the degradation o f type I collagen and other non-collagenous proteins (Vaananen et al. 2000). Integrin av p 3 plays a role in the regulation o f OC migration and
12 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 become 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 OC’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 —* HCCV + 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
demineralization
expel in
H+, thereby bone
regulating
resorption.
This
extracellular acidification continuous
release
of
for
bone
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 of resorption lacunae (Schlesinger et al. 1997; Rousselle and Heymann 2002). HCO 3 7 C F exchange at the cell’s non-resorptive surface keeps the
13 intracellular pH stable. The combined actions o f the vacuolar-ATPase proton pump and chloride/proton antiporter C1C-7 clustered at high density in the RB achieve a pH o f resorption lacunae approximating 4.5. Finally, the digested products o f collagen and other bone matrix are endocytosed from the resorption space and transported to the functional secretory domain at the top o f the polarized OCs where they are released into the extracellular environment (Teitelbaum 2000; Krieger et al. 2004). Figure 7 shows the mechanism o f osteoclastic bone resorption. Functional Secretory Domain
Basolateral Domain
Ruffled Border
Sealing Zone
Bone Resorption Lacunae (pH ~4.5) V-ATPase o) (h+) Acidified Vesicles
^ ■ 1 a vp3 Integrin 4 ^ Nucleus
0 HCOjVCI' Exchanger o Proteolytic Enzymes
Figure 7. M echanism o f osteoclastic bone reso rp tio n . Once activated, the OCs move to the resorptive bone area where they form a sealing zone to digest the underlying bone. Both H+ and Cl” are expelled into the resorption place by highly permeable membrane RB. In addition, OCs expel H+ into the resorption space by vacuolar-ATPase proton pumps. The inorganic mineral is degraded by the low pH, in concert with the activation o f matrix hydrolytic enzymes that degrade organic components, thereby leading to resorption o f the underlying bone. Taken from (Qin et al. 2012).
14 2.3.4 Regulation o f osteoclast formation, 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 (TN 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 the regulation of OC differentiation and activation by M-CSF/RANKL/OPG.
8
presents
15
Differentiation
RANKL
OB/stromal cell Figure 8. Regulation of OC differentiation and activation by M-CSF/RANKL/OPG. RANKL and M-CSF are potent inducers o f OC formation. The formation o f OCs can be induced by up-regulating RANKL and M-CSF expression on the surface o f OBs, marrow and stromal cells. RANKL and M-CSF bind to their receptors, RANK and c-fms, on the surface o f OC precursors to induce OC formation. OPG interferes with the binding o f RANKL to RANK thereby prevents OC differentiation and activation.
3. Osteoclast apoptosis 3.1 Cell apoptosis 3.1.1 Difference between apoptosis and necrosis Apoptosis and necrosis are two major ways for a cell to die. The term apoptosis is taken from a combination o f two Greek words: apo means “from” and ptosis means “falling”, which refers to the falling o f leaves from a tree. Apoptosis is the naturally occurring process o f programmed cell death. In an adult human, 50-70 billion cells undergo apoptosis each day (Reed 2006). Apoptotic cells are characterized by membrane blebbing, formation o f apoptotic bodies, chromatin condensation, and DNA fragmentation. The morphology o f apoptotic cells changes as their cellular components decompose and condense. During apoptosis, the cytoskeleton breaks up, and this results in the membrane to bulge outward
16 and bleb formation. The cells then break down into small fragments called apoptotic bodies, which are engulfed and destroyed by phagocytes without inflammatory response (Edinger and Thompson 2004; Kroemer et al. 2005). In contrast, necrosis is a different form o f cell death, which is usually detrimental and can be fatal to the organism. Necrosis is characterized by the disruption o f the cellular membrane and the swelling o f cytoplasm, leading to disintegration o f organelles. The cellular contents are released in cells with severe necrosis, which causes an inflammatory reaction and damages the surrounding cells (Zong and Thompson 2006; Golstein and Kroemer 2007). Figure 9 presents the differences between necrosis and apoptosis.
Membrane blobbing
Call contents re-packaged
(CP Q
.)/
Apoptosis
?© *;
(O f Normal Coll
N ocrosis
Osmotic pressure causes swelling
Call lysis
Figure 9. Schem atic representation o f necrosis an d apoptosis. Cellular apoptosis is generally characterized by chromatin condensation, DNA fragmentation and formation o f the apoptotic bodies. Necrosis begins with cell swelling that is reversible. It will proceed to irreversible injury if the causal damage continues. Necrosis includes rupture o f the plasma and organelle membranes, loss o f intracellular content, ultimately the cells being in the complete disintegration o f organelles (Taken from http://www.vce.bioninia.com.au/aos-3-hereditv/cell-reDroduction/cell-death.htmI).
17 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 (McStay 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.
18 •
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 extemalization 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
-10
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
19 BH3 domain proteins (e.g., Bim, Bad, Bid and Puma) and pro-apoptotic multidomain (BH1-3) proteins (e.g., Bax and Bak) (Cory et al. 2003). Anti-apoptotic members contain Bcl-2, BcI-xL, Bcl-w, Mcl-1 and A l (Gross et al. 1999; Adams 2003), which prevent mitochondrial outer membrane permeabilization (Tait and Green 2010). Caspase-9 is activated by cytochrome c released from mitochondria due to the imbalance o f pro- and anti- apoptotic Bcl-2 family members. Activated caspase - 8 and caspase-9 eventually lead to the activation and cleavage o f executor caspases, such as caspase-3, leading to DNA fragmentation (Strasser et al. 2000; Cory and Adams 2002). Figures 10 and 11 show an overview o f the extrinsic and intrinsic apoptosis pathways as well as the Bcl-2 family members, respectively.
Extrinsic pathway Fa*LorTfUit Fas or TRAIL-R
DD I FADD
|
DED
TRADD
Mitochondria Apoptosis
Figure 10. Overview o f extrinsic an d intrinsic apoptosis pathw ays. The extrinsic apoptotic pathway is triggered by the binding o f death receptors with their cognate ligands (Fas binds FasL, and TRAIL receptor (TRAIL-R) binds TRAIL), resulting in the activation o f caspase- 8 . In contrast, the intrinsic apoptotic pathway is triggered by cytotoxic stress, such as chemotherapy and radiotherapy, leading to the release o f apoptogenic factors such as cytochrome c from the mitochondrial intermembrane space. The release o f these factors activates caspase-9. The activation o f both caspase - 8 and -9 in turn induces caspase-3 activation. Eventually, caspase-3 lead to apoptotic cell death.
20
Bim, Bad Bid, Bik Bmf, Puma Hrk, Noxa
r BH3-only proteins >, f
Pro-apoptotic proteins v BH1-3
CM O CQ
Anti-apoptotic Proteins (BH1 -4 proteins)
[
Bax Bak Bok
r Bcl-2 Bcl-xL Bcl-w Mcl-1
Figure 11. Pro-apoptotic and anti-apoptotic members of Bcl-2 family.
3.3 Osteoclast apoptosis and extrinsic/intrinsic apoptosis pathways Many factors, including hormones, cytokines, growth factors and 1,25(OH)2-Vitamin D 3 are able to cause or prevent OC apoptosis (Hughes et al. 1996; Manolagas 2000; Compston 2001; Blair and Athanasou 2004). This is associated with the alteration o f levels o f M-CSF, RANKL and OPG. For example, differentiated OCs undergo rapid apoptosis under certain conditions such as the deprivation o f trophic factors (e.g., M-CSF (Akiyama et al. 2003) and RANKL (Ikeda et al. 2008)). It has been shown that both 1,25(OH)2-Vitamin D 3 and PTH prevent OCs from apoptosis, which is associated with increased expression o f RANKL and reduced levels o f OPG. It has been shown that OC apoptosis triggered by M-CSF deficiency is correlated with up-regulated expression o f Bim. M-CSF also up-regulates the expression o f the endogenous caspase inhibitor protein, an X-linked inhibitor o f apoptosis (Kanaoka et al. 2000). TG F-pl induces apoptosis o f human OCs by up-regulating Bim (Houde et al. 2009), and estrogen promotes apoptosis o f murine OCs through TGF-P (Hughes et al. 1996). These results
21
suggest that OC apoptosis involves Bcl-2 family members in the intrinsic pathway. Previous studies also indicate that the Fas-FasL system is implicated in apoptosis o f mature OCs (Wu et al. 2003; Recchia et al. 2004). Both the TRAIL and FasL pathways are involved in apoptosis o f human OCs. Indeed, the OC-like cells differentiated from umbilical cord blood monocytes express TRAIL and Fas receptors (Roux et al. 2005). TRAIL-R 1 and TRAIL-R2 contains a “death domain” which are able to mediate apoptosis but TRAIL-R3 and TRAIL-R4 are decoy receptors that are unable to induce apoptosis (Roux et al. 2005). This is in accordance with other independent findings that TRAIL-induced apoptosis is detected in human OCs obtained from peripheral blood mononuclear cells (PBMCs) (Colucci et al. 2007). Hence, both extrinsic and intrinsic pathways participate in OC apoptosis (Figure 12).
ftA N K t
X-linked inhibitor of apoptosis
Pro-apoptotic Bim
Anti-apoptotic Mcl-1
Fas/FasL TRAILR/TRAIL -------- \ — ---------- 1-----------
OC apoptosis
Figure 12. Pro-apoptotic and anti-apoptotic pathways involved in OC apoptosis.
22
3.4. Signaling pathways involved in osteoclast apoptosis Several signaling pathways are known to be implicated in the regulation o f OC apoptosis, which
includes phosphatidylinositol-3-kinase
(PI3K)/Akt,
MAPK/ERK
and NF-kB
pathways (Figure 13). PI3K coordinately activates the MAPK-ERK kinase (MEK)/ERK and Akt/NF-KB pathways to maintain OCs survival (Gingery et al. 2003). MAPK/ERK can be activated by phosphorylation via the dual specific kinase MEK. Moreover, c-Src (a tyrosine-kinase) inhibits OC apoptosis through regulation o f PI3K/Akt and ERK1/2 signaling pathways, which can support OC survival (Lee et al. 2001; Xing et al. 2001; Recchia et al. 2004). It was reported that OC apoptosis was induced when RelA/p65 nuclear translocation was blocked or NF-kB DNA binding activity was inhibited (Abbas and Abu-Amer 2003; Penolazzi et al. 2003). Therefore, PI3K, Akt, MAPK, ERK and NF-kB are involved in OC apoptosis and survival, and the regulation o f these signal pathways may be beneficial in inhibiting bone loss via targeting OC apoptosis.
—
f g Q p
--------
n f -k B
OC apoptosis Figure 13. Overview of PI3K/Akt, MEK/ERK and NF-kB signaling pathways in OC apoptosis.
23 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 (Moon 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 (PDK )-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 (Mo 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
24 growth deficiency (Cho et al. 2001; Garofalo et al. 2003). Akt3 is highly expressed in the brain and testis, but weakly in other adult tissues than A ktl and Akt2. Even though the functions o f Akt3 are still not fully understood, Akt3-deficient mice have smaller brains. Both Aktl and Akt2 are abundantly expressed in OCs, and double knockout mice o f Akt 1/2 displayed severely impaired bone development (Peng et al. 2003). It has been shown that A ktl is a crucial mediator in promoting the differentiation and survival o f OCs (Kawamura et al. 2007). In addition to the description in Figure 13, inhibition o f PI3K/Akt causes apoptosis o f mature OCs (Kim et al. 2009). Thus, PI3K7Akt pathway inhibitors may be beneficial in reducing bone resorption in metabolic bone diseases through induction o f OC apoptosis.
RTKs or GPCRs
Rac
mTOR
Gsk3p
BAD
P27
FOXO
p21
Figure 14. Overview of PI3K/Akt signaling pathway. Activated PI3K by RTKs or GPCRs translocates to the membrane and converts PIP2 to PIP3. Next, PIP3 recruits Akt and PDK1 to the cell membrane where Akt is activated and phosphorylated. Activation o f Akt modulates various downstream signaling pathways, thereby playing an important role in multiple cellular processes, such as cell motility and invasion, growth, metabolism, survival and proliferation. Taken from (Zhu and Nelson 2012). 3.4.2 ERK1/2 signaling pathway Four MAPK cascades have been identified, including ERK1/2, c-Jun N-terminal kinase (JNK), p38 and ERK5 cascades. Each family o f MAPK kinases consists o f a set o f three
25 tiers o f protein kinases termed MAPK kinase kinase (MAP3K or MEKK), MAPK kinase (MAPKK or MEK) and MAPK. MAP3K are often activated by a small G protein or adaptors. MAP3K activation leads to phosphorylation and activation o f a MEK, which then stimulates MAPK activity through dual phosphorylation on threonine and tyrosine residues in their activation loop (Wada and Penninger 2004). Figure 15 shows all four MAPK cascades. The ERK1/2 is the most studied MAPK family in OC survival and apoptosis, although JNK mediates an anti-apoptotic effect o f RANKL in OCs, whereas p38 MAPK-mediated signals are required for OC differentiation but not for its survival (Li et al. 2002; Ikeda et al. 2008). No reports regarding the role o f ERK5 in regulating OC apoptosis are available. Hence, I focus on the discussion o f ERK1/2 activation and its involvement in OC apoptosis in the following paragraph.
Growth factors/UW inflammatory cytokines
/\ *
Figure 15. F o u r M A PK cascades. MAPK cascades regulate intracellular signals triggered by extracellular or intracellular stimuli. MAP3Ks phosphorylate MAPKKs, which result in the phosphorylation o f MAPKs. There are four families o f mammalian MAPKs: ERK5, p38, JNKs and ERK1/2. The activated MAPKs shuttled into the nucleus, where they phosphorylate various substrate proteins, ultimately leading to regulation o f various cellular activities.
26 ERK1/2 is activated by MEK 1/2 via phosphorylation on both Thr202/Tyr204 and T hrl85/T yrl87 residues. ERK1/2 regulate hundreds o f cytoplasmic (e.g., ribosomal protein S6 kinases (RSKs) and MAPK-interacting kinase) and nuclear (e.g., mitogen- and stress-activated protein kinases, c-fos, Elkl and SP-1) proteins through the phosphorylation (Lu and Xu 2006; Roskoski 2012). Therefore, ERK1/2 controls a variety o f cellular processes in conjunction with scaffolding proteins including P-arrestin-1 and p-arrestin-2. ERK1/2 is reported to participate in both cellular survival and apoptosis, depending on the signal and the cell system studied (Iryo et al. 2000). In general, the ERK1/2 signaling pathway supports OC survival (Lee et al. 2001; Lee et al. 2002; Gingery et al. 2003). Further study indicates that the mechanism for ERK1/2 in inhibiting OC apoptosis is associated with Mcl-1-related intrinsic pathway (Kuo et al. 2012). However, there is a study showing that ERK1/2 inhibition reduces the number o f apoptotic OCs, which suggests the pro-apoptotic effect o f ERK1/2 signal (Recchia et al. 2004). The discrepancies among these studies remain elusive, which need further investigation. Figure 16 shows role o f MEK1/2-ERK1/2 in OC apoptosis.
Stress/ Mitogens
MEK1/2
I
0 ERK1/2
ERK1/2 activation
apoptosis?]
Figure 16. Role o f MEK1/2-ERK1/2 in OC apoptosis.
ERK1/2 inhibition
27 3.4.3 N F - kB signaling pathway 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 NF-kB:IkB complex from translocating to the nucleus. In mammalian cells, the NF-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
6
million people) present bone and joint 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 treatments 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 H 2 (PGH 2 ), 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).
32
Arachidonic acid
COX-1 & 2
PGG,
COX-1 & 2
PGH2
PGIS
PGDS
V
*
Receptors
v v s — -v/
14-PGD
13,14-dihydro-15-keto-PGD:
PGJ
Isomerization with albumin
(DK-PGD2)
15-deoxy-A12>14-PGJ 2 (15dPGJ2) Figure 20. PG D 2 m etabolites. Modified from (Pettipher and Hansel 2008) .
4.3 PGD 2 and its receptors Among these PGs, PGD 2 is a key mediator in various pathophysiological processes and diseases including vasodilatation (Cheng et al. 2006), pain (Eguchi et a l 1999), sleep (Hayaishi 2002), bronchoconstriction (Brannan et al. 2006) and asthma (Oguma et al. 2008). PGD 2 acts via two distinct GPCRs: DP and CRTH2 receptors (Boie et al. 1995; Hirai et al. 2001). The characteristics o f these two receptors are shown in Table 1. The current available selective agonists and antagonists for DP and CRTH2 receptors are described in Table 2.
35
Table 1. Characteristics of DP and CRTH2 receptors
DP receptor Gas (Boie et al. 1995)
CRTH2 receptor Gai/o and Gaq (Sawyer et al. 2002; Nagata and Hirai 2003)
al nervous system, bone, retina, lungs, intestine, lature and nasal mucosa (Boie et al. 1995; Gerashchenko et al.
Thymus, bone, brain, spleen, heart, and digestive
Wright et al. 2000; Gervais et al. 2001; Nantel et al. 2004;
system (Sawyer et al. 2002; Durand et al. 2008)
d et al. 2008) Eosinophils, OBs, OCs, basophils, macrophages, ells, OBs, OCs, dendritic cells, basophils, leukocytes, monocytes and Th2 cells (Sawyer et al. 2002; Gosset et al killer cells, epithelial cells (Nantel et al. 2004; Gallant et al. Durand et al. 2008)
al. 2003; Gallant et al. 2005; Durand et al. 2008; Tajima et al. 2008) Decreases RANKL expression, osteoclastogenesis and
;ases OPG production, bone resorption, osteoclastogenesis, cAM P formation; increases O Bs chemotaxis, OC •cular pressure and venous vasodilatation; stimulates migration, eosinophil and Th2 cell motility and Ca2+ dyl cyclase; increase Ca2+, cAMP, mucin secretion, arterial concentration; modulate eosinophil m orphology and ;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)
degranulation (Gervais et al. 2001; Hirai et al. 2001; Monneret et al. 2001; Gallant et al. 2005; Durand et al. 2008)
36 T able 2. PG D 2 receptors agonists an d antagonists
A ffinity
A ffinity
DP (nM )
C R T H 2 (nM )
pg d 2
1.7 ± 0 .3
2.4 ± 0.2
DP and CRTH2 agonists
BW 245C
0.4 ±0.1
> 80000
DP agonist
>6000
2.9 ± 0 .3
CRTH2 agonist
1.7
ND
DP antagonist
1200
0.6
CRTH2 antagonist
Com pound
D esignation
13,14-dihydro-15-keto-PGD2 (DK-PGD2) BW A868C (Giles et al. 1989) CAY 10471 (Ulven and Kostenis 2005)
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 of diacylglycerol (DAG), inositol triphosphate (IP3), and intracellular calcium, leading to activation of protein kinase C (PKC). Figure 21 shows PGD2-induced G protein coupling-dependent signal transduction via DP and CRTH2 receptors.
37
DP
CRTH2
cAMP
cAMP
PKC Figure 21. PG D 2 -induced G protein coupling-dependent signaling via its receptors. DP and CRTH2 receptors are two specific receptors o f PGD 2 . Binding o f activated DP receptor by PGD 2 to Gas-type G proteins causes activation o f downstream signals: AC, cAMP and PKA. Activated CRTH2 receptor by PGD 2 couples to Gaj-type that leads to inhibition o f AC and cAMP, while CRTH2 couples to Gaq-type G proteins resulting in increases o f PLC, DAG, IP3, Ca2+ and PKC.
4.4.2 G protein coupling-independent signal transduction A recent study demonstrated that PGD 2 induced expression o f MUC5B, a major airway mucin gene, via DP-dependent ERK and MAPK/RSK1 signals in epithelial cells through G protein-dependent pathway (Choi et al. 2011). Interestingly, GPCRs can elicit signals through the interaction with scaffolding proteins, which is independent o f G-protein coupling (Luttrell and Lefkowitz 2002; Mathiesen et al. 2005; Defea 2008). p-arrestin is one
of
these
types
of
scaffolding
proteins,
which
can
mediate
G-protein
coupling-independent signal transduction (DeWire et al. 2007; Golan et al. 2009). In mammals, the arrestins are a family o f four proteins (arrestin-1 to -4), which can be grouped into two subfamilies according to their structure and function: visual or sensory (arrestin-1 and -4) and non-visual (arrestin-2 and -3) (Gurevich and Gurevich 2006). In mammals, arrestin-1 and -4 are mainly localized to photoreceptors, whereas arrestin-2 (also
38 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 MAPK/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 (Mathiesen 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/MEK/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
39 wide variety o f functions involved in physiological processes and in pathogenesis o f diseases. For example, in the brain, PGD 2 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 PGD 2 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 PGD 2
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). Asthma is a chronic inflammatory disease o f the airways, which is characterized by bronchospasm, reversible airflow obstruction, and variable and relapsing symptoms (Fireman 2003). PGD 2 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 PGD 2 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 two distinct types o f PGD2 synthases: hematopoietic PGD synthase (H-PGDS) and lipocalin-type synthase (L-PGDS), which catalyze PGH 2 to generate PGD 2 . 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 PGD 2 as compared to PGE 2 levels (Moghaddami et al. 2013). Furthermore, an animal study reveals that PGD 2 level is increased during the development o f collagen-induced arthritis, and that treatment with PGD 2 and DP agonist BW245C significantly lowers the inflammatory response and joint 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
40 effects (Fattahi and Mirshafiey 2012). These findings indicate the protective role o f PGD 2 in inflammatory arthritis. 4.6 PGD 2 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, PGD 2 or its metabolites (e.g., A12-PGJ 2 and 15dPGJ2) cause apoptosis in granulocytes (Ward et al. 2002), neuroblastoma cells (Kondo et al. 2002), non-small cell lung carcinoma cells (Wang and Mak 2011) and human leukemia cells (Chen et al. 2005). Interestingly, PGD 2 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 PGD 2 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 PGD 2 induces apoptosis o f non-small cell lung carcinoma cells through its metabolite 15dPGJ2, which is not associated with either DP or CRTH2 (Wang and M ak 2011). These findings demonstrate that PGD 2 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 PGD 2 in apoptosis. For example, PGD 2 induces apoptosis o f cardiac myocytes via Fas/FasL-dependent extrinsic pathway, whereas cytochrome odependent pathway associates with the induction o f apoptosis o f non-small cell lung carcinoma cells by PGD 2 ’s metabolite 15dPGJ2(Wang and Mak 2011; Qiu et al. 2012). Numerous signal pathways are associated with the regulation o f PGD 2 in cellular apoptosis. For instance, PGD 2 inhibits the apoptosis o f human Th2 cells through activation o f the PI3K pathway (Xue et al. 2009), while PGD 2 metabolites induce caspase-dependent neutrophil apoptosis via inhibition o f NF-kB activation (Ward et al. 2002). Therefore, the effects o f PGD 2 are dependent on both the specific cell population and signaling pathway that is activated. However, the effects o f PGD 2 on OC apoptosis are unknown.
41 4.7 PGD 2 and osteoclast function Previous studies from our laboratory have shown that both OCs and OBs express DP and CRTH2 receptors (Durand et al. 2008). Moreover, human differentiated OCs can produce total PGs (Hackett et al. 2006). Activation o f the DP receptor reduces OPG production, while the CRTH2 receptor activation induces OBs chemotaxis and decreases RANKL expression (Gallant et al. 2005). Both cells are important to affect bone formation and resorption. Furthermore, the osteoclastogenesis in vitro increases in patients with RA due to increased percentage o f OC precursors and decreased apoptosis (Durand et al. 2011). The activation o f the DP receptor by PGD 2 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). To my knowledge, this was the first report to demonstrate the effects o f PGD 2 on human OC function. However, there is no report regarding the regulation o f PGD 2 on OC and OB apoptosis, although the effect o f PGD2on apoptosis in other cells was observed (Gervais et al. 2001; Ward et al. 2002; Xue et al. 2009). Further study on PGD 2 in regulation o f both OC and OB apoptosis will unravel its role and potential therapeutics in bone diseases where OC and OB function is abnormal.
B
DP CRTH2
Metabolites: A12-PGJ2> 15dPGJ;
Eosinophil apoptosis
Lung carcinoma cell apoptosis
Leukemia cell apoptosis
Eosinophil apoptosis
Th2 cell apoptosis
Figure 22. Regulation o f cellular apoptosis by PGD2, its metabolites and its receptors. (A) Modulation o f cellular apoptosis by PGD 2 and its metabolites; (B) Inhibition o f cellular apoptosis by PGD2 through DP and CRTH2 receptors.
HYPOTHESIS
The previous findings from my laboratory have shown that human OCs express both DP and CRTH2 receptors o f PGD 2 (Durand et al. 2008). It has been shown that PGD 2 exhibits function in regulating cellular apoptosis (Ward et al. 2002; Chen et al. 2005). However, it remains elusive whether PGD 2 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; 2) Determine the molecular mechanisms underlying these effects.
RESULTS
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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46 Resume La prostaglandine D2 (PGD2) est un mediateur lipidique synthetise a partir de l’acide arachidonique qui active directement deux types de recepteurs, DP (D-type prostanoid) et CRTH2 (chemoattractant recetor homologous molecule expressed on T-helper type 2 cells). La PGD 2 peut affecter le metabolisme osseux en influen?ant a la fois les fonctions des osteoblastes et des osteoclastes (OC), cellules impliquees aussi bien dans le remodelage osseux que dans la reparation de fracture in vivo. L’objectif de la presente etude a ete de determiner les effets de la PGD 2 sur l’apoptose des OCs humains par 1’interm ediate de ses deux recepteurs specifiques. Les OCs ont ete differencies a partir des cellules mononucleaires du sang peripherique in vitro en presence de RANKL (receptor activator for nuclear factor kB) et M-CSF (macrophage-colony stimulating factor), puis traitees avec la PGD2, ses agonistes et antagonistes specifiques. Le traitement avec la PGD 2 pendant 24 heures en presence de naproxene (10 pM), qui permet d ’inhiber la production endogene de prostaglandines, augmente de fa?on dependante de la dose le pourcentage d ’OCs apoptotiques. Ce resultat est egalement observe lorsque les OCs sont traites avec DK-PGD2, l’agoniste specifique de CRTH2, mais pas avec BW 245C, l’agoniste de DP. En absence de naproxene, l’antagoniste CAY10471 du recepteur CRTH2 reduit le taux d ’apoptose des OCs, en revanche l’antagoniste BW A 8 6 8 C du recepteur DP n ’a aucun effet. Par ailleurs, 1’induction de 1’apoptose par la PGD2 via son recepteur CRTH2 est associee a l’activation de la caspase-9, mais pas de la caspase- 8 , ce qui entraine le clivage de la caspase-3. Ces resultats montrent que la PGD 2 induit l’apoptose des OCs humains a travers l’activation de son recepteur CRTH2 et de la voie apoptotique intrinseque. M ots-cles : Osteoclastes, Apoptose, Prostaglandine D2, Recepteurs, Os
47 Prostaglandin D 2 Induces A poptosis o f H um an O steoclasts by A ctivating th e C R T H 2 receptor and th e In trin sic A poptosis P athw ay
Li Yue ab, Marianne Durand a, M. Christian Lebeau Jaco b a, Philippe Hogan a’b, Stephen McManus a, Sophie Roux a, Artur J. de Brum -Fernandesa,b
a Division o f Rheumatology, Department o f Medicine, Faculty o f Medicine, Universite de Sherbrooke, and Centre de recherche clinique Etienne-Le Bel, 3001 12e Avenue Nord, local 3858, Sherbrooke, Quebec, J1H 5N4, Canada b Department o f Pharmacology, Faculty o f Medicine, Universite de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, Quebec, J1H 5N4, Canada
*This work was presented in part at the 32nd ASBMR Annual Meeting (Toronto, Ontario, Canada, Oct. 15-19, 2010) and the 33rd ASBMR Annual M eeting (San Diego, CA, USA, Sept 16-20, 2011).
A bbreviations: PGD 2 , prostaglandin D 2 ; PGE 2 , prostaglandin E 2 ; DP, D-type prostanoid receptor; CRTH2, chemoattractant receptor homologous molecule expressed on T-helper type 2 cells; OC, osteoclast; RANKL, receptor activator for nuclear factor kB ligand, M-CSF, macrophage-colony stimulating factor; PGs; prostaglandins; AA, arachidonic acid; COXs,
cyclooxygenases;
OBs,
osteoblasts;
FBS,
fetal
bovine
serum;
DMSO,
dimethylsufoxide; PBMCs, peripheral blood mononuclear cells; TNF, tumour necrosis factor; TRAP, tartrate-resistant acid phosphatise
48 A bstract Prostaglandin D 2 (PGD 2 ) is a lipid mediator synthesized from arachidonic acid that directly activates two specific receptors, the D-type prostanoid (DP) receptor and chemoattractant receptor homologous molecule expressed on T-helper type 2 cells (CRTH2). PGD 2 can affect bone metabolism by influencing both osteoblast and osteoclast (OC) functions, both cells involved in bone remodeling and in in vivo fracture repair as well. The objective o f the present study was to determine the effects o f PGD 2 , acting through its two specific receptors, on human OC apoptosis. Human OCs were differentiated in vitro from peripheral blood mononuclear cells in the presence o f receptor activator for nuclear factor kB ligand (RANKL) and macrophage-colony stimulating factor (M-CSF), and treated with PGD2, its selective agonists and antagonists. Treatment with PGD 2 for 24 hours in the presence o f naproxen (10 pM) to inhibit endogenous prostaglandin production increased the percentage o f apoptotic OCs in a dose-dependent manner, as did the selective CRTH2 agonist compound DK-PGD 2 but not the DP agonist compound BW 245C. In the absence o f naproxen, the CRTH2 antagonist compound CAY 10471 reduced OC apoptosis rate but the DP antagonist BW A 8 6 8 C had no effect. The induction o f PGD 2 -CRTH 2 dependent apoptosis was associated with the activation o f caspase-9, but not caspase- 8 , leading to caspase-3 cleavage. These data show that PGD 2 induces human OC apoptosis through activation o f CRTH2 and the apoptosis intrinsic pathway. K eywords: Osteoclasts, apoptosis, prostaglandin D 2 , receptors, bone
Introduction Osteoclasts (OCs) participate in the pathophysiology o f several diseases associated with local or generalized bone loss, such as rheumatoid arthritis [ 1 ], osteosarcoma [2 ], osteoporosis [3], myeloma [4] and Paget’s disease o f bone [5]. Apoptosis plays a critical role in regulating the development and function o f many cells, including OCs [6 ]. The OCs undergo rapid apoptosis in the absence o f M-CSF [6 ] and RANKL [7], both OC survival factors. The induction of OC apoptosis can decrease bone resorption, thereby affecting bone remodeling, an important process involved in bone homeostasis [8 ] and bone diseases
49 [9, 10]. Understanding the mechanisms o f regulation o f OC apoptosis may not only help to improve the efficacy o f existing therapies, but also may uncover novel strategies o f drug development for the treatment o f bone pathologies. Prostaglandins (PGs) are lipid mediators synthesized from arachidonic acid (AA) through the action o f cyclooxygenases (COXs). PGs play an important role in controlling bone formation [11] and resorption [12, 13] by influencing the cross-talk between osteoblasts (OBs) and OCs [14, 15]. Modulation o f PG signaling pathways affects bone loss and fracture repair [16, 17]. We previously showed that both human OBs and OCs express the receptors o f PGD 2 , namely DP and CRTH2 receptors [18, 19]. Activation o f the DP receptor reduces osteoprotegerin production by OBs, while the CRTH2 receptor activation induces OBs chemotaxis and decreases RANKL expression [18]. Furthermore, the activation o f the DP receptor by PGD 2 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 [19] but also reduces osteoclastogenesis. These mechanisms may be relevant in humans in vivo as concentrations o f PGD 2 in the urine increase during post-fracture bone remodeling [20]. These results suggest that PGD 2 could have an anabolic effect on bone. The objective o f the present study was to determine the impact o f PGD 2 on OC apoptosis, and the pathways involved in this process.
Materials and methods Materials Fetal bovine serum (FBS) was purchased from Gibco (distributed by Invitrogen Canada, Inc., Burlington, ON, Canada). M-CSF was obtained from Peprotech, Inc. (Rocky Hill, NJ, USA). TACS Blue Label kit was purchased from R&D Systems (Minneapolis, MN, USA). PGD2, CRTH2 agonist DK-PGD2, DP agonist BW 245C, CRTH2 antagonist CAY 10471 and DP antagonist BW A 8 6 8 C were purchased from Cayman Chemical (Ann. Arbor, MI, USA); these reagents were all diluted in dimethyl sulfoxide (DMSO). PGD 2 EIA Kit was also purchased from Cayman Chemical. Caspase-3,
-8
and -9 fluorogenic substrates as well
50 as anti-actin antibody were purchased from Calbiochem (Merk, Germany). Staurosporine, caspase - 8 and -9 antibodies as well as secondary antibodies (anti-rabbit and anti-mouse) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Recombinant human IL -ip was purchased from Cedarlane (Hornby, ON, Canada). Recombinant human tumour necrosis factor (TN F)-a was purchased from Peprotech, Inc. (Hornby, ON, Canada). All other reagents were purchased from Sigma-Aldrich Canada, Ltd. (Oakville, ON, Canada).
Cell culture and treatment Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors whole blood as described before [19, 21]. The use o f human PBMCs was approved by the Ethics Review Board o f the Faculte de medicine et des sciences de la sante o f the Universite de Sherbrooke. All subjects provided an informed consent. M ononuclear cells were isolated by dextran sedimentation and Ficoll density gradient centrifugation. Cells were plated at 1.5 * 106 cells/cm 2 in 8 -well chamber glass slides or 12-well plates at 37°C and 5% CO 2 , humidified atmosphere with medium changes twice a week. The OC differentiation was induced by incubation in aM E M medium containing 10% FBS, 1% penicillin-streptomycin, M-CSF (10 ng/ml) and RANKL (50 ng/ml) for 21 days. After 21 days, the OCs were cultured for further 24 hours in 2% FBS-containing medium without M-CSF and RANKL, then treated for 24 hours with different concentrations o f PGD 2 , PGD 2 agonists (BW 245C for a DP agonist, and DK-PGD 2 for a CRTH2 agonist) in the presence o f naproxen (10 pM). Naproxen was used in the assays along with PGD 2 and its agonists to inhibit the synthesis o f endogenous prostaglandins. PGD 2 receptors antagonists (BW A 8 6 8 C, a DP antagonist, or CAY 10471, a CRTH2 antagonist) were added to the OCs cultures in the absence o f naproxen so as to further study the involvement o f the receptors in cellular apoptosis in the present o f endogenous prostaglandins.
Tartrate-resistant acid phosphatase (TRAP) staining After 21 days o f differentiation, TRAP staining was performed to identify the human OCs using a commercial kit (catalog number 387A-1KT, Sigma-Aldrich) according to the
51 manufacturer's instructions. OCs were washed with PBS, and incubated with a solution o f Naphthol AS-BI phosphoric acid and freshly diazotized Fast Garnet GBC, in the presence of tartrate at 37 °C for 35 minutes. Multinuclear (three or more nuclei) TRAP-positive cells (dark red/purple) were identified under light microscopy.
OC apoptosis determination using TACS Blue Labeling After treatment, cells on 8 -well chamber glass slides were assessed for apoptosis using the TACS Blue Label Kit following the manufacturer’s instructions [22], The cells were fixed, washed and permeabilized with an ethanolracetic acid ( 2 : 1 ) solution, and then biotinylated nucleotides were incorporated by terminal deoxynucleotidyl transferase. The biotinylated nucleotides were measured using streptavidin-horseradish peroxidase conjugate followed by the substrate, TACS Blue Label. An insoluble blue precipitate occurred in nuclei where DNA fragments underwent double-stranded breaks. Blue multinucleated (three or more nuclei) cells were counted as apoptotic OCs, and pink ones as live OCs. The stained samples were counted manually in five fields under light microscope. Staurosporine was used in all these experiments as a positive control for apoptosis; at the concentration used (1 pM for 3 hours), based on our preliminary data and previous reports, it activates both intrinsic and extrinsic apoptotic pathways [23-26].
Caspase-3, -8 and -9 activity assays Caspase activity was examined in OCs in vitro using the caspase fluorogenic substrate assay with F-2500 FL Spectrophotometer [27-30]. Staurosporine was used as a positive control o f apoptosis induction in these assays. After treatment, differentiated OCs were washed twice with PBS before being lysed for 20 minutes on ice in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8 ], 1% Igepal, 0.5% deoxycholate, 10 mM Na 4 PP, 0.1% SDS, and 0.5 mM EDTA) containing a protease inhibitor cocktail. OCs lysate proteins (30 pg) were incubated with 2 pi of Ac-DEVD-AMC (caspase-3-like substrate, 5 mM) in a reaction buffer (100 mM Hepes [pH 7.5], 20 % glycerol, and 5 mM DTT) for 2 hours at 37 °C. Caspase-3-like activity was determined at an excitation/emission wavelength pair o f 380 nm/405-500 nm. In the case o f caspase- 8 , cell lysate proteins (30 pg) were incubated with 2 pi o f Z-IETD-AFC (caspase- 8 -like substrate, 5 mM) in a reaction buffer (20 mM Hepes
52 [pH 7.5], 100 mM NaCl, 10 mM DTT, 1 mM EDTA pH 8 , and 0.1 % Igepal) for 2 hours at 37 °C. Caspase- 8 -like activity was monitored at an excitation/emission wavelength pair o f 400 nm /417-520 nm. OCs lysate proteins (50 pg) were incubated with
6
pi o f
Ac-LEHD-AFC (caspase-9-like substrate, 5 mM) in a reaction buffer (100 mM Hepes [pH 7.5], 20 % glycerol, and 5 mM DTT) for 2 hours at 37 °C. Caspase-9-like activity was surveyed at an excitation/emission wavelength pair o f 400 nm/420-520 nm.
Western blot analysis for caspase-8 and -9 protein levels After treatment, the cells were washed twice with PBS before being lysed for 20 minutes on ice in RIPA lysis buffer. Total proteins (50 pg) were separated by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membrane was blocked with 5% nonfat milk, incubated overnight at 4 °C with the primary antibody against caspase - 8 (1:250) and caspase-9 (1:250). Anti-actin antibody was used to determine the actin level as a loading control. The membranes were incubated for 1 hour at room temperature with a horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibody,
after
washing.
Bound
antibodies
were
visualized
by
an
enhanced
chemiluminescence (ECL) detection system. All bands were measured by densitometry and normalized to actin (means ±standard error o f three or four experiments) using the ImageJ software.
PGD 2 production using enzyme immunoassay The production o f PGD 2 in OC culture supernatants was assessed by the PGD 2 enzyme immunoassay (EIA) Kit (Cayman Chemical, limit o f detection: 55 pg/ml, IC 5 0 : 240 pg/ml, specificity: PGD2: 100%, PGF2: 92.4%, PGJ2: 21.6%, PGE2: 2.86%, Thromboxane B2: 2.54%, 1 l(3-PGF2 a : 1.99%, 6
8
-iso PGF2a: 1.90%, PGA2: 0.72%, 12(S)-HHTrE: 0.16%,
-keto PGF,a: 0.05%, 13,14-dihydro-15-keto PGD2: 0.02%, other PGs: 340/well), as described for a normal population in our previous publication [32].
Treatment with 2% of FBS increased the basal level of OC apoptosis After 21 days o f differentiation in the presence o f RANKL and M-CSF, further incubation in 2% FBS-containing media without RANKL and M-CSF increased the apoptosis rate o f OCs, as shown in Fig. IB and C. Compared with 10% FBS treatment (12.0 ± 1.1%),
54 treatment with 5% FBS did not change (12.9 ± 1.2%) the percentage o f apoptotic OCs, whereas 2% FBS (18.0 ± 1.5%) significantly increased the percentage o f apoptotic OCs, which was further increased in the absence o f FBS (66.1 ± 1.4%) (Fig. IB and C). All following experiments were performed with 24 hours incubation in 2% FBS so that either increase or decrease in the apoptosis rates could be detected.
a*
#
#
&
FB S
Figure 1. TR A P staining an d TA C S B lue Labeling analysis o f h u m an d ifferen tiated OCs. A, TRAP staining analysis o f human differentiated OCs obtained after 21 days o f culture in the presence o f M-CSF and RANKL. TRAP-positive cells containing three or more nuclei were considered as OCs. Arrows indicate multinucleated OCs (dark red/purple). Human OC apoptosis was induced by reduced concentration o f FBS. After 21 days o f differentiation, OCs were incubated with medium containing 0%, 2%, 5% and 10% FBS in the absence o f M-CSF or RANKL for 24 hours. B, TACS assay o f OCs in the medium containing 2% FBS in the absence o f M-CSF and RANKL for 24 hours: multinucleated cells with blue nuclei (open arrow) were counted as apoptotic whereas multinucleated cells with pink nuclei (solid arrow) were counted as alive. C, effect o f different concentrations o f FBS on apoptosis in in v//ro-differentiated OCs using the TACS Blue Label Kit. Data are means ± SE, *p