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The Practical Guide toBone Cell signalling: in sickness and in health
Aymen Idris MSc. PhD.
Institute of Genetics and Molecular Medicine University of Edinburgh
The Practical Guide toBone Cell signalling: in sickness and in health
Aymen Idris MSc. PhD.
Institute of Genetics and Molecular Medicine University of Edinburgh
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Bone diseases are mainly due to an imbalance of osteoclastic bone resorption and bone formationBone diseases are mainly due to an imbalance of osteoclastic bone resorption and bone formation
Bone resorption
OsteoClast
Bone formation
OsteoBlast
Adipocytes
Postmenopausal osteoporosis
T - Cells
Rheumatoid Arthri tis
Bone Metastases
Tumour Cells
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osteoclas tsosteoclas ts
Tumour CellsTumour Cells
osteobla stsosteobla sts
AdipocytesAdipocytes
� How to isolate / generate / maintain bone cells?
� How to study bone cell signalling in vitro / ex vivo?
� How to test novel agents (e.g. ABD56) with therapeutic potential use for the treatment of bone diseases?
� How to identify the molecular target or mechanism of action?
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ABD56 & Bone Cell SignallingABD56 & Bone Cell Signalling
Adipocytes
Osteoclast
Pre-osteoclast
Osteoblast
ABD56ABD56
ABD56ABD56
ABD56ABD56
ABD56ABD56
ABD56ABD56 BMC
Mature OB
OB precursors
Mature OC
TNFααααRANKL
M-CS F
Bone
IL-1
M-CS F RM-CS F
Vitamin D3
IL-1
RANK-LRANK
OC precursors
Adipocytes
Pre-osteoblast
Bone marrowstem cells
ABD56ABD56
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Methods of assessing bone cell signallingMethods of assessing bone cell signalling
The osteoClastThe osteoClast
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osteoBlast
Bone marrow cells
3 month oldmice
BM cells
Method for studying osteoclast formation in vitroBone Marrow - Osteoblast Co-culture
Method for studying osteoclast formation in vitroBone Marrow - Osteoblast Co-culture
OsteoBlast
2 days oldmice
collagenasedigestion
Relavent protocolBone Research ProtocolsHumana Press; Helfrich M. & Ralston S.
Relavent protocolBone Research ProtocolsHumana Press; Helfrich M. & Ralston S.
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BM-OB co-culture
Bone marrow cells
3 month oldmice
Method for studying osteoclast formation in vitroBone Marrow - Osteoblast Co-culture
Method for studying osteoclast formation in vitroBone Marrow - Osteoblast Co-culture
BGP Vitamin C
stromal BM / pre-osteoblast
7 days
BM cells
2 days
Relavent protocolCao et al, JBMR, 2005.Relavent protocolCao et al, JBMR, 2005.
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Isolate and plate BM cells
Refresh mediumVitamin D3 (10ng/ml)
plate osteoblast cellsVitamin D3 (10nM)
Isolate OB
Method for studying osteoclast formation in vitroBone Marrow - Osteoblast Co-culture
Method for studying osteoclast formation in vitroBone Marrow - Osteoblast Co-culture
Treat – Vitamin D3 IL1ββββ± agents
Fix & Terminates culture
Day 3
Day 4
Day 6
Day 8
TRAcPstainingTRAcPstaining ResorptionResorption
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Osteoclast Polarisation, Survival & Function
Idris e t al . Bon e 2004
Bone marrow stem cells
Bone marrow stem cells
OsteoblastOsteoblast
OsteoblastOsteoblastABD56ABD56
Day 8 -9
Veh
icle
AB
D56
Phalloidin stainingPhalloidin staining ResorptionResorptionTRAcPstainingTRAcPstaining
Osteoclast Formation and FunctionBone Marrow - Osteoblast Co-cultureOsteoclast Formation and Function
Bone Marrow - Osteoblast Co-culture
Vitamin D3
IL1ββββ
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Osteoclast Polarisation, Survival & FunctionBone marrow stem cells
Bone marrow stem cells
OsteoblastOsteoblast
OsteoblastOsteoblastABD56ABD56
Day 8 -9
Osteoclast Formation and FunctionBone Marrow - Osteoblast Co-cultureOsteoclast Formation and Function
Bone Marrow - Osteoblast Co-culture
Phalloidin stainingPhalloidin staining
DA PI stainingDA PI staining
Vitamin D3
IL1ββββ
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Bone marrow stem cells
Bone marrow stem cells
FunctioningosteoClast
FunctioningosteoClast
OsteoblastOsteoblast
MonocyteMonocyte MacrophageMacrophage Pre-osteoclastprecursor
Pre-osteoclastprecursor
T - CellsT - Cells B - CellsB - Cells
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Bone marrow stem cells
Bone marrow stem cells
M-CSFM-CSF
M-CSF dependant macrophage
M-CSF and RANKL generated OsteoClasts
RANKLRANKLM-CSFM-CSF
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Isolate BM cellsPlate cellsM-CSF
Macrophage
Method for studying osteoclast formation in vitroBone marrow culture: M-CSF generated macrophage
Method for studying osteoclast formation in vitroBone marrow culture: M-CSF generated macrophage
Treat M-CS F± agents
Treat M-CS F± agents
Treat M-CS F± agents
Plate cells on T/C- platesRANKL & M-C SF
Osteoclast
Fix & Terminates culture
Fix & Terminates culture
TRAc P+
Multinucl eated OC
TRAc P+
Mononucleated cells
Treat RANKL & M-CS F± agents
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Isolate BM cells
Plate cells on T/C-platesRANK-L & M-CSF
Fix & Terminates culture
Fix & Terminates culture
Method for studying osteoclast formation in vitroBone marrow culture: RANKL generated osteoclasts
Method for studying osteoclast formation in vitroBone marrow culture: RANKL generated osteoclasts
Treat RANK-L & M-CSF± agents
Macrophage
M-CSF (12.5ng/ml)RANKL (50ng/ml)
OsteoclastsTotal BM cells
(7 days)
OsteoclastsTotal BM cells
(7 days)
OsteoclastsNon-adherent BM cells
OsteoclastsNon-adherent BM cells
Non-adherent cellsM-CSF (12.gng/ml)RANKL (50ng/ml)
(7 days)
BGPVitamin C
BM stromal cells(2 - 3 days)
BM stromal cells(2 - 3 days)
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RANKL generated osteoclasts:Total BM cell versus M-CSF generated macrophage
RANKL generated osteoclasts:Total BM cell versus M-CSF generated macrophage
Total BM - M-CSF & RANKLTotal BM - M-CSF & RANKL
0100200300400500600700800900
veh Noradrenaline
OC
L co
unt
Aitkenet al. ABB 2008Aitkenet al. ABB 2008
Macrophage - M-CSF & RANKLMacrophage - M-CSF & RANKL
60%
0
50
100
150
200
250
300
350
400
veh Noradrenaline
OC
L co
unt
45%300
200
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Osteoclast Survival & Death
Idris e t al . B B RC 2008V
ehic
leA
BD
56
DAPI stainingDAPI stainingTRAcPstainingTRAcPstaining Tunnel stainingTunnel stainingABD56ABD56
Day 4 -5
Bone marrow stem cells
Bone marrow stem cells
RANKLRANKL
M-CS FM-CS F
RANKLRANKL
M-CS FM-CS F
Osteoclast ApoptosisRANKL & M-CSF generated osteoclasts
Osteoclast ApoptosisRANKL & M-CSF generated osteoclasts
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RANKL induced osteoclast formation
Osteoclast FormationRANKL & M-CSF generated osteoclasts
Osteoclast FormationRANKL & M-CSF generated osteoclasts
RANKL
RANKLABD56 Idris e t al . B B RC 2008
Bone marrow stem cells
Bone marrow stem cells
ABD56ABD56
Day 4 -6
RANKLRANKL
M-CS FM-CS F
RANKLRANKL
M-CS FM-CS F
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Osteoclastprecursor
Osteoclastprecursor
Bone marrow stem cells
Bone marrow stem cells
FunctioningosteoClast
FunctioningosteoClast
RANK LRANK L
M-CSFM-CSF
Osteoclast formation and survival are critically dependent on cytokine-induced NFκκκκB signallingOsteoclast formation and survival are critically dependent on cytokine-induced NFκκκκB signalling
RANK
RANKL
Nucleus
P
IKKγγγγ
IKK�
P
IKKαααα
NFκκκκB
NFκκκκB
P
IκκκκB
P
IκκκκBNFκκκκB
TRAF6
TAK1 DAB2
mRNA
TRAcPRANKTCRG 1CalcitoninIntegrin
TRAcPRANKTCRG 1CalcitoninIntegrin
Western Blot
Western Blot
IMMU NO
ELISA
IMMU NO
ELISA
rtPCR
qPCR
rtPCR
qPCR
IP / WB
IMMU NO
IP / WB
IMMU NO
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Osteoclast formation and survival are critically dependent on cytokine-induced NFκκκκB signalling
Osteoclast formation and survival are critically dependent on cytokine-induced NFκκκκB signalling
RANKL
WT KO
Pettit et al, Am J Path (2001)
Li et al, Proc Natl Acad Sci USA (2000)
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Nucleus
RANKL
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Osteoclast formation and survival are critically dependent on cytokine-induced NFκκκκB signalling
Osteoclast formation and survival are critically dependent on cytokine-induced NFκκκκB signalling
NFκκκκB
NFκκκκB
TRAF6
TRAF6KO
wt
Lomaga et al, Genes Dev (1999)
Iotsova et al, Nature (1997 )+/+ -/-
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Methods to study cytokine induced signaling in osteoclastsWestern Blot & Immunoprecipitation
Methods to study cytokine induced signaling in osteoclastsWestern Blot & Immunoprecipitation
60 minStimulate cellsRANKLRANKLRANKLRANKLvehiclevehicle
3minlyse cellcollect
RANK
RANKL
TAK1
TRAF6
DAB2
0 minStarve celltreat
ABD56ABD56vehiclevehiclevehiclevehicle
Generate cell
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RANK
RANKL
TAK1
TRAF6
DAB2
Methods to study cytokine induced signaling in osteoclastsWestern Blot & Immunoprecipitation
Methods to study cytokine induced signaling in osteoclastsWestern Blot & Immunoprecipitation
add anti-RANK Ab add agarose beads(ball & chain)
spin
pellet
spin
supernatant
Denatured protein
pelletsupernatant
SpinHeat
Collect
run on gel
RANK
TRAF6
transfer, probe & visualise
TRAF6
Anti-TRA F6Ab
anti-IgGHR P
ChemiluminescentSubstrate
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RANK
RANKL
TAK1
TRAF6
DAB2
Methods to study cytokine induced signaling in osteoclastsWestern Blot & Immunoprecipitation
Methods to study cytokine induced signaling in osteoclastsWestern Blot & Immunoprecipitation
add anti-RANK Ab add agarose beads(ball & chain)
spin
pellet
spin
supernatant
Denatured protein
pelletsupernatant
SpinHeat
Collect
transfer, probe & visualise
TRAF6
Anti-TRA F6Ab
anti-IgGHR P
ChemiluminescentSubstrate
http://www.ce lls ignal.com /support/p rotocols
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RANK
TRAF6 TRAF6TRAF6
anti-IgG HR PAlexa Fluor
nucleus
Methods to study cytokine induced signaling in osteoclastsImmunostaining
Methods to study cytokine induced signaling in osteoclastsImmunostaining
Anti-TRA F6
RANKL
control
TRAF6(red)
Idr is , 2008
Idr is , 2008
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Methods to study RANKL-induced NFκκκκB nuclear translocation
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IκκκκB
RANK
Nucleus
Methods to study RANKL-induced NFκκκκB nuclear translocation
Methods to study RANKL-induced NFκκκκB nuclear translocation
NFκκκκB
anti-IgG HR PAlexa Flou
Anti-NFκκκκ BV
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p65 NFkB DAPI
Idris e t al . EJP . 2008
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p65 NFκκκκB – mono-nucleated cells
p65 NFκκκκB: osteoclasts versus macrophagep65 NFκκκκB: osteoclasts versus macrophage
RA
NK
Lco
ntro
l
p65 NFkB DAPI
RA
NK
Lco
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l
p65 NFkB DAPI
p65 NFκκκκB – Osteoclasts
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Method for studying RANKL-induce d signalling rtPCR & qPCR
Method for studying RANKL-induce d signalling rtPCR & qPCR
rtPCRcDNA
M-CS F & R ANKLmature Osteoclast
M-CS F & R ANKLMulti -nucleated cells
Day 3 Day 5 Day 7
Isolate mRNA
M-CS Fmacrophage
M-CS F & R ANKLMono-nucleated cells
Day 0
qPCR
0
20
40
60
MacM-CSF
MacM-CSFRANKL
TR
AcP
80
100
Day 1 Day 3 Day 6
TRAcPmRNA expression
osteoclastM-CSFRANKL
**+
osteoclastM-CSFRANKL
*
Day 0Roche Universal ProbeLibrary
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Methods of assessing bone cell signallingMethods of assessing bone cell signalling
The osteoBlastThe osteoBlast
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C57 BL’6mice
Method for studying osteoblast differentiation and functionBone Marrow Stromal Cells
Method for studying osteoblast differentiation and functionBone Marrow Stromal Cells
BGPVitamin C
FunctioningosteoBlast
BM cellsBone nodule assay
Alazarin Red
Calvarial OBBone nodule assay
Alazarin Red
2 days oldC57 BL’6
mice
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Method for studying osteoblast differentiation and functionCalvarial Osteoblast
Method for studying osteoblast differentiation and functionCalvarial Osteoblast
Isolate OB
Plate cells on plates
TreatTerminates
2 wee ks
Plate cells on T/C- plates
Refresh medium
Refresh medium
Terminates (nodule assay)
Day 6
Mature OBDay 4
Mature OBDay 6
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Osteoclastprecursor
Osteoclastprecursor
Bone marrow stem cells
Bone marrow stem cells
FunctioningosteoClast
FunctioningosteoClast
Osteoblast from CB1 knockout mice do not support osteoclast formation normally
Osteoblast from CB1 knockout mice do not support osteoclast formation normally
OsteoblastOsteoblast
Idris et al. Cell Met. 2009
OsteoblastBone marrow
Bone marrow-Osteoblast co-culture
wtwt
wt CB1 KO
RA
NK
L
0
500
1000
1500
2000
2500
3000
**
OsteoblastBone marrow
CB1 KOwt
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Method for studying osteoblast differentiation and signalling
Method for studying osteoblast differentiation and signalling
Idris et al. Endocrinology 2008
0
40
80
120
160
200
vehicle PTHABD56
AL
K P
HO
Sac
tivit
y
****
PTH induced osteoblast differentiation PTH induced signalling
p-MEK
MEK
p-ERK
ERK
PTH
ABD
56
PTH
vehi
cle
PP
Raf
Nucleus
P
ERK
MEK-1/2
A P-1 family
PT H
ββββ γγγγαααα
PTH R
PTH
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Method for studying osteoblast-osteoclast cross talkMethod for studying osteoblast-osteoclast cross talk
Bone marrow stem cells
Bone marrow stem cells
OsteoblastOsteoblastBMCBMC
RANKL
RANKL
IL1ββββ NFκκκκ B
IL1ββββ
Veh Veh A B0
5000
1000 0
1500 0
2000 0
2500 0
RAN
KL
**
+$
qPCR
40
6050
30
40
6050
30
veh veh AM
B
pIκκκκB
Kd
IL1ββββ
actin
WesternBlot
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Method for studying osteoblast signaling ex vivo & in vivo
Method for studying osteoblast signaling ex vivo & in vivo
Bone nodule formation
vehicle
Idris et al. CTI 2008
ALN(0.5µµµµ M)
Transfer, probe & visualiseRun on gel
Lysedenature protein
2 days oldmice
ALN
collagenase digestionosteoBlast
isolatecalvaria
ALN 0.1mg/Kg ControlM
Osteoblasts ex vivokD605040
30
20unprenylatedRap1A
Actin
Idris et al. CTI 2008
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Methods of assessing bone cell signallingMethods of assessing bone cell signalling
The adipocyteThe adipocyte
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Bone Cell: originBone Cell: origin
Bone marrowstem cells
BGPVit C
Adipocytes
Insulin
M-CS F RM-CS F
Vitamin D3
BMC
Mature OB
OB precursors
Adipocytes
Mature OC
TNFααααRANKL
M-CS F
Bone
IL-1
IL-1
RANK-LRANK
OC precursors
Osteoblast
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Edinburgh
London
LondonDay 8
Isolate BM
Method for studying adipocyte singalling in vitroBone marrow culture: adipocytes
Method for studying adipocyte singalling in vitroBone marrow culture: adipocytes
7 days
Day 6
BM stromalcellsDay 6
Day 14
BM stromalcellsDay 14
Refresh mediumInsulin (1µg/ml)
Refresh mediumInsulin (1µg/ml)
Refresh mediumInsulin (1µg/ml)
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blob
blob
blob blob blob
ParisDay 10
Paris
London
Edinburgh
Spanish borderDay 14
Hendaye
Method for studying adipocyte singalling in vitroBone marrow culture: adipocytes
Method for studying adipocyte singalling in vitroBone marrow culture: adipocytes
Day 18
Day 22
Day 26
adipocyte
Refresh mediumInsulin (1µg/ml)
Refresh mediumInsulin (1µg/ml)
Refresh mediumInsulin (1µg/ml)
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Method for studying adipocyte singalling in vitroBone marrow culture: adipocytes
Method for studying adipocyte singalling in vitroBone marrow culture: adipocytes
BilbaoDay 16 Bi lbao
Paris
London
Edinburghadipocyte
Terminate
Day 30
Day 33
Refresh mediumInsulin (1µg/ml)
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Method for studying adipocyte singalling in vitroBone marrow culture: adipocytes
Method for studying adipocyte singalling in vitroBone marrow culture: adipocytes
blobDay 3
Day 13
adipocyte
Terminate
Refresh mediumAdipogenic medium
Insulin (1µg/ml)IBMX (0.5µ Μµ Μµ Μµ Μ ) – phosphodiestrase inhibitorTrog (5µµµµ M) – PPARγγγγ agonistDex (0.25µµµµ M) - GlucocorticoidIndo (0.1µµµµ M) - NSAID
Isolate BM
Refresh mediumAdipogenic medium
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Method for studying adipocyte singalling in vitroAdipocytes – Mouse primary versus 3T3-L1
Method for studying adipocyte singalling in vitroAdipocytes – Mouse primary versus 3T3-L1
Insulin induced adipocyte differentiation
Phase contrast microscopyunstaine d
control Insulin1 µµµµ g/ml
Phase contrast microscopy Oil Re d Sta ining
control Insulin1 µµµµ g/ml
Fluorescence microscopy Nile Red Staining
control Insulin1 µµµµ g/ml
Prim
ary
Mou
se A
dipo
cyte
s3
-4 w
eeks
Phase contrast microscopyunstaine d
control Insulin1 µµµµ g/ml
Fluorescence microscopy Nile Red Staining
control Insulin1 µµµµ g/ml
Fluorescence microscopy Oil Re d Sta ining
control Insulin1 µµµµ g/ml�
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AcknowledgementAcknowledgement
University of EdinburghBone Research Group
University of AberdeenBone Research Group
ECTS/AMGEN Bone Biology Fellowship
Washington University (St. Louis) Dr. Patrick Ross
Galapagos SASU (France)Dr. Patrick Mollat
