Wnt-Cadherin Interactions in Bone
Pierre MARIEINSERM U 606 & University Paris Diderot
Hôpital Lariboisière, Paris, [email protected]
http://www.idf.inserm.fr/site/u606/
Lariboisière Hospital, Paris, France
The Cadherin SuperfamilyTYPE I (Classical Cadherins)
β CATp120CT
E-CADHERIN (EPITHELIAL)N-CADHERIN (NEURAL)P-CADHERIN (PLACENTAL)R –CADHERIN (RETINAL)
Identical cadherins interact in a homophilic, Ca2+-dependent way:Form adherens junctions between cells
β CATp120CT
Ca 2+Ca 2+Ca 2+Ca 2+
HAV
CellMembrane
Functions of Cadherins
- Cell aggregation and sorting during tissue morphogenesis
- Development and maintenance of cell polarity
- Cell migration
- Cell proliferation
- Cell differentiation
- Cell survival
Cell 1
Cell 2
CADHERINS AND CANCER
Ca 2+Ca 2+Ca 2+
Mutations in the extracellular domain induce:
Gain-of-function : High affinity for cellsResistance to apoptosisHigh mobility/invasion
OR
Loss-of-function: Decreased affinity for cellsAbnormal regulation of receptortyrosine kinases
CADHERINS PLAY A ROLE IN OSTEOCLASTS
E-CAD
Cadherin-6
Osteoclasts
• Osteoclasts express E-Cadherin and Cadherin-6, but not N-Cadherin or P- Cadherin
• Inhibition of E-CAD in osteoclasts reducesthe fusion of osteoclast precursorsas well asTRAP activity
• Inhibition of Cadherine-6 reducesthe interaction between osteoblasts and osteoclastsas well as osteoclast differentiation
Mbalaviele et al., 1995; 1998
Cadherin Expression in Osteoblasts
- Ostoblasts express N-cadherin,Cadherin-11 and E-Cadherin in vitroand in vivo.
- In vivo, N-cadherin is expressed atall stages of bone formation, although at various level of expression.
Marie, JCP, 2002; Mbalavielle et al., 2006
Cadherin Expression in Human Cranial Suture
N-CAD
E-CAD
N-CAM
Marie, JCP 2000
Ab Controls Cranial suture
Mesenchymal Cells
N-CADR-CADP-CAD
Marie, JCP, 2002
N-cadherin expression occurs concomitantlywith osteoblast differentiation
ADIPOCYTES
Mesenchymal Cells
N-CADR-CADP-CAD
N-CAD CAD 11
Marie, JCP, 2002
N-cadherin expression occurs concomitantlywith osteoblast differentiation
CHONDROBLASTSADIPOCYTES
Mesenchymal Cells
N-CADR-CADP-CAD
N-CAD CAD 11
N-CAD
N-cadherin expression occurs concomitantlywith osteoblast differentiation
N-CAD
Marie, JCP, 2002
CHONDROBLASTSADIPOCYTES
Mesenchymal CellsPreosteoblasts Osteoblasts
N-CADR-CADP-CAD
N-CAD CAD 11
N-CAD CAD 11R-CAD, P-CAD
N-CAD
N-CAD CAD 11
N-cadherin expression occurs concomitantlywith osteoblast differentiation
N-CAD
Marie, JCP, 2002
CHONDROBLASTSADIPOCYTES
Mesenchymal CellsPreosteoblasts Osteoblasts
OSTEOCYTES
APOPTOSIS
N-CADR-CADP-CAD
N-CAD CAD 11
N-CAD CAD 11R-CAD, P-CAD
N-CAD N-CAD
N-CAD
N-CAD CAD 11
N-cadherin expression occurs concomitantlywith osteoblast differentiation
N-CAD
Marie, JCP, 2002
ROLE OF CADHERIN 11 IN OSTEOGENESISCadherin 11 expression promotes mesenchymal cell differentiation into osteo/chondroblast lineages (Kaii et al., 2004)
Invalidation of Cadherin 11 in mice induces:
- Delayed cranial suture mineralisation
- Decreased long bone mass
Kawaguchi et al., 2001
Role of N-Cadherin in Chondrogenesis
• Involved in mesenchymal condensation and chondrogenesis (Tuan et al., )
• TGFβ1 activates MAPK signaling and N-cadherin-mediated cell- cell adhesion in mesenchymal stem cells, and thereby promotes chondrogenic differentiation (Tuli et al., 2003)
MSCs
MesenchymalCondensation
Early chondrogenesis
Delise and Tuan, 2003
TGFβ1+
N-CAD
Role of N-cadherin in osteogenesis
In vivo expression of a dominant- negative N-cadherin in osteoblasts induces:
- delayed peak bone mass- reduced osteoblast differentiation- increased adiposity(Castro et al., 2004)
Heterozygous N-cadherin null mice show:
- accentuated ovariectomy-induced bone loss-altered osteogenesis(Lai et al., 2006)
N-cadherin knock-out is lethal (Radice et al, 1997)
Role of N-Cadherin in Osteoblast Differentiation
• Involved in osteoblast differentiation in vitro(Ferrari et al., 2000)
• Involved in BMP2-induced osteoblast differentiation in vitro (Cheng et al., 1998; Hay et al., 2000)
• Involved in FGFR2-induced osteoblast differentiation in Apert craniosynostosis (Lemonnier et al., 2001)
Regulation of N-Cadherin in Osteoblasts
PKCα
N-CADHERIN
BMP-2
FGF-2FGFR-2
TNFα, IL-11,25D
CELLAGGREGATION
OSTEOBLASTDIFFERENTIATION
BONEFORMATION
PTH
TGFβ
Marie, JCP, 2002
Frizzeld
Dsh
Tcf/Lef
Wnt
P
LRP5/6
APCAxin GSK3b
ALP, COLIAI, Runx2Osteoblast differentiationBone formation
Wnt Signaling
β-Catenin
Osteoblast Gene Transcriptionand Bone Formation
β-cate
nin
Cadherin
TCF/LEF
β-cate
nin
CADHERIN-β-CATENIN INTERACTIONIN BONE
0 7 14
0
0.1
0.2
0.3
0.4
0.5
N-CADHERIN OVEREXPRESSION DELAYS ALP ACTIVITY AND MATRIX MINERALISATION
IN MC3T3 CELLS
0 7 14
nMol
PNP/
mg
Prot
ein/
min
Control cells
N-Cadcells
Days
DaysControl cellsN-Cad cells
ALP ACTIVITY STAINING
ALP ACTIVITY Control cells
N-Cadcells
ALIZARIN RED STAINING
*
*
* :p < 0.05 vs Ct Haÿ et al., Mol Cell Biol. 2009
N-CADHERIN OVEREXPRESSION INHIBITS OSTEOBLAST MARKER EXPRESSION
IN MC3T3 CELLS
0
1
2
ALP
Runx2
Osterix OC
OPG
COL1A1
Rel
ativ
e m
RN
Aab
unda
nce
(N-C
AD
/FLA
G C
ells
)
* **
*
* :p < 0.05 vs Ct
N-Cad + Wnt3a
ALP Runx2 Osterix OC COL1A WIP10
1
2
3
4
5
11
12
13
N-CADHERIN OVEREXPRESSION INHIBITS OSTEOBLAST MARKER EXPRESSION INDUCED
BY WNT3A IN MC3T3 CELLS
Rel
ativ
e m
RN
Aab
unda
nce
(trea
ted/
cont
rol)
*
*
**
* *
* : p<0.05 vs control
Flag + Wnt3a
0
1
10
100
CONTROL +Wnt3A 15%A
rbitr
ary
units
Wnt Responsive ElementTransactivation Assay
N-CADHERIN OVEREXPRESSION INHIBITS β-CATENIN NUCLEAR TRANSLOCATION INDUCED BY WNT3A
Control cells N-CAD cells
* : p<0.05
*
β-Catenin Immunostaining
Control cells N-Cad cells
Control
+Wnt3A * *
Haÿ et al., Mol Cell Biol. 2009
N-CADHERIN INHIBITS β-CATENIN NUCLEAR TRANSLOCATIONAND INDUCES ITS DEGRADATION
IP β
Catenin / WB Ubiquitin
Control N-Cad MC3T3
Ct +Wnt Ct +Wnt
Nuclear β
catenin
Control N-Cad MC3T3
Ct +Wnt Ct +Wnt
Membranous β
catenin
Control N-Cad MC3T3
Ct +Wnt Ct + Wnt
Ub
Haÿ et al., Mol Cell Biol. 2009
N-CadLRP5
N-Cad
N-Cad
2250
IP: Ig
GFla
g
IP: Ig
GFla
g
IP: LRP5IB: N-Cad
IP: N-Cad IB: LRP5
IgGIgG
Kd
140176
Flag
N-Cad
Anti-N-Cad MergeAnti-LRP5
IgG
LDLr
IP: N-Cad
Flag N-Cad IP:IgG
Flag N-Cad
Total Lysates
N-Cad
IB:
N-cadherin Interacts Specificallywith LRP5 in Osteoblasts
DACN
FRZWNT
LRP 5 / 6
GSK3
β-Catenin
DEGRADATIONNucleus
OsteoblastDifferentiation
N-CADHERIN / LRP5/6 INTERACTION ANTAGONIZES WNT SIGNALING
+
LRP 5 / 6
Axin
Haÿ et al., Mol Cell Biol. 2009
* : p<0.05 VS WT Mice Wt mice Hetero TG
N-CADHERIN TRANSGENIC MICE SHOW DECREASED TRABECULAR BONE DENSITY, REDUCED BONE VOLUME
AND TRABECULAR THICKNESS
Trabecular BV/TV
0
8
16
%
*
Trabecular Thickness
0
0.08
0.16
mm
Trabecular Separation
0
0.2
0.4
0.6
mm
WT Transgenic
FemurFemale
μCT Analysis
*
WT Transgenic
0
2
4
6
0
100
200
300
400
Bone Formation Rate(mm3/mm2/year)
Mineral AppositionalRate (μm/day)
WT TgM F
WT TgM F M F M F
OsteoidSurface (%)
05
1015202530
WT Tg
OsteoblastSurface (%)
05
10152025
WT TgWT Tg
M F M F
MineralizingSurface (%)
*
0
4
8
12
16
WT TG
Vertebrae
** *
* * * *
N-CADHERIN TRANSGENIC MICE SHOW DECREASED TRABECULAR BONE FORMATION
Haÿ et al., Mol Cell Biol. 2009
ALP STAINING
ALP
Act
ivity
(pm
olpn
p/m
im/m
g pr
otei
n)
0
10
20
30
40
50
+Wnt3a
a
b
a
WT
TG
-Wnt3a
WT
Tg
0 7Days
Rel
ativ
em
RN
AA
bund
ance
(Tre
ated
/ Con
trol
)
1
2
3
4
Runx2 Col1 Ostx ALP OC
a a aa
a
WT
TG
N-Cadherin Transgenic Mice ShowDelayed Osteoblast Differentiation in vivo
p<0.05 vs WT Mice
SUMMARY: N-CADHERIN/LRP5/6 INTERACTIONANTAGONIZES WNT SIGNALING AND IMPAIRS
BONE FORMATIOIN AND BONE MASS
LRP5/6
Wnt3a
β-catenin
Axin
Transcription,Bone formation, Bone mass
Dsh
TCF/LEF
β-catenin
APC
Frz
GSK3
Haÿ et al., Mol Cell Biol. 2009
SUMMARY: N-CADHERIN/LRP5/6 INTERACTIONANTAGONIZES WNT SIGNALING AND IMPAIRS
BONE FORMATIOIN AND BONE MASS
β-Catenindegradation
LRP5/6
Wnt3a
N-Cadherin
β-cateninβ-catenin
Axin
Transcription,Bone formation, Bone mass
Dsh
LRP5/6
TCF/LEF
Axin
β-catenin
Wnt3a
Dsh
PUb
APC
APC Axin
Frz Frz
UPβ-Cat
enin
e
GSK3
GSK3
Haÿ et al., Mol Cell Biol. 2009
Role of N-cadherin in strain-induced osteoblast differentiation
-Strain reduces N-cadherin binding to beta-catenin and thereby induces beta-catenin translocation: activation of Wnt signaling- Strain increases Wnt5a and its receptor Ror2, thereby activating RhoA and Runx2.
Arnsdorf et al. PLoS ONE. 2009
β-CateninDegradation
N-Cadherin
β-catenin
Osteoblast Gene Transcriptionand Bone Formation
LRP5/6
Axin
Wnt3a
Dsh
GSK3
P Ub
APC Axin
UP
N-Cadherin
Signals
β-cate
nin
N-Cadherin
N-Cadherin
A B C
TCF/LEF
β-cate
nin
Marie PJ. IBMS BoneKEy 2009 6: 150-156
Summary: N-Cadherin-Wnt Interactions in Osteoblasts