Wnt-Cadherin Interactions in Bone

Pierre MARIEINSERM U 606 & University Paris Diderot

Hôpital Lariboisière, Paris, Francepierre.marie@inserm.fr

http://www.idf.inserm.fr/site/u606/

Lariboisière Hospital, Paris, France

What Are Cadherins?

Cell-cell adhesion molecules

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

Role of Cadherins in Bone

CADHERINS PLAY A ROLE IN OSTEOCLASTS

E-CAD

Cadherin-6

Osteoclasts

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

How Cadherins Can Interactwith Wnt Signaling

to Regulate Bone Homeostasis ?

Cadherins Interact With Catenins

P

Frizzled

Dsh

APCAxin GSK3b

β-Catenin

Wnt Signaling

UB

UBUB UB

Proteasome

degradation

Frizzeld

Dsh

Tcf/Lef

Wnt

P

LRP5/6

APCAxin GSK3b

ALP, COLIAI, Runx2Osteoblast differentiationBone formation

Wnt Signaling

β-Catenin

CADHERIN-β-CATENIN INTERACTION

α-catenin

Actin

β-Catenin

Cadherins

Interactions Wnt-Cadherins

Nelson & Nusse, Science 2004

Osteoblast Gene Transcriptionand Bone Formation

β-cate

nin

Cadherin

TCF/LEF

β-cate

nin

CADHERIN-β-CATENIN INTERACTIONIN BONE

N-Cadherin Antagonizes Wnt SignalingIn Osteoblasts

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

How β-catenin is controlledby N-cadherin?

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

What is the role ofN-cadherin-Wnt interaction in vivo ?

* : 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

Questions ?