Ian M Clark, PhD

Biomedical Research Centre,

University of East Anglia,

Norwich, NR4 7TJ.

United Kingdom.

What destroys cartilage in osteoarthritis and how can we stop it?"

Osteoarthritis

• > 6 million people in UK with moderate to severe OA

• predominantly age > 45 with major morbidity in age > 60

• 3 million GP visits for OA in yr 2000

• approx. £3 billion in lost productivity

NORMAL OSTEOARTHRITIC

Arthritis: The Big Picture. Arthritis Research Campaign report May 2002

Changes in cartilage during OA

• Biochemical changes– decrease in aggrecan– damage to the type II

collagen network• Morphological changes

– fibrillations/pitting– softening and loss of

cartilage thickness• Enzymatic changes

NORMAL OSTEOARTHRITIC

Collagen

Aggrecan

Synovial fluid

Bone

Cartilage

Cross section of cartilage

Cartilage

Metalloproteinases and their inhibitors

Matrix metalloproteinases23 human enzymes

(Collagenases MMP-1, -8, -13, -2, -14?)

ADAMTSs19 human enzymes

(Aggrecanases ADAMTS-1, -4, -5, -8, -9, -15?)

TIMPs4 human inhibitors

(M)MP TIMPbreakdown build up

Cartilage turnover

breakdown

(M)MPbuild up

TIMP

Cartilage degradation

The balancing act of cartilage turnover…

Which metalloproteinases are expressed by cartilage? Does

this change in disease?

Kevorkian, Davidson, Swingler

Hip replacement

‘In Europe, a joint is replaced due to osteoarthritis every 1.5 minutes.’ Wieland et al (2005)

2006/7 in the UK, one hip or knee replaced every 4 minutes

(Mr Simon Donell, Consultant Orthopaedic Surgeon, NNUH)

Cartilage samples

Osteoarthritis (OA) femoral head from THR for osteoarthritis (n=18, age 38-81)

‘Normal’ femoral head from THR following fracture to the neck of femur ‘NOF’ (n=15, age 52-93)

• ‘Taqman’ quantitative real-time RT-PCR(steady state mRNA levels measured)

• primer/probe sets for23 human MMPs, 19 human ADAMTSs, 4 TIMPs

(designed across >1 exon)

(products verified by sequencing)

Assay

Real time PCR

TIMP

OA

N

1 2 3 4

Not detected CT=40

Low expression CT 36-39.

Moderate expression CT 31-35.

High expression CT= 26-30

Very high expression CT< 25

MMP

OA

N

1 2 3 7 8 9 10 1211 13 14 15 16 17 19 20 21 23 24 25 26 27 28

ADAMTS

OA

N

1 2 3 4 5 6 7 8 9 10 12 13 14 15 16 17 18 19 20

Expression profile of MMP, ADAMTS and TIMP family in normal vs. OA cartilage (mean Ct values)

Genes that are up-regulated in OA

OA Normal0.00

0.25

0.50

0.75

1.00

AD

AM

TS

-16/

18S

(arb

itra

ry u

nit

s)

OA Normal0

50

100

150

200

250

300

350

MM

P-1

3/18

S(a

rbit

rary

un

its)

OA Normal0

5

10

15

20

25

30

35

40

45

MM

P-2

8/18

S(a

rbit

rary

un

its)

MMP-13 MMP-28 ADAMTS-16

P< 0.001

MMP ADAMTS TIMP

MMP-2 ADAMTS-2 TIMP-3MMP-9 ADAMTS-12 MMP-16 ADAMTS-14

OA Normal0.0

2.5

5.0

7.5

MM

P-1

/18S

(arb

itra

ry u

nit

s)

OA Normal0

500

1000

1500

2000

2500

MM

P-3

/18S

(arb

itra

ry u

nit

s)

OA Normal0

1

2

AD

AM

TS

-1/1

8S(a

rbit

rary

un

its)

MMP-1 MMP-3 ADAMTS-1

Genes that are down-regulated in OA

MMP ADAMTS TIMP

MMP-10 ADAMTS-5 TIMP-1 ADAMTS-9 TIMP-4 ADAMTS-15

P< 0.001

Momohara et al. Arth Rheum (2004) 50:4074

MMP28 expression in upregulated in RA cartilage

Summary

• First expression profile to assay all MMPs, ADAMTSs and TIMPs in cartilage

• MMP-28 and ADAMTS-16 expression is significantly increased in end-stage OA

Aspartate (21)

Cysteine (154)

Metallo- (191)

Serine (176)

Threonine (28)

The human degradome570 proteases

How can we stop cartilage degradation?

Dietary factors

Association between osteoarthritis and obesity suggests a possibility of an

association with diet

Observational Study

• St Thomas’ Twins UK registry

• Matched co-twin case control design compares discordant exposure- disease status

• Radiographic OA determined at the hand hip and knee

• Lumbar and cervical degeneration assessed by MRI

Dietary intake

• Food frequency questionnaire (EPIC format)• Prior analysis indicated 5 discrete patterns of

intake, that were used as the main variables in the analysis– A: Fruit and vegetable pattern– B: High alcohol pattern– C: Traditional English– D: Dieting pattern score– E: Low meat pattern score

Teucher et al 2008

Peripheral Joints

A B C D E A B C D E A B C D E

Dietary pattern

Od

ds

rati

oHip Knee Hand

Fruit and vegetable pattern at the hip

Allium

veg

etab

les

Cruci

fero

us v

eget

able

s

Gre

en le

afy

vege

tabl

es

Yello

w v

eget

able

s

Oth

er v

eget

able

sCitr

us fr

uit

Non-c

itrus

frui

t

Chips

and

roas

t pot

atoe

s

Od

ds

rati

o

Comment

• Matched analysis indicates that these findings are less likely to be confounded by other lifestyle factors

• However, the effects are small effects and (despite the dietary pattern approach) not robust to multiple testing

Allium vegetables

Cruciferous vegetables

Laboratory studiesOrganosulphur compounds derived from garlic oil

Sulforaphane1-isothiocyanato-(4R)-(methylsulfinyl) butane

glucoraphaninglucoraphanin

myrosinasemyrosinase

glucoraphanin

glucoraphanin

myrosinasemyrosinase

SFNSFN

Plant injuryPathogen attackChewingFood prep

Histone acetylation

SW1353 cell line

AcH3

Total H3

AcLys

AcH3

Total H3

AcLys

46kDa MMP-1

43kDa MMP-13

SFN (µM) 0 5 10 15 30 IL-1/OSM + + + + +

Chondroprotection – sulforaphane

Chondroprotection – diallyl disulphide

Control

IL1

IL1/

OSM 4 8 16 32 4 8 16 32 4 8 16 320

1

2

4

5

6

IL-1 IL-1/OSM

DADS (M)

Rel

ativ

e M

MP

-13

Exp

ress

ion

(arb

itra

ry u

nit

s)

ControlIL

1

IL1/

OSM 4 8 16 32 4 8 16 32 4 8 16 320.0

0.4

0.8

1.2

1.6

10

40

70

DADS (M)

IL-1 IL-1/OSM

Hyd

roxy

pro

line

Lo

ss (

%)

Mechanism?

Keap1Keap1

Nrf2Nrf2

UbUb

Cytoprotective genes

SPN

Nrf2Nrf2

Nrf2Nrf2

ARE

HO-1GSH

NQO1- Anti-inflammatory effects

Haem oxygenase-1 expressionprimary human articular chondrocytes

Nrf2 dependent

****

***

***

SFN does not regulate MMP1 expression via Nrf2

Nrf2 independent

Summary• Epidemiology shows a protective association between

allium intake and osteoarthritis

• In chondrocytes, SFN and DADS can attenuate the expression of metalloproteinase genes in a dose dependent manner

• This is likely not through the inhibition of histone deacetylases and not via Nrf2

• Both compounds induces HO-1 which is itself chondroprotective

• Both compounds can abrogate cartilage destruction in the BNC model in vitro

• At least SFN can abrogate MMP expression and cartilage destruction in human cartilage in vitro

Conclusions

• A complete understanding of protease expression and activity in the joint will allow drug development in this area

• Dietary bioactives may represent an alternative strategy in the prevention or treatment of osteoarthritis

• A thorough understanding of disease association, mechanism(s), metabolism and pharmacodynamics is needed to underpin this premise

Acknowledgements

Clark labKirsty CulleyRose DavidsonOrla JuppJanine MorrisUrsula RodgersSarah SnellingTracey Swingler

Institute of Orthopaedics, NNUHSimon DonellClare DarrahAdele Cooper

School of Medicine, UEAYongping BaoAedin CassidyAlex MacGregor

Kings College LondonFrances WilliamsTim Spector

MMP-28

Kevorkian, Rodgers

• expressed strongly in keratinocytes in vitro and in vivo

• induced by TNF

Saarialho-Kere et al. (2002) J Invest Dermatol 119:14-21

Expression of MMP-28 in skin

Epilysin (MMP-28) induces EMT in A549 cells

Illman, S. A. et al. J Cell Sci 2006;119:3856-3865

Control

MMP-28

EA mutant

Expression of MMP-28 in HeLa cells(pcDNA4-FLAG, transient transfection)

Western blot, Anti-FLAG

Conditioned mediumVector MMP-28-FLAG

75

50

37

25

Pro

CTD

755037

Cell lysateVector MMP-28-FLAG

Pro

75

50

37

Extracellular matrixVector MMP-28-FLAG

ProActive

Pre Pro Catalytic Zn HaemopexinFu

Expression of MMP-28 in SW1353 cells(pcDNA4-FLAG, stable transfection)

Conditioned medium V MMP28

Cell lysate V MMP28

Extracellular matrix V MMP28

Pro

CTD

Pro ProActive

Western blot, Anti-FLAGPre Pro Catalytic Zn HaemopexinFu

Activation of proMMP-28 require furin activity

Western blot, anti-FLAG antibody

Furin inhibitor = Decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone

Extracellular matrix

Vector only MMP-28-FLAG

- + - + furin inhibitor

75

50

37

ProActive

Expression of MMP-28 in SW1353 cellsImpact on other metalloproteinases

0

0.5

1

1.5

2

2.5

3

3.5

Arb

itra

ry U

nit

sM

MP

28/1

8S

1 2 1 2 1 2 1 2 Vector only Wild type EA mutant Pro-cat

0

0.5

1

1.5

2

2.5

3

3.5

*******

***

**

1 2 1 2 1 2 1 2 Vector only Wild type EA mutant Pro-cat

Arb

itra

ry U

nit

sM

MP

2/18

S

Expression of MMP-28 in SW1353 cellsImpact on other metalloproteinases

0

0.5

1

1.5

2

2.5

3

3.5

*******

***

**

1 2 1 2 1 2 1 2 Vector only Wild type EA mutant Pro-cat

Arb

itra

ry U

nit

sM

MP

2/18

S

Vector only Wild type EA mutant Pro-cat

Gelatin zymography

Wild-type EA mutant Pro-cat Vector only

Wild-type EA mutant Pro-cat Vector only

Permeabilized

Non-permeabilized

Expression of MMP-28 in SW1353 cellsImmunocytochemistry

Wild-type

EA mutant Pro-cat

Vector only

Expression of MMP-28 in SW1353 cellsActin cytoskeleton (phalloidin)

Expression of MMP-28 in SW1353 cellsAdhesion and migration

0

0.5

1

1.5

2

2.5

3

3.5

4

Fo

ld c

ha

ng

e

****

**** ********

***

1 2 1 2 1 2 1 2 Vector only Wild type EA mutant Pro-cat

Adhesion

Type II collagen

Migration

0

0.5

1

1.5

2

2.5

3

3.5

Fo

ld c

ha

ng

e

********

*

****

***

****

1 2 1 2 1 2 1 2 Vector only Wild type EA mutant Pro-cat

0

5

10

15

20

25

30

35

40

45

50

μm

/ho

ur

****

****

****

****** **

1 2 1 2 1 2 1 2 Vector only Wild type EA mutan Pro-cat

Adhesion

Fibronectin

Summary• recombinant proMMP-28 is a secreted protein, but full-length MMP-28 is also associated with the cell surface

• activation of proMMP-28 is via a pro-protein convertase with the ‘active’ form predominantly bound to ECM

• expression of ‘active’ MMP-28 induces MMP-2 expression and activity

• expression of MMP-28 alters cytoskeleton

• MMP-28 expression increases adhesion to type II collagen and fibronectin, but only decreases migration on the former

•MMP-28 expression alters the cell proteome