View
234
Download
1
Category
Tags:
Preview:
Citation preview
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
Recommended