![Page 1: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/1.jpg)
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?"
![Page 2: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/2.jpg)
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
![Page 3: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/3.jpg)
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
![Page 4: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/4.jpg)
Collagen
![Page 5: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/5.jpg)
Aggrecan
![Page 6: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/6.jpg)
Synovial fluid
Bone
Cartilage
Cross section of cartilage
Cartilage
![Page 7: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/7.jpg)
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
![Page 8: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/8.jpg)
(M)MP TIMPbreakdown build up
Cartilage turnover
breakdown
(M)MPbuild up
TIMP
Cartilage degradation
The balancing act of cartilage turnover…
![Page 9: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/9.jpg)
Which metalloproteinases are expressed by cartilage? Does
this change in disease?
Kevorkian, Davidson, Swingler
![Page 10: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/10.jpg)
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
![Page 11: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/11.jpg)
(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)
![Page 12: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/12.jpg)
• ‘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
![Page 13: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/13.jpg)
Real time PCR
![Page 14: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/14.jpg)
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)
![Page 15: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/15.jpg)
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
![Page 16: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/16.jpg)
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
![Page 17: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/17.jpg)
Momohara et al. Arth Rheum (2004) 50:4074
MMP28 expression in upregulated in RA cartilage
![Page 18: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/18.jpg)
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
![Page 19: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/19.jpg)
Aspartate (21)
Cysteine (154)
Metallo- (191)
Serine (176)
Threonine (28)
The human degradome570 proteases
![Page 20: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/20.jpg)
How can we stop cartilage degradation?
Dietary factors
![Page 21: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/21.jpg)
Association between osteoarthritis and obesity suggests a possibility of an
association with diet
![Page 22: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/22.jpg)
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
![Page 23: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/23.jpg)
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
![Page 24: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/24.jpg)
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
![Page 25: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/25.jpg)
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
![Page 26: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/26.jpg)
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
![Page 27: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/27.jpg)
![Page 28: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/28.jpg)
Allium vegetables
Cruciferous vegetables
![Page 29: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/29.jpg)
Laboratory studiesOrganosulphur compounds derived from garlic oil
![Page 30: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/30.jpg)
Sulforaphane1-isothiocyanato-(4R)-(methylsulfinyl) butane
glucoraphaninglucoraphanin
myrosinasemyrosinase
glucoraphanin
glucoraphanin
myrosinasemyrosinase
SFNSFN
Plant injuryPathogen attackChewingFood prep
![Page 31: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/31.jpg)
Histone acetylation
SW1353 cell line
AcH3
Total H3
AcLys
AcH3
Total H3
AcLys
![Page 32: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/32.jpg)
46kDa MMP-1
43kDa MMP-13
SFN (µM) 0 5 10 15 30 IL-1/OSM + + + + +
Chondroprotection – sulforaphane
![Page 33: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/33.jpg)
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 (
%)
![Page 34: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/34.jpg)
Mechanism?
Keap1Keap1
Nrf2Nrf2
UbUb
Cytoprotective genes
SPN
Nrf2Nrf2
Nrf2Nrf2
ARE
HO-1GSH
NQO1- Anti-inflammatory effects
![Page 35: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/35.jpg)
Haem oxygenase-1 expressionprimary human articular chondrocytes
Nrf2 dependent
****
***
***
![Page 36: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/36.jpg)
SFN does not regulate MMP1 expression via Nrf2
Nrf2 independent
![Page 37: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/37.jpg)
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
![Page 38: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/38.jpg)
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
![Page 39: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/39.jpg)
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
![Page 40: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/40.jpg)
![Page 41: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/41.jpg)
MMP-28
Kevorkian, Rodgers
![Page 42: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/42.jpg)
• 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
![Page 43: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/43.jpg)
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
![Page 44: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/44.jpg)
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
![Page 45: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/45.jpg)
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
![Page 46: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/46.jpg)
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
![Page 47: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/47.jpg)
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
![Page 48: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/48.jpg)
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
![Page 49: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/49.jpg)
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
![Page 50: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/50.jpg)
Wild-type
EA mutant Pro-cat
Vector only
Expression of MMP-28 in SW1353 cellsActin cytoskeleton (phalloidin)
![Page 51: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/51.jpg)
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
![Page 52: Ian M Clark, PhD Biomedical Research Centre, University of East Anglia, Norwich, NR4 7TJ. United Kingdom. What destroys cartilage in osteoarthritis and](https://reader035.vdocuments.us/reader035/viewer/2022081506/56649ead5503460f94bb4e08/html5/thumbnails/52.jpg)
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