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Osteoarthritis
Assorted effects of TGF-ß and chondroitinsulfate on p38and ERK 1/2 activation levels in human articular chondrocytesstimulated with LPS
J. Holzmann, N. Brandl, A. Zeman, R. Schabus, S. Marlovits, R. Cowburnand M. Huettinger
Center of Physiology and Pathophysiology, A-1090 Vienna, Waehringerstr. 10/13, Austria
keeping the balance...
anabolic statecartilage synthesis
catabolic statecartilage degradation
inflammation
activateMacrophages
NSAID
inflammatory cytokines signalthrough SAPK (p38 / JNK)
the bad guys:
MatrixMetalloProteinases (MMPs) &
MMP-1 (Interstitial Collagenase)
MMP-2 (Gelatinase A)
MMP-3 (Stromelysin 1)
MMP-9 (Gelatinase B)
MMP-13 (Collagenase-3)
cleaves triple-helical fibrillar collagen II
cleaves triple-helical fibrillar collagen II
cleaves gelatin(part. hydrolysed collagen)
cleaves gelatin(part. hydrolysed collagen)
cleaves collagen IVactivates proMMPs
Aggrecanases
Aggrecanase 1 & 2 cleaves aggrecan
ind
uce
d in
res
pon
se to
cyt
oki
nes
a
nd g
row
th fa
cto
rs
working hypothesis
IL-1IL-6TNF-alpha
enhanced MMP& Aggrecanase expression
degradation of Matrixrelease of CS
Feedback signaling of endproducts
moderation of MMP& Aggrecanase expression
synthesisof new Matrix
pharmacological doses of CS: • enhance feedback signaling• providing building blocks for aggrecan
synthesis
CS
?
• is CS able to downregulate the MMP transcription levels in LPS stimulated chondrocytes as a model for osteoarthritis?
• what are the effects of CS on the MAPK members p38 and ERK?
• is there a cross reaction between TGF-ß and CS ?
aim of the study
LPS
given that clinical studies showed a benefit when CS is taken orally…
methods
Cells were grown to confluency in DMEM containing 10 % FCS and incubated with LPS, CS, TGF-ß1
Cell lysis and protein quantitation using BCA Assay Kit
Immunoblot using phosphospecific antibodies against pERK1/2 and p-p38
Cell lysis and cDNA synthesis using random hexamer Primers and reverse Transcriptase
Expression levels of MMPs were determined by RT-qPCR.
pERK1/2 and p-p38 activation levels MMP expression levels
HAC…human articular chondrocytesfrom patients with no history of OAundergoing joint replacement because of femoral neck fracture
> 3 passage => „fibroblast like“
resultseffects on non stimulated chondrocytes
0
0,2
0,4
0,6
0,8
1
1,2
1,4
MMP2 MMP3
fold
ch
ang
e
0
5
10
15
20
25
30
35
40
MMP13
fold
cha
nge C4S
TGF-ß
CS+TGF-ß
30 min incubation
p-p38
p-ERK 2p-ERK 1
*
*** ***
TGF-ß =>induction of MMP13expressionthrough SMAD &MAPKpathway
*
control
72 h 72 h
0
2
4
6
8
10
12
14
MMP1 MMP 2 MMP 3 MMP 13
Rel
ativ
e ac
tivity
resultseffects of LPS
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
p-p38 p-ERK 1 p-ERK 2
Rel
ativ
e ac
tivity
30 min
72 h******
**
**
**
p-p38
p-ERK 2p-ERK 1
30 min 72 h
30 min 72 h
control
control
LPS strongly activates p38LPS transiently activates ERK 1/2
LPS increases transcriptionof MMP1, MMP3 & MMP13
**
72 h
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
MMP1 MMP2 MMP3 MMP13
fold
ch
an
ge
to
LP
S
CS
TGF-ß1
CS + TGF-ß1
resultseffects of TGF-ß and CS in stim. chondrocytes
87,5
*
-200
-100
0
100
200
300
400
% ch
ange
to L
PS tr
eatm
ent
CS
TGF-ß1
TGF-ß1 + CS
p-p38 p-ERK1 p-ERK2 p-p38 p-ERK1 p-ERK2
30 min 72 hCS moderates p-p38 like TGF-ß
CS counteractsthe effect of TGF-ßon ERK 1/2 at 30´
LPS
control
LPS
CS moderatesthe transcription ofMMP13 by 30%
******
***
*****
**
***
*****
***
***
**
**
******
72 h
resultssummary
CS has no effect on p38 / ERK and MMP expression in non stimulated „healthy“
dedifferentiated chondrocytes (but maybey in diff. chondrocytes?)
LPS induces a catabolic state which is characterised by a p38 / ERK activation
and enhanced MMP 1 & MMP 13 expression
CS and TGF-ß are both able to moderate the p38 activation
CS and TGF-ß show contrary effects on ERK activation and MMP13 expression
=> TGF-ß further stimulated MMP13 expression
=> CS suppressed MMP13 expression
may serve an explanation, on the cellular level, for the beneficial effects found in clinical studies with pharmacologic application of chondroitinsulfate
discussion
What are the molecular targets of CS?
membran receptor / cytokine trapping?
How is CS able to modulate TGF-ß effects?
signal crosstalk / cytokine trapping?
Nagarajan Selvamurugan et al. J Biol Chem. 2004 Apr 30;279(18)
TGF-beta enhanced MMP13 expression through crosstalkbetween p38, ERK and SMAD pathway
-200
-100
0
100
200
300
400
% c
hang
e to
LP
S tre
atm
ent
CS
TGF-ß1
TGF-ß1 + CS
0
0,5
1
1,5
2
2,5
MMP 2 MMP 3 MMP 13
fold
ch
an
ge
to
LP
S
CS
TGF-ß1
CS + TGF-ß1
discussion
p-p38 p-ERK1 p-ERK2 p-p38 p-ERK1 p-ERK2
• only transient downregulation of ERK• delayed & alleviated response to TGF-ß in the presence of CS
discussion
glycosaminoglycans (GAG) can bind cytokines like TGF-ßcytokines are then resistent to degradations therefore matrix can act as a reservoir
equlibrium between the TGF-ß bound to matrix and free GAG
supply of free CS could also act as a binding partner „trap“ TGF-ß
heparan / chondroitin sulfate negatively modulates TGF-beta1 responsiveness by decreasing the ratio of TGF-beta1 binding to TbetaR-II and TbetaR-I, facilitating endocytosis and rapid degradation of TGF-beta1
Chen et al. J Biol Chem. 2006 Apr 28;281(17)
summary
soluble chondroitinsulfate modulates signalling events in chondrocytes concurrent with
• p38 and transiently ERK1/2 downregulation• MMP-13 downregulation
may serve an explanation, on the cellular level, for the beneficial effects found in clinical studies with pharmacologic application of chondroitinsulfate.
• the molecular targets of CS has to be elucidated
• CS possibly modulates TGF-beta responsiveness by a combination of „cytokine trapping“ and enhanced TGF-betaRI + II degradation