Assorted effects of TGF-ß1 and chondroitinsulfate on
p38 and ERK 1/2 activation levels in
human articular chondrocytes stimulated with LPS
Johann Holzmann, Nina Brandl, Adolf Zemann and Manfred Huettinger
Zentrum für Physiologie & Pathophysiologie, Inst. Med. Chemie, Waehringerstrasse 10/13, A-1090 Vienna, Austria
johann.holzmann@meduniwien.ac.at
Background
Results
Inadequate cellular response of chondrocytes to stress frequently
terminates in Osteoarthritis (OA). The central pathophysiologic event is
the degradation of the extracellular matrix (ECM) by Matrix metalloproteinases (MMP). A complex interplay between a number of Cytokines
control the balance of synthesis and degradation of the ECM.
Lipopolysaccharides (LPS) are known to cause a catabolic state in
chondrocytes by activation of MAPK Members and thereby enhancing
expression of MMPs.
Chondroitinsulfate (CS) is a degradation product of the ECM and
typically biologic systems utilize a mechanism where end products signal
to the start reactions to trim the dynamics of the reaction chain.
Significant effects were observed when cells were stimulated with LPS,
invigorating catabolic metabolism in chondrocytes. LPS effects, however,
were profoundly modulated by TGF-ß1, CS and both applied in
combination. Most prominent, the silencing of p-p38 stress signal by CS
was superimposable to that of TGF-ß1. TGF-ß1 raised phospho-ERK1/2
levels three-fold over LPS induced levels. In contrast, CS treatment, alone
or combined with TGF-ß1, reduced phosphorylation significantly below LPS
induced levels. Finally, LPS induced MMP-13 mRNA levels were further
enhanced by TGF-ß1 while suppression resulted with CS.
4,5
Keeping the balance
72 h
Relative activity
3
2,5
2
1,5
1
Several factors (Mechanical trauma,
ageing,...) lead to an overstraining
and consequently to an inbalance
between synthesis and degradation
of the ECM.
Certain cytokines are known to
regulate the delicate adjusted
balance. MAPK Members p38 and
ERK play an important role in
mediating the signal from the
cellmembrane to the nucleus.
6
4
2
0
0
p-ERK 2
MMP 2
MMP 3
MMP 13
Effects of TGF-ß1 and CS on stim. HACs
30 min
400
72 h
CS
TGF-ß1
300
TGF-ß1 + CS
200
100
0
-100
-200
p-p38
p-ERK1
p-ERK2
p-p38
p-ERK1
p-ERK2
TGF-ß1 (10 ng/ml) and CS (25 µg/ml) both downregulate p-p38 activity
levels but differentially modify the 30’ activity levels of ERK1/2 in LPS
stimulated human articular chondrocytes.
Cells were grown to confluency
in DMEM containing 10 % FCS
and incubated with LPS, CS,
TGF-ß1
Cell lysis and cDNA synthesis
using random hexamer Primers
and reverse Transcriptase
2,5
8
8
CS
TGF-ß1
Relative activity
Methods
Cell lysis and protein
quantitation using BCA Assay Kit
**
8
LPS (1µg/ml) differentially stimulates the activtiy of MAPkinases (p38 and
ERK1/2) and the expression levels of matrix metalloproteinases 2, 3 and 13
in human articular chondrocytes
% change to LPS treatment
?
10
0,5
p-ERK 1
**
12
3,5
p-p38
CS
14
30 min
4
Relative activity
Here we tested the hypothesis whether CS can fulfill such a feedback
control and modulates cell signalling and MMP expression.
TGF-ß1 and/or soluble CS was added to human articular chondrocytes
(HACs) and activation of p38 and ERK1/2 was determined by immunoblot
analysis. Expression levels of mRNA of MMP 2, 3 and 13 were determined
by real-time-PCR
Effects of LPS
7,52
CS + TGF-ß1
1,5
1
*
0,5
0
MMP 2
MMP 3
MMP 13
CS (25 µg/ml) significantly downregulates MMP 13 expression levels in
LPS stimulated chondrocytes after 24 h, while TGF-ß1 or both in
combination further enhance expression.
Immunoblot using
phosphospecific antibodies
against pERK1/2 and p-p38
Expression levels of MMPs were
determined by RT-qPCR. IODvalues are normalized to GAPDH
and presented as relative to control
values.
Determination of relative change
to control (Integrated Density)
pERK1/2 and p-p38 activation levels
MMP expression levels
Discussion
The results show that soluble chondroitinsulfate modulates signalling
events in chondrocytes concurrent with MMP-13 downregulation. The
effects observed suggest a feedback signalling mechanism cross talking
with TGF-ß1 signal pathways and may serve an explanation, on the cellular
level, for the beneficial effects found in clinical studies with pharmacologic
application of chondroitinsulfate.