PowerPointPrsentation

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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_at_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 metallo- proteinases (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. 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
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.
Effects of LPS


Keeping the balance
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
CS ?
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.
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
Cell lysis and cDNA synthesis using random
hexamer Primers and reverse Transcriptase
Immunoblot using phosphospecific antibodies
against pERK1/2 and p-p38
Expression levels of MMPs were determined by
RT-qPCR. IOD-values 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