Lee, E.-J.

Dvir-Ginzberg, M.

Gabay, O.

Gagarina, V.

Hall, D.J.

Tipografía de Noticiero de Vigo

Galiciana

Noticiero de Vigo : diario independiente de la mañana: Ano XXI Número 5505 - 1905 outubro 6

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215 THE CHROMATIN MODIFYING ENZYME SIRT1 REPRESSES THE ARTHRITIS-ASSOCIATED MMP13 EXPRESSION IN HUMAN ARTICULAR CHONDROCYTES 

E.-J. Lee

M. Dvir-Ginzberg

O. Gabay

V. Gagarina

D.J. Hall

Crossref

215 THE CHROMATIN MODIFYING ENZYME SIRT1 REPRESSES THE ARTHRITIS-ASSOCIATED MMP13 EXPRESSION IN HUMAN ARTICULAR CHONDROCYTES

S122 Poster Presentations
knee joints with TGF-beta alone or TGF-beta + BMP-2 three times
on alternate days (200 ng/knee) and measured proteoglycan (PG)
content in cartilage.
In addition, we evaluated the signiﬁcance of a shift in Smad
balance in human OA cartilage by exposure of the explants to
TGF-beta or a combination of TGF-beta + ALK5 inhibitor SB-
505124. RNA was isolated for Q-PCR analysis.
Results: Overexpression of BMP-2 in murine knee joints, thereby
stimulating the Smad1/5/8 signaling route, decreased Smad2/3P
expression in cartilage and increased staining of VDIPEN and
NITEGE epitopes (MMP- and ADAMTS-mediated cartilage break-
down respectively). In vitro, chondrocyte exposure to BMP-2 re-
sulted in up regulation of aggrecan, collagen type II, collagen type
X and MMP13 expression. Thus, elevating Smad1/5/8 signaling is
associated with concomitant reduction of Smad2/3 signaling and
OA-like alterations.
After in vivo exposure to TGF-beta, a mild decrease in PG content
(8%) in tibial cartilage was found. However, combining TGF-beta
with BMP-2 resulted in 21% decrease in PG content. PG staining
was lost in the deep zones of tibial cartilage, just above the
tide-mark.
To investigate the effect of reduced Smad2/3 activation on overall
Smad signaling we blocked ALK5 activity (Smad2/3 route) in
TGF-beta activated chondrocytes with SB-505124. Besides the
expected decrease in Smad2/3P, Smad1/5/8P was enhanced,
thereby contributing to a shift in Smad signaling balance.
Human OA cartilage exposure to TGF-beta inhibited MMP-13
expression. This inhibition was totally blocked by SB-505124. This
indicates that in chondrocytes inhibition of Smad2/3P activation
enhances MMP-13 expression.
Conclusions: Our data indicate that the Smad2/3 and Smad1/5/8
balance determines chondrocyte function. Either up regulation
of the Smad1/5/8 pathway or down regulation of the Smad2/3
route, both we have observed in OA cartilage, appears associated
with increased chondrocyte MMP-13 expression. This ﬁts with
published observations that Smad1/5/8 stimulates while Smad2/3
signaling blocks chondrocyte terminal differentiation.
These results indicate that factors changing the balance to dom-
inant Smad1/5/8 signaling, for instance changes in TGF beta
superfamily ligands or receptors, will affect chondrocyte differ-
entiation and MMP-13 expression and might play a role in OA
development.
215
THE CHROMATIN MODIFYING ENZYME SIRT1 REPRESSES
THE ARTHRITIS-ASSOCIATED MMP13 EXPRESSION IN
HUMAN ARTICULAR CHONDROCYTES
E.-J. Lee, M. Dvir-Ginzberg, O. Gabay, V. Gagarina, D.J. Hall
NIH, Bethesda, MD
Purpose: Osteoarthritis (OA) is a degenerative joint disease of
the articular cartilage resulting in the depletion of collagens and
proteoglycans due in part to accelerated turnover and inadequate
repair. Maintenance of healthy articular cartilage in human adults
relies on the optimal expression of a number of unique extracel-
lular matrix genes, such as aggrecan and collagen type II, by
the chondrocytes in this tissue. MMP13 is a proteolytic enzyme
that degrades the extracellular matrix, including the cartilage-
speciﬁc component, type II collagen. MMP13 is thought to play
a critical role in cartilage degradation in OA. SirT1 is an NAD-
dependent histone deacetylase that lengthens organism life span
by reducing the severity of age-related diseases. Since OA is an
age-associated disease, it is pertinent to explore the role SirT1
plays in MMP expression in human adult chondrocytes from OA
patients. Here we show that MMP13 expression and activity is
downregulated by SirT1 in human chondrocytes.
Methods: Human chondrocytes (hChs) were isolated from the
knees of osteoarthritic patients undergoing total knee arthroplasty,
supplied by the National Disease Research Interchange (NDRI),
Philadelphia, PA. Cells were cultured in monolayer. Chondrocyte
transfections were carried out using the Amaxa Nucleofector tech-
nology. Cells were processed for protein or RNA extraction followed
by immunoblotting or RT-PCR according to standard procedures,
using the indicated antibodies and primer pairs.
Results: MMP13 levels were assessed in OA human chondro-
cytes stably expressing SirT1. Immunoblot assays conﬁrmed SirT1
overexpression and an increase in SirT1 activity in cells express-
ing wild type SirT1 indicates that the ectopic SirT1 is active
within these chondrocytes. The SirT1-expressing cells display sig-
niﬁcantly reduced MMP13 RNA and protein levels compared to
control cell lines. The activity of MMP13 was also decreased
in SirT1-overexpressing cells. These results demonstrate that
MMP13 gene expression is suppressed by SirT1. Treatment of
chondrocytes with the SirT1 inhibitor nicotinamide (NAM), led to a
signiﬁcant increase in MMP13 mRNA levels and MMP13 protein
levels. When SirT1 protein levels were also reduced by SirT1
siRNA, it led to increased MMP13 expression. Treatment of cells
with IL1beta led to induction of MMP13, yet SirT1 was partially
able to block this IL1beta-mediated induction.
To explore the basis by which Sirt1 represses MMP13 transcrip-
tion, we focused on the Lef1 transcription factor, a component of
the Wnt-signaling pathway. Lef1 has been demonstrated to posi-
tively regulate MMP13 transcription in chondrocytes. We ﬁnd that
SirT1 represses LEF1 levels and as a consequence represses
transcription from Lef1 target promoters.
Conclusions: We ﬁnd that longevity factor SirT1 is a potent re-
pressor of MMP13 gene expression, an enzyme involved in joint
destruction in OA, an age-associated disease. The repression
of MMP13 occurs even in the presence of the cytokine IL1beta.
MMP13 is a critical player in joint destruction in OA. The underlying
mechanism by which SirT1 represses MMP13 appears to involve
the Lef1 transcription factor a known regulator of MMP13 tran-
scription. That Lef1 is repressed by SirT1 is consistent with recent
data indicating that Wnt/β-catenin signaling pathway is involved in
IL-1beta-mediated cartilage degeneration. Taken together, these
data show that SirT1 represses the expression of genes known to
play a critical role in the onset of cartilage pathology.
216
P188 ALONE OR IN COMBINATION WITH OP-1 IS
EFFECTIVE IN REDUCING CARTILAGE DEGENERATION
AND PROMOTING CELL SURVIVAL IN THE MODEL OF
ACUTE TRAUMA TO HUMAN ANKLE CARTILAGE: THE
MECHANISM OF ACTION
S. Bajaj, K. Sampath, C. Pascual-Garrido, F. Hirschfeld,
A.A. Hakimiyan, L. Rappoport, M.A. Wimmer, T.R. Oegema Jr.,
S. Chubinskaya
Rush Univ. Med. Ctr., Chicago, IL
Purpose: There is a clear need to recognize the risk of OA follow-
ing joint injury, and to develop and implement strategies to prevent
posttraumatic OA. The main objectives of this continuing study
were to investigate the effect and understand the mechanism of
action of P188 surfactant alone or in combination with osteogenic
protein-1 (OP-1) on cartilage degeneration and cell survival in
acute trauma to human ankle cartilage.
Methods: We developed a model of open joint acute trauma in
which injury is created to human ankle cartilage ex vivo by the
application of a 4 mm cylindrical impactor with stress of 30 MPa.
This type of injury is limited to damage on the articular surface. To
investigate the effect of therapeutic interventions, sixteen normal
tali from human organ donors were impacted using a 4mm indenter


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215 THE CHROMATIN MODIFYING ENZYME SIRT1 REPRESSES THE ARTHRITIS-ASSOCIATED MMP13 EXPRESSION IN HUMAN ARTICULAR CHONDROCYTES

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