Download tnf-alpha stimulated activation of mmp

TNFα STIMULATED ACTIVATION OF MMP-2 IN NUCLEUS PULPOSUS CELLS OCCURS
THROUGH EGR-1 MEDIATED TRANSCRIPTION OF MT1-MMP
*
Séguin, C A; *,**Pilliar, R M; +*,**Kandel, R A
CIHR BioEngineering of Skeletal Tissues Team, *Mount Sinai Hospital, Toronto, Canada;
**
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Canada.
[email protected]
INTRODUCTION
Degeneration of the intervertebral disc (IVD) involves a shift in the
balance between catabolic and anabolic processes leading to changes in
tissue architecture and function[1]. TNFα is a proinflammatory cytokine
expressed within the nucleus pulposus (NP) of degenerate non-herniated
IVDs, whose expression correlates with age and the degree of disc
degeneration. Although implicated in the intitiation of discogenic pain,
the role of TNFα in IVD degeneration remains poorly understood. We
have developed a culture system that permits the formation of NP tissue
in vitro, thus enabling investigations into the mechanisms regulating
tissue degradation [2]. We have demonstrated that when exposed to low
levels of TNFα, NP cells demonstrate increased gene expression of
MMP-1, -3, -13 and ADAMTS-4 and -5, suggesting that TNFα
stimulation of NP cells is a good model to study mechanisms regulating
disc degeneration in vivo [3]. Among the enzymes known to be activated
by TNFα, MMP-2 (gelatinase-A) is thought to contribute to the
progression of degeneration and to the induction of neovascularization
that occurs in the early stages of disc degeneration. In this study, we
investigated if TNFα functionally activates MMP-2 and the regulatory
mechanisms underlying TNFα-mediated MMP-2 activation in NP cells.
MATERIALS AND METHODS
NP Tissue Formation: The NP was dissected from bovine caudal spines
and NP cells were isolated as previously described[2]. NP cultures were
maintained for 2 wk to allow tissue formation similar to the native NP.
NP Tissue Treatments: Cultures were treated with TNFα (25 ng/ml,
Sigma) for 48 h in the presence of mitogen activated protein kinase
(MAPK) inhibitors SB220225 (0-20 uM), SP600250 (0-20 uM),
PD98059 (0-50 uM) or U0126 (0-10 uM)(Calbiochem).
Gelatin Zymography: Conditioned medium was separated on 7.5% gels
containing 0.1% gelatin by SDS-PAGE. Gels were incubated in
renaturing buffer (30 min) and overnight in developing buffer.
Gelatinase activity was visualized by Coomasie blue staining.
RT-PCR Analysis: Total RNA was isolated from in vitro-formed tissues
by Trizol extraction, reverse-transcribed (Superscript II, Invitrogen), and
relative gene expression was examined by PCR.
Immunoblot Analysis: Cell lysates were isolated from NP tissues
following disruption of tissues by Dounce homogenization in RIPA
buffer. 25 ug of protein was separated on 7.5% SDS-PAGE,
electroblotted to nitrocellulose, and probed with antibodies reactive with
ERK1/2 or Egr-1 (Cell Signaling), MMP-2 or MT1-MMP (Chemicon).
Electrophoretic Mobility Shift Assays (EMSA): Nuclear extracts were
prepared following disruption of tissues by Dounce homogenization.
10ug of protein were used in EMSA with radiolabelled oligonucleotides
containing the Egr-1 consensus or bp -303 to -284 of the MT1-MMP
promoter. For supershift reactions, antibodies against Egr-1 were
included. Complexes were resolved by electrophoresis on 4%
polyacrylamide gels and visualized by autoradiography.
Transfections and Luciferase Reporter Analysis: Transient
transfections of NP cells were performed using Fugene-6 (Roche) and
2ug of either pGL3-Basic vector or the MT1-MMP(-300)-promoter
luciferase reporter construct [4] along with 0.1 ug pRL-SV40 to control
for transfection efficiency. Luciferase expression was measured using
the Dual Luciferase System (Promega).
RESULTS
Gelatinolytic activities corresponding to both latent and active forms of
MMP-2 were present in the medium of TNFα-treated cultures by 48 h
and were not present in untreated cultures. The role of MAPK pathways
in the induction of MMP-2 activation was investigated using
pharmacological inhibitors. Whereas inhibition of JNK or p38 did not
alter MMP-2 activity, inhibition of ERK prevented the TNF -induced
activation of MMP-2.
As TNF functionally activated MMP-2, the effects of TNF on levels of
MMP-2 and its potential regulators MT1-MMP, and TIMP-2 were
determined. TNF up-regulated MT1-MMP gene expression in a timedependent manner. Changes in mRNA were associated with increased
levels of MT1-MMP protein in NP cells following 24h. There were no
significant changes in MMP-2 or TIMP-2 mRNA or protein levels.
A GC-rich sequence in the MT1-MMP promoter containing consensus
sites for Egr-1 can regulate gene expression [4]. In NP tissues, TNF
induced a rapid and transient increase in Egr-1 mRNA by 0.5h which
returned to baseline following 8h. A corresponding increase in levels of
Egr-1 protein was detected, which peaked by 2h of TNF treatment and
returned to basal levels by 24h. Selective blockade of the ERK pathway
prevented TNF -induced Egr-1 gene expression suggesting Egr-1 as an
effector protein in the ERK-mediated MMP-2 activation. EMSAs were
conducted to assess the DNA-binding activity of Egr-1 and complex
formation on the MT1-MMP promoter. Activation of Egr-1 and binding
to the MT1-MMP promoter were detected by 2h of TNF treatment and
returned to constitutive levels by 8h. ERK inhibition prevented Egr-1
activation and complex formation at the MT1-MMP promoter. The
complex on the MT1-MMP promoter was confirmed to contain Egr-1 by
competition with excess unlabelled oligonucleotides, and supershift
reactions containing monoclonal Egr-1 antibodies.
Figure1:Analysis of NP cell gene expression following TNFα treatment
Transcriptional activity of the MT1-MMP promoter was assessed in NP
cells expressing the basal promoter sequence coupled to the luciferase
reporter gene (MT1-MMP(-300)-promoter luciferase)[4]. As suggested
by PCR analysis, TNFα induced a significant increase in transcriptional
activation of the MT1-MMP promoter. In keeping with the regulatory
role of Egr-1, transcriptional induction of MT1-MMP was completely
prevented by selective ERK inhibition.
DISCUSSION
As MMP-2 is implicated in the pathogenesis of IVD degeneration, the
current study determined the intracellular mechanisms responsible for
the induction of MMP-2 activity in a TNFα-induced model of NP
degeneration. We demonstrate that in NP cells, TNFα induced MMP-2
gelatinase activity is dependent on a transient ERK-dependent increase
in Egr-1 production, which was followed by an increase in the
production of MT1-MMP. We confirmed that up-regulation of MT1MMP gene expression was mediated by ERK MAPK and Egr-1
transcriptional regulation of the MT1-MMP promoter by luciferasereporter assays. These findings suggest that TNFα induces the activation
of both MMP-2 and MT1-MMP in NP tissues in vitro and in this way
may contribute to TNFα-induced NP tissue degradation. As regulation
of these enzymes is important for maintenance of IVD homeostasis in
vivo, understanding the mechanisms governing their activation may be
essential to delineate and eventually prevent the process of IVD
degeneration.
REFERENCES: [1] Buckwalter. (1995) Spine 20(11) : 1307. [2]
Séguin et al. (2004) Spine 29(12):1299. [3] Séguin et al. (2005) Spine (in
press). [4] Haas et al. (1999) J.Biol.Chem.274(32):22679.
ACKNOWLEDGEMENTS: Supported by the Arthritis Society of
Canada and NSERC Canada.
52nd Annual Meeting of the Orthopaedic Research Society
Paper No: 0012