The document examines the effects of interleukin-1 (IL-1) and oncostatin M (OSM) on the expression of matrix metalloproteinase (MMP), ADAM, and ADAM-TS genes in human chondrocytes. The study finds that IL-1 and OSM synergistically induce expression of the collagen-degrading enzymes MMP-1, MMP-8, MMP-13, and MMP-14 as well as the aggrecan-degrading enzyme ADAM-TS4. In particular, MMP-1, MMP-3, and MMP-13 expression is induced early, while MMP-8 expression occurs later. IL-1 and OSM also synergistically induce MMP

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Koshy PJ, Lundy CJ, Rowan AD, Porter S, Edwards
DR, Hogan A, Clark IM, Cawston TEThe
modulation of matrix metalloproteinase and
ADAM gene expression in human chondrocytes
by interl...
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2. ARTHRITIS & RHEUMATISM
Vol. 46, No. 4, April 2002, pp 961–967
DOI 10.1002/art.10212
© 2002, American College of Rheumatology
The Modulation of Matrix Metalloproteinase and
ADAM Gene Expression in Human Chondrocytes by
Interleukin-1 and Oncostatin M
A Time-Course Study Using Real-Time Quantitative
Reverse Transcription–Polymerase Chain Reaction
P. J. T. Koshy,1
C. J. Lundy,2
A. D. Rowan,1
S. Porter,2
D. R. Edwards,2
A. Hogan,2
I. M. Clark,2
and T. E. Cawston1
Objective. Previous studies have reported elevated
levels of interleukin-1 (IL-1) and oncostatin M (OSM)
in rheumatoid joints, as well as the synergistic degra-
dation of human articular cartilage by this cytokine
combination. The present study was undertaken to
investigate the ability of IL-1 and OSM to modulate
gene expression of matrix metalloproteinase (MMP),
ADAM, and ADAM-TS (ADAM with thrombospondin
motifs) family members in human chondrocytes.
Methods. T/C28a4 human chondrocytes were
stimulated for 2–48 hours with IL-1 and/or OSM. Total
RNA was harvested, reverse transcribed, and assessed
by real-time polymerase chain reaction for the expres-
sion of various MMP, ADAM, and ADAM-TS messenger
RNAs (mRNA). Results were normalized to 18S ribo-
somal RNA.
Results. IL-1 and OSM synergized to markedly
induce the expression of the collagenases MMP-1,
MMP-8, and MMP-13 as well as MMP-3, an activator of
proMMPs. Expression of mRNA for MMPs 1, 3, and 13
was induced early, whereas that of MMP-8 mRNA
occurred late. Gene expression of MMP-14, an MMP
that degrades collagen and activates proMMP-13, was
elevated by this combination. IL-1 and OSM also syn-
ergized to induce gene expression of the aggrecanase
ADAM-TS4, but not ADAM-TS5.
Conclusion. These data indicate that the potent
cartilage-degrading properties of the combination of
IL-1 and OSM are potentially mediated by a synergistic
induction of the aggrecan-degrading enzyme ADAM-
TS4 and the collagen-degrading enzymes MMP-1,
MMP-8, MMP-13, and MMP-14, although differences
in the magnitude of response and in the time course of
induction were observed. A role for MMPs 3 and 14 in
the activation of proMMPs may also be implicated.
Articular cartilage is a highly specialized tissue
that covers the surface of synovial joints, allowing its
smooth articulation. Cartilage is synthesized and main-
tained by chondrocytes, and is composed primarily of
water, proteoglycan (principally aggrecan), and collagen
(1). Type II collagen fibrils are arranged to form a
fibrillar network within which are trapped aggrecan
molecules that pull water into the tissue. These two
components provide cartilage with its tensile strength
and the ability to resist compression. The degradation of
cartilage is one of the key features of the arthritides and
results in disability.
The degradation of cartilage in vitro and in vivo is
believed to be mediated by neutral endopeptidases of
the metalloproteinase class of enzymes (2). Two families
in this class that have been implicated are the matrix
metalloproteinase (MMP) and ADAM families (2,3).
Supported by the Arthritis Research Campaign and the
Medical Research Council of Great Britain.
1
P. J. T. Koshy, BSc, PhD, A. D. Rowan, BSc, PhD, T. E.
Cawston, BSc, PhD: University of Newcastle, Newcastle upon Tyne,
UK; 2
C. J. Lundy, BSc, PhD, S. Porter, BA, MSc, DipACC, D. R.
Edwards, BA, PhD, A. Hogan, BSc, PhD, I. M. Clark, BSc, PhD:
University of East Anglia, Norwich, UK.
Address correspondence and reprint requests to A. D.
Rowan, BSc, PhD, Department of Rheumatology, Cookson Building,
The Medical School, University of Newcastle, Framlington Place,
Newcastle upon Tyne NE2 4HH, UK. E-mail: A.D.Rowan@ncl.ac.uk.
Submitted for publication June 11, 2001; accepted in revised
form December 5, 2001.
961

3. Recently, two members of an ADAM-related family
known as ADAMs with thrombospondin motifs
(ADAM-TS) (4), ADAM-TS4 and ADAM-TS5 (initial-
ly named ADAM-TS11), were the first identified en-
zymes (5,6) possessing the ability to cleave aggrecan at
what was shown (7) to be the physiologically and patho-
logically relevant site. Collagen-degrading enzymes have
been identified within the MMP family. These include
the collagenases (MMP-1, MMP-8, and MMP-13), gela-
tinase A (MMP-2), and MMP-14 (a membrane-type
MMP) (8). MMPs are synthesized as proenzymes that
require proteolytic processing into the active form (2).
Important in this critical step may be activator enzymes
such as MMP-3 and MMP-14, which are able to activate
one or more of the procollagenases (8).
We previously reported that the combination of
interleukin-1 (IL-1) and oncostatin M (OSM) promotes
synergistic cartilage degradation, stimulating the repro-
ducible loss of proteoglycan and collagen from bovine,
porcine, and human cartilage ex vivo (9). In addition,
both IL-1 levels and OSM levels are elevated in rheu-
matoid arthritis (RA) synovial fluid (9,10). In this study,
real-time quantitative reverse transcription–polymerase
chain reaction (RT-PCR) was used to investigate the
ability of IL-1 in combination with OSM to modulate the
expression of MMP, ADAM, and ADAM-TS family
members in the T/C28a4 human chondrocyte cell line, in
order to identify mediators of IL-1/OSM–induced carti-
lage degradation.
MATERIALS AND METHODS
Recombinant human IL-1␣ and OSM were used as
previously described (9). Cytokines were stored at Ϫ70°C, and
were diluted into culture medium immediately prior to use.
Tissue culture reagents and all other chemicals have been
described previously (9).
Cell culture and RNA extraction. Human immortal-
ized chondrocytes (T/C28a4) (11) were grown to 70% conflu-
ence and serum-starved overnight as previously described (9).
Cells were stimulated with medium containing test reagents for
2–48 hours. Following stimulation, cells were lysed in RNeasy
lysis buffer (Qiagen, Crawley, UK) and total cellular RNA was
prepared according to the manufacturer’s instructions. Iso-
lated RNA was stored at Ϫ80°C until required for further
experiments.
Reverse transcription. Total RNA (1 ␮g) was reverse
transcribed in a 20-␮l reaction using 2 ␮g of random hexamers
and Superscript II reverse transcriptase (Life Technologies,
Paisley, UK) according to the manufacturer’s instructions.
Design of primers and probes for TaqMan PCR.
Oligonucleotide primers and fluorescent-labeled TaqMan
probes were designed using Primer Express 1.0 software
(Applied Biosystems, Warrington, UK). Sequences for the
MMP primers and probes are under the copyright of Applied
Biosystems. Sequences for the ADAM and ADAM-TS primers
and probes are shown below: ADAM-10 forward primer
5Ј-AGCGGCCCCGAGAGAGT-3Ј, reverse primer 5Ј-
AGGAAGAACCAAGGCAAAAGC-3Ј, probe 5Ј-FAM
(6-carboxy-fluorescein)–ATCAAATGGGACACATGAGAC-
GCTAACTGC–TAMRA (6-carboxy-N,N,NЈNЈ-tetramethyl-
rhodamine)–3Ј; ADAM-17 forward 5Ј-GAAGTGCCA-
GGAGGCGATTA-3Ј, reverse 5Ј-CGGGCACTCACTGCT-
ATTACC-3Ј, probe 5Ј-FAM-TGCTACTTGCAAAGGCGT-
GTCCTACTGC-TAMRA-3Ј; ADAM-TS1 forward 5Ј-
GGACAGGTGCAAGCTCATCTG-3Ј, reverse 5Ј-TCTAC-
AACCTTGGGCTGCAAA-3Ј, probe 5Ј-FAM-CAAGC-
CAAAGGCATTGGCTACTTCTTCG-TAMRA-3Ј; ADAM-
TS4 forward 5Ј-CAAGGTCCCATGTGCAACGT-3Ј, reverse
5Ј-CATCTGCCACCACCAGTGTCT-3Ј, probe 5Ј-FAM-
CCGAAGAGCCAAGCGCTTTGCTTC-TAMRA-3Ј; AD-
AM-TS5 forward 5Ј-TGTCCTGCCAGCGGATGT-3Ј, reverse
5Ј-ACGGAATTACTGTACGGCCTACA-3Ј; probe 5Ј-FAM-
TTCTCCAAAGGTGACCGATGGCACTG-TAMRA-3Ј.
In order to control against amplification of genomic
DNA and to ensure that the PCR signal was generated from
complementary DNA (cDNA), primers were placed within
different exons, close to intron–exon boundaries. BLASTN
searches were conducted on all primer/probe nucleotide se-
quences to ensure gene specificity. The identity of PCR
products was confirmed by direct sequencing of amplicons.
The 18S ribosomal RNA gene was used as an endogenous
control to normalize for differences in the amount of total
RNA in each sample. TaqMan 18S ribosomal RNA primers
and a 5Ј-VIC–labeled probe were used according to the
manufacturer’s instructions (Applied Biosystems).
TaqMan PCR. Relative quantitation of gene expres-
sion was performed using the Applied Biosystems ABI Prism
7700 sequence detection system (TaqMan). PCR reactions for
all samples were performed in duplicate in 96-well optical
plates using 5 ng of cDNA, 12.5 ␮l 2ϫ TaqMan Universal PCR
mastermix (Applied Biosystems), 100 nM probe, 200 nM of
each primer, and water to a 25 ␮l final volume. Thermocycler
conditions comprised an initial holding at 50°C for 2 minutes,
then 95°C for 10 minutes. This was followed by a 2-step
TaqMan PCR program consisting of 95°C for 15 seconds, and
60°C for 60 seconds for 40 cycles.
Statistical analysis. During PCR, the TaqMan probe
emits a fluorescence signal that increases in intensity in direct
proportion to the amount of specific amplified product. The
ABI Prism 7700 instrument measures the cycle-to-cycle
changes in fluorescence in each sample. Data are initially
expressed as a threshold cycle (CT), defined as the point at
which an increase in fluorescence above a baseline signal can
first be detected. The fewer cycles it takes to reach the CT, the
greater the initial template copy number. The CT values
generated were used to calculate relative input amounts of
template cDNA, using the standard curve method as described
by the manufacturer (1997 User Bulletin no. 2; Applied
Biosystems). The input cDNA levels measured corresponded
directly to the levels of RNA reverse transcribed. Thus, data
are presented graphically as relative levels of messenger RNA
(mRNA) for each primer/probe set. Direct comparisons be-
tween levels cannot be made between different primer/probe
sets.
962 KOSHY ET AL

4. RESULTS
Effect of IL-1␣ in combination with OSM on the
expression of MMP genes. The expression of the colla-
genases MMP-1, MMP-8, and MMP-13 was highly in-
duced in T/C28a4 human chondrocytes following stimu-
lation with IL-1␣ in combination with OSM (Figure 1).
This cytokine combination also highly induced MMP-3
mRNA expression (Figure 2A). Maximal induction of
mRNA for MMPs 1, 3, and 13 was observed early (8–12
hours), whereas MMP-8 mRNA was induced late (max-
Figure 1. The effect of interleukin-1 (IL-1) and oncostatin M (OSM) on the gene expression of
matrix metalloproteinase 1 (MMP-1), MMP-8, and MMP-13 in human chondrocytes. T/C28a4
chondrocytes were treated for 2–48 hours with medium alone (control) (diagonally hatched bars),
IL-1␣ (1 ng/ml) (open bars), OSM (10 ng/ml) (horizontally hatched bars), and IL-1␣ (1 ng/ml) in
combination with OSM (10 ng/ml) (solid bars). Following treatment, total RNA was isolated from
cells and reverse transcribed, and the resulting cDNA was used in separate real-time quantitative
polymerase chain reaction assays with specific primers and probes for MMP-1 (A), MMP-8 (B), and
MMP-13 (C). Results were normalized to 18S ribosomal RNA and presented graphically as relative
mRNA levels (see Materials and Methods).
MODULATION OF MMP, ADAM, AND ADAM-TS IN CHONDROCYTES BY IL-1 AND OSM 963

5. imal at Ն48 hours) (Figures 1A–C and 2A). A smaller
synergistic induction of MMP-14 was also observed
following IL-1␣/OSM stimulation (Figure 2C). Expres-
sion of mRNA for MMPs 1, 3, 8, 13, and 14 was also
induced by IL-1␣ alone (Figures 1 and 2). In addition,
MMP-9 gene expression was induced by IL-1␣, but both
basal and IL-1␣–induced levels were inhibited by the
addition of OSM (Figure 2B). No clear modulation of
MMP-2, MMP-15, MMP-16, or MMP-17 gene expres-
sion was observed (data not shown).
Figure 2. The effect of IL-1 and OSM on the gene expression of MMP-3, MMP-9, and MMP-14
in human chondrocytes. T/C28a4 chondrocytes were treated for 2–48 hours with medium alone
(control) (diagonally hatched bars), IL-1␣ (1 ng/ml) (open bars), OSM (10 ng/ml) (horizontally
hatched bars), and IL-1␣ (1 ng/ml) in combination with OSM (10 ng/ml) (solid bars). Following
treatment, total RNA was isolated from cells and reverse transcribed, and the resulting cDNA was
used in separate real-time quantitative polymerase chain reactions with specific primers and probes
for MMP-3 (A), MMP-9 (B), and MMP-14 (C). Results were normalized to 18S ribosomal RNA
and presented graphically as relative mRNA levels (see Materials and Methods). See Figure 1 for
definitions.
964 KOSHY ET AL

6. Figure 3. The effect of IL-1 and OSM on the gene expression of ADAM-10 and ADAM with
thrombospondin motifs family members (ADAM-TS1, ADAM-TS4, and ADAM-TS5) in human
chondrocytes. T/C28a4 chondrocytes were treated for 2–48 hours with medium alone (control)
(diagonally hatched bars), IL-1␣ (1 ng/ml) (open bars), OSM (10 ng/ml) (horizontally hatched
bars), and IL-1␣ (1 ng/ml) in combination with OSM (10 ng/ml) (solid bars). Following treatment,
total RNA was isolated from cells and reverse transcribed, and the resulting cDNA was used in
separate real-time quantitative polymerase chain reactions with specific primers and probes for
ADAM-10 (A), ADAM-TS1 (B), ADAM-TS4 (C), and ADAM-TS5 (D). Results were normalized
to 18S ribosomal RNA and presented graphically as relative mRNA levels (see Materials and
Methods). See Figure 1 for definitions.
MODULATION OF MMP, ADAM, AND ADAM-TS IN CHONDROCYTES BY IL-1 AND OSM 965

7. Effect of IL-1␣ in combination with OSM on the
expression of ADAM and ADAM-TS genes. ADAM-10
mRNA expression appeared to be moderately up-
regulated by IL-1␣/OSM between 12 hours and 24 hours
poststimulation (Figure 3A). No clear modulation of
ADAM-17 gene expression was observed (data not
shown). A minor, but consistent, induction of ADAM-
TS1 mRNA was seen following IL-1␣ stimulation be-
tween 12 hours and 24 hours, but was not enhanced by
costimulation with OSM (Figure 3B). IL-1␣ alone and
OSM alone failed to increase ADAM-TS4 gene expres-
sion, but these cytokines in combination synergized to
up-regulate its expression over 4–24 hours (Figure 3C).
IL-1␣ enhanced ADAM-TS5 expression over 4–48
hours; however, the addition of OSM appeared to
partially inhibit this increase (Figure 3D).
DISCUSSION
Aggrecan and collagen loss from cartilage results
in the loss of functionality of the tissue and joint
disability. It is therefore important to identify the en-
zymes that are potentially involved in the turnover of
these matrix components, and the cytokines that regu-
late the production of these proteases. We have previ-
ously reported that IL-1 and OSM, both present at
elevated levels in RA synovial fluid (9,10), are a potent
combination in promoting cartilage degradation (9). In
this study, we have used real-time PCR to investigate the
ability of IL-1 in combination with OSM to modulate the
gene expression of aggrecan- and collagen-degrading
enzymes, as well as that of other family members, in the
T/C28a4 human chondrocyte cell line. The T/C28a4 cell
line has previously been characterized for its use as a
model of primary chondrocytes (11). Consistent with the
data reported in the present study, we have previously
shown qualitatively by Northern blotting that IL-1/OSM
synergistically induced the expression of MMP-1 mRNA
in both primary human articular chondrocytes and the
T/C28a4 cell line (12).
The present study clearly demonstrates the abil-
ity of IL-1 and OSM to synergize to potently induce the
mRNA expression of the collagenases MMP-1, MMP-8,
and MMP-13. Interestingly, there appeared to be a
differential regulation of these genes with respect to
time. Expression of mRNA for MMPs 1 and 13 in-
creased early, rapidly peaking at ϳ12 hours before
decreasing slowly. These results using real-time RT-
PCR are consistent with previous data, in which time-
course studies assessing MMP-1 expression have quali-
tatively shown by Northern blotting that IL-1/OSM–
induced MMP-1 expression had clearly decreased by 48
hours following a maximal increase between 8 hours and
24 hours (12). In contrast with the early induction of
MMP-1 and MMP-13 shown in the present study,
MMP-8 mRNA levels were induced slowly and were still
increasing by 48 hours. These data may implicate
MMP-1 and MMP-13 in the early phase of chondrocyte-
mediated cartilage collagen breakdown, with MMP-8
having a significant role at a later stage. These observa-
tions suggest similarities, but also real differences, in the
way these genes are regulated.
MMPs are synthesized as proenzymes that re-
quire activation (2). Thus, the marked induction of
MMP-3 mRNA, concurrent with that of MMP-1 and
MMP-13 and prior to that of MMP-8 mRNA, may be of
significance since this enzyme is able to activate pro-
forms of all 3 collagenases (8). In addition, the gene
expression of MMP-14, an enzyme shown to possess
both collagen-degrading activity and the ability to acti-
vate proMMP-13 (8), was moderately up-regulated by
the combination of IL-1 and OSM. MMP-14 is a mem-
ber of the membrane-type MMPs that can undergo
activation within the Golgi by furin-type enzymes during
cellular export. Thus, it can be secreted and expressed at
the cell surface in the active form (13). MMP-14 may,
therefore, play an important role in IL-1/OSM–induced
cartilage destruction, either directly via pericellular col-
lagen degradation or indirectly via the activation of
procollagenases.
IL-1 alone was also able to up-regulate gene
expression of MMPs 1, 3, 8, 13, and 14. However,
costimulation with OSM resulted in the synergistic in-
duction of these mRNA. In contrast, whereas IL-1
stimulated MMP-9 mRNA expression, OSM inhibited
both basal and IL-1–induced expression. IL-1/OSM
failed to modulate the gene expression of the gelatinase
MMP-2 and that of the membrane type MMPs, MMP-
15, MMP-16, and MMP-17. The marked differences
observed in the effect of IL-1 and OSM on the transcrip-
tional regulation of the MMP family suggest that a
comparison of the respective gene promoter regions may
be revealing. For example, previous studies have shown
significant differences between the promoters of the
highly inducible MMP-1 and the less responsive MMP-2
gene (see ref. 14 and references therein). It is uncertain,
however, what effect this cytokine combination has on
the posttranscriptional regulation of these genes.
The effect of IL-1 and OSM on the regulation of
genes encoding the aggrecan-degrading enzymes
ADAM-TS4 and ADAM-TS5 (5,6) was also investi-
gated. Our data show that although IL-1 alone failed to
966 KOSHY ET AL

8. increase ADAM-TS4 mRNA levels, the combination of
IL-1 and OSM synergistically induced the expression of
this gene. In contrast, ADAM-TS5 expression was ele-
vated by IL-1, but IL-1–induced expression was partially
inhibited by OSM. This differential regulation may be
explained by a close examination of the promoter re-
gions of these genes. These data perhaps implicate
ADAM-TS4 as the aggrecanase responsible for the
synergistic degradation of aggrecan that occurs following
IL-1/OSM stimulation. ADAM-TS5, however, may be
important in IL-1–induced aggrecan loss, and may also
contribute to this process in IL-1/OSM–stimulated car-
tilage. Compared with ADAM-TS4 and -TS5, ADAM-
TS1 mRNA regulation showed an intermediate profile.
Its expression was induced by IL-1 but was not further
modulated by the addition of OSM. ADAM-10, but not
ADAM-17, mRNA levels were moderately increased
following IL-1/OSM treatment. ADAM-10 is known to
possess the ability to process pro–tumor necrosis factor
␣ (TNF␣) to its active form (15). Thus, the generation of
active TNF␣ may further promote a proinflammatory
cycle of events.
Several limitations of this type of study must be
noted. First, the results show relative levels of mRNA
expression and thus, although the effect of different
treatments on the levels of expression of a given gene
can be assessed, comparisons between the levels of
different mRNA cannot be made. For example, the
levels of one collagenase mRNA may be far in excess of
another, even though both are equally inducible by
proinflammatory cytokines. For a determination of ab-
solute levels of mRNA, standards for all mRNA would
be required. Second, mRNA expression does not neces-
sarily reflect protein expression and activity. However,
despite the limitations, mRNA measurements are po-
tentially a good indicator of important downstream
mediators of the processes under investigation.
In summary, this study has made the following
novel observations. First, a marked differential regula-
tion of the genes assessed was demonstrated. This was
observed with respect to differences in gene inducibility
(e.g., MMP-1ϾMMP-14ϾMMP-2), temporal expression
(e.g., MMP-8 versus MMP-1 and MMP-13), and respon-
siveness to cytokine stimulation (e.g., ADAM-TS4 ver-
sus ADAM-TS5). Comparative studies of these gene
promoters are in progress to identify the mechanisms
underlying the apparent differences in the transcrip-
tional regulation of these genes. Second, this study has
identified a number of potential mediators of IL-1/OSM–
induced cartilage destruction. All 3 human collagenases
MMP-1, MMP-8, and MMP-13, as well as MMP-14, may
be implicated in IL-1/OSM–induced collagen degradation,
while a role for ADAM-TS4 was suggested in the break-
down of aggrecan. The data also suggested that MMPs 3
and 14 and ADAM-10 may be important in IL-1/OSM–
induced cartilage breakdown, perhaps via the activation of
proMMPs and proTNF␣, respectively.
Thus, the combination of IL-1 and OSM, both of
which are up-regulated in the rheumatoid joint, has been
shown to induce the chondrocyte expression of genes
encoding enzymes that can degrade the principal com-
ponents of articular cartilage.
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MODULATION OF MMP, ADAM, AND ADAM-TS IN CHONDROCYTES BY IL-1 AND OSM 967