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
V79C cells were derived from V79 cell line by through chronic oxidative stress that was found to be radio-resistant. These cells had demonstrated transformation–like stable changes and could be used as model system to study oxidant induced carcinogenesis. Our objective was to understand mechanism of radiation-resistance in these cells. Apoptotic cell death was inhibited in these cells as visualized microscopically by Hoechst staining and nucleosomal ladder formation in agarose gel. Release of cytochrome c in cytoplasm and Apoptosis Inducing Factor in the mitochondrial and nuclear fraction of cells were determined by Western blotting. The caspase 9 and caspase 3 activities in these cells were estimated from fluorimetric assays. These results revealed that the radiation resistance was due to inhibition of caspase-dependent apoptotic death pathways although Apoptosis Inducing Factor mediated pathway remained unaffected. These findings may aid in understanding the mechanism of radiation resistance in tumors arising from oxidative stress.
Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
V79C cells were derived from V79 cell line by through chronic oxidative stress that was found to be radio-resistant. These cells had demonstrated transformation–like stable changes and could be used as model system to study oxidant induced carcinogenesis. Our objective was to understand mechanism of radiation-resistance in these cells. Apoptotic cell death was inhibited in these cells as visualized microscopically by Hoechst staining and nucleosomal ladder formation in agarose gel. Release of cytochrome c in cytoplasm and Apoptosis Inducing Factor in the mitochondrial and nuclear fraction of cells were determined by Western blotting. The caspase 9 and caspase 3 activities in these cells were estimated from fluorimetric assays. These results revealed that the radiation resistance was due to inhibition of caspase-dependent apoptotic death pathways although Apoptosis Inducing Factor mediated pathway remained unaffected. These findings may aid in understanding the mechanism of radiation resistance in tumors arising from oxidative stress.
Activation of surrogate death receptor signalling triggers peroxynitrite depe...Saurabh Shekhar
includes information about cisplatin resistance cancer cells and their execution through peroxynitrite triggered apoptosis due to death signaling receptors basedon the findings of research article published in cell death and diseases.
Exchange traded forwards for Large Processors & Corporate BuyersNCDEX Ltd.
Challenges Faced by Large Processors & Corporate Buyers
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Sur RxJava, on trouve facilement des guides pour démarrer, et la documentation de référence couvre bien l'ensemble des opérateurs. Mais les exemples d'utilisations concrètes sont plus rares. Dans cette présentation, vous apprendrez comment Hawkular Metrics utilise RxJava pour implémenter:
- une interface REST avec JAX-RS
- le stockage des métriques dans Apache Cassandra
- les calculs d'aggrégats
- l'interaction avec les autres composants Hawkular via JMS
Power Brands – award winning beverage industry experts who can help you develop a beverage and marketing strategy for your new or existing beverage brand. Considered by many to be the leading beverage consultant in the industry, Power Brands can help you understand your beverage development, manufacturing and marketing needs. With an expert beverage consultant at your side, you will be better equipped for success in the ever expanding beverage business. Contact us and talk to a beverage consultant now, our premier consulting services can help you grow your beverage business. The initial call is free and will help you to better understand your options.
Determination and comparison rate of expression markers of osteoblast derived...IJERD Editor
Nowadays high accident rates, fractures leading to permanent bone disorders and the impossibility of bone transplant have made scientists to look for new methods of repairing injured bones. Considering the application of stem cells in bone tissue engineering, there exists the necessity to investigate various culture methods and suitable fields and scaffolds. Thus, we decided to induce adipose-derived stem cells into osteoblast cells in two systems of pellet culture and monolayer and compare osteogenic markers. Methods: Stem cells have been separated via mechanical and enzymatic methods and cultured in monolayer and pellet culture models with osteogenic medium. Then, RNA was separated from differentiated cells, complementary DNA (cDNA) was synthesized and amplified. Polymerase chain reaction (PCR) product was transferred to electrophoresis gel. The intensity of the bands was measured by Image-J software and analyzed by SPSS.
ABSTRACT- Coronary artery disease (CAD) is suspected as a leading cause of mortality in developed countries. Due
to cholesterol and fat deposit plaque is forming into the inner walls of the arteries of the heart, which leads to narrowing
of blood vessels of heart and reduce the blood flow rate into heart. Proprotein convertase subtilisin-like kexin type 9
(PCSK9) is one of the candidate gene that regulate lipoprotein retention pathway of CAD development. It is a newly
discovered serine protease that plays a key role in LDL-C homeostasis by mediating LDL receptor (LDLR). The LDL
receptor is breakdown through a post transcriptional mechanism and induces the production of very low-density
lipoprotein in the fasting state. The aim of this study was to investigate the frequency of single nucleotide
polymorphism (SNP) of PCSK9 gene of 155 CAD patients and 102 ages matched healthy controls. Serum lipids
including total cholesterol (TC), triglycerides (TG), HDL, LDL, and VLDL were analyzed. PCR-RFLP analysis was
carried out to genotype regions carrying Eam 1104I restriction site in the PCSK9. Gene considering significant
difference in serum TC, TG, HDL-C, LDL-C and VLDL-C levels (P<0.001, <0.0001) of patients and control samples.
In CAD patients, G allele frequency is less than A allele frequency. G allele is responsible for decreasing the
LDL: HDL ratio which shows evidence in having its protecting effect on the occurrence of CAD in West Bengal Population.
Key-words- CAD, PCSK9, SNP, Eam1104I, Polymorphism, West Bengal population
Models of Human Diseases Conference (2010) Tetrahymena model by Dr. R. Pearl...Medical Education Advising
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Dr. R.E. Pearlman, York University
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Targeted RNA Sequencing, Urban Metagenomics, and Astronaut GenomicsQIAGEN
Dr. Chris Mason’s presentation will cover the QIAseq Targeted RNA Panel beta testing, Qiagen’s Sample-to-Insight solution for Metagenomics sequencing as demonstrated in the Metasub project, as well as Chris latest endeavor the, “Twins Study”, which will be a comprehensive genomic study of the impact of spaceflight on the human body. Astronaut Scott Kelly will be studied during the course of his year-long stay at the ISS as will his twin brother, Mark Kelly, a former NASA astronaut who will remain on earth.
http://www.npr.org/sections/thetwo-way/2015/03/27/395536140/nasa-to-study-a-twin-in-space-and-his-brother-on-earth
DNA damage repair Neil3 gene Knockout in MOLT-4iosrjce
RNAi is superannuated cellular mechanism that protect organism against viruses that replicate
through double- stranded RNA. RNAi can be used to diminish gene expression from plasmid expressing and
inserted sequence repeat. A stable harpin would be expressed after the vector was integrated into the genome.
In this paper a shiRNA expressing vector for Neil3 was designed and developed which is capable of replication
in MOLT-4. This shiRNA vector had the ability to arose the RNAi pathway, and reduce the gene expression of
Neil3. This was assessed by using pSilence 4.1CMV as a vector, and Gapdh as positive control.
Human mesenchymal stem cells (MSC) are important tools for several cell-based therapies. However, their
use in such therapies requires in vitro expansion during which MSCs quickly reach replicative senescence. Recent studies
on the other hand, have implicated telomerase in the cellular response to oxidative damage, suggesting that telomerase has
a telomere-length independent function that promotes survival. Here, we studied the DNA damage accumulation
and repair during in vitro expansion as well as after acute external oxidative exposure of control MSCs and MSCs that
overexpress the catalytic subunit of telomerase (hTERT MSCs). We showed that hTERT MSCs at high passages
have a significant lower percentage of DNA lesions as compared to control cells of the same passages. Additionally, less damage was accumulated due to external oxidative insult in the nuclei of hTERT overexpressing cells as compared to the
control cells. Moreover, we demonstrated that oxidative stress leads to diverse nucleus malformations, such as multillobular
nuclei or donut-shaped nuclei, in the control cells whereas hTERT MSCs showed significant resistance to the formation
of such defects. On the basis of these results, we propose that hTERT enhancement confers resistance to genomic damage due to the amelioration of the cell’s basic antioxidant machinery.
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