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Transforming growth factor exerts opposite effects from interleukin-1 on cultured rabbit articular chondrocytes through reduction of interleukin-1 receptor expression.

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44
TRANSFORMING GROWTH FACTOR p EXERTS
OPPOSITE EFFECTS FROM INTERLEUKIN-1P ON
CULTURED RABBIT ARTICULAR CHONDROCYTES
THROUGH REDUCTION OF
INTERLEUKIN-1 RECEPTOR EXPRESSION
F. REDINI, A. MAUVIEL, S. PRONOST, G. LOYAU, and J.-P. PUJOL
Objective. The aim of the study was to determine
whether transforming growth factor P (TGFP) modulates the effects and the receptor expression of interleukin-1 (IL-I) in rabbit articular chondrocytes (RAC).
Methods. Collagen, glycosaminoglycan, and collagenase production, together with '251-labeled IL-1
binding, were analyzed in RAC cultures.
Results. TGFP reduced both IL-1 effects on
matrix metabolism and IL-1 receptor expression.
Conclusion. TGFP acts as an antagonist of the
effects of IL-1through down-regulation of its receptor
expression.
Interleukin-1 (IL-1) has been shown to exert
deleterious effects on cartilage matrix, promoting metalloprotease release (1) and inhibiting collagen and
proteoglycan synthesis (2,3). Its role in joint destruction such as that in osteoarthritis and rheumatoid
arthritis has been largely documented. By contrast,
transforming growth factor p (TGFp) appears to be a
potential candidate for stimulating matrix formation,
and therefore producing effects that are opposite to
those of IL-1 (4). Because of its presence in bone and
cartilage (5,6), TGFp could play a role in the process
of repair of cartilage matrix. Indeed, TGFP has been
shown to increase the expression of most of the
From the Laboratoire de Biochimie du Tissu Conjonctif,
Service de Rhumatologie, CHU Cate de Nacre, Caen, France.
Supported by INSERM (CRE 900706 and CJF 91-06).
F. RCdini, PhD; A. Mauviel, PhD (current address: Department of Dermatology, Thomas Jefferson University, Philadelphia,
PA); S. Pronost, PhD Student; G. Loyau, MD; J.-P. Pujol, PhD.
Address reprint requests to J.-P. Pujol, PhD, Laboratoire
de Biochimie du Tissu Conjonctif, CHU CBte de Nacre, Niv 3,
14033 Caen Ctdex, France.
Submitted for publication February 4, 1991; accepted in
revised form August 25, 1992.
Arthritis and Rheumatism, Vol. 36, No. 1 (January 1993)
extracellular matrix components in a number of cell
types, including articular chondrocytes (7), and is also
able to inhibit the synthesis of metalloproteases while
enhancing that of the TIMP (tissue inhibitor of metalloproteases) (8).
Given that IL-1 and TGFp are likely to act in
concert as parts of a cytokine network which modulates chondrocyte activity, it is of interest to study
their effects on in vitro cultures when they are added in
various combinations. Here, we show that TGFp
overrides the effects of IL-lP when introduced in
rabbit articular chondrocyte (RAC) cultures either
before or together with the monokine. Since there is
evidence that TGFP could modulate the effects of
cytokines such as IL-1 at the level of receptor expression (9,10), we determined whether this mechanism
could play a role in the effects observed in our experimental model.
MATERIALS AND METHODS
IL-IP and TGFP. Human platelet-derived TGFPl
was obtained from R & D Systems (Minneapolis, MN) and
human recombinant IL-1P was a gift from Dr. D. Lando,
Roussel-Uclaf (Romainville, France).
Chondrocyte culture. Chondrocytes were prepared
from 3-week-old rabbits (1 1) and cultured in Dulbecco's
modified Eagle's medium (DMEM) containing 10% heatinactivated fetal calf serum (FCS), at 37"C, in a 5% CO,
environment. The experiments were performed on primary
cultures to avoid dedifferentiation, using a seeding density of
2 x lo' cells/cm*. By the end of the experiments (6-7 days),
the cultures had reached confluency, and no significant
variation in cell number could be observed at the moment of
final labeling.
Binding assay with 'zI-labeled IL-I@. Confluent cultures in 2-cm2 dishes, with and without exposure to TGFP (5
ng/ml) for 3 days, were washed once with DMEM plus 1%
TGFP REDUCES IL-1R EXPRESSION ON RAC
FCS and twice with binding medium (DMEM, 0.1% bovine
serum albumin [BSAI, and 25 mM HEPES, pH 7.5). Cultures were then incubated at 4°C with various concentrations
of '251-labeled I L - l p (specific activity 79.2 pCilpg; New
England Nuclear, Boston, MA) in 0.2 ml (final volume).
Nonspecific binding was determined in the presence of a
50-fold excess of unlabeled ligand. Optimal binding of -1'2
labeled IL-1p was observed at 4 hours.
At the end of the incubation period, the free ligand
was removed, and the cell layer was extensively washed
with binding medium. The bound material was then extracted with lysis buffer (phosphate buffered saline, 25 mM
HEPES, 0.1% BSA, 10% glycerol, and 1% Triton X-100,pH
7.5) at 0.5 ml per dish. Scatchard analysis was performed
with the EBDNLIGAND computer program.
Cross-linking experiments. The 75-cm2 flasks were
incubated with 800 pM '2SI-labeled IL-1p and washed as
above. Binding buffer (5 ml; 40 mM HEPES, 128 mM NaCI,
5 mM KCl, I .2 mM CaCl,, 5 mM MgSO,, 5 mg/ml BSA, pH
7.4) was added, and the cells were incubated with 50 mM
disuccinimidyl suberate (DSS) for 15 minutes at 4"C, under
gentle rotatory shaking. The cells were then scraped in
0.25M sucrose, 10 mM Tris HC1, 1 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride (PMSF), and 10 &ml leupeptin, pH 7, pelleted at 5,OOOgfor 10 minutes, and lysed in cell
solubilization buffer (10 mM Tris HCI, 1 mM EDTA, 1%
Triton X-100, 1 mM PMSF, 50 pg/ml leupeptin, and 100
p g h l trypsin inhibitor, pH 7).
The cell debris was removed by centrifugation at
10,OOOg for 10 minutes. Supernatants were concentrated to
70 pl under speed-vac and mixed to an equal volume of
twice-concentrated electrophoresis sample buffer (12),
heated at 100°C for 2 minutes, and used for electrophoresis.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. SDS-polyacrylamide gels (6%) were prepared and run according to the
method of Laemmli (12). Gels were vacuum-dried, exposed
to Kodak AR-XOmat film (Eastman Kodak, Rochester,
NY), and stored for 1 4 weeks at -70°C.
Assay of newly synthesized collagen. 3H-proline labeling was done on day 6 of the experiments, under the same
protocol as above, using 2-cm2 dishes. The medium used
was 0.5 ml of DMEM plus 10% FCS containing 100 pg/ml
ascorbic acid, 50 pg/ml /3-aminopropionitrile, and 2 pCi/ml
of 3H-proline (20-30 Ci/mmole; CEA Saclay, France). After
24 hours, the labeled collagen was quantified in both the
medium and the cell-layer fractions as collagenase-digestible
material (2).
Assay of the newly synthesized glycosaminoglycans
(GAG). The same culture protocol was used for these
studies, except that 2 pCi/ml of "S-sulfate (25 Ci/mg;
Amersham, Buckinghamshire, UK) was used. Medium and
cell-layer fractions were assayed for labeled GAGS using
pronase digestion and precipitation with cetylpyridinium
chloride, as previously described (11).
Assay of collagenolytic activity. For these experiments, medium was replaced on day 5 with 2% FCScontaining DMEM for 16 hours, and then with serum-free
medium for 24 hours. Collagenase activity was assayed on
trypsin-activated medium, using 3H-acetylated collagen
(kindly provided by Dr. C. Lafuma, Laboratoire de Biochimie du Tissu Conjonctif, Creteil, France) (13).
45
Cell-layer protein assay. Parallel dishes treated in the
same way were used to quantify the cell-layer protein
content after solubilization with O.2M NaOH (14).
Statistical analysis. All the results presented are the
mean 5 SD of 3 experiments. Each experiment was performed on triplicate or quadruplicate samples. Student's
r-test was used to evaluate the differences between the
means of each group.
RESULTS
TGFP and IL-IP concentrations (1-25 ng/ml
and 500 pg/ml, respectively) were chosen based on
previous studies from our laboratory showing that 500
pg/ml IL-1p markedly enhanced matrix degradation
and inhibited matrix biosynthesis, and that 1-25 ng/ml
of TGFP stimulated collagen and GAG production by
more than 100% in cultured RAC (7,ll).
TGFP prevention of the effects of IL-lP on
matrix metabolism by RAC. Collagen biosynthesis. As
can be seen in Figure lA, when IL-1p was added to the
cells for the last 3 days of culture, following a previous
3-day incubation in control medium, the collagen
production was significantly reduced, as we have
reported previously (2). Addition of TGFP alone (5
ng/ml) to the cultures from day 1 to day 4 induced a
slight, but not significant, increase in collagen biosynthesis. This suggests that the stimulative effect of
TGFP on RAC collagen synthesis previously observed
in 24-hour incubations (7) does not persist up to 7 days
when the factor is present in the first 4 days and then
removed from culture.
Pretreatment of the cells with TGFP over the
first 3-day period prior to incubation with IL-lp alone
led to enhancement of total 3H-proline incorporation
into newly synthesized collagen, by 243%, 326%, and
420%, respectively, for 1,5, and 25 ng/ml of TGFp, as
compared with cultures with IL-lp alone. The collagen synthesis was also stimulated compared with
control cultures containing only culture medium, by
51% and 86%, respectively, for 5 and 25 ng/ml of
TGFP.
Glycosaminoglycan biosynthesis. Incubation of
RAC with IL-lP during the last 3-day period caused a
marked decrease in proteoglycan synthesis, by 38%
(Figure IB). When TGFP alone was added to the cell
cultures for the first 3-day period, no significant increase in total GAG synthesis was seen at the end of
the 7-day period. This confirms the notion that the
continuous presence of TGFp is required for stimulation to be present at the end of the incubation period.
When the cells were first treated with TGFP for
3 days, followed by IL-lp during the second incuba-
REDINI ET AL
46
-
...
h
... ...
Y
-
..... .
I
_.
u
day
1-4
day
4-7
0
1
5 25
- - -
-
0
1
+
+
5 2 5 TGF-O (nglml)
+ + IL-1B (500 pg/ml)
Figure 1. Effect of TGF-P pretreatment on IL-l-induced effects on
RAC matrix metabolism. A, Collagen synthesis was measured in
RAC cultures which were incubated (24 hours after seeding) for 3
days in 1 ml of 10% FCS-containing DMEM (control medium), with
or without TGF-p (1-25 nglml). The medium was removed, and 1 ml
of control medium with or without I L - l p (500 pg/ml) was added for
another 3-day incubation. On day 6, paminopropionitrile (SO pg/
ml), ascorbic acid (100 pglml), and 2 pCi/ml 3H-proline were added
for the last 24 hours, and the amount of radiolabeled collagen was
measured as described in Materials and Methods. B, GAG synthesis
was measured in RAC cultures, as in A, except that on day 6, 2
pCi/ml '%-sulfate was added for the last 24 hours, and the amount
of radiolabeled GAG was measured. C, Collagenase secretion was
measured in RAC cultures, as in A, except that the medium was
replaced on day 5 with 2% FCS-containing DMEM for 18 hours and
then with serum-free medium for 24 hours, and the collagenase
activity in the culture medium was determined. Values are the mean
and SEM of quadruplicate dishes.
= P < 0.01 versus controls;
= P < 0.001 versus controls; "' = P < 0.001 versus
IL-l-stimulated test samples. See Figure 2 for definitions.
+++
+*
tion period, the production of GAG was higher than
that of the cultures treated with IL-lP alone over the
last 3 culture days. In that case, the amounts of
radioactive GAG were increased by 48%, 188%, and
224%, respectively, for 1,5, and 25 ng/ml of TGFP, as
compared with IL-1P alone.
Collagenase assay. As expected, the collagenolytic activity present in the medium of cultures treated
with IL-lP for the last 3 days of the experiment was
markedly increased, by 250% (Figure 1C). When the
cells were first treated with TGFP for another 3 days
and then with IL-1P for 3 days, the collagenolytic
activity was reduced, in a dose-dependent manner,
compared with cultures incubated with IL-1P alone
(-44% and -85%, respectively, for 5 and 25 ng/ml of
TGFP). For the highest concentration of TGFP used,
collagenolytic activity decreased to control levels. The
presence of TGFP alone for the first 3-day period did
not modify the collagenase levels present in the culture
medium.
Opposite effects of IL-lP and TGFP on matrix
metabolism by RAC. Another set of experiments was
carried out to determine whether TGFP can exert
effects opposite to those of IL-lp when added alone
after the cytokine or together with IL-IP during the
last 3-day period.
Collagen biosynrhesis. The experimental protocol for radiolabeling was the same as that for the first
set of experiments. No modification of collagen synthesis could be detected when IL-1P was added to the
cultures for the first 3 days of the experiment and then
replaced by control medium (Figures 2 A, I and A, 11).
The synthesis of collagen was not altered at the end of
the 3-day culture with IL-1P (not shown). The addition
of TGFP alone during the last 3 days led to increased
'H-proline incorporation into the newly synthesized
collagen, by 17%, 66%, and 53%, respectively, for 1,
5 , and-25 ng/ml of TGFP, as compared with control
cultures (Figure 2 A, I).
Since TGFP alone, without IL-1 preincubation,
causes a dose-dependent increase in collagen biosynthesis when added to the cells for the last 3-day period
(17%, 34%, and 49%, respectively, for 1, 5, and 25
ng/ml), this experiment may simply reflect the effects
of TGFP alone. 1L-1 itself had no effect under these
conditions.
Long-term treatment (6 days) with IL-lp induced an important inhibition of total collagen synthesis, by 71% (Figure 2 A, 11). The presence of TGFP
together with IL-1P in the last 3 days completely
reversed the effect of the cytokine at concentrations of
5 and 25 ng/ml, an increase of 51% and 58%, respectively, compared with control cultures and 403% and
TGFp REDUCES IL-1R EXPRESSION ON RAC
47
1
U
c, 1
...
1-4
- - - -
4-7
0
1
5
25
*
*
+
0
1
5 25
+
Figure 2. Effect of transforming growth factor P (TGF-P) added after (I) or together with (11) interleukin-I (IL-I), on rabbit articular
chondrocyte (RAC) matrix metabolism. A, Collagen synthesis was measured in RAC cultures which were treated (24 hours after seeding) with
IL-1 (500 pg/ml) for 3 days and with TGF-P (1-25 ng/ml) for the last 3 days, or for 6 days with 1L-1 and for the last 3 days with TGF-/3 + IL-1.
On day 6, paminopropionitrile (50 d m l ) , ascorbic acid (100 pg/ml) and 2 pCi/ml .)H-proline were added for a 24-hour incubation. The amount
of radiolabeled collagen was measured as described in Materials and Methods. B, Glycosaminoglycan (GAG) synthesis was measured in RAC
cultures, as in A, except that on day 6, 2 pCi/ml "S-sulfate was added for a 24-hour incubation, and the amount of radiolabeled GAG was
measured. C , Collagenase production was measured in RAC cultures, as in A, except that the medium was replaced on day 5 with 2% fetal calf
serum (FCShontaining Dulbecco's modified Eagle's medium (DMEM) for 18 hours and then with serum-free medium for 24 hours, and the
collagenase activity in the culture medium was determined. Values are the mean and SEM of quadruplicate dishes.
= P < 0.01 versus
controls;
= P < 0.001 versus controls; "' = P < 0.001 versus appropriate IL-1-stimulated test samples.
+++
+
48
427%, respectively, compared with IL-lp-treated
cells. The concentration of 1 ng/ml of TGFp was able
only to counteract the effect of IL-Ip (Figure 2 B, 11).
Glycosaminoglycan biosynthesis. When chondrocyte cultures were first treated with IL-1p for 3
days and then re-fed with control medium for 3 additional days, the 35S-sulfate radioactivity incorporated
into GAG during the last 24 hours was not significantly
af€ected (Figure 2 B, I). Treatment of the cells with
increasing concentrations of TGFP after IL-lp led to a
marked augmentation of the GAG synthesis, by 59%,
60%, and 75%, respectively, for 1, 5, and 25 ng/ml of
TGFp, compared with IL-Ip alone. As observed with
collagen biosynthesis, this result simply reflects the
effects of TGFp alone, without preincubation with
IL- 1, since the growth factor increased GAG biosynthesis by 3%, 17%, and 51%, respectively, for 1,5, and
25 ng/ml, and IL-1p itself had no effect on GAG
synthesis.
When IL-lp was present over the 6 days of
culture, it caused a dramatic decrease in GAG biosynthesis, by 71% (Figure 2 B, 11). This, taken together
with the findings of the first set of experiments, shows
that the effect of IL-1 is reversible, as was the case
with TGFp above. When TGFp was associated with
IL-Ip during the last 3 days of the experiment, the
growth factor effect overrode the inhibitory action of
the cytokine (increased by 85%, 177%, and 173%,
respectively, for 1,5, and 25 ng/ml of TGFP compared
with IL-1p alone), although never achieving the level
of GAG biosynthesis in the controls (medium alone).
Collagenase assay. When the cells were treated
with IL-Ip and then re-fed with fresh control medium,
the collagenolytic activity in the medium was increased by 48% compared with control cultures (Figure 2 C, I). The addition of TGFp after IL-1p decreased the collagenase expression (compared with
that with IL-lp alone) for 1, 5 , and 25 ng/ml TGFp
(reduced by 42%, 9%, and 90%, respectively). Surprisingly, the 5 ng/ml concentration was less inhibitory
than the other 2 concentrations in the 3 independent
experiments performed.
A 6-day treatment with IL-1p caused a dramatic
increase (534%) in collagenolytic activity (Figure 2 C,
11). The addition of 1 ng/ml of TGFp during the last 3
days, together with IL-lp, did not override the stimulatory effect of the cytokine (increase of 516%), but
the highest concentrations of the growth factor reduced, and even reversed, this effect (73% and 70%
decreased at 5 and 25 ng/ml of TGFP, as compared
with control cultures). It must be noted that TGFp
either alone or present in the cultures for the last 3
REDINI ET AL
days or for all 6 days of the incubation period, did not
alter the collagenase level.
TGFP modulation of IL-lP receptor expression.
Treatment of RAC with 5 n g h l of TGFP for 3 days
caused a decrease in the specific binding of '251-labeled
IL-1p (by 50%) (Figure 3A). A plot of specific binding
of IL-lp, as a function of radioligand concentration,
indicated that the binding was dose dependent and
saturable (Figure 3B). Pretreatment of RAC with
TGFp decreased their subsequent specific binding of
labeled IL-1p (Figure 3C). Scatchard analysis supports
the existence of one class of IL-1 receptor (IL-1R) on
rabbit articular chondrocytes, with an average of 3,500
receptors per cell and a K, (affinity constant) of 88 pM.
Treatment with TGFp decreased the number of IL-1R
to l,800/cell(49% decrease) with no significant change
in the affinity (Kd = 79 pM). The decrease in the
number of receptors per cell was consistently observed in 3 separate experiments. These results clearly
demonstrated that the inhibition of binding of 1251labeled IL-1p to TGFp-treated cells was related to a
half-fold decrease in IL-1R number, without changes
in the receptor affinity.
Interestingly, in regard to the experiments described above, the TGFpinduced down-regulation of
IL-1R expression was still observed after 3 days
following treatment with the factor. This finding is
consistent with previous reports (9,lO) and clearly
supports the hypothesis that a reduction in the number
of IL-1R constitutes one of the mechanisms which
could account for the blocking effects of TGFp against
the effects of IL-1 on matrix metabolism in RAC.
12'I-labeled IL-1p was cross-linked to the IL1PR proteins with DSS, the labeled proteins were
separated by SDS-PAGE, and revealed by autoradiography (results not shown). '251-labeledIL- 1p specifically labeled 2 major protein bands with approximate
molecular weights of 80 kd and 35 kd. The former
protein is similar to the major cross-linked band of 80
kd identified in RAC by Harvey et a1 (10). The second
protein band could be another IL- I-binding protein or
a breakdown product of the 80-kd IL-1R.
DISCUSSION
We demonstrate here that TGFp is capable of
modulating the effects induced by IL-1 on articular
chondrocyte metabolism. The growth factor exerts an
antagonistic action on IL-1 activity, which is displayed
on both matrix-degrading and matrix-synthesizing
mechanisms. We also provide the first evidence that
the blocking effect of TGFp on the monokine action is
exerted when TGFp alone is added before IL-lp, as
TGFp REDUCES IL-1R EXPRESSION ON RAC
day
-
1-4
d ~ y4-7
+
+
TGF-fl (5ng/rnl)
TGF-fl
(5 ng/ml)
0.03
5P
0.02
~
0
~~
1
2
Bound (pM)
3
Figure 3. Effect of TGF-p on 12SI-labeledIL-lp binding. A, Effect
on total 1251-labeledIL-lp binding was measured in RAC (plated in
24-well cluster plates and grown to confluency) incubated for 3 days
with or without 5 ng/ml TGF-@and for the last 3 days with or without
5 ng/ml TGF-p. Cells were rinsed once with 1% FCScontaining
DMEM and twice with binding medium, treated with 'Z51-labeledI L l p
(30 pM) for 4 hours at 4°C (with constant mixing), and bound radioactivity was determinedby extracting the cell layer with a lysis buffer (see
Materials and Methods). Specific binding in untreated and T G F - P
treated cells was calculated by subtracting from total binding the
amount of binding in the presence of a 50-fold excess of unlabeled
ligand. Results are specific "%labeled IL-lp binding. Values are the
mean and SD of 3 determinations from 3 independent experiments.
*** = significant decrease compared with binding in the absence of
TGF-p treatment. B and C, 12sI-labeledIL-lp dose-response binding in
control and TGF-&treated chondrocytes was measured in RAC cultures, as in A. '2SI-labeledIL-lp binding was determined by adding
various concentrations of labeled IL-1 in the presence of a 50-fold
excess of unlabeled l i d (B).Values are the average of 3 determinations; this experiment was performed 3 times and gave the same
pattern of results. The dissociation constant (KJ was calculated by
Scatchard analysis (C), assuming a single binding site. B/F = bound
free (see Figure 2 for other definitions).
well as when the 2 factors are present together.
Indeed, pretreatment of RAC with TGFP (25 ng/ml)
for 3 days blocked further effects of IL-1, i.e., inhibi-
49
tion of matrix synthesis and increase in collagenase
production.
Our data are consistent with those in a recent
report showing that TGFP was able to inhibit the
IL- 1-induced increase in neutral proteinase activity in
cultures of RAC (10). However, it was reported that
TGFP must be continuously present with IL-1 to exert
its antagonistic effect. Our data clearly provide evidence that pretreatment of RAC with TGFP alone was
sufficient to prevent subsequent action by IL-1. It
must be noted that in their experiments, Harvey et al
(10) preincubated the cultures with TGFp for only 24
hours instead of 3 days as in our study. It is likely that
differences in experimental conditions, such as the
sequence of addition of the factors, the concentrations
chosen, and the duration of the experiments, may
account for some of the differences in the results
obtained.
There are several mechanisms which could
mediate the opposing action exerted by TGFP on the
IL-1 effects on RAC. Here, we have found evidence
that this TGFP property could be exerted through
down-regulation of IL- 1 R expression, which confirms
previous data (9,lO). IL-1R have been identified in a
variety of cells including macrophages, fibroblasts, T
cells, and chondrocytes. Most of the studies indicate
the existence of a single class of IL-1R. Our study
shows that RAC display only one class of high-affinity
receptors for IL-lp, with a Kd of 88 x 10-'*M. The
reported number of receptors ranges from 250 to 5,000
per cell for a variety of cell types. Our findings also
confirm also that RAC in culture contain approximately 3,500 IL-1R per cell. Using affinity-labeling
methods, we found that IL-1 binding to RAC is inhibited in TGFPtreated cells through a decrease in IL-1R
expression. Interestingly, we demonstrated that the
down-regulation of IL-1R expression by TGFP is still
observed 3 days after removal of the growth factor.
This surprising result contrasts with the data published
by Harvey and coworkers (lo), who reported that the
effect on IL-1 binding exerted by TGFP was reversed
at 24 hours after removal of TGFP. One possible
mechanism could be that the TGFP-induced increase
in extracellular matrix deposition may mask the IL-1binding sites at the cell surface.
In addition to the TGFPinduced decrease in
IL-1R expression, there are other mechanisms
whereby the factor could exert its opposing effects.
For example, it might interfere with the signaling
pathway of the monokine. Unfortunately, there is still
uncertainty regarding the intracellular signaling mechanisms involved in the action of IL-1. There is clear
evidence, however, that the cytokine causes a rapid
REDINI ET AL
50
increase in protein phosphorylation in a variety of
cells, one of which is the small heat-shock protein (hsp
27) (15). Although the function of hsp 27 is largely
unknown, its striking phosphorylation in cells responding to a number of agents suggests that it may
play a role in intracellular signaling. It is possible that
TGFP interferes with the protein kinase responsible
for hsp 27 phosphorylation, which has been recently
characterized as a cytosolic protein of 45 kd. In that
case, both down-regulation of IL-1R expression and
alteration of the IL- 1-signaling pathway would be
reduced by TGFP. The relative importance of each
mechanism would depend on the experimental conditions, namely, on whether RAC cultures are exposed
to TGFP prior to IL- 1 treatment or vice versa. Further
understanding of the TGFP-induced changes in IL-1
action will require greater knowledge of the signaling
pathway of TGFP itself, which is still ill-defined.
Taken together, these in vitro data suggest that
TGFP could play a role in limiting cartilage degradation through inhibition of the biological activity of
IL-1. Since TGFP is present in cartilage and bone (5,6)
and is expressed by cultured chondrocytes, these
findings may have some relevance to the role of this
factor in the process of repair of damaged cartilage.
However, the exact in vivo part played by TGFP in the
metabolism of articular cartilage, namely, in the repair
attempts exhibited by the tissue in early stages of
osteoarthritis, requires further investigation.
ACKNOWLEDGMENTS
We thank Dr. D. Lando (Roussel-Uclaf, Romainville, France) for kindly providing human recombinant IL-I p
and Dr. C. Lafuma (Laboratoire de Biochimie du Tissu
Conjonctif, Creteil, France) for the generous gift of 3Hacetylated collagen.
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expressions, growth, reduction, transforming, factors, effect, opposite, rabbits, interleukin, culture, receptov, articular, chondrocyte, exerts
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