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Microenvironment regulation of extracellular signalregulated kinase activity in chondrocytesEffects of culture configuration interleukin-1 and compressive stress.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 3, March 2003, pp 689–699
DOI 10.1002/art.10849
© 2003, American College of Rheumatology
Microenvironment Regulation of
Extracellular Signal–Regulated Kinase Activity in Chondrocytes
Effects of Culture Configuration, Interleukin-1, and Compressive Stress
Kelvin W. Li, Aaron S. Wang, and Robert L. Sah
chondrocyte monolayers exhibited a pattern that was
distinct from that in other culture systems, suggesting
that the extracellular matrix plays an important regulatory role in modulating the response to extracellular
stimuli. Since IL-1 and dynamic loading have distinct
effects on chondrocyte biosynthesis, signaling pathways
other than ERK participate in the chondrocyte responses to these stimuli.
Objective. To compare extracellular signal–
regulated kinase (ERK) activity in response to
interleukin-1 (IL-1) in chondrocytes under various culture configurations designed for the study of cartilage
biology and repair, and also in response to dynamic load
for chondrocytes in cartilage.
Methods. Isolated bovine articular chondrocytes
were maintained in serum-supplemented medium under
4 culture configurations: high-density monolayer, attached to a cut surface of cartilage, within tissueengineered constructs, or within intact cartilage explants. Samples were subjected to a change of medium
with or without IL-1. Cartilage explants were also
subjected to dynamic compression.
Results. In chondrocyte monolayers, both basal
and IL-1–stimulated ERK activities were similarly elevated at 0.5 hours after medium change, diminishing by
74% after 16 hours. In contrast, chondrocytes in other
culture configurations exhibited lower basal levels of
ERK activity and a moderate activation of ERK in
response to IL-1 that was sustained over the 16-hour
treatment time. The dynamic component of loading of
cartilage explants led to a 5-fold activation of ERK,
compared with free-swelling controls, that was indistinguishable from the effects of IL-1.
Conclusion. ERK signaling in response to IL-1 in
Articular cartilage, the load-bearing, low-friction,
wear-resistant material in skeletal joints, is actively
maintained by a population of chondrocytes that is
sparsely distributed throughout a dense extracellular
matrix (1). Under normal conditions, the chondrocytes
mediate matrix remodeling through a balance between
synthesis and degradation of matrix components. This
balance is regulated by chemical and mechanical factors
that make up the cellular microenvironment. In vitro
studies have shown that chondrocyte metabolism is
regulated by cytokines (2), growth factors (3), matrix
adhesion (4,5), and mechanical loading (6,7). In vivo, the
coordinated influences of these cues regulate the biologic behavior of chondrocytes and, thus, the state of
cartilage health. In healthy joints, dynamic compression
associated with joint loading is necessary for the maintenance of cartilage homeostasis (6,8). In degenerative
conditions, tissue homeostasis is disrupted, perhaps partially due to the effects of interleukin-1 (IL-1), an
inflammatory cytokine that is found in elevated levels in
osteoarthritic joints (9). In cultures of isolated chondrocytes and cartilage explants, IL-1 induces a number of
degradative processes, including inhibition of matrix
(10) or DNA (11) synthesis and induction of matrix
metalloproteinases (12). The signal transduction mechanisms mediating the opposing regulatory effects of IL-1
Supported by the NIH, the National Science Foundation, the
National Aeronautics and Space Administration, and the Beckman
Foundation.
Kelvin W. Li, PhD, Aaron S. Wang, BS, Robert L. Sah, MD,
ScD: Whitaker Institute of Biomedical Engineering, University of
California, San Diego.
Address correspondence and reprint requests to Robert L.
Sah, MD, ScD, Department of Bioengineering, Mail Code 0412,
University of California, San Diego, 9500 Gilman Dr., La Jolla, CA
92093-0412. E-mail: rsah@ucsd.edu.
Submitted for publication July 1, 2002; accepted in revised
form December 9, 2002.
689
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and dynamic compression in normal cartilage and in
cartilage repair situations remain to be elucidated.
There is considerable overlap in the signal transduction pathways of various chemical and mechanical
extracellular stimuli. Exposure of isolated chondrocytes
to cytokines (13–15), growth factors (13,16), matrix
adhesion (17,18), or mechanical stress (19–21) individually activates extracellular signal–regulated kinase
(ERK), providing opportunities for interaction between
these major regulatory cues. ERK is a member of the
ubiquitous family of mitogen-activated protein kinases
(MAPKs) that transmit stimuli from outside the cell to
the nucleus and play important roles in a variety of cell
processes by influencing transcriptional regulation (22).
In chondrocytes, activation of signaling cascades involving ERK has been implicated in the regulation of
proliferation (16), dedifferentiation (23), apoptosis (17),
and biosynthesis of matrix molecules (19,24) and protease inhibitors (15). In most of these studies
(13,14,16,19), activation of ERK was transient, reaching
a peak after 15–30 minutes, but usually dissipating
within a few hours. These studies on ERK signaling
pathways in chondrocytes have often used isolated cells
cultured in monolayer (13–17,19,20,23,24). This may be
problematic, because the differentiated chondrocyte
phenotype is typically unstable in monolayer culture
over time (25) and can be modulated by morphology
(26,27) as well as specific cell–matrix interactions (5,28).
Since the responses of chondrocytes depend
greatly on their culture conditions, a number of experimental models have been introduced to study various
aspects of cartilage repair (Figure 1A). To simulate a
clinical scenario in which chondrocytes are transplanted
into a focal cartilage defect and come into contact with
host cartilage, chondrocytes were seeded at a high
density and allowed to attach to a cartilage surface
(29,30) (Figure 1B). These transplanted chondrocytes in
vitro maintained a stable cartilage phenotype during
short-term culture, as indicated by deposition of macromolecules characteristic of cartilage (29). To fabricate
replacement tissue for implantation, chondrocytes were
formed into cartilaginous tissue after recovery from
culture in alginate using the alginate-recovered chondrocyte (ARC) method (31) (Figure 1C), a procedure
designed to use phenotypically stable chondrocytes (26).
In addition, intact cartilage explants are in common use
experimentally because, when maintained in medium
supplemented with serum, they provide a stable, wellcharacterized in vitro model system for the study of
chondrocytes within their native extracellular matrix
(32) (Figure 1D). Relatively little information is avail-
LI ET AL
Figure 1. A, Schematic of cartilage repair and in vitro models. The
models were used to study the regulation of chondrocytes involved in
B, integrative repair following chondrocyte transplantation, C, bulk
repair following implantation of a cartilaginous construct, and D, tissue
homeostasis in the host cartilage.
able on the regulation of ERK in chondrocytes in any of
these experimental systems.
This study was designed to test the hypothesis
that the microenvironment of chondrocytes affects the
way that ERK signaling is regulated. The first objective
was to directly compare the magnitude and time course
of ERK activation when chondrocytes were exposed to
exogenous IL-1 in different in vitro models used in the
study of cartilage repair. Specifically, the culture configurations compared were cultures of isolated chondrocytes in monolayer, isolated chondrocytes transplanted
to cartilage, isolated chondrocytes precultured in alginate and then formed into constructs, and cartilage
explants. The second objective was to examine the
differential regulation of ERK signaling in cartilage
explants by IL-1 and static and dynamic loading, stimuli
that have different effects on matrix synthesis.
MATERIALS AND METHODS
Materials. Materials for harvest and culture of chondrocytes and cartilage explants were obtained as previously
described (29–31,33). In addition, recombinant human IL-1␣
was obtained from R&D Systems (Minneapolis, MN). Leupeptin, aprotinin, and sodium orthovanadate were obtained
from Sigma (St. Louis, MO). Phenylmethylsulfonyl fluoride
was obtained from Calbiochem (La Jolla, CA). Rabbit polyclonal anti–ERK-2 antibody (C-14) and rabbit normal IgG
were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA), and rabbit polyclonal anti–phosphorylated ERK-1/2
REGULATION OF ERK IN CHONDROCYTES
(anti–p-ERK-1/2) antibody (no. 9101) was purchased from
Cell Signaling Technology (Beverly, MA). Activated recombinant rat ERK-2 standards were from Stratagene (La Jolla,
CA). Microfiltration plates (96-well) with polyvinylidene fluoride (MultiScreen HV) and phosphocellulose (MultiScreen
PH) filters were purchased from Millipore (Bedford, MA).
Protein A–Sepharose 4 Fast-Flow and enhanced chemiluminescence kits were obtained from Amersham Pharmacia
Biotech (Piscataway, NJ). We purchased 2-mercaptoethanol
from Bio-Rad (Hercules, CA). Materials for in vitro kinase
reactions were obtained as previously described (34), except
that ␥32P-ATP was from ICN Biomedicals (Costa Mesa, CA)
and myelin basic protein (MBP) was from Invitrogen (Carlsbad, CA).
Sample preparation and culture. To generate samples
for comparative studies, 4 different systems of chondrocyte
cultures were prepared using cartilage harvested under aseptic
conditions from the femurs of 1–3-week-old calves. Isolated
chondrocytes were obtained by sequential enzymatic digestion
from full-thickness cartilage slices with pronase and collagenase (30). Some of these chondrocytes were plated at high
density (200,000 cells/cm2) onto tissue-culture plastic and
incubated at 37°C in an atmosphere of 5% CO2 with medium
(Dulbecco’s modified Eagle’s medium [DMEM], 10 mM
HEPES buffer, 0.1 mM nonessential amino acids, 0.4 mM
L-proline, 2 mM L-glutamine, 100 units/ml penicillin, 100 ␮g/ml
streptomycin, and 0.25 ␮g/ml amphotericin B) supplemented
with 10% fetal bovine serum (FBS). For this and all other
culture systems, the volume of the medium used was at least 1
ml per 106 cells per day in culture. Chondrocyte monolayers
were maintained in culture for either 1 or 4 days.
Isolated chondrocytes were also used in combination
with cartilage substrates to form an in vitro transplantation
model, as described previously (29,30). Briefly, calf articular
cartilage discs (6.4 mm diameter ⫻ 0.25 mm thick) were
harvested from patellofemoral groove cartilage (after the
removal of the articular surface), lyophilized overnight to lyse
endogenous chondrocytes, and then stored at ⫺20°C until they
were rehydrated for use as nonviable cartilage substrates. Cells
(80,000) were seeded onto each cartilage substrate (250,000
cells/cm2) and allowed to attach undisturbed for 2 hours. The
cartilage substrates, laden with transplanted cells, were incubated in medium (described above) supplemented with 10%
FBS and 25 ␮g/ml ascorbate for either 1 or 4 days.
Other chondrocytes, following isolation from cartilage,
were formed into cartilaginous constructs using the ARC
method (31). Briefly, chondrocytes were resuspended at 4 ⫻
106 cells/ml in 1.2% alginate and polymerized in 102 mM CaCl2
to form spherical beads. Alginate bead cultures were maintained at 37°C in an atmosphere of 5% CO2 for 8 days in a 1:1
ratio of DMEM:Ham’s F-12 medium, supplemented with 0.1
mM nonessential amino acids, 0.4 mM L-proline, 2 mM
L-glutamine, 100 units/ml penicillin, 100 ␮g/ml streptomycin,
0.25 ␮g/ml amphotericin B, 10% FBS, and 25 ␮g/ml ascorbate.
The alginate beads were dissolved using 55 mM sodium citrate
in 0.15M NaCl, and the released cells, along with their
associated matrix coat, were seeded (5 ⫻ 106 cells/cm2) in
culture well inserts (6.5 mm diameter) with a base of porous
(0.4 ␮m) polyester. These cultures were maintained in
DMEM-based medium (described above) including 10% FBS
and 25 ␮g/ml ascorbate for 12 days and then released as
691
cohesive cartilaginous discs (⬃1.3 mm thick) and maintained
in free-swelling culture for an additional day.
Cartilage explant discs (3 mm diameter ⫻ 1 mm thick)
were harvested from the middle and deep zones of patellofemoral calf cartilage as previously described (33). Explants
were maintained in free-swelling culture in medium (250 ␮l
medium per explant) including 10% FBS and 25 ␮g/ml ascorbate for 6 days.
Biochemical and biomechanical treatments. Effect of
culture configuration on ERK activation by IL-1. To compare
the effects of exogenous IL-1 on chondrocytes in the various
culture systems, samples were maintained in culture for the
durations specified and then subjected to changes in medium,
some of which included IL-1. Samples were analyzed for basal
ERK activity by using standard culture medium (including
FBS) for the change, whereas the additional effect of IL-1 was
determined by switching to medium supplemented with 5
ng/ml IL-1, a concentration shown to induce a number of
degradative processes in bovine cartilage explants (35). IL-1␣
was chosen for these experiments since bovine chondrocytes
are more responsive to the ␣-isoform than to the ␤-isoform
(36). Fresh batches of medium were prepared in advance and
preincubated at 37°C in an atmosphere of 5% CO2 for at least
1 hour prior to changes in medium. Stimulation durations were
0.5 and 16 hours to allow assessment of transient and sustained
ERK activation, respectively, since chondrocyte monolayers
were previously shown to respond to IL-1 stimulation with
rapid MAPK activation that dissipated within an hour (13,14).
As a control study to determine the effects of changing
medium on chondrocytes in monolayer, some cultures were
switched to fresh medium containing either 10% FBS or 0.01%
bovine serum albumin, while others were subjected to a
simulated change of medium in which conditioned culture
medium was removed for a few seconds and then pipetted back
into the original sample well. These chondrocyte monolayers
were analyzed for ERK activity either immediately before
changing medium or after 0.5 or 16 hours.
Effect of static and dynamic compression on cartilage
explants. While some cartilage explant discs underwent IL-1
stimulation, others were transferred from free-swelling culture
into independent loading chambers within a standard incubator and subjected to static or dynamic unconfined compression
between nonporous platens, as described previously (33). The
culture medium included 10% FBS and 25 ␮g/ml ascorbate.
For statically loaded explants, a compressive stress of 84 kPa
was applied and held, reaching mechanical equilibrium at a
strain of ⬃15% (relative to the initial cut thickness) after 5
hours. For dynamically loaded explants, a time-varying stress
waveform peaking at 280 kPa was applied to induce small
oscillatory strains (⫾2.4%) of 0.01 Hz around the static
compression level (33). The static loading protocol inhibits
both cell proliferation (33) and glycosaminoglycan biosynthesis
in calf cartilage (33,37), whereas the dynamic loading protocol
stimulates glycosaminoglycan biosynthesis (33,37) while inhibiting cell proliferation (33). As with the IL-1 treatments,
treatment durations for loaded samples were either 0.5 or 16
hours.
Quantification of activated ERK. Following various
treatments, samples in the different culture configurations
were prepared for the extraction of cellular proteins by 1 of 2
methods. Chondrocytes either in monolayer culture or trans-
692
planted onto cartilage were rinsed briefly with ice-cold phosphate buffered saline (PBS) containing 100 ␮M sodium orthovanadate and then incubated with lysis buffer (20 mM Tris,
pH 7.6, 3 mM EDTA, 3 mM EGTA, 100 ␮M sodium orthovanadate, 10 ␮g/ml leupeptin, 2 mM dithiothreitol, 10
␮g/ml aprotinin, 1 mM p-nitrophenyl phosphate, 1 mM phenylmethylsulfonyl fluoride, 250 mM NaCl, 20 mM
␤-glycerophosphate, and 0.5% Nonidet P40 [NP40]) (38) for
15 minutes at 4°C on an Adams Nutator (Becton Dickinson,
Franklin Lakes, NJ). Lysate solutions were collected into
microfuge tubes either by vigorous pipetting (for chondrocytes
in monolayer) or after scraping the cartilage surfaces with a
flat-ended spatula (for transplanted cells). In the case of
transplanted chondrocytes, 10 disc substrates, each seeded
with 80,000 transplanted cells initially, were subjected to
similar treatments, and their lysed cells were pooled to generate a single sample for later analysis.
For either explant cultures or ARC constructs, treatments were terminated by replacing culture medium with
ice-cold PBS containing sodium orthovanadate, followed by
rapid transfer of the samples into liquid nitrogen. For explants
under mechanical compression, the medium was replaced with
the PBS rinse by infusion while maintaining the applied load,
and the explants were flash-frozen within 5 minutes. Frozen
samples were stored at ⫺70°C until they were powdered for
extraction, as described previously (38). Briefly, frozen samples
were powdered using a liquid nitrogen–immersible mortarand-pestle (Fisher Scientific, Fair Lawn, NJ), resuspended in
20 mM Tris (pH 7.6), 3 mM EDTA, 3 mM EGTA, and
protease inhibitors (500 ␮l/explant), and ground at 4°C for 30
seconds into particulates using a drill-mounted tissue grinder
(Fisher Scientific). Solutions were lysed by the addition of
NaCl, ␤-glycerophosphate, and NP40 for the final concentrations described above.
To extract cellular proteins, crude lysates from all
terminated cultures were incubated for an additional 30–60
minutes at 4°C on the Nutator and then centrifuged for 20
minutes at 16,000g. Pellets were routinely analyzed for DNA to
provide a normalization factor for sample analysis. Pellets were
solubilized overnight with proteinase K and then analyzed for
DNA (using Hoechst 33258) and for cell content (assuming 7.7
pg DNA/cell) (30). The clarified supernatants, after digestion
with proteinase K, contained a relatively small amount of the
total recovered DNA (e.g., 5 ⫾ 2%, n ⫽ 12, and 1 ⫾ 2%, n ⫽
12 for monolayer and explant samples, respectively). Clarified
supernatants (“extracts”) were stored at ⫺70°C until use.
Extracts were analyzed by a multiwell ERK activity
assay. ERK-2 was first purified from cell extracts by immunoprecipitation. Extracts were transferred into 96-well filter
plates (MultiScreen HV) in volumes corresponding to 200,000
cells (50–90 ␮l) and incubated with a 20 ␮l slurry containing
2.5 ␮l fully swollen protein A–Sepharose and 2 ␮g anti–ERK-2
rabbit polyclonal antibody (pAb). After overnight incubation
at 4°C on an orbital shaker (500 revolutions per minute),
unbound material was removed by vacuum filtration through
the filter bottoms, leaving immunocomplexes bound within the
Sepharose resin. After 4 rinses (200 ␮l each) with kinase
reaction buffer (13 mM Tris HCl, 0.7 mM EGTA, 10 mM
MgCl2, 3.3 mM ␤-glycerophosphate, 130 ␮M Na3VO4, 130 ␮M
dithiothreitol, 0.13 ␮M protein kinase A inhibitor peptide, 1.3
␮M protein kinase C inhibitor peptide, 1.3 ␮M calmidazolium
LI ET AL
chloride, and 8.3 ␮M ATP, pH 7.5), a multiwell in vitro
reaction was initiated (34). Briefly, kinase reaction buffer,
supplemented with ␥32P-ATP (17 ␮Ci/ml, 4,000 Ci/mmole)
and 667 ␮g/ml MBP, was added for a total volume of 60 ␮l per
well. After 30 minutes, reaction solutions were quenched with
the addition of 60 ␮l of 75 mM phosphoric acid, and a portion
(40 ␮l) of the quenched solution was filtered through phosphocellulose microfiltration plates (MultiScreen PH) and
rinsed 5 times with phosphoric acid and 3 times with 70%
ethanol to bind proteins and remove free radiolabel.
The extent of substrate phosphorylation was assessed
by liquid scintillation counting of membranes for 32P radioactivity, providing an index of functional kinase activity. To
generate calibration curves for standards, the functional activity of activated recombinant rat ERK-2 was routinely analyzed
by adding known amounts to representative extracts prior to
immunoprecipitation. To measure the background signal of
the assay, sham immunoprecipitations were performed by
substituting 2 ␮g normal rabbit IgG for anti–ERK-2 pAb.
Samples were analyzed in duplicate, and the average counts
per minute reading for each sample was related to the activity
of activated ERK standards by first subtracting the corresponding background readings and then dividing by the slope
of the appropriate calibration curve.
To visualize the amount of total ERK-2 and p-ERK1/2, some extracts were additionally analyzed by immunoblotting. Portions of extracts, corresponding to 100,000 cells, were
separated on 10% polyacrylamide minigels by electrophoresis
and then transferred to nitrocellulose. Membranes were
probed with a pAb against the activated form of ERK-1/2 for
3 hours at a dilution of 1:1,000. Secondary antibody reactivity
was visualized by enhanced chemiluminescence and scanned
with a flatbed scanner (Epson 1240U; Epson, Long Beach,
CA). After storage at 4°C, membranes were stripped by
incubation in 2% sodium dodecyl sulfate, 62.5 mM Tris HCl
(pH 6.7), and 100 mM 2-mercaptoethanol at 50°C for 30
minutes and reprobed for total ERK-2 using anti–ERK-2 pAb.
Statistical analysis. To determine the effects of IL-1 or
compression in each culture configuration, data were analyzed
by analysis of variance (ANOVA), where the main factors were
stimulation type and duration. In the case of chondrocytes
plated either on plastic or on cartilage, the incubation time
(either 1 or 4 days in culture) represented a third main
ANOVA factor. When a significant difference was detected,
Tukey’s test was used to compare groups. Data are expressed
as the mean ⫾ SEM.
RESULTS
Validation of multiwell ERK activity assay.
While previous multiwell assay methods made use of
antibodies immobilized onto a well bottom for the
purpose of kinase purification (34), antigen capture was
found to improve when the antibodies resided within a
Sepharose resin. Since this Sepharose-based immunoprecipitation had not been previously described, it was
important to verify that the results from the multiwell
ERK activity assay used in this study were both sensitive
REGULATION OF ERK IN CHONDROCYTES
Figure 2. Typical calibration curves for multiwell extracellular signal–
regulated kinase (ERK) activity assay. Activated ERK standards were
immunoprecipitated in either lysis buffer (䊐) (LB), extracts of monolayer chondrocytes (F) (mono), or extracts of cartilage explants (‚)
(exp). Immunoprecipitates, sham immunoprecipitations () (sham IP,
prepared using nonspecific IgG), and activated ERK standards added
directly into reaction wells (ƒ) (direct reactions) were all analyzed for
functional ERK activity.
to and specific for the activated ERK in the analyzed
extracts.
When recombinant rat p-ERK standards were
added to various lysis buffers and immunoprecipitated
with antibodies for ERK-2 prior to in vitro kinase
reactions, regression analysis indicated that the amount
of 32P radioactivity associated with MBP was linearly
proportional to the amount of p-ERK added, up to the
maximum 16 ng studied (Figure 2). Here, the sensitivity
of the assay, given by the slope of the linear fit, varied
somewhat with the buffer in which the immunoprecipitation was performed, ranging from 444 to 607 cpm/ng
p-ERK (R2 ⫽ 0.93–0.99, P ⬍ 0.001; n ⫽ 12) when the
immunoprecipitation buffer consisted of extracts of cartilage explants, extracts of monolayer chondrocytes, or
fresh lysis buffer. Measured radioactivities were ⬃5-fold
higher when p-ERK standards were added directly into
reactions (forgoing the immunoprecipitation step), with
a slope of 2,608 cpm/ng p-ERK (R2 ⫽ 0.96, P ⬍ 0.001;
n ⫽ 12), suggesting that the capture efficiency of immunoprecipitation (determined as the ratio of sensitivities)
ranged from 17% to 23%. In contrast, results with sham
immunoprecipitations were insensitive to the addition of
p-ERK standards, with a slope of 2.5 cpm/ng p-ERK
(R2 ⫽ 0.74, P ⫽ 0.14; n ⫽ 4), indicating that observed
693
radioactivities were the result of specific antigen capture
by anti–ERK-2 pAb.
Effect of culture configuration on ERK activation
by IL-1. Some chondrocyte monolayers were maintained
in serum-supplemented medium until they were stimulated by switching to medium with or without IL-1. In
this culture system, the effects on ERK activity were
indistinguishable whether the chondrocyte monolayers
were stimulated on day 1 (data not shown) or on day 4
(Figure 3) following cell plating (P ⫽ 0.7). In both cases,
the inclusion of IL-1 had no additional effects (P ⫽
0.15). Thirty minutes after monolayers underwent
changes of medium including serum, ERK activity was
high, with the measured activity equivalent to that of
4.4 ⫾ 0.3 ng (mean ⫾ SEM) p-ERK per 200,000 cells.
The response in the initial half-hour appeared to be a
transient activation of ERK due to the medium change,
since 16 hours of continuous exposure led to a 74%
reduction in ERK activity to 1.2 ⫾ 0.2 ng p-ERK per
Figure 3. Activation of extracellular signal–regulated kinase (ERK)
in chondrocyte monolayers. Isolated chondrocytes were plated at high
density on tissue-culture plastic and maintained in culture in Dulbecco’s modified Eagle’s medium (DMEM) ⫹ 10% fetal bovine serum
(FBS). On day 4, the culture medium was replaced with DMEM
including the supplements indicated. Cultures were terminated following either a 0.5- (■) or a 16-hour (䊐) treatment period. Extracted
cellular proteins were analyzed by A, Western blot for both activated
and total ERK or by B, multiwell in vitro assay for functional ERK
activity (n ⫽ 7 or 8 samples from 4 animals per culture condition).
Values are the mean and SEM. ⴱⴱⴱ ⫽ P ⬍ 0.001, 0.5 hours versus 16
hours. IL-1 ⫽ interleukin-1; p-ERK ⫽ phosphorylated ERK.
694
LI ET AL
Figure 4. Activation of ERK in chondrocytes transplanted to cartilage. Chondrocytes were
passaged at high density onto a cut cartilage surface and maintained in culture in DMEM ⫹
ascorbate (asc) ⫹ 10% FBS for 1 day (A and B) or 4 days (C and D) before the culture medium
was replaced with DMEM including the supplements indicated. Cultures were terminated
following either a 0.5- (■) or a 16-hour (䊐) treatment period. Extracted cellular proteins were
analyzed by Western blot for both activated and total ERK (A and C) or by multiwell in vitro assay
for functional ERK activity (B and D) (n ⫽ 2 samples from 2 animals per culture condition).
Values are the mean and SEM. ⴱ ⫽ P ⬍ 0.05. See Figure 3 for other definitions.
200,000 cells. This was comparable with the ERK activity level detected immediately before medium change
(1.7 ⫾ 0.1 ng per 200,000 cells [n ⫽ 3] for time 0
controls; not shown). On the other hand, both changing
to basal medium (without serum) and simulated medium
changes using conditioned medium resulted in relatively
low ERK activity after 0.5 hours (1.1 ⫾ 0.4 [n ⫽ 5] and
1.7 ⫾ 0.5 [n ⫽ 3] ng p-ERK per 200,000 cells, respectively),
suggesting that the initially high levels of p-ERK were not
nonspecific responses to fluid turbulence generated by
changing medium.
The attachment of chondrocytes to a cartilage
substrate altered their ERK activation response to serum and IL-1 supplementation, and this was apparent
after only 1 day in culture. In chondrocytes transplanted
to cartilage, regulation of ERK activity by different
medium formulations depended on the culture duration
(P ⬍ 0.01) prior to medium exposure. For transplanted
cells, overall, the inclusion of IL-1 in the medium had a
significant effect (P ⬍ 0.05 by ANOVA) when it was
introduced into culture on day 4 (P ⬍ 0.05 for interaction), and the effects did not vary between 0.5 and 16
hours of exposure (P ⫽ 0.4 by ANOVA). When transplanted chondrocytes were incubated on cartilage for
only 1 day, a subsequent change of medium led to ERK
activity equivalent to 3.4 ⫾ 0.3 ng p-ERK per 200,000
cells (n ⫽ 8) (Figures 4A and B) at both 0.5- and 16-hour
time points, regardless of the presence of IL-1 (P ⫽ 1.0).
On day 4, the basal response to the replacement of
normal (serum-supplemented) culture medium was
lower, with 0.74 ⫾ 0.14 ng p-ERK per 200,000 cells (n ⫽
4), making the activation of ERK by IL-1 evident in
comparison, with a 3.6-fold increase (P ⬍ 0.05) (Figures
4C and D) that was sustained for at least 16 hours.
In cartilaginous constructs fabricated from
ARCs, changing medium did not seem to elicit an
increase in ERK activity unless the medium was supplemented with IL-1 (P ⬍ 0.001) (Figure 5). Exposure of
constructs to fresh medium maintained low basal levels
of ERK activity (0.58 ⫾ 0.19 ng p-ERK per 200,000
cells), whereas inclusion of IL-1 in the medium resulted
in a 3-fold activation of ERK (to 1.71 ⫾ 0.17 ng p-ERK
per 200,000 cells), and the results did not change detectably between 0.5 and 16 hours (P ⫽ 0.4).
REGULATION OF ERK IN CHONDROCYTES
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Effect of static and dynamic compression on
cartilage explants. Static and dynamic compression of
cartilage explants also had marked and differential
effects on ERK activation, compared with free-swelling
control explants (P ⬍ 0.001) (Figure 6A–D). Although
treatment time (overall P ⫽ 0.5) had a slight interaction
with treatment (P ⫽ 0.03), post hoc testing indicated
that the ERK activities resulting from each treatment
were indistinguishable between 0.5 and 16 hours (P ⫽
0.9, 0.3, 0.9, and 0.9 for FBS, IL-1, static loading, and
dynamic loading treatment groups, respectively). The
low level of static compression applied here did not
affect ERK signaling in explants compared with freeswelling basal controls (P ⫽ 0.3), but the application of
dynamic load led to a sustained activation of 0.76 ⫾ 0.09
ng p-ERK per 200,000 cells (n ⫽ 14), which was 5.4
times greater than that in basal controls (P ⬍ 0.001) and
2.5 times greater than that in statically loaded samples
(P ⬍ 0.001). Furthermore, the ERK responses to IL-1
and dynamic loading in explants were indistinguishable
from one another (P ⫽ 0.9).
DISCUSSION
Figure 5. Activation of ERK in alginate-recovered chondrocyte
(ARC) constructs. ARCs were incubated in pellet culture to form a
cohesive cartilaginous construct over a 13-day period. Constructs were
exposed to a medium change with the medium formulations indicated,
and cultures were terminated following either a 0.5- (■) or a 16-hour
(䊐) treatment period. Extracted cellular proteins were analyzed by A,
Western blot for both activated and total ERK or by B, multiwell in
vitro assay for functional ERK activity (n ⫽ 3 or 4 samples from 2
animals per culture condition). Values are the mean and SEM. ⴱⴱⴱ ⫽
P ⬍ 0.001. asc ⫽ ascorbate (see Figure 3 for other definitions).
Cartilage explants also had a low basal level of
ERK activity that was sensitive to IL-1 exposure (P ⬍
0.001) (Figures 6A and B) but not affected by stimulation time (P ⫽ 0.5). In free-swelling culture with serumsupplemented medium, cartilage explants exhibited
basal ERK activity equivalent to 0.14 ⫾ 0.03 ng p-ERK
per 200,000 cells (n ⫽ 14) at both 0.5- and 16-hour time
points (P ⫽ 0.9). Alternatively, the introduction of
medium including IL-1 increased the level of ERK
activity by 5-fold, reaching a peak at 0.85 ⫾ 0.12 ng
p-ERK per 200,000 cells by 16 hours, although treatment
time did not markedly affect the ERK response (P ⫽
0.3).
In this study, the effects of the biochemical and
mechanical microenvironment of chondrocytes on ERK
regulation were analyzed by comparing responses in
various culture conditions. ERK signaling in cartilage
explants (Figure 6) was distinguished from that in monolayer cultures of chondrocytes (Figure 3) by 4 general
characteristics: 1) lower activity under basal conditions
(after a medium change), 2) differential responsiveness
to serum and IL-1, 3) lower peak activity when stimulated, and 4) sustained activity when stimulated.
Among the 4 culture systems examined, monolayer cultures were unique in having a transient phase in
ERK activation (Figure 3), and the other ex vivo models,
which expose chondrocytes to an increasingly extensive
matrix structure, either exhibited (Figure 5) or acquired
over time (Figure 4) many of the ERK activation
characteristics of cartilage explants. One day following
chondrocyte transplantation to cartilage, cells exhibited
persistently high levels of ERK activity (Figures 4A and
B), but over time, ERK activity decreased and became
susceptible to regulation by IL-1 (Figures 4C and D).
Likewise, the trends in ERK regulation were similar in
well-developed cartilaginous constructs and cartilage
explants, with overall levels of ERK activity being 2
times greater in the constructs (Figures 5A and B).
These results suggest that the biochemical composition
of the immediate microenvironment surrounding chon-
696
LI ET AL
Figure 6. Activation of ERK in cartilage explants. Calf cartilage explant disks were stimulated by
free-swelling culture in different culture media (A and B) or subjected to static (␴stat) or dynamic
(␴dyn) mechanical compression (C and D). Cultures were terminated following either a 0.5- (■) or
a 16-hour (䊐) treatment period. Extracted cellular proteins were analyzed by Western blot for both
activated and total ERK (A and C) or by multiwell in vitro assay for functional ERK activity (B
and D) (n ⫽ 7 or 8 samples from 4 animals per culture condition). Values are the mean and SEM.
ⴱⴱⴱ ⫽ P ⬍ 0.001. asc ⫽ ascorbate (see Figure 3 for other definitions).
drocytes, as dictated by culture conditions, influences
how soluble stimuli initiate signaling pathways. Furthermore, sustained activation of ERK in cartilage explants
was a motif that also occurred in response to dynamic,
but not static, mechanical loading (Figures 6C and D),
with an effect that was identical to the activation by IL-1.
Direct comparison of ERK activation between
different culture systems required a number of considerations, including normalization to provide a basis for
comparison between sample types. We normalized ERK
activity by the amount of DNA (cell number) that was
recovered during extraction, since extracted total protein, used commonly as a normalization factor
(13,14,34), was likely to vary markedly among the culture systems. Qualitative inspection indicated that equivalent amounts of total ERK were made available for
analysis, because immunoblots for total ERK-2 showed
similar levels of total ERK-2 when extracts from different culture systems were analyzed in volumes corresponding to 100,000 cells (Figures 3A, 4A, 4C, 5A, 6A,
and 6C).
To allow comparison with existing studies on the
response of cartilage to IL-1 (39,40) and compression
(30,37), cartilage from calves was used as the cell source
for the various culture systems investigated here. Also
for comparison’s sake, serum was included in the culture
media, and it exhibited interactive effects with IL-1 in
these experiments. Chondrocytes in the monolayer culture system were characterized by a transient phase of
ERK activation in response to a medium change that
included either FBS or IL-1 (Figures 3A and B), but the
effects were not additive when FBS and IL-1 were
combined, even though the chondrocytes had been
cultured with FBS prior to stimulation (Figures 3A
and B).
Temporary ERK activation by IL-1 has been
observed both in human chondrocytes that had been
serum starved (13) and in rabbit chondrocytes in serumsupplemented culture (14). Fluid-induced shear has also
been reported to transiently increase ERK activation in
chondrocyte monolayers either with (19) or without
serum (19,20) included in the flow medium. However,
the transient ERK activation associated with changing to
medium that included serum in this study (Figures 3A
REGULATION OF ERK IN CHONDROCYTES
and B) could not be attributed to direct mechanical
stimulation alone or to the abolishment of nutrient
boundary layers, since changing to either serum-free or
conditioned medium did not elicit the same response.
Furthermore, neither significant depletion of serum
components during culture nor accumulation of metabolic waste was likely, since transient ERK activation
was evident even when increased medium volumes were
used for culture either before or during IL-1 stimulation
(data not shown). Further studies may clarify the mechanisms underlying this transient activation of ERK in
serum-supplemented monolayer cultures.
In culture configurations that maintained the
presence of a significant matrix structure, hindered
transport may have been partially responsible for the
relatively low peak levels of ERK activity in the chondrocyte response to serum and IL-1. The lower degree of
ERK activation in such cultures may reflect an ability of
the dense cartilage matrix to buffer chondrocytes from
sudden changes in their chemical and mechanical environment. The extracellular matrix presents a hindrance
to mass transport, with slowing of exchange kinetics and
chemical partitioning (41,42). Thus, with more matrix,
both IL-1 and fresh serum components would require
more time to diffuse into the tissue and reach receptor
targets on cell surfaces, and would also equilibrate at
lower local steady-state concentrations. For molecules
similar in size to IL-1, the time constant for diffusion of
IL-1 through the cartilage tissue is ⬃7 hours and the
partition coefficient is 0.3–0.7 (41). Experimental observations confirm that IL-1 is able to effectively penetrate
and regulate cartilage within that time frame (43). Such
a partition coefficient would still result in a local,
steady-state concentration of IL-1 in the explants above
the threshold concentration (1 ng/ml), leading to maximal ERK activation in isolated rabbit chondrocytes (14).
Thus, while the matrix does not completely protect
endogenous chondrocytes from exposure to IL-1, it may
physically dampen its effects.
Furthermore, direct chondrocyte–matrix interactions appeared to markedly modulate ERK activity.
Attachment to cartilage for 1 day eliminated the transient phase of ERK activation from the chondrocyte
response to serum and IL-1 exposure (Figure 4A),
suggesting that the initial adhesion to cartilage exerts an
immediate influence prior to the pericellular accumulation of matrix de novo. Chondrocytes may interact with
cartilage matrix through a number of receptors. The
initial firm adhesion of chondrocytes to a cut surface of
cartilage is mediated by engagement of ␤1 and av␤5
integrin cell surface receptors (44). In intact articular
697
cartilage, chondrocytes are embedded in matrix and use
integrins to attach to matrix components (45). Integrinmediated signaling events include the activation of ERK
in many cell types (46), including chondrocytes (17,18).
Although ligation of some integrins alone leads to an
activation of ERK that is typically transient, synergistic
responses are mediated by growth factors (47,48), mechanical loading (49–51), and cytokines (52) to activate
signaling cascades, including sustained ERK activation
in some cases (47). In the culture systems studied here,
a similar type of cooperativity may occur between attachment to extracellular matrix molecules and chemical
(or mechanical) stimuli. Differences in the ERK activity
and responses observed between the in vitro culture
configurations examined here suggest that the adhesion
of chondrocytes to components in the extracellular
matrix modulates the subsequent cellular response to
cytokines, affecting both the time course and the magnitude of ERK activation.
The regulation of ERK activation within cartilage
found here (Figure 6) is generally consistent with, and
extends, previous observations. Transient activation of
ERK by IL-1 was observed in human cartilage explants,
but the effect required incubation in an atmosphere of
low oxygen tension (53). In bovine cartilage explants, the
application of static compression was previously found
to induce activation of ERK in a compressionamplitude–dependent manner (21). Application of
⬎25% static compression activated ERK, while 15%
compression, the degree of static compression resulting
from the loading applied here, did not lead to a significant ERK response. The superimposed effect of dynamic compression on ERK activation was not investigated previously. These findings indicate that ERK
signaling is operative within cartilage explants in response to a range of chemical and mechanical perturbations.
The extent and time course of ERK activation in
chondrocytes has certain implications. ERK signaling
pathways have typically been associated with the regulation of cell proliferation (22). Despite the initially
confluent seeding density, chondrocytes transplanted to
cartilage initially undergo significant proliferation in this
model system (30). This proliferation pattern coincides
with the basal ERK activity in transplanted chondrocytes, where proliferation and ERK activity are both
relatively high during culture on days 1 and 2 (Figures
4A and B). On the other hand, confluent monolayer
chondrocytes in this study did not proliferate detectably when cultured on plastic (data not shown),
despite the high peak level of ERK activity associated
698
LI ET AL
with each medium change. This is consistent with the
hypothesis that only sustained ERK activation induces
cell division (54).
The ERK signaling pathway does not appear to
be the sole determinant of aggrecan synthesis within
intact cartilage for the chemical and mechanical stimuli
examined here. In articular chondrocytes, activation of
ERK may have functional implications regarding aggrecan biosynthesis (19,24). However, in this study, similar
patterns of ERK activation were observed whether
chondrocytes in explants were exposed to IL-1 (10,39,40)
or to the selected dynamic loading protocol (33,37),
which have opposing effects on biosynthesis. Also, lowlevel static loading, such as IL-1, inhibits biosynthesis
(33,37), but had little marked effect on ERK activity.
Thus, other signaling pathways may govern biosynthesis
of a variety of matrix molecules, either independently or
in an interactive manner with ERK.
11.
12.
13.
14.
15.
16.
ACKNOWLEDGMENTS
The authors thank Dr. Shu Chien, Dr. John Y.-J. Shyy,
and Ms Suli Yuan for guidance on ERK activity assays, Dr.
Joan Heller Brown and Dr. Valerie Sah for assistance with
cartilage extractions, and Dr. Michael DiMicco for discussion
of transport issues and technical advice.
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