From the Institute of Anatomy, Freie
Universität Berlin, Königin-Luise-Strasse 15, D-14195
Berlin, Germany, ¶ University Medical Center Benjamin Franklin,
Department for Trauma Surgery, Freie Universität Berlin,
Hindenburgdamm 30, D-12200 Berlin, Germany,
Trauma and
Reconstructive Surgery, Charité, Campus Virchow-Klinikum,
Augustenburger Platz 1, D-13353 Berlin, Germany
Received for publication, December 1, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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We previously have reported that the
mitogen-activated protein kinase (MAPK) pathway is stimulated by
adhesion of human chondrocytes to anti- Apoptosis, or programmed cell death, plays a key role in
embryogenesis, immunological competence and tissue homeostasis for cell
removal and can distinguished biochemically and morphologically from
cell necrosis, which is a passive, energy-independent form of cell
death. Chondrocyte degradation and death occurs in enchondral ossification as well as in age-associated athropathies such as osteoarthritis (1, 2). Chondrocyte apoptosis, can be induced in
vitro by a variety of agents, such as nitric oxide, oxygen radical
scavengers (3), tumor necrosis factor (4), and interleukin-1 It is known that many cell types including chondrocytes require
integrin mediated interactions with the extracellular matrix to
survive, differentiate, and proliferate (6, 7). Cells undergo a
specific cell death or apoptosis in the absence of specific matrix
components (8). Interaction between chondrocytes and cartilage matrix
components or anti- It is well known that one of the early reactions occurring in the cell
after the damage of its DNA is the activation of
poly(ADP-ribose)polymerase (PARP), a nuclear enzyme present in
eukaryotes. Several lines of evidence show that PARP is involved in
different cellular functions including DNA repair (11, 12), DNA
replication (13), and programmed cell death (14, 15). During apoptosis,
clumps of heterochromatin and nuclear fragmentation appear and PARP is
one of the earliest proteins to be specifically cleaved from 116 kDa to
two fragments of ~85 and 25 kDa (16).
A group of cysteine proteases called "caspases" play a central role
in apoptosis. They are synthesized as proenzymes, containing an
N-terminal prodomain. Activation occurs by cleaving this prodomain at a
specific aspartic acid cleavage site that is localized between the
prodomain and each subunit of the caspase. One caspase can process and
activate the other in a cascade reaction beginning by initiator
caspases, which interact with specific adaptor proteins. Initiator
caspases activate effector caspases (e.g. caspase-3, -6, and
-7) either directly or indirectly that cleave a number of structural
and regulatory proteins including DFF45 (DNA fragmentation factor)/inhibitor of caspase-activated DNases, PARP, lamine, and cytokeratines (17).
Mitogen-activated protein kinase (MAPK) p44 (Erk1) and p42 MAPK (Erk2)
are important mediators of cellular responses to intracellular signaling proteins. MEK1 and MEK2 regulate the activity of Erk1/2 by
phosphorylating threonine and tyrosine residues, and further upstream
MAPK kinase kinases (Raf) regulate the activity of MEK by
phosphorylation of two serine residues. The activated Erk1/2 then
translocates into the nucleus and regulates the activities of several
nuclear transcription factors (18-22).
Furthermore, it is known that the MEK/Erk signaling pathway is involved
in the cell growth and differentiation in many cell types. However, the
role of MEK/Erk signaling pathway in chondrocyte function and
differentiation is at present not fully understood. Therefore, in this
study, we examined the essential role of Erk pathway on chondrocyte
differentiation and survival by treating chondrocytes with the
inhibitor U0126, which has been shown to specifically block
mitogen-activated protein kinase/Erk kinase (MEK), the kinase upstream
of Erk (23). Upon treatment with U0126, chondrocytes undergo apoptosis,
suggesting that a functional death-mediating signaling cascade is
associated with the extracellular matrix protein. Inhibition of MEK
with inhibitors is a relatively new approach to clarify the role of
these kinases in cellular functions.
Antibodies--
Polyclonal anti-phospho-p42/p44 Erk1/2
antibodies and U0126 were purchased from Promega (Mannheim, Germany).
Polyclonal anti-active caspase-3 antibody and monoclonal anti-PARP
antibody were purchased from Becton Dickinson (Heidelberg, Germany).
Anti-pan Erk1/2 antibodies were purchased from Transduction
Laboratories (Heidelberg, Germany). Secondary antibodies conjugated
with alkaline phosphatase were purchased from Roche Molecular
Biochemicals (Mannheim, Germany).
Collagen type II, trypsin, pronase, and collagenase were purchased from
Sigma (Munich, Germany). IGF-I was purchased from Biomol (Hamburg,
Germany). Dual-system-APAAP-complex was purchased from DAKO (Hamburg, Germany).
Chondrocyte Culture--
Primary cultures of chondrocytes were
prepared from human cartilage as described previously in detail (7).
Briefly, human articular cartilage specimens (from femoral heads
obtained during joint replacement surgery for femoral neck fractures)
were collected in Ham's F-12 medium. Cartilage slices were digested
with 1% pronase and then with 0.2% collagenase. After rinsing in
growth medium (Ham's F-12/Dulbecco's modified Eagle's medium: 50/50,
10% fetal calf serum, 25 µg/ml ascorbic acid, 50 µg/ml
gentamicin), a single cell suspension was obtained. The cells were
cultured in alginate beads as described previously in detail (24). To
dissolve the alginate for subsequent separation of the cells, the
alginate beads were placed in 55 mM sodium citrate in 0.15 M NaCl.
IGF-I Stimulation and Cell Scoring--
Coverslips coated with
collagen type II (500 µg/ml in 0.02 N acetic acid at 4 °C
overnight), were washed three times with phosphate-buffered saline, and
then incubated with serum-free medium at 37 °C for 1 h prior to
use. Human chondrocytes isolated from alginate beads were washed three
times with serum-free medium. After counting, the cells were diluted to
1.5 × 106/ml in serum-free medium, cultured on
prepared coverslips for 30 min, and then stimulated with IGF-I (100 ng/ml). A specific positive effect of collagen type II and IGF-I has
been shown, on the stabilization, differentiation, survival, and
adhesion of chondrocyte in vitro (7). After washing, the
attached chondrocytes were treated with U0126 (1 µM) or
left untreated. The cultures were investigated after 1, 2, 4, 6, 8, 10, 12, and 14 h by light microscopy. The number of cells was
determined by scoring cells from three different microscopic fields.
These assays were performed in triplicate, and the results are provided
as mean values with standard deviations from three independent experiments.
Transmission Electron Microscopy--
The U0126-treated or
untreated cultures as described above were fixed in 1% glutaraldehyde
and 1% tannic acid in 0.1 M phosphate buffer, pH 7.4. Subsequently, they were post-fixed in a 2% OsO4 solution.
After dehydration in the ascending alcohol series, the specimens were
embedded in Epon. Ultrathin sections were contrasted with 2% uranyl
acetate and lead citrate and investigated under a transmission electron
microscope Zeiss EM10.
Immunohistochemistry--
A detailed description of the
technique used for the following experiments has been published
previously (25). Briefly, the cells were fixed for 10 min. After
counting, the cells were diluted to 1.5 × 106/ml in
serum-free medium, plated on dishes coated with collagen type II
stimulated with IGF-I and then treated with U0126 (0.1, 1, 10 µM) for 1 h or left untreated. After rinsing, the
cells were immunolabeled as follows: 1) incubation with serum (1:20 in
Tris buffer) at room temperature for 10 min; 2) incubation with primary
antibodies (anti-active caspase-3 and normal rabbit IgG 1:30) in a
moist chamber overnight at 4 °C; 3) rinsing; 4) incubation with
mouse-anti-rabbit IgG antibodies (1:50) at room temperature for 30 min;
5) rinsing; 6) incubation with dual system bridge antibodies (1:50) for
30 min at room temperature; 7) rinsing; 8) incubation with dual
system-APAAP-complex (1:50) for 30 min at room temperature; 9) rinsing;
10) new fuscin staining for 30 min at room temperature. Subsequently,
the specimens were washed and dried, covered with Kaisers
glycerin/gelatin and examined under a Zeiss Axiophot 100 light microscope.
Western Blot Analysis--
A detailed description of the culture
technique used for the following experiments has been published
previously (7). Briefly, chondrocytes were harvested from alginate
cultures and washed three times with serum-free medium. After counting,
the cells were diluted to 1.5 × 106/ml in serum-free
medium, plated on dishes coated with collagen type II, and then treated
with U0126 (0.1, 1, 10, 100, 1000 µM) for 1 h or
left untreated. After rinsing with phosphate-buffered saline, cells
were extracted with lysis buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% Triton X-100, 1 mM sodium
orthovanadate, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 0.01% aprotinin, 4 µg/ml pepstatin
A, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride) on ice for 30 min. For immunoblotting, equal amounts of total
proteins were separated on 10% or 7.5% SDS-polyacrylamide gel
electrophoresis gels under reducing conditions. Proteins were transferred onto nitrocellulose. Membranes were blocked with 5% (w/v)
skimmed milk powder in phosphate-buffered saline/0.1% Tween 20 overnight at 4 °C and incubated with primary antibodies diluted in
blocking buffer for 1 h at room temperature. After five washes in
blocking buffer, membranes were incubated with alkaline
phosphatase-conjugated secondary antibody diluted in blocking buffer
for 30 min at room temperature. Membranes were finally washed five
times in blocking buffer, twice in 0.1 M Tris, pH 9.5 containing 0.05 M MgCl2 and 0.1 M
NaCl; specific binding was detected using nitro blue tetrazolium and
5-bromo-4-chloro-3-indoyl-phosphate (p-toluidine salt;
Pierce) as substrates and quantitated by densitometry. Protein
determination was done with the bicinchoninic acid system (Pierce)
using BCA as a standard.
Statistical Analysis--
The results are expressed the
means ± S.D. of a representative experiment performed in
triplicate. Data shown are representative of three independent
experiments. The means were compared using Student's t test
assuming equal variances. Statistical significance was at
p < 0.05.
Effects of U0126 on Chondrocyte Adhesion to Collagen Type
II--
Human chondrocytes were cultured on collagen type II-coated
Petri dishes, serum starved, stimulated with IGF-I and then either treated with U0126 or left untreated for the indicated times. The
chondrocytes grown on collagen type II without U0126 exhibited a round
to oval shape and numerous small ridge-like or cuspidal surface
processes from the beginning of cultivation onwards (Fig. 3A). The chondrocytes grown on collagen type II with U0126
showed cell detachment, loss of cell processes, and membrane shrinkage. The number and density of the chondrocytes cultivated on collagen type
II and in the absence of U0126 was significantly higher than those
cultivated on collagen type II and treated with U0126. After a culture
period of 1, 2, 4, 6, 8, 10, 12, and 14 h, the total number of
treated chondrocytes grown on collagen type II was reduced by about
13% (p = 0.06), 35% (p = 0.009), 42%
(p = 0.01), 55% (p = 0.008), 64%
(p = 0.006), 66% (p = 0.002), and 71%
(p = 0.005) compared with those cultivated on collagen
type II and in the presence of IGF-I (Fig.
1).
Electron Microscopy of U0126-induced Changes in
Chondrocytes--
Fig. 2A shows an
untreated human chondrocyte cultured on collagen type II and stimulated
with IGF-I. Typical cartilage cells were mainly round to oval,
contained a well developed rough endoplasmic reticulum, a large Golgi
apparatus, and other organelles or structures, such as mitochondria and
small vacuoles. The nucleus was large and round, with minimal
heterochromatin. The cells formed a pericellular freshly matrix,
which was closely attached to the cell membrane. Cytoplasmatic
processes were present. In contrast, a representative chondrocyte from
an U0126-treated culture (Fig. 2B) had a cellular morphology
typical of apoptosis, with nuclear changes including chromatin
condensation into dense areas along the nuclear membrane and nuclear
fragmentation. The cellular membrane was irregular (bleb formation),
and cytoplasmic vacuoles (dilatation of mitochondria and endoplasmic
reticulum) can be seen.
Induction of Chondrocyte Apoptosis by U0126--
Human
chondrocytes cultured on collagen type II with exposure to IGF-I
undergo apoptosis after treatment with U0126. Immunohistochemical analysis with antibodies against cellular factors involved in apoptosis
showed that treated chondrocytes were active caspase-3 positive (Fig.
3). In addition, to verify that the
U0126-induced increase of chondrocyte apoptosis is
dose-dependent, serum-starved human chondrocytes cultured
on collagen type II were treated with various concentrations of U0126
(0.1, 1, 10 µM) and immunolabeled with anti-active
caspase-3 antibodies. The results showed a marked dose-dependent increase in induction of activated caspase-3
in chondrocytes cultured on collagen type II after 1 h (Fig. 3,
B-D). In contrast, control cultures of
chondrocytes treated with IgG maintained their nuclear morphology
throughout the culture period (Fig. 3A). The total number of
caspase-3 positive chondrocytes grown on collagen type II, treated with
IGF-I was increased in the presence of 0,1 µM U0126 by
about 27% (p = 0.0076), of 1 µM U0126 by
55% (p = 0.094), and of 10 µM U0126 by
about 74% (p = 0.095) compared with those cultivated
on collagen type II, treated with IGF-I and in the absence of
U0126.
U0126 Changes in the Activity of Extracellular Signal-regulated
Kinase 1 and 2--
It has been shown that a reduction in the Erk
signaling pathway stimulates the apoptotic pathway in different cell
types (26). For this reason, we examined whether the inhibition of
Erk1/2 activity plays a role in transmitting the apoptotic signals in human chondrocytes. Anti-pan Erk1/2 antibody recognizes both inactive and active forms of Erk1/2 and indicates the expression level of total
Erk1/2. Anti-phospho-Erk1/2 recognizes only phosphorylated Erk1/2 and
indicates the activation of Erk1/2. Western blot analysis with
anti-activated Erk1/2 from chondrocytes cultured on collagen type II
stimulated with IGF-I in the presence of 1 µM U0126
demonstrated a significant decrease in Erk phosphorylation (44-kDa band
completely abolished), compared with chondrocytes cultured on collagen
type II stimulated with IGF-I and in the absence of U0126 (Fig.
4B). In contrast, Western blot
analysis with a pan-Erk antibody, which recognizes both the
phosphorylated and nonphosphorylated forms of Erk1 and 2, did not
change in response to U0126 treatment (Fig. 4A).
Densitometric analysis of the results from immunoblotting performed in
triplicate from adhesion of IGF-I-treated chondrocytes to collagen type
II with antibodies against phospho-Erk1/2 showed that in the presence
of 0.1 µM U0126 the relative Erk1/2 proteins expression
had fallen by 43%/51% (p = 0.002/p = 0.01), in the presence of 1 µM U0126 the relative Erk1/2
proteins expression had fallen by 89%/99,9% (p = 0.0063/p = 0.0048), and in presence of 10 µM U0126 the relative Erk1/2 proteins expression had
fallen by 97%/99.9% (p = 0.0019/p = 0.0031) (compared with adhesion of untreated chondrocytes to collagen
type II).
U0126 Treatment Leads to PARP Cleavage--
Cleavage of the
116-kDa polypeptide PARP to its characteristic 85-kDa fragment is
considered as a marker of apoptosis (27, 28). Immunoblot analysis with
antibody against PARP reveals that PARP proteolysis increased in
chondrocytes cultured on collagen type II stimulated with IGF-I and
treated with various concentrations of U0126 (Fig.
5). Densitometric analysis of the results
showed a significant increase in proteolysis of PARP in response to as little as 0.1 µM U0126. PARP cleavage activity increased
with increasing concentration of U0126 in culture. Taken together, these results indicate that the proteolysis of PARP by U0126 is dose-dependent.
Signaling by integrins or transmembrane G proteins leads to
activation of the Ras-MAP kinase signaling pathway. Erk1/Erk2, a
downstream kinase of MAPK pathway, regulates the expression of various
transcription factors. Activation of MAPK pathway may be a mechanism by
which integrins regulate gene expression (6). This study addresses the
important role of the Ras-MAP kinase signaling pathway in the
stimulation of human chondrocyte differentiation. In a previous study
(7), we could show that activation of MAPK pathway regulates the
activity of a number of intracellular signaling proteins through
phosphorylation. We demonstrated that collagen binding integrins and
activated IGF-I receptor coimmunoprecipitate with intracellular
signaling adaptor proteins such as Src-homology collagen, and this
common target forms the Src-homology collagen/Grb2/Erk-complex leading
to Ras-mitogen-activated protein kinase signaling pathway activation
(7). We suggested that this mechanism most likely prevents chondrocyte
dedifferentiation to fibroblast-like cells and chondrocyte death.
To investigate the consequences of inhibition of MAP kinase pathway, we
used a specific inhibitor of the MAPK signaling pathway, U0126, which
can inhibit the phosphorylation and activation of the Erk1/Erk2 in a
dose-dependent manner. We demonstrated that specific
inhibition of Erk1/Erk2 resulted in activation of caspase-3 and
cleavage of PARP in human chondrocytes in vitro. Therefore the specific inhibition of Ras-mitogen activated kinase leads to
apoptosis because activation of caspase-3 and cleaving of PARP are
common features of apoptosis (27, 28).
After 1 h of treatment with U0126, we found caspase-3 labeling in
the nucleus of chondrocytes. In fact, it is suggested according to
recent experiments that pro-caspase-3 is localized in mitochondria and
cytoplasm but activated caspase-3 is localized in cytoplasm and nucleus
(29, 30). Caspase-3 has been reported to be translocated to the nucleus
during apoptosis (31). It is known that activated caspase-3 cleaves
PARP into an NH2-terminal 24-kDa fragment containing the
DNA-binding domain and an about 85-kDa COOH-terminal fragment containing the automodification and catalytic domains, and PARP cleavage seems to be characteristic of later "irreversible" stages of apoptosis (32). This was used to distinguish apoptotic and necrotic
cell death (15). According to other investigators (32), the PARP
cleavage product was accompanied by the reduction of intact PARP.
Caspase-3, in its activated form is the main cause of apoptotic
modification, such as PARP degradation. Intact PARP inhibits
endonucleases that cleave DNA during apoptosis (33).
At the moment, one can only speculate about the particular mechanisms
leading to caspase-3 activation. As mentioned above Erk1/Erk2
translocates into the nucleus and regulates the activities of several
nuclear transcription factors. Inhibition of Erk1/Erk2 prevents
translocation of it into the nucleus, because of that the expression of
genes that code for proapoptotic proteins may increase or genes
that code for antiapoptotic proteins may be repressed. Interruption
of the MAPK pathway by inhibition of Erk1/Erk2 may also prevent
the inactivation of proapoptotic factors as previously described (34).
Indeed, they have further reported that apoptosis correlated with a
reduced activity of Erk signaling for ovarian granulosa cells.
A group of proteins regulating apoptosis are members of the Bcl2
superfamily, which comprises a large number of pro- and antiapoptotic molecules mainly located in the mitochondrial outer membrane and therefore partly inducing mitochondrial changes, e.g.
cytochrome c release (35-36). The ratio of pro- (Bax, Bak,
Bid, Bad ... ) versus antiapoptotic (Bcl2,
Bclxl, Bclw ... ), Bcl2 superfamily members determines how cells respond to death signals (35). In
osteoarthritic cartilage the expression of Bcl2 is lower than in
healthy cartilage (37). Bcl2 is also down-regulated in serum-free cultured human articular chondrocytes (38) and in articular chondrocytes in transgenic mice lacking the expression of type II
collagen (39).
Several observations suggest that pathological conditions that cause
degradation of cartilage may induce apoptosis (40, 41). It seems that
chondrocyte apoptosis is involved in several cartilage diseases,
e.g. osteoarthritis (37, 40), rheumatoid arthritis (42),
chondrodysplasias, and chondrosarkomas (38). Recently it was reported
that the pathogenesis of osteoarthritis may also result from the
inappropriate response of chondrocytes to catabolic and anabolic
stimuli from mitogens, i.e. growth factors and hormones
(43). The effects of particular growth factors (e.g. IGF-I)
are mediated by MAPK pathway (7). It was found that the mRNA of a
special protein Egr-1 (early growth response-1) was significantly
down-regulated in samples from osteoarthritic cartilage. The Egr-1
protein binds to a specific GC-rich sequence in the promotor region of
many target genes hence regulating their expression (43). Several
studies have indicated that interruption of integrin/extracellular
matrix interactions especially between integrins and collagen
type II results in apoptosis of chondrocytes (10, 39).
It is important to know the apoptosis pathways of chondrocytes because
inhibition of chondrocyte apoptosis may be of therapeutic value after
cartilage injury and in arthritis. Apoptosis is also important in
physiological conditions such as chondrogenesis and during enchondral
ossification. It was recently reported (44) that growth-arrest-specific
gene 2 (gas 2) might play an important role in regulating
chondrocyte proliferation and differentiation. They observed further
that exactly at the same time when mesenchymal cells of mouse limb bud
interdigital tissues die by apoptosis, the gene product of gas
2 was cleaved by caspases and suggested that caspase-3 might be
involved in gas 2 cleavage. Expression of gas 2 is coupled to increased susceptibility to apoptosis. Furthermore, it
has been suggested that apoptosis of chondrocytes during development
may control chondrocytes number in cartilage tissue (45).
In the present study, we demonstrate that chondrocytes underwent
apoptosis after treatment with U0126, a novel inhibitor of MEK1/2,
suggesting that MAP kinase pathway has a regulatory function in
chondrocytes differentiation and survival.
1-integrin
antibodies or collagen type II in vitro. These mechanisms
most likely prevent chondrocyte dedifferentiation to fibroblast-like
cells and chondrocyte death. To investigate whether this pathway plays
an essential role for the differentiation, phenotype, and survival of
chondrocytes, we blocked mitogen-activated protein kinase/extracellular
signal-regulated kinase (Erk) (MEK), a kinase upstream of the kinase
Erk by using U0126. Exposure of chondrocytes to U0126 caused activation
of caspase-3 in a dose-dependent manner. Western blot
analysis with an antibody specific for dually phosphorylated Erk shows
that collagen type II induced phosphorylation of Erk1/2 was
specifically blocked by U0126 in a dose-dependent manner.
Immunohistochemical analysis showed that treated chondrocytes were
caspase-3 positive. In treated chondrocytes, the cleavage of 116-kDa
poly(ADP-ribose)polymerase resulted in the 85-kDa apoptosis-related cleavage fragment and was associated with caspase-3 activity. Analysis
by electron microscopy showed typical morphological signs of
apoptosis, such as crescent-shaped clumps of heterochromatin, and a
degraded pericellular matrix. Thus, these results indicate that the
MEK/Erk signal transduction pathway is involved in the maintenance of
chondrocytes differentiation and survival. These data stimulate further
investigations on the role of mitogen-activated protein kinase pathways
in human chondrocytes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(5).
1-integrin antibodies leads to a
rearrangement of cytoskeletal and signaling proteins localized at focal
adhesions and focal adhesion kinase (6-9). These stimulate docking
proteins such as Src-homology collagen. Src-homology collagen then
associates with growth factor receptor-bound protein 2, and extracellular signal-regulated kinase
(Erk)1 (7). These mechanisms
most likely prevent chondrocyte dedifferentiation to fibroblast-like
cells and chondrocytes death (7-8, 10).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effects of U0126 on chondrocyte adhesion to
collagen type II. Serum-starved human chondrocytes were cultured
on glass coverslips coated with collagen type II stimulated with IGF-I
and then treated with U0126 or left untreated in serum-free medium for
14 h. The adherent chondrocytes were quantified every hour by
scoring cells from three different microscopic fields. The attachment
assay revealed that chondrocytes cultured on collagen type II in
absence of U0126 show a significantly higher density of chondrocyte
adhesion from the beginning of cultivation compared with those
cultivated on collagen type II with treatment with U0126. The mean
values and S.D. from three independent experiments are indicated.
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Fig. 2.
Electron microscopy of chondrocytes.
A, electron microscopic demonstration of an untreated
chondrocyte (C) containing smooth surface, large nucleus
(N) with much loosely packed, despiralized and functionally
active euchromatin and little densified, functionally inactive
heterochromatin, numerous cavities of rough endoplasmic reticulum and
surrounded by a pericellular matrix sheath closely attached to the cell
membrane (arrows). Bar 0.4 µm, × 10,000. B,
after treatment with U0126 for 1 h, a representative chondrocyte
has nuclear changes with peripheral segregation and aggregation of
chromatin into dense areas (*) along the nuclear membrane, swellings
and dilatations of cell organelles (mitochondria and endoplasmic
reticulum), and bleb formation at the cell membrane
(arrows). Bar 0.4 µm, × 10,000.
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Fig. 3.
Immunodetection of active caspase-3 by APAAP
method. Serum-starved human chondrocytes were plated on dishes
coated with collagen type II stimulated with IGF-I and then treated
with various concentrations of U0126: 0, 0.1, 1, and 10 µM for 1 h. The cells were immunolabeled with
anti-active caspase-3 antibodies. The results showed a marked
dose-dependent increase in induction of activated caspase-3
(arrows) in chondrocytes (B-D). In
contrast, control cultures of chondrocytes treated with IgG maintained
their nuclear morphology throughout the culture period
(A).
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Fig. 4.
Activity profiles of Erk1/2 after treatment
with U0126. Serum-starved human chondrocytes were plated on dishes
coated with collagen type II stimulated with IGF-I and then treated
with various concentrations of U0126: 0, 0.1, 1, and 10 µM for 1 h. The cells were immunolabeled with
anti-Erk1/2 (A) and anti-phospho-Erk1/2 (B). The
results showed a significant dose-dependent decrease of
phosphorylated Erk1/2.
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Fig. 5.
Detection of PARP and its apoptotic fragment
in chondrocytes exposed to U0126. Serum-starved human chondrocytes
were plated on dishes coated with collagen type II stimulated with
IGF-I and then treated with various concentrations of U0126: 0, 0.1, 1, 10, 100, and 1000 µM for 1 h. Intact PARP and the
85-kDa fragment were analyzed by Western blotting using the monoclonal
anti-PARP antibody. After 1 h of treatment with different doses of
U0126, the 85-kDa fragment was visible, and this process was
dose-dependent.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We are indebted to Dr. A. Scheid for reviewing the manuscript. We gratefully acknowledge Ingrid Wolff's expert photographic work and Angelika Hardje's and Angelika Steuer's technical assistance.
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FOOTNOTES |
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* This work was supported by the Deutsche Forschungsgemeinschaft (DFG Grant Sh 48/2-2 and Sh 48/2-3).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We dedicate this paper to Prof. H. G. Baumgarten on the occasion of his 65th birthday and acknowledge the longstanding excellent collaboration with him.
§ To whom correspondence should be addressed: Institute of Anatomy, Free University of Berlin, Königin-Luise-Strasse 15, D-14195 Berlin, Germany. Tel.: 49-30-8445-1916; Fax: 49-30-8445-1916; E-mail: mehshaki@zedat.fu-berlin.de.
Published, JBC Papers in Press, January 16, 2001, DOI 10.1074/jbc.M010859200
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ABBREVIATIONS |
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The abbreviations used are: Erk, extracellular signal-regulated kinase; PARP, poly(ADP-ribose)polymerase; MAPK, mitogen-activated protein kinase; MEK, MAPK/Erk; IGF, insulin-like growth factor.
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