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Normal expression of type 1 insulin-like growth factor receptor by human osteoarthritic chondrocytes with increased expression and synthesis of insulin-like growth factor binding proteins.

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ARTHRITIS & RHEUMATISM
Val. 39, No. 6, June 1996, pp 968-978
0 1996, American College of Rheumatology
968
NORMAL EXPRESSION OF TYPE 1 INSULIN-LIKE GROWTH FACTOR
RECEPTOR BY HUMAN OSTEOARTHRITIC CHONDROCYTES
WITH INCREASED EXPRESSION AND SYNTHESIS OF
INSULIN-LIKE GROWTH FACTOR BINDING PROTEINS
GINETTE TARDIF, PASCAL REBOUL, JEAN-PIERRE PELLETIER, CHANGSHAN GENG,
JEAN-MARIE CLOUTIER, and J 0 H A ” E MARTEL-PELLETIER
Objective. Our previous research demonstrated
that, in contrast to normal chondrocytes, human osteoarthritic (OA) chondrocytes were hyporesponsive to
stimulation by insulin-like growth factor 1 (IGF-1). The
aim of the present investigation was to examine whether
this finding was due to an alteration in the level of
IGF receptors (IGFRs) and/or IGF binding proteins
(IGFBP).
Methods. A quantitative reverse transcriptase
polymerase chain reaction technique (RT-PCR) was
used to measure the type 1 IGFR messenger RNA
(mRNA) level, and Northern blotting was used to measure type 2 IGFR and IGFBP mRNA levels. Western
immunoblotting was used to identify and measure
IGFBP levels.
Results. There were similar levels of type 1IGFR
mRNA in normal and OA chondrocytes. The level of
type 2 IGFR mRNA, in which an increased amount of
which can interfere with the biologic effects of IGF-1,
was lower in OA chondrocytes compared with normal
chondrocytes. Articular chondrocytes produced IGFBP-2,
IGFBP-3, and IGFBP-4, and OA chondrocytes secreted
and expressed higher amounts than did normal chondrocytes. There was also an increased level of IGFBP-3
Supported by grants from The Arthritis Society.
Ginette Tardif, PhD, Pascal Reboul, PhD, Jean-Pierre Pelletier, MD, Changshan Geng, PhD, Jean-Marie Cloutier, MD, Johanne
Martel-Pelletier, PhD: University of Montreal, and Louis-Charles
Simard Research Center, Notre-Dame Hospital, Montreal, Quebec,
Canada.
Drs. Tardif and Reboul had an equal scientific input in the
realization of this study.
Address reprint requests to Johanne Martel-Pelletier, PhD,
Rheumatic Disease Unit, Notre-Dame Hospital, 1560 Sherbrooke
Street East, Montreal, Quebec, H2L 4K8, Canada.
Submitted for publication September 13, 1995; accepted in
revised form January 17, 1996.
in the OA chondrocyte lysates. IGFBPs 1,5, and 6 were
not detectable.
Conclusion. OA chondrocytes synthesize and express a larger amount of 3 IGFBPs. This observation,
along with a lack of detectable change in type 1 IGFR
mRNA level, suggests that the hyporesponsiveness of
OA chondrocytes to IGF-1 might implicate the involvement of IGFBPs in this pathologic process.
Osteoarthritis (OA) is characterized by a progressive degeneration and erosion of the cartilage, and it
results from a failure of the chondrocyte to maintain the
balance between synthesis and degradation of the extracellular matrix. The pathologic changes are related,
depending on the stage of the disease, to a combination
of both a degradation of the main components of the
matrix and/or a decrease in the synthesis and/or quality
of the matrix macromolecules (1). Among the constituents that play a role in the mechanism of cartilage repair
are growth factors, such as insulin-like growth factor 1
(IGF-1) (2,3). This growth factor enhances the synthesis
of collagen and proteoglycan (2-5). There is also evidence that it could play a role in the pathogenesis of OA.
This was shown both in vitro, where IGF-1 was found to
reduce interleukin-1 (IL-1)-stimulated cartilage degradation (6), and in vivo in an experimental model of OA,
in which therapeutically administered IGF-1 in combination with a synthetic protease inhibitor (sodium pentosan polysulfate [PPS]) produced significant improvement in several OA parameters compared with PPS
alone (7). IGF-1 alone had no effect, and the IGF-1treated cartilage was macroscopically similar to the
untreated OA cartilage (7), which is consistent with
previous data showing that arthritic chondrocytes are
hyporesponsive to IGF-1 stimulation (8,9). The exact
IGF RECEPTORS AND IGFBP IN OA CHONDROCYTES
reason for the improvement when PPS and IGF-1 were
administered together is presently unknown. However,
several hypotheses can be considered. It is possible that
because PPS is a protease inhibitor, it reduces proteoglycan breakdown in the matrix. And if, as recently
suggested, proteoglycans have the ability to bind I G F
binding protein (IGFBP) (lo), this reduced proteoglycan breakdown may, in turn, have increased t h e binding
of IGFBP. T h e r e may be other explanations for this
observation (7), and investigations of the mechanisms of
action would b e of great interest.
There are 2 types of IGF receptor (IGFR). Type
1 I G F R is a glycoprotein that is similar to, but distinct
from, the insulin receptor ( l l ) , and it has been suggested
to be responsible for the effects of IGF-1. T h e role of
type 2 I G F R is unclear. It consists of a polypeptide chain
with 99% homology to the cation-independent mannose6-phosphate receptor, which targets the lysosomal enzymes to lysosomes (12). In addition to its binding to
specific receptors, IGF-1 also binds t o IGFBPs, a family
of 6 proteins (IGFBPs 1-6). T h e IGFBPs are soluble
proteins found primarily extracellularly, with some being
membrane-bound (13). Due to their ability to bind to
IGF-1, both the receptors and the I G F B P may contribute to the markedly increased level of binding sites
observed in human OA chondrocytes (8). Furthermore,
increased levels of one or more binding proteins may
sequester additional IGF-1, making it unavailable to
exert its anabolic action.
Using affinity cross-linking, we previously showed
that human OA chondrocytes, compared with normal
chondrocytes, had a markedly increased intensity of
bands corresponding to the known IGFBPs (8). In the
present studies, we pursued this line by investigating
which elements in the I G F system are involved in the
hyporesponsiveness of O A chondrocytes to IGF-1. It is
possible that a n alteration in the I G F R may have
occurred in this pathologic tissue or that a diminution in
the bioavailability of IGF-1 involving the I G F B P may
have occurred. W e therefore investigated these two
possibilities. More specifically, we examined the possible
changes in OA chondrocytes with respect to the gene
expression of both receptor types (type 1 and type 2) and
investigated the nature, the presence, a n d the expression
level of I G F B P in these cells.
PATIENTS AND METHODS
Specimen selection. OA cartilage from tibia1 plateaus
and femoral condyles (mean i SEM age 70 i 2) were
obtained from patients undergoing knee arthroplasty. The
969
diagnosis of OA was based on findings of clinical and radiologic evaluations (14). These specimens represented moderateto-severe OA. as defined macroscopically according to the
appearance of OA cartilage. Surface fibrillation and pitting
were prominent. along with eburnation of variable size. The
specimens were full-thickness strips of tissue cut across the
surface, excluding mesenchymal repair tissue and subchondral bone.
Normal control cartilage (mean t SEM age 62 % 7)
was taken from the same location as for the OA cartilage, and
was obtained from adult cadavers within 12 hours of death.
The subjects had no history of joint disease, and each specimen
was examined macroscopically and microscopically to ensure
that only normal tissue was used.
Culture of chondrocytes. Cartilage specimens were
obtained under aseptic conditions, and samples were dissected
on ice. Chondrocytes were released from articular cartilage by
sequential enzymatic digestion at 37”C, as previously described
(8). After being released, the cells were centrifuged, washed,
seeded at high density (-lo5 cells/cm’) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL,
Gaithersburg, MD) supplemented with 10% heat-inactivated
fetal calf serum (HyClone, Logan, UT) and an antibiotic
mixture (100 units/ml of penicillin base and 100 pg/ml of
streptomycin base; Gibco BRL) at 37°C in a humidified
atmosphere of 5% COz, 95% air. Only primary cultures were
used in this study in order to ensure the chondrocyte phenotype (15,16). Twenty-four hours prior to further experiments,
the cells were incubated in serum-free medium.
Western immunoblot. Chondrocytes were incubated in
DMEM containing antibiotics for 72 hours at 37°C. The
culture medium was dialyzed (6,000-8,000-dalton cut-off,
Spectra/Phor; Spectrum Medical Industries, Los Angeles, CA)
against distilled water, the proteins were precipitated overnight
at -20°C with 5 volumes of acetone, and the pellets were
directly suspended in sodium dodecyl sulfate (SDS) gel sample
buffer. Chondrocytes were washed 3 times with cold phosphate
buffered saline (PBS), scraped in PBS, and centrifuged (1,OOOg
for 5 minutes). The pellet was solubilized directly in an SDS gel
sample buffer.
IGFBP were determined according to the procedure
described by Smith et a1 (17). For each specimen (culture
medium or chondrocyte), 20 pg of protein was electrophoresed
on a discontinuous 4-12% SDS-polyacrylamide gel (18). The
proteins were transferred electrophoretically onto a nitrocellulose membrane (Hybond C extra; Amersham Canada,
Oakville, Ontario) according to the method of Towbin et a1
(19). The efficiency of transfer was controlled by a brief
staining of the membrane with 0.1% Ponceau S (Sigma, St.
Louis, MO) in 5% acetic acid, and destaining in water before
immunoblotting.
Immunoblotting was performed according to the
method of Burnette (20), with minor modifications. The
membranes were immersed overnight in a blocking solution
consisting of 10% skim milk in PBS and washed twice for 15
minutes with PBS containing 0.05% Tween 20. Membranes
were incubated for 90 minutes in PBS containing 0.05% Tween
20 and 0 . 3 % skim milk with either anti-human IGFBP-1
(anti-HuIGFBP-1; generously provided by Dr. R. C. Baxter,
Royal Prince Hospital, Camperdown, Australia), antiHuIGFBP-2,4, or 5 (UBI. Lake Placid, NY),anti-HuIGFBP-3
970
(generously provided by Dr. C. Maack, Celtrix, Palo Alto, CA),
or anti-HuIGFBP-6 (Austral Biologicals, San Ramon, CA).
These polyclonal antibodies raised in rabbits were used at a
dilution of 1:1,000 except for the IGFBP-6, which was 1:lOO.
The membranes were then washed 3 times, for 10 minutes
each, with PBS-Tween, TBS-Tween (10 mMTris HCI, pH 7.4,
and 0.9% NaCl, in 0.05% Tween 20), and TBS alone, and
incubated for 30 minutes in TBS with 5% skim milk containing
lZ5I-labeledprotein A (500,000 counts per minuteiml, specific
activity 40 mCi/mg; Amersham). The radioactive solution was
removed and the membranes rinsed again, as described above.
The dried membranes were wrapped in Kapak Scotchpak bags
and exposed to x-ray film at -80°C overnight for 4 days,
utilizing Du Pont Cronex Lighting Plus film.
Immunoprecipitation was also performed for some of
the IGFBPs (IGFBPs 1, 5, and 6). Five hundred microliters of
the culture medium (1:2 dilution with PBS) was incubated for
2 hours at 4°C with protein A-agarose (Bio-Rad, Richmond,
CA) and nonspecific IgG (Dimension Laboratories, Mississauga, Ontario, Canada). The latter was used in order to
prevent nonspecific immunoreaction. The samples were centrifuged (10,OOOg for 5 minutes), the supernatant mixed again
with protein A-agarose, and the specific IGFBP antibody
(dilution 1500 for IGFBPs 1 and 5, and 1:50 for IGFBP-6 in
PBS), and incubated at 4°C for 18 hours. The immunoprecipitate was collected after a centrifugation, washed twice in
PBS-0.05% Tween 20, and the resulting immunoprecipitates
were subsequently analyzed.
DNA manipulations. Plasmid DNA extraction, gel
electrophoresis, DNA restriction and ligation, transformation
of Escherichia coli bacterial cells (DHSa), and Southern blotting were performed according to the methods of Sambrook et
a1 (21). The restriction enzymes, T4 DNA ligase, and the RNA
polymerases were used according to the manufacturer’s specifications (Pharmacia LKB, Uppsala, Sweden). The sequencing
of double-stranded plasmid DNA was performed in an automated DNA sequencer (AL,F DNA sequencer; Pharmacia).
RNA extraction. Total RNA was isolated from confluent cultures of primary normal and OA human chondrocytes,
as previously described (8). Briefly, the cells were lysed in a
preheated (60°C) buffer containing 20 mM sodium acetate, pH
5,0.5% SDS, and 1 mM EDTA. The lysate was then extracted
twice with preheated phenol (60°C) in 20 mM sodium acetate,
pH 5. The RNA in the resultant aqueous phase was precipitated overnight at -20°C with 2 volumes of ethanol. The pellet
was resuspended in the acetate buffer and reprecipitated with
ethanol. Following solubilization of the RNA pellet in sterile
water, RNA was quantitated spectrophotometrically and examined by agarose gel electrophoresis.
Reverse transcriptase polymerase chain reaction (RTPCR). RT-PCR assays were carried out in a Gene ATAQ
Controller (Pharmacia LKB) and performed as previously
described (22). In this experiment, 2 pg of the total RNA was
used. Each PCR cycle consisted of a denaturation step for 1
minute at 95”C, and an annealing/elongation step for 1.5
minutes at 60”C, except for the last cycle, for which the
elongation step was prolonged to 7 minutes at 60°C.
The oligonucleotide primers were prepared with a
DNA synthesizer (Cyclone model; Biosearch, Montreal, Quebec, Canada) and used at a final concentration of 200 nM. A
specific set of primers for type 1 IGFR was developed in our
laboratory. The sequences for type 1 IGFR primers were
TARDIF ET AL
5’-GAAGTATGCCGACGGCACCATCGAC-3’
(sense
primer) which corresponded to position 2058-2082 bp of the
published sequence (23), and 5‘-CTCGCTGATCCTCAACTTGTGATCC-3‘ (antisense primer) from position 2653-2677 bp.
The size of the amplified fragments was 620 bp, as expected (23).
Expression of type 1 IGFR mRNA by quantitative
RT-PCR. Construction of RhLA standard. Total RNA extracted
from human chondrocytes was reverse-transcribed and amplified as described above, and then cloned directly into the
pCRII vector (Invitrogen, San Diego, CA). The resulting
plasmid was cut with Eco RI to release the cloned type 1 IGFR
fragment; the fragment was subsequently cloned into the Eco
RI site of pBluescript I1 SK+ (Stratagene, La Jolla, CA). The
construct was cut with Sty I (Stratagene); the enzyme does not
cleave pBluescript, but cuts into the type 1 IGFR fragment at
512 bp from the 5’ end. The linear DNA was treated for 20
minutes with mung bean nuclease (Pharmacia) and religated
with T4 DNA ligase (Pharmacia). The religated plasmid was
sequenced; the nuclease had removed 6 bp, 2 being in the
recognition site of Sty I. The deletion rendered the plasmid
resistant to digestion with Sty I. RNA transcripts from the
mutated type 1 IGFR fragment were synthesized with the
Riboprobe kit (Promega, Madison, WI) and quantitated spectrophotometrically. The RNA was further diluted for use as
the internal standard in the quantitation of mRNA transcripts
by the RT-PCR technique. This standard was added at a
known concentration to the RT-PCR reaction and amplified by
the same primers as the specific target mRNA.
Quantitative RT-PCR. Type 1IGFR mRNA was quantitated by competitive PCR, as described by Gilliland et a1 (24)
and Wang et al (25). To quantitate the mRNA, a specific
number of molecules (106-108) of the internal standard was
added to the RT-PCR reaction mixture. Amplification was for
25 cycles. The amplification rates for both the standard and
target mRNA were the same, and the ratio of the products
remained constant throughout the cycles. Following the reaction, 7 pl of the mixture was digested with Sty I to distinguish
between the RNA amplified from the standard and the cellular
RNA. The PCR digestion products were separated on a 1.2%
agarose gel and stained with ethidium bromide to visualize the
bands. Band intensity was measured on the film negative with
a laser scanning densitometer (model GS 300; Hoefer Scientific Instruments, San Francisco, CA). The number of RNA
molecules in the sample was found when the ratio of the band
intensities (as determined by densitometry) of the internal
standard and to the sample was 1:l. The results were calculated as mRNA molecules and expressed as the number of
mRNA molecules per cell.
Expression of type 2 IGFR and IGFBP mRNA by
Northern blotting. Five micrograms (for IGFBP-3) or 10 pg
(for type 2 IGFR and IGFBPs 2 and 4) of total RNA was
resolved on 1.2% formaldehyde-agarose gels and transferred
electrophoretically to nylon membranes (Hybond-N; Amersham) as described previously (22).
The human type 2 IGFR, IGFBP-2, and IGFBP-3
probes were constructed by cloning the respective RT-PCRamplified fragments directly into the pCRII vector. The resulting plasmids with either type 2 IGFR or IGFBP-3 were cut
with Eco RI to release the fragments that were cloned into the
Eco RI site of pBluescript SK+. Each construct was sequenced
to verify the integrity of the probes. The primer sequences for
type 2 IGFR were 5’-GCTGTGCAGTTATACATGGG-3’
971
I LLAJ
IGF RECEPTORS AND IGFBP IN OA CHONDROCVTCc
(sense) and S'-TCGTAGTGTGTCTATGTCTC-3'
(antisense), corresponding to positions 288-307 bp and 785-804
bp, respectively (26). The size of the amplified fragment was
517 bp. The primer sequences for IGFBP-2 were 5'AATGGCGATGACCACTCAGA-3' (sense) and 5'-?TGTCACAGTTGGGGATGTG-3' (antisense), corresponding to
positions 454-473 bp and 778-797 bp of the published sequence (27). The size of the amplified fragment was 343 bp.
The primer sequences for IGFBP-3 were 5'-CCAGGAAATGCTAGTGAGTC-3' (sense) and 5'-GTC?TCCATITCTCTACGGC-3' (antisense), corresponding to positions 400-419
bp and 638-657 bp, respectively (28). The size of the amplified
fragment was 180 bp, instead of the expected 258 bp. This was
probably due to a deletion during the process. However, the
fragment was sequenced and corresponded to 180 bp of the
published sequence (28).
The IGFBP-4 probe (505 bp in pBluescript SK+) was
provided by Dr. S. Shimasaki (29) (Scripps Research Institute,
La Jolla. CA). A 780 Pst I/Xba I fragment from GAPDH
complementary DNA ((cDNA] 1.2 kb) (no. 57090; American
Type Culture Collection, Rockville. MD) was subcloned into a
pGEM-3Z vector (Promega). The latter probe served as a
control for RNA loading. since GAPDH is constitutively
expressed. In our study, both sense and antisense probes were
tested. Data revealed that the antisense probes used were
specific.
The RNA probes were transcribed from these plasmids, labeled with DIG 11-UTP (digoxigenin-ll-uridine-5'
triphosphate; Boehringer Mannheim Biochemica, Mannheim,
Germany) and were revealed using the luminescent PPD
[4-methoxy-4-(3-phosphatephenyl)-spiro-(
1,2-dioxetane-3,2'adamantane) disodium salt] reagent, as described elsewhere
(22). The membranes were then subjected to autoradiography
using Kodak XAR-5 film (Eastman Kodak, Rochester, NY) at
room temperature. The autoradiographs were scanned by laser
densitometer for mRNA measurement, and the results were
calculated as the relative expression normalized to GAPDH.
The membranes were first hybridized with one probe,
then stripped (22) and rehybridized for GAPDH, the only
exceptions being type 2 IGFR and IGFBP-4, where the
membrane was stripped twice.
Statistical analysis. Data are expressed as the mean 2
SEM. Statistical analysis was assessed by the nonparametric
Mann-Whitney test. Significant differences were confirmed
only when the P value was less than 0.05.
RESULTS
Types 1 and 2 IGFR mRNA expression. As
reported in an earlier study (8), the expression of type 1
IGFR by human chondrocytes was very weak, and a
large amount (4.5 pg) of poIy(A)+ RNA was required
to detect a signal using the Northern blot technique.
Therefore, due to the limited amount of available tissues, especially OA cartilage, the comparison of the level
of type 1 IGFR mRNA expression between individuals
was performed using a quantitative RT-PCR method.
This sensitive method required an internal RNA standard, which was added at known concentrations to the
Standard Molecules (X 106)
2645 --w
1605 -+
1198+
Internal Standard
Sample
676 --w
517-
A
MM550 -
m-
-
$
.
2
450
-
4m-
350-
;
300-
E
250-
2
mo-
0
2
150-
100
SO
OA
Normal
B
Figure 1. A, Representative quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis gel for type 1 insulin-like growth
factor receptor (IGFR) messenger RNA (mRNA) in human chondmcytes. Internal R N A standards were added at increasing concentrations
without (lanes 2,3. and 4) or with (lanes 5.6, and 7) a fixed amount of the
chondrocyte sample. All of the amplified PCR products were digested
with Sty I and run on agarose gels. The internal standard was constructed
in such a way that it couid not he digested by Sty I (higher band). However,
Sty I cuts the PCR products amplified from the sample (lower band). MW
= pGEM DNA markers; type 1 IGFR (sample) is at 620 bp. B,Histogram
of the expression of type 1 IGFR mRNA in normal (n = I ) and
osteoarthritic (OA; n = 4) human chondrocytes.
RT-PCR reaction and then amplified by the same primers as the specific target mRNA. The standard, as
described in Patients and Methods, also served as a
TARDIF ET AL
972
Normal
1
2
3
4
Osteoarthritic
5
6
1
2
3
4
5
6
TypeII IGF-R
28s -+
18s-
GAPDH
A
Normal
OA
B
Figure 2. A, Northern blot of the expression of type 2 IGFR and GAPDH mRNA in normal and OA human
chondrocytes. The positions of 28s and 18s ribosomal RNA are indicated. After an overnight incubation, the membrane
was exposed for 1 hour and 45 minutes for type 2 IGFR and 15 minutes for GAPDH. B, Histogram of the expression
of type 2 IGFR mRNA in normal (n = 6) and OA (n = 6) human chondrocytes.
transcription control for the RT reaction. This standard
was a fragment of the same size and sequence (except
for a few basepairs) as the fragment to be amplified. The
amplification rates for both fragments were the same,
and the ratio of the products remained constant
throughout the cycles. Figure 1A is representative of the
PCR-amplified fragments for both the sample and the
standard. Analysis of the results from normal control
(n = 4) and OA (n = 4) chondrocytes (Figure 1B)
demonstrated no difference in the level of steady-state
mFWA for type 1 IGFR.
Although speculative, one may postulate that
under pathologic conditions, a shift of the receptor type
may have occurred in the chondrocytes. Considering
that the recognized activity of the type 2 IGFR was
either to target lysosomal enzymes or to act as a
reservoir/clearance for IGF-2 (12), an increased amount
of this receptor may have hindered the specific biologic
effects of IGF-1. We next examined the mRNA level of
the type 2 IGFR. Because preliminary experiments
revealed reliable detection of mRNA by Northern blot,
we quantitated its level of expression in normal (n = 6)
and OA (n = 6) chondrocytes (Figure 2A). Analyzing
the mRNA level by densitometry and normalizing it to
GAPDH showed that the type 2 IGFR level was slightly
decreased in OA cells. No statistical significance was
obtained when values from OA cells were compared
with those from normal cells (Figure 2B).
IGFBP characterization, protein level, and
mRNA expression. In view of the previous results regarding the IGFR, one should suspect the probability
that endogenous factors may interfere with the hormone
973
IGF RECEPTORS AND IGFBP IN OA CHONDROCYTES
OA
Normal
kDa
1
2 3 4
5
1 2 3 4
IGF
5BP-2
53
109.8
35
42.8
29
33.3
86.8
26.8
21
20.7
-
Normal
OA
IGFBP-3
1 2 3 1 2 3
-+
-+
+
B
OA
Normal
kDa
1 2 3 4
5
1
IGF
2 3 4 5BP-3
53
35
29
21
OA
Normal
kDa
1 2 3
4
Figure 3. Western immunoblot of freshly released normal and osteoarthritic (OA) human chondrocyte (A) culture medium of insulin-like
growth factor binding protein-2 (IGFBP-2), IGFBP-3, and IGFBP-4
and (B) human chondrocyte lysate of IGFBP-3. Molecular weight
markers are in kilodaltons.
5
1 2 3 4
IGF
5BP-4
53
35
29
21
A
action of OA chondrocytes. Factors such as the IGFBP,
which are capable of competing with the IGFR for
hormone binding, are an interesting possibility. In order
to characterize which of the IGFBP were secreted by the
chondrocytes, Western immunoblotting was conducted
using specific antibodies against human IGFBPs 1-6.
IGFBPs 2, 3, and 4 were detected in the conditioned medium at the expected size; 32 kd for IGFBP-2,
a doublet of 38/42 kd for IGFBP-3, and 28 kd for
IGFBP-4 (Figure 3A). The chondrocyte lysate showed
the presence of only IGFBP-3; interestingly, only 1 band
was detected, at -38 kd (Figure 3B). IGFBPs 1, 5, and
6 could not be found either in the culture medium or in
the chondrocytes, even after immunoprecipitation. As
illustrated in Figure 3A, IGFBP-4 synthesis was not
detectable in normal cells, whereas a high level was
found in the OA cells. IGFBP-2 and 3 were detected in
2 of the 5 normal specimens, but higher levels were
TARDIF ET AL
974
found in OA cells. Of note, IGFBP-2 Western immunoblot had, in addition to the band characteristic of
IGFBP-2, a higher band which appeared to correspond
to IGFBP-3. This is likely since, according to the manufacturer (UBI), the IGFBP-2 antibody could crossreact (0.1-0.5%) with IGFBP-3. Figure 3B illustrates
the IGFBP-3 in the cell lysate, where a faint band could
be seen in one of the normal specimens and high levels
in OA samples.
We further investigated whether the changes in
the amounts of these IGFBP were associated with an
increased level of mRNA expression. Northern blot
analysis (Figure 4) revealed a definite mean increase in
the steady-state mRNA level for these IGFBP. In OA, a
1.6-fold increase in the mRNA level was recorded for
IGFBP-2, 1.8-fold for IGFBP-3, and 1.4-fold for
IGFBP-4 (Figure 4D). Statistical analysis revealed a
significant difference (P < 0.02) in IGFBP-4 levels
between OA and normal cells. Differences in IGFBP-3
levels were not statistically significant (P < 0.09).
DISCUSSION
This study extends our previous observations (S),
and suggests that changes in the level of synthesis of
IGFBP, but not in the level of type 1 IGFR, may
contribute to the hyporesponsiveness of human OA
chondrocytes to stimulation by IGF-1. We demonstrated
that chondrocytes produced 3 of the IGFBPs (IGFBPs 2,
3, and 4), which were synthesized in larger amounts by
OA cells. The level of cell-associated IGFBP-3 was also
elevated in OA. Each of the IGFBP present had a higher
steady-state mRNA level in OA than in normal cells.
OA is a degenerative joint disease in which the
balance between the synthesis and the degradation of
matrix macromolecules is disturbed, favoring the degradative process over the reparative one, and leading to a
progressive loss of cartilage. Evidence is mounting which
suggests that the aborted reparation of the damaged
cartilage, as seen in OA, may have to do more with
chondrocyte hyporesponsiveness to growth factors than
to their synthesis. This hypothesis appears to apply to
IGF-1, since although OA chondrocytes synthesized
more IGF-1 than normal chondrocytes (30,31) and
presented an increased number of IGF-1 binding sites
per cell, these diseased cells were not responsive to
IGF-1 stimulation (8).
In this study, we showed that the change in the
expression of type 1 IGFR is unlikely to be responsible
for OA chondrocyte IGF-1 resistance to stimulation,
since similar steady-state mRNA levels of this receptor
were found in normal and OA chondrocytes. This result
indicates that the reported hyporesponsiveness of OA
chondrocytes to IGF-1 ( 8 ) likely occurred at the pre- or
postreceptor levels. The sequestration of IGF-1 before
binding and involvement of IGFBP is one possibility.
Another possibility, and not exclusive of the former, is
that other components of the IGF signaling pathway
may be altered in OA chondrocytes. These may include
second messengers, such as the insulin receptor substrate I kinase, the insulin receptor substrate Iassociated phosphatidyl inositol-3 kinase activity, etc.
The existence of 2 type 1 IGFR transcripts in various
tissues and cell lines with differences in signaling properties, biologic activity, and internalization rate was
recently reported (32). Therefore, a change in the
relative abundance of these 2 forms is another possibility. The methods used in our study do not distinguish
between these 2 putative transcripts, since according to
the report (32), they differ by the presence of only 3
bases.
Moreover, and prompted by a study (33) in which
it was shown that the numbers of type 1IGFR decreased
and type 2 IGFR increased during chondrocyte differentiation, as well as the suggestion that during the OA
process, chondrocytes may to some extent be altered, we
examined whether there had been a shift in the expression of receptor types on OA chondrocytes. If that had
occurred, it may have hindered the IGF-l-specific biologic effects since, as far as the activity of the type 2
IGFR is concerned, its recognized activity is to target
lysosomal enzymes or to act as a reservoir/clearance for
IGF-2 (12,26). Unfortunately, our results did not confirm this hypothesis, since the mRNA steady-state level
of the type 2 IGFR was similar (slightly lower) in the OA
and normal chondrocytes.
IGFBP are believed to have several functions,
including the modulation of IGF binding to their receptors. In general, each cell type secretes a distinct pattern
of binding proteins and changes in their secretion are
associated with changes in certain cell functions. In
various pathologic states, the level of one or more of
these IGFBP has been found to disrupt the normal
metabolism through sequestration of the IGF (34,35).
The detection of IGFBPs 2, 3, and 4 in culture
medium is consistent with those detected in immature
chondrocytes from animals (36-38). Generally,
IGFBP-3 and IGFBP-4 are found in 2 forms, representing the glycosylated and nonglycosylated species,
whereas IGFBP-2 is nonglycosylated. In our study,
IGFBP-3 was found in the culture medium as a 38/42-kd
doublet, and as a single band of 38 kd in the cell lysate.
1
2
Normal
3
4
5
1
6
2
Osteoarthritic
3
4
5
6
18s +
IGFBP-2
18s -+
GAPDH
A
1
Normal
3
4
2
5
6
1
2
Osteoarthritic
3
4
5
6
IGFBP-3
18s18sGAPDH
B
1
2
Normal
3
4
5
6
1
2
Osteoarthritic
3
4
5
6
IGFBP-4
18s18s-
GAPDH
c
IGFBP-2
IFFBP-3
T
IGFBP-4
12
1.2
1.O
1.0
0.8
T
0.8
T
0.6
04
0.2
0.0
Normal
OA
Normal
OA
Normal
OA
D
Figure 4. Northern blot for the expression of (A) !ype 2 insulin-like growth factor binding protein-2 (IGFBP-2), (B) IGFBP-3, and (C) IGFBP-4
mRNA, along with their corresponding GAPDH mRNA, in normal and OA human chondrocytes. The position of 18s ribosomal RNA is indicated.
For IGFBP-2, the membrane was allowed to stand overnight and was exposed for 120 minutes (10 minutes for GAPDH); IGFBP-3 was exposed for
30 minutes (15 minutes for GAPDH); and for IGFBP-4, the membrane was allowed to stand overnight and was exposed for 30 minutes (15 minutes
for GAPDH). (D) Histogram of the expression of IGFBP-2, IGFBP-3, and IGFBP-4 mRNA in normal (n = 6) and OA (n = 6 ) human chondrocytes.
975
976
IGFBP-4 was identified only as the 28-kd form. It is
possible, as shown in other systems examining this
binding protein, that only 1 form prevails (39-41).
However, this may be due to the affinity of the antibody,
since according to the manufacturer (UBI), it has more
affinity for the 28-kd species.
The lack of detection of IGFBPs 1,5, and 6 in the
culture medium, even after immunoprecipitation, was
surprising since various cells, including fibroblasts and
human bone cells (42-44), were able to secrete these
binding proteins. However, our findings corroborate
those of Wang et a1 (45), who showed by in situ
hybridization of murine skeleton that IGFBP-5 and
IGFBP-6 mRNA were abundant during cartilage development but significantly declined as cartilage matured.
Moreover, in that study, the IGFBP-1 mRNA was
undetectable in cartilage at any stage of development.
However, the possibility exists that they are present only
in minute amounts, and are therefore undetectable by
our methods. Alternatively, our results may indicate that
these binding proteins were extensively degraded in
the culture medium. This is substantiated, at least for
IGFBP-5, by recent reports demonstrating that although
human dermal fibroblasts produce IGFBP-5, only minimal intact protein (31 kd) is detected in the conditioned
medium, but is present mostly as fragments of 23 kd,
which derive from IGFBP-5 proteolysis (46). Proteases
that are able to degrade binding proteins have been
reported in many systems and appear to belong to the
serine protease family and/or to the metalloproteases.
Such proteases, and more particularly the metalloproteases, are produced by human chondrocytes and in a
higher amount by OA chondrocytes (1). However, in the
culture medium, they are usually secreted in a latent
form and require enzymatic or chemical activation before they can exert their action. It may be that IGFBP-5
is degraded by a specific protease. IGFBPs 2, 3, and 4
were found undegraded in the chondrocyte culture
medium, since the antibodies we used had the ability to
recognize some of the smaller fragments (47) corresponding to the IGFBP degradation product.
Importantly, our study shows that OA chondrocytes produced a higher level of IGFBPs 2,3, and 4 than
did normal chondrocytes, and demonstrated an increased level of the steady-state mRNA for these binding proteins. However, the IGFBP genes in our cells
were not coordinated with those of either type 1 or type
2 IGFR. The result demonstrating that only IGFBP-3 is
present in the chondrocyte lysate and in a higher amount
in the OA lysate corroborates the literature which states
that this protein has the strongest tendency to associate
TARDIF ET AL
with the cell surface and is found in greater abundance
when the target cell is stimulated (13,48).
While much information has been published concerning the control of cellular secretion of these binding
proteins, their modulation of IGF-1 action on the target
cell remains incompletely studied. It is generally accepted that they sequester IGF, thereby inhibiting its
action. However, even if this appears to be constant for
IGFBP-4 and IGFBP-2 (49,50), controversy exists
whether IGFBP-3 acts as a potentiator or an inhibitor of
IGF activity (51-53). This controversy derives from the
findings of in vitro studies and is possibly related to the
experimental conditions, in which the nature of the IGF
regulation appeared to depend on the recognition of
IGFBP-3 by cell surface or matrix components. Indeed,
the recognition signals may lie in the glycosylation
and/or phospholylation state, and its subsequent IGF
action determined by the status of the exogenous
IGFBP-3 that is added.
In this study, although the exact agent(s) contributing to the IGFBP increase is unknown, several factors
which are present in higher amounts in OA tissues have
been identified as modulating the binding proteins, not
only at the protein level, but also at the mRNA level. For
example, in human fibroblasts, both IGF-1 and transforming growth factor p demonstrated their ability to
increase levels of IGFBP-3 and 4, and in the case of
IGF-1, without influencing the transcript levels
(35,543). In certain cell types, the intracellular level of
CAMPalso seemed to increase both the protein and the
mRNA expressions of IGFBP-4 (42,56). Moreover,
IL-1p and tumor necrosis factor a, cytokines known to
play a major role in this articular tissue disease, also
induced the production of some binding proteins, more
specifically, IGFBP-3 and IGFBP-4, which appeared to
occur by an increase in the mRNA level (57,58). The
understanding of the elements that are able to modulate
these IGFBPs is important in this pathologic tissue, and
experiments are currently underway to identify and
elucidate their mode of action.
ACKNOWLEDGMENTS
The authors wish to express their gratitude to Dr. R. C.
Baxter, Royal Prince Hospital, Camperdown, Australia, and
Dr. C. Maack, Celtrix Pharmaceuticals, Santa Clara, CA, for
generously providing the antibodies against IGFBP-1 and
IGFBP-3, respectively, as well as to Dr. S. Shimasaki, Scripps
Research Institute, La Jolla, CA, for the IGFBP-4 cDNA. The
expert secretarial assistance of Ms. C. Tremblay and Ms. S.
Fiori is also appreciated.
IGF RECEPTORS AND IGFBP IN OA CHONDROCYTES
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