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in Ovine Articular Chondrocytes1
Department of Medicine, Modbury Public Hospital (D.S., J.D.M.), Modbury 5092, South Australia, Australia; and the Cooperative Research Center for Tissue Growth and Repair, Child Health Research Institute, Women’s and Children’s Hospital (D.S., T.E.R., D.A.B.), North Adelaide 5006, South Australia, Australia; and the Department of Veterans Affairs, Medical Center (D.L.A.), Seattle, Washington 98108
Address all correspondence and requests for reprints to: Damir Sunic, Department of Medicine, Modbury Public Hospital, Smart Road, Modbury 5092, South Australia, Australia.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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increased the IGFBP-5 level in a
dose-dependent manner, showing maximum activity at 200 U/ml.
Furthermore, IL-1, but not IGF-I, induced IGFBP-5 messenger RNA
expression, as assessed by Northern blot analysis. Coincubation of
IGF-I with IL-1 resulted in a substantially increased IGFBP-5
protein level, suggesting a synergism between the mechanisms of action
of these two factors. Des(1–3)IGF-I and LR3IGF-I were 10
times more potent than IGF-I in stimulating proteoglycan synthesis,
indicating inhibition of IGF-I activity by endogenous IGFBPs. IL-1
reduced the IGF-I bioactivity, but had no effect on the activities of
the IGF-I analogs, thus implying that locally produced IGFBPs,
particularly IGFBP-5, which was substantially increased when IGF-I and
IL-1 were coincubated, mediated the reduction of the IGF-I activity.
Our results demonstrate that IGF-I and IL-1 synergistically increase
the level of IGFBP-5 in OAC by inhibiting the proteolysis and
stimulating the expression of IGFBP-5, respectively. Furthermore,
the attenuation of IGF-I-stimulated proteoglycan synthesis by IL-1
in OAC appears to be mediated by chondrocyte IGFBPs. We conclude that
locally produced IGFBPs, in particular IGFBP-5, may play a critical
role in the regulation of cartilage matrix degradation in inflammatory
and degenerative arthritides. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Production of IGFBPs by chondrocytes from different species has been reported (9, 10, 11, 12, 13), and their ability to inhibit IGF actions has been demonstrated (10, 12). In addition, chondrocytes isolated from osteoarthritic cartilage express and secrete higher levels of IGFBP-2, -3, -4, and -5 than chondrocytes from normal cartilage (14, 15). Furthermore, elevated levels of IGFBPs have been detected in synovial fluids from patients with osteoarthritis (OA) or rheumatoid arthritis (RA) (16, 17, 18). These findings suggest that the diminished responsiveness of chondrocytes from osteoarthritic cartilage to IGF-I is attributable to increased levels of IGFBPs (19).
Interleukin-1 (IL-1), an inflammatory cytokine, has been implicated in the pathology of inflammatory and degenerative arthritis (20, 21). An increase in the rate of proteoglycan degradation and decreased proteoglycan synthesis are among its most prominent catabolic actions in diseased articular cartilage (22, 23, 24). IL-1 can also induce cartilage nonresponsiveness to IGF-I through a mechanism that remains unknown (22, 25). Furthermore, recent studies have shown that IGFBP-3 protein levels in rat (26) and human (27) chondrocytes can be increased by this cytokine.
The aim of this study was to investigate the effects of IGF-I and
IL-1 on IGFBP production in ovine articular chondrocytes. In
addition, by using IGF analogs with reduced affinity for IGFBPs, we
aimed to establish the role for the endogenous IGFBPs in mediating the
effects of IL-1 on proteoglycan synthesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(3 x 108
U/mg) was a gift from Hoffmann-La Roche (Nutley, NJ). IGF-II was
125I labeled by the chloramine-T method, as previously
described (29). Rabbit antihuman IGFBP-2 and IGFBP-5 antisera were
purchased from Upstate Biotechnology (Lake Placid, NY).
Cell culture
Articular cartilage from ovine metacarpophalangeal joints was
aseptically dissected and incubated in DMEM containing 1 mg/ml
collagenase, 1 mg/ml hyaluronidase, 10% FBS, 150 U/ml penicillin, 150
µg/ml streptomycin, and 2.5 µg/ml fungizone overnight at 37 C.
Isolated chondrocytes were washed twice with PBS and resuspended in
DMEM supplemented with FBS, penicillin, streptomycin, and fungizone as
indicated above. Cells were plated at a density of 2.5 x
105 cells/cm2 in 24-well plates (Falcon, Becton
Dickinson Co., Lincoln Park, NJ) for detection of IGFBPs, in 48-well
plates (Costar, Cambridge, MA) for determination of proteoglycan
synthesis, and in 6-well plates (Falcon) for RNA isolation and cultured
at 37 C in humidified atmosphere of 5% CO2. After reaching
confluence, monolayers were washed with PBS and incubated in serum-free
medium for 24 h. Serum-free medium was then replaced with medium
containing 0.01% BSA and the growth factors or IL-1, and the
chondrocytes were cultured for an additional 48 h. For
proteoglycan synthesis determination, [35S]sulfate was
added in the final 24 h. Conditioned medium was collected for
IGFBP analysis, and cell layers were used for proteoglycan synthesis
assay or RNA extraction.
Western ligand blotting and immunoblotting
Conditioned medium samples (1 ml) were dialyzed overnight at 4 C
against 0.1 M acetic acid and vacuum dried. Samples were
reconstituted in 30 µl SDS sample buffer (0.0625 M Tris,
2% SDS, 5% glycerol, and 0.001% bromophenol blue, pH 6.8) and
subjected to SDS-PAGE and Western ligand blot analysis as described by
Hossenlopp et al. (30). Briefly, samples were
electrophoresed on 12.5% SDS-polyacrylamide gels under nonreducing
conditions and then electroblotted onto nitrocellulose membranes
(Schleicher and Schuell, Dassel, Germany). The membranes were probed
overnight with 5 x 105 cpm
[125I]IGF-II/membrane, and IGFBP bands were visualized by
autoradiography.
The immunoblotting was performed using the enhanced chemiluminescence (ECL) detection system supplied by Amersham (Castle Hill, Australia). The nitrocellulose membranes were blocked for 2 h at room temperature in Tris-buffered saline-Tween [TBS-T; 20 mM Tris base, 137 mM NaCl (pH 7.6), and 0.1% Tween-20] containing 3% BSA, washed in TBS-T, and incubated overnight at 4 C with TBS-T buffer containing either anti-IGFBP-2 (1:2000) or anti-IGFBP-5 (1:500) antiserum. The membranes were washed and incubated for 1 h at room temperature with goat antirabbit IgG horseradish peroxidase conjugate (Dakopatts, Copenhagen, Denmark) at a final dilution of 1:2000, followed by extensive rinsing. The nitrocellulose was then treated with the ECL reagents according to the manufacturer’s instructions, and the reactive bands were visualized by autoradiography.
Northern blot analysis
Total RNA was extracted from cultured cells using the RNAzol B
kit (Biotecx Laboratories, Houston, TX), following the protocol
supplied by the manufacturer. The isolated RNA was quantitated
spectrophotometrically, and equal amounts of RNA (15 µg/lane) were
loaded and size-fractionated onto a 1% agarose-formaldehyde gel,
transferred to a Hybond nylon membrane (Amersham), and cross-linked by
UV light. To confirm the integrity, equal loading, and even transfer of
the RNA, the gel was stained with ethidium bromide, and ribosomal RNAs
were visualized before and after the transfer. The probe used for
IGFBP-5 messenger RNA (mRNA) detection, derived from the 640-bp
EcoRI-BamHI restriction fragment of the rat
IGFBP-5 cDNA (provided by Dr. J. D’Ercole, University of North
Carolina, Chapel Hill, NC), was labeled with
[-32P]deoxy-CTP using the Amersham Mega Prime random
primer labeling kit. The hybridization was performed at 42 C for
16 h in 5 x SSPE (750 mM NaCl, 50 mM
NaH2PO4 x H2O, 6.25 mM
EDTA, pH 7.4), 45% formamide, 5 x Denhardt’s solution, 0.2%
SDS, and 100 µg/ml salmon sperm DNA. After hybridization, the
membranes were washed at 42 C with 2 x saline-sodium citrate
(SSC), 0.1% SDS, and 1 mM EDTA and then with 0.5 x
SSC, 0.1% SDS, and 1 mM EDTA, and bound radioactive
material was visualized by autoradiography. Glyceraldehyde phosphate
dehydrogenase (GAPDH) mRNA was detected using an
[-32P]UTP-labeled complementary RNA (cRNA) probe that
was prepared from a 400-bp fragment of the rat GAPDH cDNA using Promega
riboprobe kit (Promega, Madison, WI) and T7 RNA polymerase. The
membranes were stripped of the IGFBP-5 probe using 0.5% SDS,
rehybridized with the GAPDH probe at 65 C for 16 h, and washed
with 0.1 x SSC buffer at 65 C, and labeled bands were visualized
by autoradiography.
Proteolytic activity assay
Recombinant human IGFBP-5 (rhIGFBP-5) was labeled with
Na125I to a specific activity of 50–100 µCi/µg protein
using the chloramine-T method, as described previously (29). To detect
IGFBP-5 proteolytic activity in conditioned medium, samples of medium
conditioned by OAC grown under basal conditions (20 µl) were
incubated with [125I]-IGFBP-5 (20,000 cpm) in the
presence or absence of IGFs at 37 C for 6 or 24 h. The reaction
was stopped by the addition of 6.6 µl of 4 x SDS buffer.
Samples were subjected to SDS-PAGE and analyzed by autoradiography.
Proteolysis was calculated by dividing the ODs of the proteolytic
fragments by the sum of the densities of the fragments and intact
IGFBP-5. The inhibition of proteolysis was calculated by the following
formula: [(A - B)/A] x 100%, where A is proteolysis in
conditioned medium without IGFs, and B is proteolysis in conditioned
medium in the presence of IGFs.
Proteoglycan synthesis
Proteoglycan production was estimated by measuring the
incorporation of [35S]sulfate into newly synthesized
glycosaminoglycans. Confluent chondrocytes were treated with the
indicated factors for 48 h and exposed to 3 µCi
[35S]sulfate/ml for the final 24 h. The cell layers
were solubilized in 0.2 M NaOH, which was subsequently
neutralized by the addition of 3 vol PBS. The radioactivity of cetyl
pyridinium chloride-precipitable material was measured in a
scintillation counter.
Densitometry
Quantitative analyses were performed by scanning laser
densitometry on UltroScan XL, Gel Scan XL(2.1), Pharmacia LKB (Uppsala,
Sweden).
Statistical analysis
Statistical analysis was performed using Student’s t
test. The results were considered significantly different at
P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Treatment of the cells with 50 ng/ml
IGF-I resulted in the appearance of a 29/31-kDa IGFBP doublet in the
medium. Des(1, 2, 3)IGF-I was less potent than IGF-I in stimulating the
appearance of this 29/31-kDa doublet, whereas LR3IGF-I had
no effect. Several other growth factors were also tested to determine
whether this positive effect on the 29/31-kDa IGFBP was IGF specific.
As shown in Fig. 1a, bFGF, TGFß, and PDGF had no effect on the
29/31-kDa doublet at doses up to 50, 25, and 50 ng/ml, respectively.
The 35-kDa band was identified as IGFBP-2, whereas the IGF-regulated
29/31-kDa doublet was identified as IGFBP-5 using Western
immunoblotting (Fig. 1b). The mobility of the 24-kDa band corresponded
with that of the nonglycosylated form of IGFBP-4.
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for 48 h, and the
IGFBPs secreted into the conditioned medium were analyzed by Western
ligand blotting. As shown in Fig. 2, IL-1 increased the level of the 29/31-kDa IGFBP in OAC-conditioned
medium in a dose-dependent manner, reaching the maximum stimulatory
effect at 200 U/ml. The doublet was confirmed to be IGFBP-5 by Western
immunoblotting (Fig. 3a).
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on the IGFBP-5
level in OAC-conditioned medium, chondrocytes were cultured in the
presence of both factors, and the medium was analyzed by Western
immunoblotting and densitometry (Fig. 3
, a and b). Although separate
incubations of chondrocytes with 50 ng/ml IGF-I and 1000 U/ml IL-1
caused 9- and 4.5-fold stimulations of the density of IGFBP-5 bands,
respectively, coincubation of the same concentrations of IGF-I and
IL-1 resulted in a 33-fold increase in IGFBP-5 over basal levels.
This synergistic effect of IGF-I and IL-1 suggests that these two
factors acted through different, complementary mechanisms to stimulate
IGFBP-5 production.
To determine whether IGFBP-5 protein levels in conditioned medium
reflect the expression of IGFBP-5 mRNA, we carried out Northern blot
analysis of total RNA isolated from OACs treated with 50 ng/ml IGF-I,
1000 U/ml IL-1, or both factors together for 48 h. As shown in
Fig. 4, IL-1 induced an increase in
the expression of 6.0-kilobase IGFBP-5 mRNA. In contrast, neither the
basal level of IGFBP-5 mRNA expression nor the IL-1-stimulated
transcript levels was affected by IGF-I. These data demonstrate that
IGF-I and IL-1 used distinct pathways in the regulation of OAC
IGFBP-5.
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, IGFBP-5 underwent a
time-dependent limited proteolysis in cell-free conditions, degrading
into 22- and 16-kDa fragments. The degradation of IGFBP-5 was
significantly inhibited by IGF-I (39% inhibition), whereas neither
des(1, 2, 3)IGF-I nor LR3IGF-I significantly prevented IGFBP-5
breakdown (Fig. 5b).
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). All three growth factors increased
proteoglycan synthesis to the maximum level of 2.5- to 3-fold over the
control value.
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increased the IGFBP-5 level in OAC culture, particularly
when coincubated with IGF-I (as demonstrated in Fig. 3), we speculated
that increased levels of this binding protein could affect the IGF-I
bioactivity. To determine the effect of IGFBP-5 on IGF-stimulated
proteoglycan synthesis, we incubated chondrocytes with 10 and 50 ng/ml
IGF-I for 48 h in the presence or absence of rhIGFBP-5.
Recombinant IGFBP-5 (500 ng/ml) was added at the beginning of the 48-h
incubation period, and additional 500 ng/ml was added 1 h before
the addition of [35S]sulfate. As shown in Fig. 7, the addition of rhIGFBP-5
significantly reduced the stimulatory effect of IGF-I on proteoglycan
synthesis. The basal level of proteoglycan synthesis remained
unchanged, while the activities of 10 and 50 ng/ml IGF-I were reduced
by 50% and 35%, respectively.
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. Neither basal nor des(1, 2, 3)IGF-I- and
LR3IGF-I-stimulated proteoglycan production was affected by
the cytokine, whereas IGF-I-stimulated proteoglycan synthesis was
diminished by 45% (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These results are consistent with previous studies that have demonstrated IGFBP-5 proteolytic activities in fibroblast (35, 36)- and osteoblast (8)-conditioned media that are inhibited by IGF-I and IGF-II. In addition, Matsumoto et al. (13) reported that in rat articular chondrocytes, IGFBP-5 is regulated by IGF-I and IGF-II at both the transcriptional and posttranslational levels. They found that the transcriptional regulation mediated through the IGF-I receptor was a predominant mechanism by which IGF-I stimulates IGFBP-5 in chondrocyte-conditioned medium, although inhibition of the IGFBP-5 protease(s), as a result of interactions between IGFs and IGFBPs, was also involved.
IL-1, which is found in the disease-affected cartilage at elevated
levels, has been associated with articular cartilage matrix degradation
in joints affected by OA and RA (20, 23). In addition, the ability of
IL-1 to affect IGFBP production in chondrocytes has been reported
previously. Matsumoto et al. (26) found that IL-1ß
increases the secretion of 40-kDa IGFBP (presumably IGFBP-3) by rat
articular chondrocytes, whereas Olney et al. (27) reported
that IL-1 stimulates production of IGFBP-3 in human articular
chondrocytes. A recent report by Wang et al. (37)
demonstrated the ability of IL-1ß to induce IGFBP-3 gene expression
and protein production in Leydig cells. Furthermore, Olney et
al. (14) found that IGFBP-5 protein level and gene expression are
increased in OA human cartilage and chondrocytes isolated from OA
cartilage, respectively. Our results show that IL-1 increased
IGFBP-5 protein level in ovine articular chondrocyte culture in a
dose-dependent manner. Moreover, IL-1 induced the expression of
IGFBP-5 mRNA, suggesting that the IGFBP-5 regulation took place at the
gene level. The combined effect of IGF-I and IL-1 on IGFBP-5 protein
level detected in the medium conditioned by OAC exceeded the sum of
their separate effects by 2.5-fold, strongly suggesting a synergism of
their mechanisms of action. It is likely that this dramatic increase in
IGFBP-5 is a consequence of an increase in the expression of this
binding protein (induced by IL-1) and subsequent protection of the
newly synthesized IGFBP-5 from proteolysis by IGF-I.
As we and others have demonstrated, chondrocyte IGFBPs negatively affect IGF actions in vitro (11, 12). Joosten et al. (38) demonstrated that murine arthritic cartilage is nonresponsive to IGF-I, resulting in decreased proteoglycan synthesis despite the presence of functionally unaltered IGF receptors on the chondrocytes. This finding was followed by the studies of Dore et al. (19) and Tardif et al. (15), who reported increased IGFBP production by human OA chondrocytes accompanied by their nonresponsiveness to IGF-I stimulation. These findings together with recent reports of elevated IGFBP levels in synovial fluids of OA and RA patients (16, 17, 18) point to a possible involvement of IGFBPs in the pathophysiological processes in articular cartilage. Although others proposed that IL-1 could suppress IGF activity in chondrocytes by increasing locally produced IGFBPs, no direct evidence was given to support this speculation (27).
In the present study, IGF-I was 10 times less potent in stimulating
proteoglycan synthesis in OAC than its analogs with reduced affinities
for IGFBPs, des(1, 2, 3)IGF-I and LR3IGF-I, clearly
demonstrating the negative effect of the locally produced IGFBPs on
IGF-I activity. Furthermore, rhIGFBP-5 suppressed the IGF-I stimulatory
effect on proteoglycan synthesis, a result in accordance with previous
findings that showed a negative modulation of IGF activ-ity by
IGFBP-5 in osteoblasts (8, 39). IL-1, which induced IGFBP-5
expression and, in the presence of IGF-I, caused a 33-fold
increase in IGFBP-5 protein level, diminished IGF-I-stimulated
[35S]sulfate incorporation into newly synthesized
proteoglycans by 45%, whereas the activities of des(1, 2, 3)IGF-I and
LR3IGF-I remained unaffected. These data demonstrate the
negative effect of the endogenous IGFBPs and strongly suggest that the
suppressive effect of IL-1 on IGF-I-induced proteoglycan production
in OAC was mediated through increased levels of IGFBP-5. Thus, a high
level of IGFBP-5 contributes to the IGF-I sequestration preventing the
growth factor from interacting with the receptors. The IGFBP-mediated
decrease in proteoglycan synthesis could be a relevant in
vivo mechanism by which IL-1 exerts its catabolic effect and
disturbs the balance between the synthesis and degradation of cartilage
matrix macromolecules in pathological conditions.
In summary, IGFBP-5 in OAC is up-regulated by IGF-I and IL-1 by two
apparently distinct mechanisms that complement each other and act
synergistically. Furthermore, locally produced IGFBPs, in particular
IGFBP-5, mediate the IL-1 suppression of IGF-I-induced proteoglycan
synthesis.
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Footnotes |
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Received September 2, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and interleukin-6. Lymphokine Cytokine Res 12:219–223[Medline]
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