Ferguson Laboratory, Musculoskeletal Research Center, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213
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ABSTRACT |
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Chondrocytes in arthritic cartilage respond poorly to insulin-like growth factor I (IGF-I). Studies with inducible nitric oxide synthase (iNOS) knockout mice suggest that NO is responsible for part of the cartilage insensitivity to IGF-I. These studies characterize the relationship between NO and chondrocyte responses to IGF-I in vitro, and define a mechanism by which NO decreases IGF-I stimulation of chondrocyte proteoglycan synthesis. Lapine cartilage slices, chondrocytes, and cartilage from osteoarthritic (OA) human knees were exposed to NO from the donors S-nitroso-N-acetylpenicillamine (SNAP) or (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate] (DETA NONOate), by transduction with adenoviral transfer of iNOS (Ad-iNOS), or by activation with interleukin-1 (IL-1). NO synthesis was estimated from medium nitrite, and proteoglycan synthesis was measured as incorporation of 35SO4. IGF-I receptor phosphorylation was evaluated with Western analysis. SNAP, DETA NONOate, endogenously synthesized NO in Ad-iNOS-transduced cells, or IL-1 activation decreased IGF-I-stimulated proteoglycan synthesis in cartilage and monolayer cultures of chondrocytes. OA cartilage responded poorly to IGF-I; however, the response to IGF-I was restored by culture with NG-monomethyl-L-arginine (L-NMA). IGF-I receptor phosphotyrosine was diminished in chondrocytes exposed to NO. These studies show that NO is responsible for part of arthritic cartilage/chondrocyte insensitivity to anabolic actions of IGF-I; inhibition of receptor autophosphorylation is potentially responsible for this effect.
chondrocytes; arthritis; signal transduction; matrix proteoglycan synthesis; cartilage; insulin-like growth factor I; insulin-like growth factor I receptor
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INTRODUCTION |
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NITRIC OXIDE (NO)
has been identified as one mediator of cartilage dysfunction in
arthritis (5, 9). It is produced in large amounts by
chondrocytes stimulated by the cytokines found at elevated
concentrations in the synovial fluid of arthritic patients (19,
25, 32), and studies in animals have implicated NO as an
important factor in rheumatoid arthritis (RA) (21, 33). NO
is produced by chondrocytes in diseased human cartilage (1, 27,
29), and we have known for several years that NO is responsible
for a substantial portion of the inhibition of chondrocyte proteoglycan
and collagen synthesis imposed by interleukin-1 (IL-1) and certain
other cytokines (10, 12, 34, 37). The first study to show
prophylactic effects of inducible nitric oxide synthase (iNOS)
inhibitors in canine experimental osteoarthritis (OA) was recently
published (26). NO also causes chondrocyte apoptosis in
the joints of rabbits with experimentally induced OA (11).
Studies of lapine articular chondrocytes have suggested inhibition of
prolyl hydroxylase as a potential mechanism through which NO inhibits
cartilage collagen synthesis (2). We recently showed that
NO can decrease chondrocyte production of transforming growth
factor- (TGF-
), a major anabolic agent in cartilage
(35). Thus many studies have implicated NO in
pathophysiologies involving altered cartilage matrix metabolism.
There are multiple mechanisms by which NO could directly modulate chondrocyte metabolism (5). These include: 1) binding of NO to the heme group of guanylate cyclase, thus activating this enzyme and increasing concentrations of cGMP; 2) reacting with free thiols to form S-nitrosothiol compounds; 3) reacting with superoxide to form peroxynitrite, which may promote oxidative injury to the cells; 4) nitrosylation of target proteins with subsequent modulation of function; and 5) promoting ADP ribosylation of proteins, thus modifying their behavior. Which, if any, of these mechanisms are involved in NO modulation of chondrocyte function in arthritis remains to be defined.
In vitro and in vivo studies have confirmed the role of insulin-like
growth factor I (IGF-I) in cartilage growth and development and in
adult cartilage; although only moderately mitogenic, it normally
stimulates the synthesis of collagen and proteoglycans, the major
constituents of the cartilage matrix. It induces expression and
synthesis of collagen type II and proteoglycan core protein, thus
stabilizing chondrocyte phenotype (19). IGF-I is also used in conjunction with TGF- to maintain cartilage proteoglycan content in organ culture (18, 42).
However, chondrocytes in both human (7, 28) and experimentally induced arthritis (31, 41) are insensitive to the anabolic actions of IGF-I. The concomitant increases in IGF-I and the IGF binding proteins (IGFBPs) in the synovial fluid of patients with both RA and OA have prompted the working hypothesis that an imbalance in the relative amounts of IGF-I and the BPs causes chondrocyte insensitivity to IGF-I in diseased joints. These BPs act as transport proteins in plasma, prolong the half-life of IGF, provide a means of tissue localization for IGF, modulate interaction of IGF with its receptors, and, in some cases, have IGF-I independent effects on cells. Thus the maintenance of IGF actions on chondrocytes is complicated by regulation of and by IGFBPs and the proteases that degrade IGFBPs and therefore indirectly affect responses to IGF-I (reviewed in Ref. 20).
A role for NO in chondrocyte insensitivity to IGF-I is suggested by recent studies of zymosan-induced arthritis in mice with disrupted iNOS genes (38). The inflammatory response in zymosan-injected joints was similar in wild-type and iNOS knockout mice. Cartilage from wild-type mice showed the expected insensitivity to IGF-I. However, the cartilage of mice unable to produce NO maintained their ability to increase proteoglycan synthesis in response to IGF-I. This suggests that a major factor in the development of chondrocyte insensitivity to the actions of IGF-I is the increase in NO secondary to the induction of iNOS. Similar protective effects were recently reported from studies of a model of OA in these iNOS knockout mice (39).
Although these previous studies demonstrate the importance of NO in IGF-I insensitivity, they provide no evidence as to whether the effect of NO is via modulation of IGF-I/chondrocyte interactions per se or is secondary to NO modulation of the cytokines produced by the inflammatory cells present in the arthritic joint (13), which could then alter the response of chondrocytes to IGF-I. Furthermore, NO could potentially modify either IGFBP content or activity in the arthritic joint, or alter the interactions of IGF-I with the BPs. The current studies were designed to 1) determine whether chondrocyte insensitivity to IGF-I could be observed in an in vitro system, and if so, 2) utilize cartilage and chondrocytes in culture to begin an analysis of the mechanisms by which NO causes unresponsiveness to IGF-I. We used lapine cartilage and chondrocytes in organ and monolayer culture and found that exposure to NO inhibits the IGF-I-stimulated increase in proteoglycan synthesis. This is seen with NO produced exogenously from the NO donors S-nitroso-N-acetylpenicillamine (SNAP) and (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate] (DETA NONOate) or endogenously following IL-1 stimulation or adenoviral transfer of iNOS (Ad-iNOS) into chondrocytes. This effect of NO is reversible. Relevance to human disease is implied, as inhibition of NO synthesis with NG-monomethyl-L-arginine (L-NMA) during in vitro organ culture of human articular cartilage recovered from OA joints restores a brisk anabolic response to IGF-I. IGF-I-stimulated tyrosine phosphorylation of the IGF-I receptor is decreased by prior exposure of chondrocytes to NO, suggesting that chondrocyte insensitivity to IGF-I in the presence of NO is due at least in part to an action of NO on this step in the signal transduction pathway.
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METHODS |
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Materials were obtained from the following suppliers: New
Zealand White rabbits, 5-6 lbs (Myrtle's Rabbitry, Thompson
Station, TN); arthritic human cartilage harvested secondary to
arthroplasty for degenerative joint disease (National Disease Research
Interchange, Philadelphia, PA; and University Drive VA Medical Center,
Pittsburgh, PA); Eagle's minimum essential medium (MEM), FBS,
antibiotics, and other tissue culture supplies (GIBCO BRL; or
Sigma Chemical, St. Louis, MO); crude clostridial collagenase
(Worthington Biochemical, Freehold, NJ); 35S-labeled sodium
sulfate, 1 Ci/mmol (NEN, Boston, MA); human IGF-I and human platelet
TGF-1 (R & D Systems, Minneapolis, MN); human long
R3IGF-I and des(1-3)-IGF-I
(GroPep, N. Adelaide, Australia); SNAP, DETA NONOate, and
L-Sepiapterin (Alexis/L C Labs, San Diego, CA); recombinant, human IL-1
was a generous gift of Dr. Elizabeth Arner
(Du Pont Merck, Wilmington, DE); L-NMA was synthesized by Drs. Paul Dowd and Wei-Zhang (Department of Chemistry, University of
Pittsburgh, PA); antiphosphotyrosine (PY20) (Transduction Laboratories, Lexington, KY); antibody to the IGF-I receptor (IGF-IR
) (C-20; Santa
Cruz Biotechnology, Santa Cruz, CA); ECL Western blotting detection reagents (Amersham Pharmacia Biotech, Arlington Heights, IL).
All other reagents were obtained from Sigma Chemical.
Rabbit and human articular cartilage culture. Articular cartilage was shaved from the knee and shoulder joints of New Zealand White rabbits. The slices were distributed into 48-well plates (10-15 mg wet wt/well determined at the initiation of culture). They were maintained in Ham's F-12 medium containing 10% FBS for 20 h before the medium was changed to MEM with 0.5% FBS. Fresh medium with agonists and antagonists was added and after 24 h, aliquots of conditioned medium (CM) were collected for determination of nitrite and proteoglycan synthesis measured as described below. Human femoral condyle cartilage specimens were obtained within 24 h of surgery (surgery was to replace the knee due to degenerative joint disease) and were handled in the same fashion as the lapine specimens.
Rabbit articular chondrocyte culture.
Monolayer cultures of lapine articular chondrocytes isolated by the
method of Green (per Stadler et al., Ref. 32) were maintained in 6- or 24-well plates. The cells were grown in Ham's F-12 medium supplemented with 10% FBS and antibiotics and used without subculture. At confluence, the cell layers were washed with PBS, and the medium was
changed to MEM supplemented with antibiotics and 0.5% FBS. Agonists and antagonists were added at the times and concentrations indicated in the specific protocols and/or the legends of Figs. 1-9, and conditioned medium was collected for assays.
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Nitrite determination. NO production was determined as the nitrite concentration in conditioned medium by using a spectrophotometric assay based on the Griess reaction (2). We have previously documented that ~51% of the NO generated by chondrocytes in culture accumulates as nitrite over a wide range of NO production (32).
Proteoglycan synthesis. Proteoglycan synthesis was determined in chondrocytes or cartilage slices cultured for 30 h in low-serum medium with agonists and antagonists as noted. The cells were labeled with 35S-labeled sodium sulfate, 20 µCi/ml, for the final 8 h of incubation. Aliquots of CM and cell or cartilage extracts (4 M guanidine HCl, 4°C, 48 h) were eluted on Sephadex G-25M in PD-10 columns with 4 M guanidine hydrochloride solution, and the radioactivity in newly synthesized proteoglycans was determined by scintillation counting.
Transduction of chondrocytes with Ad-iNOS.
To facilitate evaluation of the effects of NO independent of other
actions of IL-1 on the cell, chondrocytes transduced with an adenoviral
vector carrying the human iNOS gene (Ad-iNOS) were used. The adenoviral
vector, supplied by Dr. Imri Kovesdi of GenVec (Rockville, MD), is a
first generation, A1A3
serotype 5 adenovirus into which human iNOS cDNA has been cloned. Briefly, an adenovirus transfer plasmid containing a human
cytomegalovirus promoter driving a cDNA encoding human iNOS and an
artificial splice sequence was cotransfected into cells of the 293 cell
line (CRL 1573; ATCC, Rockville MD), with the large
Cla I fragment of adenovirus DNA. Intracellular
recombination of the plasmid with the Cla I fragment
generated a full-length recombinant adenoviral genome. Recombinant
adenovirus was plaque purified and screened for nitrite by the Griess
reaction. This CsCl gradient purified stock with a titer of
1010 pfu/ml was prepared by Dr. Paul Robbins at the
University of Pittsburgh School of Medicine Human Gene Therapy Center.
Western analysis of IGF-I receptor tyrosine phosphorylation.
IGF-I-stimulated tyrosine phosphorylation was evaluated as described by
Chow et al. (4). Briefly, chondrocyte monolayers were
treated as described in the legends to Figs. 1-9, the medium was
removed, and the cells were washed with ice-cold Gey's balanced salt
solution containing 0.1 mM Na3VO4. The cells
were lysed in a buffer containing 164 mM NaCl, 50 mM Tris, pH 7.4, 1%
Nonidet P-40 (NP-40), 1 mM EDTA, 1 mM NaF, 1 mM phenylmethylsulfonyl
fluoride (PMSF), 1 mM Na3VO4, and 1 µg/ml
each of aprotinin, leupeptin, and pepstatin. The cells were incubated
with shaking at 4°C for 30 min, scraped, transferred to
microcentrifuge tubes, and stored at 80°C until assayed. The
lysates were centrifuged at 14,000 g for 5 min, and the
supernatants were assayed for protein content (Bradford dye binding
kit; Bio-Rad, Richmond, CA). Equal amounts of solubilized protein were
reacted with 20 µl of 50% protein A-Sepharose, and the supernatants
were immunoprecipitated at 4°C overnight with 2 µg/ml antibody to
the IGF-I receptor
-chain. The antibody was adsorbed to protein
A-Sepharose and washed three times with cold buffer containing 0.5%
NP-40, 1% Triton X-100, 150 mM NaCl, 20 mM Tris (pH 7.4), 2 mM EGTA, 2 mM EDTA, 0.2 mM PMSF, and 0.2 mM orthovanadate. Samples were heated for
5 min at 95°C in Laemmli buffer with 5%
-mercaptoethanol,
separated by SDS-PAGE (7.5% acrylamide gels), and transferred to
nitrocellulose membranes (Hybond ECL, Amersham). The blots were blocked
with 3% BSA, probed with anti-phosphotyrosine antibody (1:1,000),
washed, and reacted with anti-mouse peroxidase, and the protein bands containing phosphotyrosine were visualized with ECL and
autoradiography. The identity of the 105-kDa phosphorylated band as the
-subunit of the IGF-I receptor was confirmed with specific
antibody to this protein.
Statistics. Experiments were done at least three times, and data are presented as means ± SE. Each incubation condition was represented by duplicate or triplicate wells, with replicates from each individual culture averaged and used as one value for purposes of statistical analysis. The significance of differences between mean values was determined by t-test.
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RESULTS |
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Figure 1 shows that basal proteoglycan synthesis was decreased by ~30% in cartilage slices from the knee and shoulder joints of rabbits exposed to NO generated by SNAP. The concentration of nitrite in control conditioned media was 3.4 ± 0.3 vs. 47 ± 4 µM in media from slices exposed to SNAP. IGF-I-stimulated proteoglycan synthesis was significantly decreased at all concentrations of IGF-I tested.
As chondrocytes in their normal milieu in cartilage may behave
differently from those in monolayer, the effects of NO on primary cultures of isolated chondrocytes were characterized. Figure
2 shows that similar results were seen in
chondrocytes maintained in monolayer culture. SNAP and DETA NONOate
generated NO did not significantly decrease basal proteoglycan
synthesis in these studies, but did depress the ability of IGF-I to
increase matrix proteoglycan synthesis. In contrast, the stimulation of
chondrocyte proteoglycan synthesis by either 10 or 50 pM TGF- was
not affected by NO.
The generation of NO by donors such as SNAP and DETA NONOate is different from that following induction of iNOS. Thus chondrocytes were transduced with Ad-iNOS to allow generation of NO within the cell in a pattern that more closely mimics the in vivo chondrocyte exposure to NO. As shown in Fig. 3, although the conditioned media nitrite (24 ± 4 µM) was less than that seen following exposure to SNAP, stimulation of proteoglycan synthesis in response to IGF-I was almost completely eliminated at all concentrations tested. These changes cannot be attributed to differences in cell numbers, either when comparing cultures transduced with iNOS with and without L-NMA (2.0 ± 0.3 × 105 vs. 1.98 ± 0.2 × 105 cells/well) or those cultures with and without 50 ng/ml IGF-I (1.85 ± 0.3 × 105 vs. 2.0 ± 0.3 × 105 cells/well). All cultures were transduced with the same adenoviral preparation, and L-NMA was added to inhibit NO synthesis, thus controlling for potential effects of the transduction procedure per se on IGF-I-stimulated proteoglycan synthesis. Furthermore, similar results have been seen using L-N5-(1-iminoethyl)-ornithine · 2HCl (L-NIO) (data not shown), another iNOS inhibitor, to repress NO synthesis in transduced cells. Thus the difference between the response to IGF-I with and without L-NMA can be attributed to the presence of NO.
Data from cells transduced with iNOS show that the effect of NO is both time and concentration dependent. Figure 4 shows the dose-dependent inhibition of IGF-I-stimulated proteoglycan synthesis by NO. The multiplicity of infection with Ad-iNOS during the overnight transduction was 0, 33, 67, and 100 pfu/cell, to yield populations of cells generating variable concentrations of NO/nitrite. In this series of experiments, the control values for proteoglycan synthesis ranged from 29 ± 1 pmol [35S]sulfate incorporated into proteoglycan when nitrite was 3 µM, to 25 ± 1 pmol when CM nitrite was 13.3 µM or 23 µM. Synthesis of NO to produce CM concentrations of ~10 µM nitrite (NO synthesized at a rate to produce 55 nmol nitrite/24 h, per 106 cells; calculated from the data in Fig. 4) caused a 50% inhibition of IGF-I-stimulated proteoglycan synthesis. The relationship between medium nitrite concentrations and the inhibition of IGF-I-stimulated proteoglycan synthesis is thus not linear over the range of NO synthesis studied. Rather, there appears to a modest effect when NO increased from 3-8 µM and a striking decrease in sensitivity to IGF-I when NO increased from 8-13 µM, with little further decrease in response when NO increased from 13-25 µM.
Figure 5 shows that NO effects on chondrocyte responsiveness to IGF-I are reversible. In this series of experiments, chondrocytes were transduced with Ad-iNOS, and 1 mM L-NMA was added at different times after transduction with the virus. Proteoglycan synthesis was evaluated 120 h after transduction; however, L-NMA was present to inhibit NO synthesis for variable periods of time as noted on the x-axis. Thus the "0 h" time point is from cells that were maintained continuously for 5 days with NO synthesis uninhibited; the "120 h" time point is from chondrocytes transduced and maintained in the presence of L-NMA, i.e., in which NO synthesis was continuously inhibited. CM nitrite averaged 20 ± 1.4 µM/24 h in CM of cells without L-NMA and 4 ± 1 µM/24 h in CM of cells in which NO synthesis was suppressed. Basal proteoglycan synthesis was significantly decreased in the cells continuously exposed to NO (compare iNOS, time 0, to iNOS, any time after addition of L-NMA, P < 0.05); however, there was no difference in the basal rate in cells cultured with L-NMA for 24 through 120 h. Inhibition of NO synthesis for 72 h or longer effectively restored the IGF-I response to normal levels; there was no difference in the rate of stimulated proteoglycan synthesis between cells with NO synthesis continuously suppressed or with L-NMA added 96 or 72 h before IGF-I and subsequent analysis of matrix protein synthesis. However, if NO synthesis was suppressed for <72 h, then a time-dependent inhibition of the response to IGF-I was noted (27% inhibition at 48 h; 50% inhibition at 24 h; 100% inhibition if NO synthesis was allowed to proceed without interruption for 5 days). These studies show that the NO inhibition of chondrocyte response to IGF-I is not due to any irreversible alterations in the cells but rather is modulated by time of exposure to NO.
Previous studies have shown that IL-1 inhibition of chondrocyte
proteoglycan synthesis is effected by both NO-dependent and independent mechanisms (12, 35, 37). Figure
6 shows that IL-1 modestly but
significantly (30%) decreased basal proteoglycan synthesis, which was
not relieved by the addition of L-NMA, suggesting that this
effect was not dependent on NO. Nonetheless, the low concentrations of
NO generated by these chondrocytes (12 µM) are sufficient to blunt
the anabolic response to IGF-I, because L-NMA inhibition of
NO synthesis restored the IL-1 inhibited response to IGF. Similar
results were seen when NO synthesis was inhibited by L-NIO,
another iNOS inhibitor (data not shown). A submaximal concentration of
TGF- alone (10 pM) modestly increased proteoglycan synthesis and was
minimally affected by IL-1 either with or without L-NMA.
However, this low concentration of TGF-
acted synergistically with
IGF-I to increase proteoglycan synthesis by 450%. This synergistic stimulation was significantly blunted by IL-1 and restored when NO
synthesis was inhibited with L-NMA.
Cartilage from OA patients generates NO at low levels. Thus preliminary
experiments were done to determine if culture with L-NMA to
blunt chondrocyte NO production in these tissues could affect the
response to IGF-I. Figure 7 shows data
from cartilage slices taken from three different patients with
degenerative joint disease in the course of total joint replacement.
Conditioned media nitrite concentrations were 3.5 ± 0.44 vs.
1.03 ± 0.12 µM/10 mg wet wt in the medium from slices
cultured with 0.75 mM L-NMA. L-NMA had little
effect on the basal level of proteoglycan synthesis. The response to
IGF-I was minimal in the cartilage incubated without L-NMA,
confirming previous data showing insensitivity to IGF-I in OA (7,
28). However, in the cartilage slices cultured with
L-NMA to block endogenous production of NO, IGF-I increased proteoglycan synthesis some twofold. The response to exogenous TGF-
was also evaluated, and the increment effected by culture with
L-NMA was insignificant compared with the restoration of IGF-I responsiveness.
Much of the insensitivity to IGF-I in arthritis is currently attributed to changes in IGF-I BPs (20, 41); thus a series of experiments was done to test whether the acute effects of NO could be acting via modulation of these factors. IGF-I analogs that do not complex with the BPs are available. Figure 8 shows that the response to two of these analogs, long R3IGF-I and des(1-3)-IGF-I, is inhibited to the same extent as the response to IGF-I. Addition of IL-1, which further increases NO and also activates chondrocyte responses similar to those during the inflammatory phase of arthritis, further decreased both basal and IGF-I-stimulated proteoglycan synthesis. This inhibition was similar regardless of whether the agonist was IGF-I or the BP-insensitive analogs long R3IGF-I or des(1-3)-IGF-I.
The results of Western analysis of tyrosine phosphorylation of the
IGF-I -receptor are shown in Fig. 9. SNAP or DMSO vehicle was added
to chondrocytes 40 min before IGF-I, and the cells were washed and
lysed 5 or 10 min later (Fig.
9A); iNOS-transduced cells
were lysed at 2 and 5 min after stimulation with IGF-I (Fig. 9B). Basal tyrosine phosphorylation was not detected;
however, a strong band was seen at ~100 kDa, which corresponds to the
molecular weight of the
-subunit of the IGF-I receptor.
Previous exposure to NO generated by SNAP decreased this signal by 80%
in lapine chondrocytes. Similar results are seen in the lysates from
chondrocytes transduced with iNOS, cultured with or without
L-NMA, stimulated with IGF-I, and terminated after 2 and 5 min activation (NO inhibited tyrosine phosphorylation of the IGF-I
receptor by 92% at 2 min and by 80% after 5 min). Exposure of human
femoral condyle chondrocytes to SNAP also caused a modest inhibition of
IGF-I receptor tyrosine phosphorylation at 2 and 5 min after
stimulation (data not shown).
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DISCUSSION |
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These studies show that NO inhibits IGF-I-stimulated proteoglycan
synthesis by chondrocytes in monolayer or organ culture in a dose- and
time-dependent fashion. They confirm previous observations that NO,
under some conditions, also depresses basal chondrocyte proteoglycan
synthesis, but they suggest that the inhibition of response to IGF-I is
a separate phenomenon that occurs at lower concentrations of
NO. This is observed in Fig. 4, where a striking inhibition of
IGF-I-stimulated proteoglycan synthesis is seen at concentrations of NO
that minimally affect basal synthesis. Furthermore, NO from SNAP or
DETA NONOate does not decrease basal proteoglycan synthesis by
chondrocytes in monolayer, although the response to IGF-I, but not
TGF-, is significantly inhibited. This is also suggested by the data
in Fig. 6, showing that IL-1 depression of basal proteoglycan
synthesis is independent of NO, yet inhibition of NO production in IL-1
activated cells is necessary to restore IGF-I-stimulated synthesis to
control levels. Thus these experiments enable discrimination between NO
effects on basal vs. IGF-I-stimulated proteoglycan synthesis. Our data
support the hypothesis, inferred from the studies with iNOS knockout
mice, that NO will adversely affect the ability of IGF-I to maintain proteoglycan synthesis in vivo (18, 38). Furthermore, we
have identified a potential mechanism for NO inhibition of IGF-I
actions in the diminished IGF-I receptor tyrosine phosphorylation in
tissues exposed to NO.
A synergistic effect on proteoglycan synthesis was observed when both
IGF-I and TGF- were present. This is consistent with the
observations by Yaeger et al. (42) that these growth
factors act synergistically to increase aggrecan gene expression in
human articular chondrocytes and by Chopra and Anastassiades
(3) of synergistic stimulation of proteoglycan synthesis
in bovine chondrocytes. The inhibition of this synergy by IL-1 and the
restoration of responsiveness by the NO synthesis inhibitor
L-NMA have not been previously documented. These studies
support an important role for NO in modulating cartilage
matrix proteoglycan synthesis in vivo, where the cells are
exposed to multiple agonists and antagonists and the net response will
determine whether matrix components are maintained or lost.
Perhaps the most convincing evidence for the relevance of our
observations to human disease is the L-NMA restoration of
IGF-I responsiveness of damaged articular cartilage obtained from the femoral condyles of human knees at the time of arthroplasty (Fig. 7).
This material was marginally responsive to anabolic activation by
either IGF-I or TGF-. These data are in contrast with the earlier
report by Lafeber et al. (16) that cartilage from OA knees
is hyperresponsive to TGF-
. However, their analysis was done after
96-h stimulation by TGF-
, at a time when the induction of other
autocrine factors that could affect proteoglycan synthesis would be
apparent. The inability to respond to IGF-I quite likely contributes to
the continuing loss of cartilage in OA, in conjunction with the loss
through enhanced catabolism (5, 9). Similar to the results
seen with IL-1-activated cells, L-NMA minimally affects the
response to TGF-
; however, the response to IGF-I is effectively
restored under these culture conditions. These results coupled with the
minimal effects on basal proteoglycan synthesis suggest that, in human
OA, inhibition of responsiveness to IGF-I is a major pathophysiological
effect of NO.
It is unlikely that NO affects chondrocyte responsiveness to IGF-I by altering plasma membrane receptor expression or affinity. Dore et al. (7) identified type 1 IGF-I receptors on normal and OA human chondrocytes and found that the numbers of IGF-I receptors on cells from arthritic tissue were actually increased. The IGF-I affinity of the receptors was somewhat lower in arthritic chondrocytes; however, the deficit in response was not overcome by high concentrations of IGF-I. More recently, Tardif et al. (36) found identical levels of type 1 IGF receptor mRNA in normal and human OA chondrocytes. These studies suggest that receptor number and affinity are not major contributors to chondrocyte insensitivity to IGF-I. The concentration of IGF-I in the synovial fluid of both OA (36) and RA (15) joints is actually increased; however, most studies suggest that this is paralleled by an increase in the concentration of IGFBPs. This imbalance in the relative amounts of IGF-I and the BPs is hypothesized to cause chondrocyte insensitivity to IGF-I in diseased joints (14, 19). This hypothesis is strengthened by the observations that IL-1 stimulates increases in the synthesis of IGFBPs by chondrocytes of several species, including humans (24). Thus we investigated the possibility that acute effects of NO on IGF-I actions on chondrocytes could be effected by alterations in IGF/IGFBP interactions. Modification of IGF-I by substitution of an Arg for the Glu at position 3 (hence "R3") and the addition of a 13-amino acid extension peptide (hence "long") at the NH2 terminus or deletion of the NH2-terminal tripeptide Gly-Pro-Glu [des(1-3)-IGF-I] causes a marked decrease in the binding to IGFBPs (43). Thus, if NO is acting by increasing expression of IGFBPs or by enhancing their affinity for IGF-I, then the response to long R3IGF-I and des-IGF-I should not be affected by NO. The results shown in Fig. 8 suggest that the mechanism of NO inhibition of response to IGF-I does not involve IGF-I BPs, at least not under the acute conditions of these studies. Even in cells exposed to IL-1, which has been previously shown to increase BP synthesis (24), the response to IGF-I and the BP-insensitive analogs were identically inhibited by NO. However, we cannot exclude the possibility that alterations in BPs may contribute to NO modulation of IGF actions under in vivo conditions where chronic stimulation and the interaction of multiple agonists determines chondrocyte functions.
Insensitivity to activation by IGF-I in OA is not overcome by high
concentrations of IGF-I (7, 28, 31), suggesting that NO
may have direct effects on IGF-I signaling in chondrocytes. Thus we
evaluated the initial step in the signaling cascade subsequent to IGF-I
interaction with chondrocytes, tyrosine phosphorylation of the IGF-I
receptor. The results shown in Fig. 9 suggest that NO interferes with
IGF-I actions at this point; regardless of whether the NO exposure was
from SNAP or from Ad-iNOS, the tyrosine phosphorylation of the receptor
was diminished. The specific mechanism responsible for this action
remains to be defined. The IGF-I type 1 receptor is a heterotetrameric
complex composed of two extracellular -subunits and two
intracellular
-subunits. The
- and
-subunits are held together
by disulfide bonds. Association of these four components is necessary
for further signal transmission (30). There is precedent
for NO altering receptor function in other tissues. For example,
Estrada et al. (8) reported that NO reversibly inhibits
the epidermal growth factor receptor tyrosine kinase in a
cGMP-independent manner. NO-dependent S-nitrosylation of critical thiol groups downregulates the activity of the
N-methyl-D-aspartate receptor in neurons
(17).
The single study showing NO inhibition of IGF-I actions in chondrocytes was reported by Clancy et al. (6). They concluded that NO inhibited the response of bovine chondrocytes to the anabolic actions of IGF-I via increases in cGMP. These effects were seen when the cells were grown on a fibronectin-coated (but not plastic or albumin) surface. They determined that NO disruption of a fibronectin-induced intracellular assembly of an activation complex [comprised of integrin, actin, rhoA, and focal adhesion kinase (FAK)] was responsible for the effects of NO. However, our studies clearly show that NO inhibits IGF-I-stimulated proteoglycan synthesis in chondrocytes adherent to plastic. Although the effects of NO are apparent in the absence of an exogenous fibronectin substrate, we cannot exclude the possibility that the cells have deposited a matrix that could potentially participate in the NO modulation of chondrocyte responses to IGF-I. Our data suggest that NO may alter other signal transduction molecules in addition to disrupting the adhesion kinase signaling complex to effect chondrocyte insensitivity to the anabolic actions of IGF-I. The specific mechanisms by which NO decreases IGF-I receptor autophosphorylation in chondrocytes is currently under investigation. Whether NO is acting in part through increases in cGMP or via NO-dependent S-nitrosylation or nitrosylation of critical tyrosines in signal transduction proteins will be determined.
In summary, our studies support the hypothesis that NO is responsible
for at least part of chondrocyte insensitivity to the anabolic actions
of IGF-I in OA and inflammatory conditions. These effects can be
mimicked by exposure to exogenous NO from the NO donors SNAP and DETA
NONOate, by transduction of normal cells with iNOS, and activation of
chondrocytes with IL-1. The decrease in IGF-I-stimulated tyrosine
phosphorylation of the IGF-I receptor -subunit following exposure to
NO provides a means by which NO may diminish the chondrocyte anabolic
response to this growth factor. Further studies will be necessary to
define the specific mechanisms by which NO alters IGF-I receptor
tyrosine phosphorylation.
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ACKNOWLEDGEMENTS |
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This work was supported in part by Department of Veterans Affairs, the Western Pennsylvania Chapter of the Arthritis Foundation, and the Ferguson Foundation.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. K. Studer, Dept. of Orthopaedic Surgery, C-313 PUH 200 Lothrop St., Pittsburgh, PA 15213 (E-mail: rstuder{at}pitt.edu).
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.
Received 20 December 1999; accepted in final form 27 April 2000.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Amin, AR,
DiCesae PE,
Vyas P,
Attur M,
Tzeng E,
Billiar TR,
Stuchin SA,
and
Abramson SB.
The expression and regulation of nitric oxide synthase in human osteoarthritis-affected chondrocytes: evidence for up-regulated neuronal nitric oxide synthase.
J Exp Med
182:
2097-2102,
1995[Abstract].
2.
Cao, M,
Westerhausen-Larson A,
Niyibizi C,
Kavalkovich K,
Georgescu CF,
Rizzo CF,
Hebda PA,
Stefanovic-Racic M,
and
Evans CH.
Nitric oxide inhibits the synthesis of type II collagen without altering Col2A1 mRNA abundance: prolyl hydroxylase as a possible target.
Biochem J
324:
305-310,
1997[ISI][Medline].
3.
Chopra, R,
and
Anastassiades T.
Specificity and synergism of polypeptide growth factors in stimulating the synthesis of proteoglycans and a novel high molecular weight anionic glycoprotein by articular chondrocyte cultures.
J Rheumatol
25:
1578-1584,
1998[ISI][Medline].
4.
Chow, JC,
Condorelli G,
and
Smith RJ.
Insulin-like growth factor-I receptor internalization regulates signaling via the Shc/mitogen-activated protein kinase pathway, but not the insulin receptor substrate-1 pathway.
J Biol Chem
273:
4672-4680,
1998
5.
Clancy, RM,
Amin AR,
and
Abramson SB.
The role of nitric oxide in inflammation and immunity.
Arthritis Rheum
41:
1141-1151,
1998[ISI][Medline].
6.
Clancy, RM,
Rediske M,
Tang X,
Nijher N,
Frendel S,
Phillips M,
and
Abramson SBO
Nitric oxide disrupts fibronectin-induced assembly of a subplasmalemmal actin/Rho A/focal adhesion kinase signaling complex.
J Clin Invest
100:
189-1796,
1997
7.
Dore, S,
Pelletier JP,
DiBattista JA,
Tardif G,
Brazeau P,
and
Martel-Pelletier J.
Human osteoarthritic chondrocytes possess an increased number of insulin-like growth factor 1 binding sites but are unresponsive to its stimulation.
Arthritis Rheum
37:
253-263,
1994[ISI][Medline].
8.
Estrada, C,
Gomez C,
Martin-Nieto J,
DeFrutos T,
Jimenez A,
and
Villalobo A.
Nitric oxide reversibly inhibits the epidermal growth factor receptor tyrosine kinase.
Biochem J
326:
369-376,
1997[ISI][Medline].
9.
Evans, CH,
Watkins SC,
and
Stefanovic-Racic M.
Nitric oxide and cartilage metabolism.
Methods Enzymol
269:
75-88,
1996[ISI][Medline].
10.
Fukuda, K,
Kumano F,
Takayama M,
Saito M,
Otani K,
and
Tanaka S.
Zonal differences in nitric oxide synthesis by bovine chondrocytes exposed to interleukin-1.
Inflamm Res
44:
434-437,
1995[ISI][Medline].
11.
Hashimoto, S,
Takahashi K,
Amiel D,
Coutts RD,
and
Lotz M.
Chondrocyte apoptosis and nitric oxide production during experimentally induced osteoarthritis.
Arthritis Rheum
41:
1266-1274,
1998[ISI][Medline].
12.
Häuselmann, HJ,
Oppliger L,
Michel BA,
Stefanovic-Racic M,
and
Evans CH.
Nitric oxide and proteoglycan biosynthesis by human articular chondrocytes in alginate culture.
FEBS Lett
352:
361-364,
1994[ISI][Medline].
13.
Hill, JR,
Corbett JA,
Kwon G,
Marshall CA,
and
McDaniel ML.
Nitric oxide regulates interleukin 1 bioactivity released from murine macrophages.
J Biol Chem
271:
22672-22678,
1966
14.
Johes, JI,
and
Clemmons DR.
Insulin-like growth factors and their binding proteins: biological actions.
Endocr Rev
16:
3-34,
1995[ISI][Medline].
15.
Kanety, H,
Shimon I,
Ehrenfeld M,
Israeli A,
Pariente C,
and
Karasik A.
Insulin-like growth factor I and its binding proteins 3 and 4 are increased in human inflammatory synovial fluid.
J Rheumatol
23:
815-818,
1996[ISI][Medline].
16.
Lafeber, FPJG,
Van der Kraan PM,
Huber-Bruning O,
Van den Berg WB,
and
Bijlsma JWJ
Osteoarthritic human cartilage is more sensitive to transforming growth factor than is normal cartilage.
Br J Rheumatol
32:
281-286,
1993[ISI][Medline].
17.
Lipton, SA,
Choi YB,
Pan ZH,
Lei SZ,
Chen HSV,
Sucher NJ,
Loscalzo J,
Singel DJ,
and
Stamler JS.
A redox-based mechanism for the neuroprotective and neurodestructive effects of nitric oxide and related nitroso-compounds.
Nature
364:
626-632,
1993[ISI][Medline].
18.
Luten, FP,
Hascall VC,
Nissley SP,
Morales TK,
and
Reddi AH.
Insulin-like growth factors maintain steady-state metabolism of proteoglycans in bovine articular cartilage explants.
Arch Biochem Biophys
267:
416-425,
1988[ISI][Medline].
19.
Maier, R,
Bilbe G,
Rediske J,
and
Lotz M.
Inducible nitric oxide synthase from human articular chondrocytes: cDNA cloning and analysis of mRNA expression.
Biochim Biophys Acta
1208:
145-150,
1994[ISI][Medline].
20.
Martel-Pelletier, J,
DiBattista JA,
Lajeunesse D,
and
Pelletier JP.
IGF/IGFBP axis in cartilage and bone in osteoarthritis pathogenesis.
Inflamm Res
47:
90-100,
1998[ISI][Medline].
21.
McCartney-Francis, N,
Allen JB,
Mizel DE,
Albina JE,
Xie OW,
Nathan CF,
and
Wahl SM.
Suppression of arthritis by an inhibitor of nitric oxide synthase.
J Exp Med
178:
749-754,
1993[Abstract].
22.
Morales, TI.
Transforming growth factor- and insulin-like growth factor-1 restore proteoglycan metabolism of bovine articular cartilage after depletion by retinoic acid.
Arch Biochem Biophys
15:
190-198,
1994.
23.
Nita, A,
Ghivizzani SC,
Galea-Lauri J,
Bandara G,
Georgescu HI,
Robbins PD,
and
Evans CH.
Direct gene delivery to the synovium.
Arthritis Rheum
39:
820-828,
1996[ISI][Medline].
24.
Olney, RC,
Wilson DM,
Mohati M,
Fielder PJ,
and
Smith RL.
Interleukin-1 and tumor necrosis factor- increase insulin-like growth factor-binding protein-3 (IGFBP-3) production and IGFBP-3 protease activity in human articular chondrocytes.
J Endocrinol
146:
279-286,
1995[Abstract].
25.
Palmer, RMJ,
Hickery MS,
Charles IG,
Moncada S,
and
Bayliss MT.
Induction of nitric oxide synthase in human chondrocytes.
Biochem Biophys Res Commun
193:
398-405,
1993[ISI][Medline].
26.
Pelletier, JP,
Jovanovic D,
Fernandes JC,
Manning P,
Connor JR,
Currie MG,
DiBattista JA,
and
Martel-Pelletier J.
Reduced progression of experimental osteoarthritis in vivo by selective inhibition of inducible nitric oxide synthase.
Arthritis Rheum
41:
1275-1286,
1998[ISI][Medline].
27.
Pelletier, JP,
Mineau F,
Ranger P,
Tardif G,
and
Pelletier JM.
The increased synthesis of inducible nitric oxide inhibits IL-1ra synthesis by human articular chondrocytes: possible role in osteoarthritic cartilage degradation.
Osteoarthritis Cartilage
4:
77-84,
1996[ISI][Medline].
28.
Posever, J,
Phillips FM,
and
Pottenger LA.
Effects of basic fibroblast growth factor, transforming growth factor-1, insulin-like growth factor-1, and insulin on human osteoarthritic articular cartilage explants.
J Orthop Res
13:
832-837,
1995[ISI][Medline].
29.
Sakuraia, H,
Kohsaka H,
Liu MF,
Higashiyama H,
Hirata Y,
Kanno K,
Saito I,
and
Miyasaka N.
Nitric oxide production and inducible nitric oxide synthase expression in inflammatory arthritides.
J Clin Invest
96:
2357-2363,
1995[ISI][Medline].
30.
Sasaoka, T,
Ishiki M,
Sawa T,
Ishihara H,
Takata Y,
Imamura T,
Usui I,
Olefsky JM,
and
Kobayashi M.
Comparison of the insulin and insulin-like growth factor 1 mitogenic intracellular signaling pathways.
Endocrinology
137:
4427-4434,
1996[Abstract].
31.
Schalkwijk, J,
Joosten LAB,
van den Berg WB,
and
van de Putte LBA
Chondrocyte non-responsiveness to insulin-like growth factors 1 in experimental arthritis.
Arthritis Rheum
32:
894-900,
1989[ISI][Medline].
32.
Stadler, J,
Stefanovic-Racic M,
Billiar TR,
Curran RD,
McIntyre LA,
Georgescu HI,
Simmons RL,
and
Evans CH.
Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide.
J Immunol
147:
3915-3920,
1991
33.
Stefanovic-Racic, M,
Meyers K,
Meschter C,
Coffey JW,
Hoffman RA,
and
Evans CH.
Comparison of the nitric oxide synthase inhibitors methylarginine and aminoguanidine as prophylactic and therapeutic agents in rat adjuvant arthritis.
J Rheumatol
22:
1922-1928,
1995[ISI][Medline].
34.
Stefanovic-Racic, M,
Möllers MO,
Miller LA,
and
Evans CH.
Nitric oxide and proteoglycan turnover in rabbit articular cartilage.
J Orthop Res
15:
442-449,
1997[ISI][Medline].
35.
Studer, RK,
Georgescu HI,
Miller LA,
and
Evans CH.
Nitric oxide inhibits TGF- production by chondrocytes: implications for matrix synthesis.
Arthritis Rheum
42:
248-257,
1999[ISI][Medline].
36.
Tardif, G,
Reboul P,
Pelletier JP,
Geng C,
Cloutier MJ,
and
Martel-Pelletier M.
Normal expression of type 1 insulin-like growth factor receptor by human osteoarthritic chondrocytes with increased expression and synthesis of insulin-growth factor binding proteins.
Arthritis Rheum
39:
968-978,
1996[ISI][Medline].
37.
Taskiran, D,
Stefanovic-Racic M,
Georgescu HI,
and
Evans CH.
Nitric oxide mediates suppression of cartilage proteoglycan synthesis by interleukin-1.
Biochem Biophys Res Commun
200:
142-148,
1994[ISI][Medline].
38.
Van de Loo, FAJ,
Arntz OJ,
van Enckevort FHJ,
van Lent PLEM,
and
van den Berg WB.
Reduced cartilage proteoglycan loss during zymosan-induced gonarthritis in NOS2-deficient mice and in anti-interleukin-1-treated wild-type mice with unabated joint inflammation.
Arthritis Rheum
41:
634-646,
1998[ISI][Medline].
39.
Van den Berg, WB,
van de Loo R,
Joosten LAB,
and
Arntz OJ.
Animal models of arthritis in NOS2-deficient mice.
Osteoarthritis Cartilage
7:
413-415,
1999[ISI][Medline].
40.
Verschure, PJ,
Joosten LAB,
van de Loo FAJ,
and
van den Berg WB.
IL-1 has no direct role in the IGF-1 non-responsive state during experimentally induced arthritis in mouse knee joints.
Ann Rheum Dis
54:
976-982,
1995[Abstract].
41.
Verschure, PJ,
Van Noorden CJF,
van Marle J,
and
van den Berg WB.
Articular cartilage destruction in experimental inflammatory arthritis: insulin-like growth factor-1 regulation of proteoglycan metabolism in chondrocytes.
Histochem J
28:
835-857,
1996[ISI][Medline].
42.
Yaeger, PC,
Masi TL,
Buck de Ortiz JL,
Binette R,
Tubo R,
and
McPherson JM.
Synergistic action of transforming growth factor- and insulin-like growth factor-I induces expression of type II collagen and aggrecan genes in adult human articular chondrocytes.
Exp Cell Res
237:
318-325,
1997[ISI][Medline].
43.
Yateman, ME,
Claffey DC,
Cwyfan-Hughes SC,
Frost VJ,
Wass JAH,
and
Holly JMP
Cytokines modulate the sensitivity of human fibroblasts to stimulation with insulin-like growth factor-I (IGF-I) by altering endogenous IGF-binding protein production.
J Endocrinol
137:
151-159,
1993[Abstract].