Nitric oxide inhibits chondrocyte response to IGF-I: inhibition of IGF-IRbeta tyrosine phosphorylation

R. K. Studer, E. Levicoff, H. Georgescu, L. Miller, D. Jaffurs, and C. H. Evans

Ferguson Laboratory, Musculoskeletal Research Center, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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-beta (TGF-beta ), 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-beta 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta 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-1beta 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-IRbeta ) (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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Fig. 1.   Exogenous NO generated by 0.1 mM S-nitroso-N-acetylpenicillamine (SNAP) inhibits insulin-like growth factor I (IGF-I)-stimulated proteoglycan synthesis by lapine cartilage slices. Cartilage slices were prepared as described in METHODS. 0.1 mM SNAP or DMSO vehicle (0.5%) were added 40 min before IGF-I at the concentrations indicated. Twenty-four hours later, conditioned media samples were taken for analysis of NO production as nitrite, [35S]sulfate was added at 20 µCi/ml, and culture continued for 8 h. Conditioned media and guanidine HCl extracts of the cartilage slices were analyzed for incorporation of 35S into proteoglycans, and the data are reported as the sum of conditioned media (CM) and extract cpm/10 mg wet wt of tissue. CM nitrite concentration was 47 µM (SNAP + IGF-I) or 3.4 µM (IGF-I); , IGF-I; , SNAP + IGF-I. Values are means ± SE of n = 3-5 experiments.



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Fig. 2.   NO inhibits IGF-I-stimulated proteoglycan synthesis by lapine chondrocytes in monolayer culture. Chondrocytes were isolated and grown to confluence as described in METHODS. Medium serum was decreased to 0.5% for 24 h before addition of agonists and antagonists. Then, 0.1 mM SNAP or (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate] (DETA NONOate) was added 40 min before 50 ng/ml IGF-I or transforming growth factor-beta (TGF-beta , 10 or 50 pM). Twenty-four hours later CM samples were taken for analysis of NO, [35S]sulfate was added at 20 µCi/ml, and incubation continued for 8 h. Media and cell extracts were analyzed for incorporation of 35S into proteoglycans, and data are given as the sum of CM and cell extract pmol sulfate incorporated/well (1.5 × 105 cells). CM nitrite concentrations were 29 ± 4 µM (SNAP), 55 ± 1 µM (DETA NONOate), or 2.4 ± 0.2 µM (control). Values are means ± SE of n = 3-7. *P < 0.05 compared with control.



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Fig. 3.   Endogenously generated NO in Ad-iNOS-transduced cells inhibits IGF-I-stimulated proteoglycan synthesis. Confluent chondrocytes in monolayer culture were transduced with Ad-iNOS as described in METHODS. NG-monomethyl-L-arginine (L-NMA) was present continuously where noted. Twenty-four hour after transduction, IGF-I was added at the concentrations indicated. Twenty-four hours later, CM samples were taken for analysis of NO synthesis, [35S]sulfate was added, and culture continued for 8 h. Data are presented as the sum of CM and chondrocyte extract cpm/well. CM nitrite concentration was 24 ± 4 µM (iNOS-transduced cells) or 4 ± 1 µM (iNOS-transduced cells + L-NMA). Values are means ± SE of n = 7.



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Fig. 4.   NO dose dependently inhibits IGF-I-stimulated chondrocyte proteoglycan synthesis. Chondrocytes were treated as described in Fig. 3 with variable multiplicity of infection of Ad-iNOS to generate a range of NO synthesis. IGF-I was added at 50 ng/ml. Proteoglycan synthesis is expressed as the IGF-I-stimulated increase above basal (pmol [35S]sulfate incorporated into proteoglycans) during the 8-h pulse labeling period. Values are means ± SE of n = 6.



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Fig. 5.   NO inhibition of IGF-I-stimulated proteoglycan synthesis is reversible. Chondrocytes were transduced with Ad-iNOS, and 1 mM L-NMA was added at various times after transduction; the "120 h" data point is from cells transduced in the presence of L-NMA and maintained continuously with L-NMA to inhibit NO synthesis. The "96 h" data point is from transduced cells cultured with L-NMA for the final 4 days, the "72 h" data point is from cells with iNOS inhibited for the final 3 days, the "48 h" data point is from cells with iNOS inhibited for the last 2 days, and the "24 h" data point is from cells with iNOS inhibited for the final day of the experiments. The 0 time point is data from cells in which iNOS synthesis of NO was not inhibited. Twenty-four-hour accumulation of NO in the absence of L-NMA was 20 ± 1.4 µM nitrite; addition of L-NMA reduced it to 4 ± 1 µM. Values are means ± SE of n = 4-6.



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Fig. 6.   Interleukin-1beta (IL-1beta ) induction of NO synthesis inhibits growth factor-stimulated chondrocyte proteoglycan synthesis. Chondrocytes were grown to confluence, medium serum was reduced to 0.5% for 24 h, and IL-1beta (2 ng/ml) was added. L-NMA, 0.5 mM, was present where noted. IGF-I (50 ng/ml) and/or TGF-beta (10 pM) were added 6 h after IL-1beta . Twenty-four hours later, CM samples were taken for determination of nitrite, and [35S]sulfate was added for determination of proteoglycan synthesis. Addition of growth factors did not alter NO synthesis; values of CM nitrite were 2.4 ± 0.3 µM (vehicle), 12 ± 1 (IL-1), and 3.1 ± 0.4 (IL-1 + L-NMA). Values are means ± SE of n = 4. *P < 0.05 compared with vehicle. #P < 0.05 compared with IL-1.



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Fig. 7.   L-NMA restores human cartilage responsiveness to IGF-I. Full-thickness cartilage slices were prepared from specimens removed from human femoral condyles during arthroplasty for degenerative joint disease within 24 h of the surgery. They were maintained in MEM with 10% FBS for 5 days with or without 1 mM L-NMA. Medium serum was decreased to 0.5% at 24 h before addition of 50 ng/ml IGF-I or 50 pM TGF-beta . Proteoglycan synthesis was evaluated 48 h later. Values are means ± SE of n = 3-4. *P < 0.05 compared with control (vehicle and + L-NMA) and IGF-I (vehicle).



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Fig. 8.   IGF-binding protein-independent analog stimulation of proteoglycan synthesis is inhibited by NO. Chondrocytes were transduced with Ad-iNOS as described in METHODS. L-NMA (1 mM) was present continuously in the control group. IL-1 was added to some wells 24 h after transduction, and 50 ng/ml IGF-I, 10 ng/ml human long R3IGF-I, or des-IGF added after 6 h. Conditioned media samples for determination of NO synthesis were taken, and proteoglycan synthesis was evaluated 24 h later. CM nitrite concentrations were as follows: iNOS + L-NMA, 4.5 ± 1.4 µM; iNOS, 23 ± 4 µM; and iNOS + IL-1, 37 ± 3.5 µM. Values are means ± SE of n = 6. *P < 0.05 compared with iNOS + L-NMA. #P < 0.05 compared with iNOS alone.



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Fig. 9.   IGF-I receptor tyrosine phosphorylation is inhibited in chondrocytes exposed to SNAP or transduced with iNOS. Chondrocytes were grown to confluence in 6-well plates and treated as follows. A: 0.1 mM SNAP, followed in 40 min by IGF-I, 50 ng/ml; cells were lysed 5 and 10 min later. B: transduced with Ad-iNOS, with or without 1 mM L-NMA, and stimulated with IGF-I 24 h later. Cells were lysed 2 and 5 min after addition of IGF-I. Western analysis of tyrosine phosphorylation of the IGF-I receptor beta -unit was done as described in METHODS. Results are from representative experiments.

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, A1-A3- 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.

Transduction of chondrocytes was done as follows: monolayers of chondrocytes were washed with Gey's Balanced Salt Solution, and 1 × 107 pfu of virus in 0.2 ml MEM containing 0.1% BSA, with or without 1 mM L-NMA added to each well. After overnight incubation, the cells were washed, and culture was continued for 24 h in MEM, 0.5% FBS, with or without L-NMA, agonists were added, and conditioned media for determination of NO production were collected 24 h later. Proteoglycan synthesis was also evaluated at this time. Transduction efficiency was determined by parallel studies using the same concentration of virus but containing the lacZ marker gene. These preparations were stained with 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-Gal) (23), and the percentage of transfected cells was determined by counting the fraction of blue-stained cells. When the number of plaque-forming units (pfu) of virus was varied over the range used to determine the dose response of the NO inhibition of IGF-I-stimulated proteoglycan synthesis (Fig. 4), the percentage of blue (transduced cells) was 32 ± 2, 61 ± 3, or 76 ± 2% (n = 9).

The accumulation of nitrite in the CM of iNOS-transduced cells parallels that seen in IL-1beta -activated chondrocytes (9, 32, 35) during the times posttransduction evaluated in the current studies. The maximum rate of NO production occurred during the first 24 h (25 ± 4 µM), was maintained during 24-48 h (23 ± 1.4 µM), and declined to 16 ± 1 µM from 48 to 72 h after transduction. As previously published, NO synthesis declines more rapidly in IL-1-activated cells; however, the pattern is similar during the 24- to 48-h period investigated in these studies.

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 beta -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% beta -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 beta -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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
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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-beta 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-beta 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-beta 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-beta 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 beta -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 beta -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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta , 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-beta 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-beta . These data are in contrast with the earlier report by Lafeber et al. (16) that cartilage from OA knees is hyperresponsive to TGF-beta . However, their analysis was done after 96-h stimulation by TGF-beta , 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-beta ; 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 alpha -subunits and two intracellular beta -subunits. The alpha - and beta -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 beta -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.


    ACKNOWLEDGEMENTS

This work was supported in part by Department of Veterans Affairs, the Western Pennsylvania Chapter of the Arthritis Foundation, and the Ferguson Foundation.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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 beta  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-beta 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-alpha 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-beta 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[Abstract/Free Full Text].

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-beta 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-beta 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].


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