Chondrocyte response to growth factors is modulated by p38 mitogen-activated protein kinase inhibition
© Studer et al., licensee BioMed Central Ltd. 2004
Received: 24 July 2003
Accepted: 16 October 2003
Published: 7 November 2003
Inhibitors of p38 mitogen-activated protein kinase (MAPK) diminish inflammatory arthritis in experimental animals. This may be effected by diminishing the production of inflammatory mediators, but this kinase is also part of the IL-1 signal pathway in articular chondrocytes. We determined the effect of p38 MAPK inhibition on proliferative and synthetic responses of lapine chondrocytes, cartilage, and synovial fibroblasts under basal and IL-1-activated conditions.
Basal and growth factor-stimulated proliferation and proteoglycan synthesis were determined in primary cultures of rabbit articular chondrocytes, first-passage synovial fibroblasts, and cartilage organ cultures. Studies were performed with or without p38 MAPK inhibitors, in IL-1-activated and control cultures. Media nitric oxide and prostaglandin E2 were assayed.
p38 MAPK inhibitors blunt chondrocyte and cartilage proteoglycan synthesis in response to transforming growth factor beta; responses to insulin-like growth factor 1 (IGF-1) and fetal calf serum (FCS) are unaffected. p38 MAPK inhibitors significantly reverse inhibition of cartilage organ culture proteoglycan synthesis by IL-1. p38 MAPK inhibition potentiated basal, IGF-1-stimulated and FCS-stimulated chondrocyte proliferation, and reversed IL-1 inhibition of IGF-1-stimulated and FCS-stimulated DNA synthesis. Decreases in nitric oxide but not prostaglandin E2 synthesis in IL-1-activated chondrocytes treated with p38 MAPK inhibitors are partly responsible for this restoration of response. Synovial fibroblast proliferation is minimally affected by p38 MAPK inhibition.
p38 MAPK activity modulates chondrocyte proliferation under basal and IL-1-activated conditions. Inhibition of p38 MAPK enhances the ability of growth factors to overcome the inhibitory actions of IL-1 on proliferation, and thus could facilitate restoration and repair of diseased and damaged cartilage.
Keywordschondrocytes interleukin-1 nitric oxide p38 mitogen-activated protein kinase transforming growth factor beta
Proinflammatory cytokines are responsible for much of the pathophysiology of both osteoarthritis and rheumatoid arthritis . Activation of p38 mitogen-activated protein kinase (MAPK) has been implicated in the catabolic and anti-anabolic actions of both IL-1 and tumor necrosis factor alpha . These cytokines are also induced in mechanically stressed [3, 4] and damaged cartilage. The signal pathways they activate, including p38 MAPK, may thus influence the course of cartilage repair. It is therefore important to understand the consequences of p38 MAPK inhibition on cartilage/chondrocyte responses to the anabolic effectors, which stimulate the repair processes of proliferation and cartilage matrix protein synthesis.
Members of the pyridinyl imidazole class of compounds that inhibit p38 MAPK have been developed, and their potential as therapeutic agents in inflammation, arthritis, septic shock, and myocardial injury is currently being explored . One of these compounds, SB 203580 (SB), is a potent inhibitor of cytokine production in mice and rats, and decreases paw inflammation in collagen-induced arthritis in mice . A second related compound, SB 242235, decreases adjuvant-induced arthritis in rats . SB also inhibits IL-1 induction of inducible nitric oxide synthase (iNOS) in bovine chondrocytes , and thus blocks nitric oxide (NO) synthesis. This effect may also protect cartilage from the damaging actions of NO .
p38 MAPK was recently identified, however, as part of the signal transduction pathway effecting transforming growth factor beta (TGF-β) stimulation of aggrecan gene expression by the chondrogenic cell line ATDC5 [10, 11]. The relationship between TGF-β signaling through p38 MAPK and the Smad family was characterized in C2C12 cells. The conclusion was that the nuclear target of p38, ATF-2, becomes phosphorylated in response to TGF-β and forms a complex with Smad 4 . Similar signal synergy studies have not been carried out for chondrocytes. However, given the important anabolic and anticatabolic  actions of TGF-β, any maneuver that modifies responses to TGF-β and other anabolic growth factors could have critical consequences for maintenance and repair of cartilage. These studies were thus initiated to determine whether p38 MAPK inhibition affects chondrocyte responses to TGF-β, insulin-like growth factor 1 (IGF-1), and serum, and also whether p38 MAPK inhibition reverses the anti-anabolic actions of IL-1 on proliferative and synthetic responses of rabbit articular chondrocytes, cartilage, and synovial fibroblasts.
Materials and methods
Materials were obtained from the following suppliers: New Zealand White rabbits, 5–6 lb (Myrtle's Rabbitry, Thompson Station, TN, USA); modified Eagle's medium (MEM), fetal calf serum (FCS), antibiotics, other tissue culture supplies, and protease inhibitor cocktail for use with mammalian cells (Sigma Chemical, St Louis, MO, USA); DuoSet IC for phospho-p38alpha (R&D Systems, Minneapolis, MN, USA); protein assay reagent (Bio-Rad, Hercules, CA, USA); type 1 collagenase and trypsin (Worthington Biochemical, Freehold, NJ, USA); 35S-sodium sulfate, 1 Ci/mmol (NEN, Boston, MA, USA); [methyl-3H]thymidine and prostaglandin E2 (PGE2) enzyme immunoassay kits (Amersham Pharmacia Biotech, Piscataway, NJ, USA); human TGF-β1, human IGF-1, recombinant human IL-1β (R&D Systems, Minneapolis, MN, USA); Sc-58125 (Cayman Chemical, Ann Arbor MI, USA); SB 203580 (SB) and SB 202190, hydrochloride (Calbiochem, San Diego, CA, USA); and N-monomethyl-L-arginine (L-NMA) was synthesized by Dr Paul Dowd and Dr Wei-Zhang (Department of Chemistry, University of Pittsburgh, PA, USA). All other reagents were obtained from Sigma Chemical.
Rabbits were euthanized using a protocol approved by the IACUC of the Pittsburgh, Pennsylvania VA Healthcare System. Chondrocytes were isolated from knee and shoulder joints of mature New Zealand white rabbits and the cells were cultured as previously described . Cartilage slices from the same joints were used in some experiments. Fibroblasts were cultured from synovial membranes of lapine knee joints using a procedure similar to that for chondrocytes as previously reported . Cells were grown to 80% confluence in Falcon Multiwell, 48-well plates, and medium serum was reduced to 1% 24 hours before addition of dimethylsulfoxide vehicle at < 0.5% or the specific p38 MAPK inhibitors SB 203580 (SB) or SB 202190, hydrochloride, at 1 μM. The inhibitor concentrations used were kept below 2 μM as effects not related to p38 MAPK inhibition have been seen in other cell types with higher concentrations . Seven hours later, 50 ng/ml IGF-1, 50 pM TGF-β or 5–10% FCS was added. Proteoglycan synthesis or proliferation was determined 24 hours after addition of growth factors.
Phosphorylated p38 MAPK was determined as an index of activation using a commercially available kit (R&D systems) on cell lysates collected 30 min after activation with IL-1 (2 ng/ml) and 60–120 min after addition of TGF-β (100 pM). Chondrocytes were grown to confluence in six-well plates, the serum reduced for 24 hours, fresh medium added, and the cells lysed after activation with IL-1 or TGF-β. After treatment the cells were washed twice with phosphate-buffered saline, lysed, the lysates analyzed as per kit instructions and the results normalized to the average protein content of 33 μg/ml (determined on 10-fold dilution of lysates as per Bio-Rad protein assay instructions).
Proteoglycan synthesis was measured as the incorporation of 35S-sulfate (6 hour pulse label) into molecules separated from unincorporated label using PD-10 columns as described for this laboratory . Proliferation was measured as the incorporation of [3H]thymidine during a 2 hour pulse label into trichloroacetic acid precipitated material. NO was assayed as the nitrite concentration in conditioned media (CM) using the Griess reaction, and CM PGE2 was assayed using the ELISA kit from Amersham Pharmacia Biotech.
Chondrocytes transduced with an adenoviral vector carrying the human inducible nitric oxide synthase gene (Ad-iNOS) were used in some studies to facilitate evaluation of the effects of NO independent of other actions of iNOS inducing cytokines on the cell. The adenoviral vector, previously described  with a titer of 1010 pfu/ml, was prepared by Dr Paul Robbins (University of Pittsburgh School of Medicine Human Gene Therapy Center). Transduction of chondrocytes was carried out as follows: monolayers of chondrocytes were washed with Gey's Balanced Salt Solution, and 1 × 107 pfu virus in 0.2 ml Dulbecco's modified Eagle's medium (DMEM) containing 0.1% bovine serum albumin, with or without 1 mM L-NMA added to each well. The transduction efficiency was 76% under these conditions . The cells were washed after overnight incubation, and the culture continued for 24 hours in MEM, 0.5% fetal bovine serum, with or without L-NMA, agonists added, and conditioned media for determination of NO production collected 24 hours later. Proliferation was also evaluated at this time.
Experiments were performed at least three times, and data are presented as mean ± standard error. Statistically significant differences (P < 0.05) were determined using Student's t test.
IL-1 induces iNOS and cyclooxygenase-2 (COX-2) in chondrocytes. The products of these enzymes, NO and PGE2, have been shown to reduce chondrocyte proliferation [20, 21]. SB inhibition of p38 MAPK decreased NO in CM from IL-1-activated cells from 7.5 ± 0.41 μM (IL-1) to 4.7 ± 0.26 μM (IL-1 + SB), a significant 50% inhibition of the increase above control values of 2.4 ± 0.35 μM (vehicle) and 1.97 ± 0.51 μM (SB). A series of experiments was initiated to evaluate the ability of NO alone to modulate chondrocyte proliferation in the absence of other factors present in IL-1-activated cells.
We also tested the effects on lapine chondrocyte proliferation of exogenous PGE2 at concentrations generated in the previous experiments. Consistent with the results in Fig. 9, there were no significant effects of PGE2 in this concentration range (0.1–6 ng/ml) on chondrocyte proliferation (data not shown).
Cartilage/chondrocyte metabolism may also be affected by synovial hyperplasia and by the products secreted by the synovial fibroblasts . We therefore evaluated the effects of inhibition of p38 MAPK on basal, growth factor-stimulated, and IL-1-activated proliferation of lapine synovial fibroblasts (passage 1). The effects of p38 MAPK inhibition in lapine synovial fibroblasts were modest in comparison with those found in chondrocytes. SB had no effect on basal proliferation and the 29% stimulation in the presence of FCS is less than that seen in chondrocytes. SB had no effect on proliferation in the presence of IGF-1 or TGF-β. SB did significantly stimulate proliferation of IL-1-activated fibroblasts under both basal (1629 ± 115 dpm/well versus 2970 ± 803 dpm/well) and FCS-stimulated conditions (8014 ± 449 dpm/well versus 10372 ± 1104 dpm/well). IL-1 did not increase NO synthesis in these preparations, and there were only modest changes in PGE2 synthesis under the conditions evaluated (data not shown).
Summary of effects of p38 MAPK inhibition on proteoglycan synthesis
IL-1 + SB*
The net effect of p38 MAPK inhibition on matrix protein (proteoglycan) synthesis will thus depend on the growth factor milieu effecting cartilage homeostasis. The partial reversal of IL-1 inhibition in cartilage, and the lack of an effect on the response to the complex mix of factors contained in FCS, suggests that p38 MAPK inhibition in vivo would positively affect cartilage proteoglycan synthesis.
Summary of effects of p38 MAPK and COX-2 inhibition on chondrocyte proliferation
Lapine chondrocyte proliferation
IL-1 + SB*
IL-1 + L-NMA*
IL-1 + Sc-58125*
p38 MAPK inhibition had little effect on lapine fibroblast proliferation under basal and IL-1-activated conditions, precluding a concomitant synovial hyperplasia and potential increases in catabolic factors thereby secreted.
The data suggest a significant component of IL-1 inhibition of rabbit chondrocyte proliferation is effected through p38 MAPK-mediated actions. The role of p38 MAPK in the regulation of proliferation has been extensively studied ; however, the relationship varies with cell type. For example, p38 MAPK activation is linked with increased proliferation in vascular smooth muscle cells , and is necessary for the fibroblast growth factor 2 stimulation of fibroblasts , but it arrests proliferation of thymocytes . The current studies show that, in lapine chondrocytes, inhibition of p38 MAPK enhances basal and growth factor-stimulated proliferation, and can restore proliferation in IL-1-activated cells. These data suggest that, in this cell type, p38 MAPK activation is associated with decreased DNA synthesis.
L-NMA inhibition of NO synthesis in IL-1-activated cells did increase IGF-1-stimulated and FCS-stimulated proliferation, but not as effectively as SB under some conditions. This suggests that some, but not all, of the potentiation of proliferation by SB in IL-1-activated cells may be secondary to the decrease in NO synthesis when p38 MAPK is blocked. The NO dose response (Fig. 6) shows that lapine chondrocyte proliferation is sensitive to NO over the concentration range found in the conditioned media of IL-1-activated cells, and is modulated by SB. The effects of NO in the context of IL-1-activated chondrocytes where multiple factors are altered may be different from that in cells where NO synthesis is enhanced in isolation from these factors. The data do suggest, however, that the diminution of NO synthesis by p38 MAPK inhibitors may contribute to their ability to blunt the anti-anabolic actions of IL-1. This pathway may or may not be relevant to human disease, as Badger and colleagues  found that SB 242235, another selective p38 MAPK inhibitor, did not decrease IL-1 induction of iNOS and NO synthesis in human chondrocyte cultures. However, p38 MAPK activation by NO has been demonstrated in several cell types [28, 29] and has been linked with NO induction of heme oxygenase 1 in HeLa cells . The possibility that some of the pathophysiologic actions of NO in human chondrocytes may be mediated via p38 MAPK activation has not been evaluated, and thus remains a potential point of therapy by inhibitors of p38 MAPK in cytokine-activated human cartilage/chondrocytes.
SB also inhibited IL-1-stimulated increases in PGE2 synthesis/accumulation (Fig. 8). However, Sc-58125 inhibition of COX-2 and the resulting decreases in PGE2 had little effect on chondrocyte proliferation (Fig. 9). This suggests that the SB inhibition of PGE2 production in IL-1-treated and IL-1 + TGF-β-treated cells contributes minimally to the restoration of proliferation in rabbit chondrocytes. The effects of prostaglandins on chondrocyte proliferation have been variable. For example, Blanco and Lotz  concluded that NO inhibition of normal human chondrocyte proliferation was effected by concomitant changes in PGE2. Lowe and colleagues  showed that exogenous PGE2 had a dose-dependent, biphasic effect on rat chondrocytes with suppression at the lower concentrations tested (0.1 μM, or 35 ng/ml) and stimulation at higher concentrations (5 μM, or 1760 ng/ml). Schwartz and colleagues  found that PGE2 from 0.007 to 15 ng/ml increased the cell number and [3H]thymidine incorporation in chick costochondal cartilage cells.
Our data suggest that normal rabbit articular chondrocyte proliferation is relatively insensitive to the range of CM PGE2 attained subsequent to IL-1 activation. The relationship between chondrocyte proliferation and PGE2 thus seems highly species dependent. We recently reported that human chondrocyte proliferation is inhibited by PGE2 concentrations found in CM following IL-1 activation . The mechanisms by which p38 MAPK inhibition restores proliferation in IL-1-activated/stressed human chondrocytes may thus be different from those described for lapine preparations (current studies) or for bovine preparations .
These studies were initiated, in part, to determine whether inhibition of p38 MAPK would blunt the actions of TGF-β on chondrocytes. In the case of proliferation, this was not found to be so. Under basal conditions, SB did not modify the modest TGF-β-stimulated increases in DNA synthesis; in IL-1-activated cells, the response to TGF-β was actually enhanced. In contrast, both SB 203580 (SB) and SB 202190 blunted the ability of TGF-β to stimulate proteoglycan synthesis in chondrocytes in a monolayer culture and in situ in a cartilage organ culture. Ridley and colleagues  showed that even low concentrations of SB inhibited proteoglycan synthesis in bovine nasal septum cartilage in organ cultures in the presence of 10% FCS. We did not see a similar inhibition in rabbit cartilage. Furthermore, stimulation of proteoglycan synthesis by IGF-1 in lapine chondrocytes in monolayer culture was also unaffected by SB. Perhaps the difference between these results is related to species differences in the relative importance of the p38 MAPK pathway in maintaining proteoglycan synthesis. Regardless, our data do suggest that the signal pathways that effect TGF-β stimulation of chondrocyte proliferation and chondrocyte proteoglycan synthesis differ; the pathway activating proteoglycan synthesis appears to involve p38 MAPK, while that activating proliferation does not.
The data showing only minor modulation of IL-1-inhibited proteoglycan synthesis by SB (Figs 2 and 3) are consistent with prior studies of bovine cartilage . However, even the ability to modestly reverse the anti-anabolic effects of IL-1 may be therapeutic under conditions of mild inflammation as is often seen in osteoarthritis, and in some stages of the repair of injured cartilage. Although proteoglycan synthesis responses to TGF-β are blunted, the responses to IGF-1 and FCS remain intact in the presence of p38 MAPK inhibitors. Coupled with the ability to reverse effects of low concentrations of IL-1 and the minimal effects on synovial fibroblasts, this suggests that p38 MAPK inhibition could have a positive effect on cartilage maintenance and repair.
Although activation of p38 MAPK has been observed in tissues from arthritic joints  and from mechanically stressed cartilage [2, 3], and has been shown to be involved in IL-1 inhibition of collagen synthesis  and IL-1 induction of collagenases , this is the first report showing effects of basal levels of p38 MAPK activity on chondrocyte proliferation. Inhibition of p38 MAPK potentiates basal and stimulated proliferation. It thus appears to have a regulatory function in lapine chondrocytes under both normal and cytokine-activated conditions. p38 MAPK inhibition can partially reverse IL-1 inhibition of proteoglycan synthesis, and thus could contribute to maintenance of matrix proteins in cytokine-activated and stressed cartilage. Whether similar effects of p38 MAPK inhibitors on chondrocyte responses to cytokines and growth factors are found in human cartilage/chondrocyte preparations should be evaluated.
adenoviral vector carrying the human inducible nitric oxide synthase gene
Dulbecco's modified Eagle's medium
enzyme-linked immunosorbent assay
fetal calf serum
insulin-like growth factor 1
inducible nitric oxide synthase
mitogen-activated protein kinase
modified Eagle's medium
plaque forming units
transforming growth factor beta.
The Department of Veterans Affairs and the Ferguson Orthopaedic Fund supported this work.
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