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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

Previous studies have shown the influence of subchondral bone osteoblasts (SBOs) on phenotypical changes of articular cartilage chondrocytes (ACCs) during the development of osteoarthritis (OA). The molecular mechanisms involved during this process remain elusive, in particular, the signal transduction pathways. The aim of this study was to investigate the in vitro effects of OA SBOs on the phenotypical changes in normal ACCs and to unveil the potential involvement of MAPK signaling pathways during this process.

Methods

Normal and arthritic cartilage and bone samples were collected for isolation of ACCs and SBOs. Direct and indirect coculture models were applied to study chondrocyte hypertrophy under the influence of OA SBOs. MAPKs in the regulation of the cell–cell interactions were monitored by phosphorylated antibodies and relevant inhibitors.

Results

OA SBOs led to increased hypertrophic gene expression and matrix calcification in ACCs by means of both direct and indirect cell–cell interactions. In this study, we demonstrated for the first time that OA SBOs suppressed p38 phosphorylation and induced ERK-1/2 signal phosphorylation in cocultured ACCs. The ERK-1/2 pathway inhibitor PD98059 significantly attenuated the hypertrophic changes induced by conditioned medium from OA SBOs, and the p38 inhibitor SB203580 resulted in the up-regulation of hypertrophic genes in ACCs.

Conclusion

The findings of this study suggest that the pathologic interaction of OA SBOs and ACCs is mediated via the activation of ERK-1/2 phosphorylation and deactivation of p38 phosphorylation, resulting in hypertrophic differentiation of ACCs.

Explanations concerning the cause of osteoarthritis (OA) have long focused on the destruction of articular cartilage, the activating factors of which were thought to be triggered as a result of repetitive loading (1). Pathologic changes of cartilage in OA are associated with changes in the cellular phenotype of articular cartilage chondrocytes (ACCs) to a state of terminal differentiation (2, 3). However, the long-term molecular events that are responsible for this transition are not well understood.

Recent studies suggest that the subchondral bone plays a major role in OA cartilage changes, an indication of active communication between the subchondral bone and the cartilage in the progression of OA (4, 5). Bone anabolic factors, such as osteocalcin, osteopontin, and alkaline phosphatase (ALP) are all up-regulated in OA subchondral bone osteoblasts (SBOs) as compared with normal SBOs, supporting the notion of a dysfunction of osteoblast behavior (6–8). It has been shown in animal models of OA that a thickening of subchondral bone precedes cartilage changes (9, 10), and it has further been demonstrated that in vivo factors produced by OA SBOs increase glycosaminoglycan release from the cartilage (11) and can influence cartilage-specific gene expression (12). It was demonstrated by the application of a coculture model of bovine explant subchondral bone and cartilage that excision of subchondral bone from articular cartilage resulted in increased chondrocyte death, thus demonstrating the important role of subchondral bone in maintaining joint homeostasis (13). However, the molecular mechanisms, and, in particular, the signaling pathways, by which normal and OA SBOs regulate the articular cartilage phenotype remain unknown.

Activation of the 3 major classes of MAPKs (ERK-1/2, JNK, and p38 MAPK) has been detected in chondrocytes (14). MAPKs are known to be responsible for the conversion of a vast number of extracellular stimuli into specific cellular responses, including chondrocyte proliferation and differentiation (15, 16). The requirement of MAPK signaling pathways, in particular, p38 and ERK-1/2, during various phases of endochondral ossification has also been demonstrated in several studies (17, 18). MAPK signaling pathways have been shown to play a distinct role in aspects of cartilage biology, such as cartilage matrix synthesis and homeostasis (19, 20). The role of MAPK signaling in skeletal development and in the biology of cartilage points toward a possible association of altered MAPK signaling and OA. Indeed, alterations in these signaling pathways are reported to play a prominent role in chondrocyte dysfunction as a part of OA pathogenesis and disease progression (21).

Since the OA SBOs are reported to alter the cartilage phenotype, it is possible that these alterations in ACCs may occur via MAPK regulation. However, no studies to date have explored the role of MAPK signaling factors in the cell–cell interactions of SBOs and ACCs. The present study was designed to investigate MAPK signaling pathways in the hypertrophic changes of normal ACCs induced by OA SBOs using both direct and indirect coculture systems.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Articular cartilage sample collection and determination of phenotype.

Ethical approval for this project was granted by the Queensland University of Technology and the Prince Charles Hospital Ethics Committees, and informed consent was given by all subjects involved. ACCs from OA patients (n = 5) were sourced from the main defective area of the medial compartment cartilage in which there were degenerative changes. The mean ± SD age of the OA patients in this study was 65.20 ± 5.94 years. ACCs from normal subjects (n = 3) were obtained from trauma patients, where knee tissue was available. Normal subjects were healthy adults with a mean ± SD age of 53.56 ± 10.76 years, no clinical signs or symptoms of joint, metabolic, or hormonal diseases (osteoporosis), and no history of medications that might affect cartilage or bone metabolism. To eliminate the possibility that these samples had early OA, those showing any evidence of cartilage changes were excluded. These changes included softening of the hyaline articular cartilage, thinning and fibrous dislocation, ulcerations of the cartilage, and light sclerosis of the subchondral bone.

Cartilage features were classified according to the Mankin scale (22), based on histologic assessment of Safranin O–and hematoxylin and eosin–stained sections. Chondrocytes from the cartilage tissues were isolated according to a previously described method (23). Only early-passage ACCs (passages 0–2) showing strong expression of type II collagen (COL2) and aggrecan (AGG) were used for subsequent experiments. To determine phenotype changes in ACCs, chondrogenic (COL2, AGG) and hypertrophic (ALP, COL10, core- binding factor α1 [CBFA1]) marker genes were measured according to their messenger RNA (mRNA) expression.

Subchondral bone sample collection and determination of phenotype.

Bone specimens were taken within 5 mm of the subchondral bone plate. SBOs from OA patients (n = 5) were sourced from weight-bearing sites, where the cartilage was degraded and showed prominent subchondral bone erosion and density. These samples were obtained from patients with advanced OA who were undergoing primary total knee replacement surgery. The mean ± SD age of the OA patients in this study was 65.20 ± 5.94 years. SBOs from normal subjects (n = 3) were collected from patients undergoing surgery for fracture repair who had no evidence of bone erosion or cartilage degeneration, as judged according to criteria established by the American College of Rheumatology (24). Normal subjects were healthy adults with a mean ± SD age of 53.56 ± 10.76 years, no clinical signs or symptoms of joint, metabolic, or hormonal diseases (osteoporosis), and no history of medications that might affect cartilage or bone metabolism.

After removing the overlying cartilage, SBOs were isolated according to the method described by Beresford et al (25, 26). Isolated normal and OA SBOs were differentiated in osteogenic medium supplemented with 10% fetal bovine serum (In Vitro Technologies), 50 units/ml of penicillin, 50 μg/ml of streptomycin, 10 nM dexamethasone, 10 mM β-glycerophosphate, and 50 μg/ml of ascorbic acid. The cells were then used for characterization of bone cell phenotype, as determined by the expression of the bone markers alkaline phosphatase and osteocalcin, as well as by staining with 1% alizarin red solution after 2 weeks of osteogenic induction.

Chondrocyte pellet culture.

Cell culture systems known to preserve the chondrocyte phenotype were used in the coculture studies. ACCs (2 × 105 cells) were resuspended in serum-free chondrogenic medium (serum-free high-glucose Dulbecco's modified Eagle's medium [DMEM; Invitrogen]) supplemented with 10 ng/ml of transforming growth factor β3 (TGFβ3; Bio Scientific), 10 nM dexamethasone, 50 mg/ml of ascorbic acid, 10 mg/ml of sodium pyruvate, 10 mg/ml of proline, and an insulin–transferrin–selenium supplement (final concentration of the supplement 10 μg/ml of insulin, 5.5 μg/ml of transferrin, 5 ng/ml of sodium selenite, 0.5 mg/ml of bovine serum albumin, and 4.7 μg/ml of linoleic acid) and then centrifuged at 600g for 20 minutes to form a pellet. Pellets were allowed to differentiate for 2 weeks under 3-dimensional conditions in 15-ml Falcon tubes containing chondrogenic medium, which was replenished every 2–3 days. After 2 weeks of chondrogenesis, ACC pellets were cocultured with normal or OA SBOs as described below.

High-density micromass culture.

High-density micromass droplets were prepared as described previously (17). Briefly, ACCs were trypsinized, resuspended in growth medium at a final cell density of 2.5 × 107 cells/ml, spotted as droplets of 10 μl/well in 24-well culture plates, and incubated at 37°C for 2 hours to allow cell attachment to the plate. Micromasses were cultured for 1 week in chondrogenic medium. After 1 week, micromasses containing ACCs were cultured with the conditioned medium generated from normal or OA SBOs as described below.

Direct coculture.

ACC pellets were prepared as described above, placed directly upon the monolayer of normal or OA SBOs (75,000 cells/well) in 24-well plates, and cocultured for another 2 weeks in high-glucose DMEM supplemented with 1% fetal calf serum (FCS), 0.5% L-glutamine, 50 units/ml of penicillin, 50 μg/ml of streptomycin, 50 μM ascorbic acid, 10 nM dexamethasone, and 10 mM β-glycerophosphate. After 14 days of coculture, the ACC pellets were washed 3 times in phosphate buffered saline, fixed in 4% paraformaldehyde for 10 minutes, and stained with 1% alizarin red or 0.5% Alcian blue to assess the effect of normal or OA SBOs on ACC matrix deposition. RNA and protein were also extracted from some of the pellets. The culture system selected for this coculture study was modified from the previously described protocols for the formation of a chondro-osseous rudiment in micromass cultures (27).

Indirect coculture and preparation of SBO conditioned medium.

Passage 2 SBOs from normal and OA subchondral bone (2.5 × 105 cells) were cultured in high-glucose DMEM supplemented with 1% FCS, 0.5% L-glutamine, 25 units/ml of penicillin, 25 μg/ml of streptomycin, 50 μM ascorbic acid, 10 nM dexamethasone, and 10 mM β-glycerophosphate in 25-cm2 flasks for 2 days. The media from these flasks was collected and centrifuged at 1,000g for 15 minutes. The supernatants were transferred to fresh tubes and mixed with an equal volume of fresh (preincubated in 37°C in the incubator) medium with the same supplements to form conditioned medium. During coculture experiments, ACC micromasses prepared as described above were grown for 1 week in conditioned medium from either normal or OA SBOs. Control ACCs were cultured in the same medium composition described above, but were not incubated with SBOs. Medium was replenished every 2 days. At the end of the coculture period, protein and total RNA were harvested from the ACCs, and some cells were fixed with 4% paraformaldehyde and stained with alizarin red and Alcian blue to assess extracellular matrix deposition.

Detection of MAPK activation.

MAPK-mediated cellular interactions were evaluated with the use of MAPK inhibitors SB203580 and PD98059 (both from Novabiochem), which inhibit the p38 and ERK-1/2 pathways, respectively. ACC micromasses were incubated with or without the MAPK inhibitors in conditioned medium prepared from normal or OA SBOs. Stock solutions of each inhibitor were dissolved in DMSO (final concentration of DMSO not exceeding 0.1% [volume/volume]). An equal amount of DMSO vehicle was added to control ACCs. Medium was replenished every 2 days. Optimal concentrations for inhibition in ACCs were found to be 10 μM for ERK-1/2 and 5 μM for p38. At these concentrations, there was no evidence of cytotoxicity, nor was cell proliferation influenced by the addition of the inhibitors. All experiments were performed in triplicate.

RNA extraction and quantitative reverse transcription–polymerase chain reaction (RT-PCR).

Total RNA was isolated with TRIzol reagent (Invitrogen), treated with DNase, and column purified using an RNeasy Mini kit (Qiagen). Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using Superscript III (Invitrogen) according to the manufacturer's instructions. PCR primers were designed based on cDNA sequences from the National Center for Biotechnology Information Sequence database using Primer Express software; primer specificity was confirmed by BLASTN searches. Quantitative RT-PCR was performed with an ABI Prism 7000 Thermal Cycler (Applied Biosystems) using SYBR Green detection reagent. Briefly, 2 μl of cDNA, 20 pmoles of gene-specific primers, and 10 μl of 1× Master Mix were used in a 20-μl reaction volume; each sample was determined in duplicate. Thermocycling conditions were as follows: 1 cycle of 10 minutes at 95°C for activation of the polymerase, 40 cycles of 10 seconds at 95°C, and 1 minute at 60°C for amplification. Dissociation curve analysis was performed to verify the absence of primer dimers and/or nonspecific PCR products. The relative expression of the genes of interest was normalized against housekeeping genes GAPDH and 18S RNA.

Western blotting.

Total protein lysates were harvested by lysing the cells with a lysis buffer containing 1M Tris HCl (pH 8), 5M NaCl, 20% Triton X-140, 0.5M EDTA, and a protease inhibitor cocktail (Roche). The cell lysate was clarified by centrifugation, and the protein concentration was determined with a bicinchoninic acid protein assay (Sigma). Ten micrograms of protein was separated by electrophoresis on a 12% sodium dodecyl sulfate–polyacrylamide gel, transferred to a nitrocellulose membrane, and blocked with a Tris–Tween buffer containing 5% nonfat milk. The membranes were incubated overnight at 4°C with primary antibodies against phospho-p38 (1:1,000 dilution; Genesearch), phospho–ERK-1/2 (1:2,000 dilution; Quantum Scientific), and tubulin (1:5,000 dilution; Quantum Scientific). The membranes were washed 3 times in Tris buffered saline–Tween and incubated for 1 hour with anti-rabbit secondary antibody at a dilution of 1:2,000. The protein bands were visualized using ECL Plus Western Blotting Detection Reagents (Amersham Biosciences) and exposed on x-ray film (Fujifilm). Immunoblots were analyzed by densitometry using ImageJ software (NIH Image, National Institutes of Health; online at: http://rsbweb.nih.gov/ij/).

Statistical analysis.

Each normal sample of ACCs was cocultured with either normal (n = 3) or OA (n = 3) SBOs, and the study was repeated in 3 normal ACC samples. Results are presented as the mean ± SD. The relative expression represents the mean of 3 combinations of chondrocytes and osteoblasts in the coculture studies. Repeated-measures analysis of variance with post hoc tests was used to assess statistical significance. P values less than or equal to 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Expression of chondrogenic and hypertrophic genes in normal and OA ACCs.

The cell proliferation study indicated that there was no difference in cell numbers between normal and OA ACC pellet cultures (data not shown). The expression of messenger RNA (mRNA) for chondrogenic and hypertrophic marker genes was compared between normal and OA ACCs. The expression of CBFA1, COL10, and ALP was significantly up-regulated in OA ACCs as compared with normal ACCs (P ≤ 0.05 for each comparison), whereas the expression of COL2 and AGG was significantly down-regulated in OA ACCs as compared with normal ACCs (P ≤ 0.05 for each comparison) (Figure 1A). These results indicated that OA ACCs had greater potential to undergo hypertrophic differentiation as compared with normal ACCs. With regard to MAPK phosphorylation, we observed that phosphorylation of p38 was down-regulated in OA ACCs as compared with normal ACCs (Figure 1B). On the other hand, phosphorylation of ERK-1/2 was significantly up-regulated in OA ACCs as compared with normal ACCs (Figure 1C).

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Figure 1. Characterization of micromass culture of articular cartilage chondrocytes (ACCs) from normal subjects and patients with osteoarthritis (OA). A, The expression of mRNA for chondrogenic and hypertrophic marker genes was compared between normal (n = 3) and OA (n = 5) ACC micromasses after 7 days of culture in hypertrophic differentiation medium containing high-glucose Dulbecco's modified Eagle's medium supplemented with 1% fetal calf serum, 0.5% L-glutamine, 50 units/ml of penicillin, 50 μg/ml of streptomycin, 50 μM ascorbic acid, 10 nM dexamethasone, and 10 mM β-glycerophosphate. The expression of CBFA1, COL10, and ALP was significantly up-regulated in OA ACCs as compared with normal ACCs. However, the expression of COL2 and AGG was significantly decreased in OA ACCs as compared with normal ACCs. Values are the mean ± SD. = P ≤ 0.05. B and C, Western blot analysis (top) was performed to determine the changes in phosphorylation of p38 (B) and ERK-1/2 (C). Tubulin was used as a loading control. Results are representative of protein bands from 3 separate experiments. The densities of the bands identified in the Western blots were quantified by densitometry using ImageJ software (bottom), and the relative densities were compared. Values are the mean ± SD. = P < 0.05 for between-group differences.

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Hypertrophic differentiation of normal ACCs in coculture with OA SBOs.

Direct coculture.

No difference in cell proliferation rates was observed in SBOs after 14 days of culture (data not shown). On day 14 of coculture, glycosaminoglycan matrix deposition was lower in the coculture groups as compared with the noncocultured ACC pellets. There was slightly lower staining intensity in the coculture with OA SBOs as compared with that in the coculture with normal SBOs (Figure 2A, top). On the other hand, mineralization in the ACC pellets was significantly enhanced in ACCs that had been cocultured with OA SBOs as compared with the noncocultured ACCs and the ACCs cocultured with normal SBOs (Figure 2A, middle). The expression of COL2 immunostaining was decreased in the coculture groups as compared with the noncocultured ACC pellets. Furthermore, the expression of COL2 in ACCs was significantly decreased in the presence of OA SBOs as compared with the normal SBOs (Figure 2A, bottom).

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Figure 2. Effects of subchondral bone osteoblasts (SBOs) from normal subjects (n = 3) and patients with osteoarthritis (OA; n = 3) on articular cartilage chondrocyte (ACC) matrix deposition and gene expression in direct coculture. A, Cartilage matrix, including glycosaminoglycans (GAGs) stained with Alcian blue and COL2, was significantly decreased and mineralization matrix stained with alizarin red was significantly increased in ACC pellets cocultured with OA (n = 3), but not normal (n = 3), SBOs. ACC pellets grown alone were used as controls (n = 3). B and C, The expression of the chondrogenic markers COL2 and AGG was down-regulated (B), but the expression of the hypertrophic markers CBFA1, COL10, and ALP was significantly up-regulated (C), in ACCs cocultured with OA SBOs. Levels of mRNA were normalized against those of GAPDH and 18S, and the relative expression is shown. Values are the mean ± SD of 3 combination studies of normal ACC pellets cocultured with OA SBOs. = P < 0.05.

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At the gene expression level, cartilage-specific genes, such as COL2 and AGG, were significantly lower in ACCs cocultured with OA SBOs than in ACCs cocultured with normal SBOs or in ACCs alone (Figure 2B). These observations were further validated by the mRNA expression of hypertrophy and mineralization marker genes in ACC pellets. The results of quantitative RT-PCR analysis indicated that OA SBOs induced a significant up-regulation of mineralization and hypertrophic markers, such as COL10, ALP, and CBFA1, as compared with the coculture group containing normal SBOs and with ACC pellets alone (Figure 2C).

Indirect coculture.

When ACC micromasses were cultured with normal or OA SBO conditioned medium, Alcian blue staining revealed that cartilage matrix deposition was attenuated by both normal and OA SBO conditioned medium; however, cartilage matrix loss was more prominent in OA SBO conditioned medium (Figure 3A, top). Conversely, matrix mineralization was greater in ACC micromasses grown for 7 days in OA SBO conditioned medium, as demonstrated by alizarin red staining (Figure 3A, bottom). Induction of the cartilage-specific genes COL2 and AGG was significantly down-regulated in the presence of both normal and OA SBO conditioned medium as compared with ACCs cultured alone, although this decrease was more prominent in ACCs grown in the presence of OA SBO conditioned medium (Figure 3B). In contrast, the expression of cartilage hypertrophy markers CBFA1, COL10, and ALP, were significantly up-regulated in the presence of OA SBO conditioned medium (Figure 3C) as compared with both normal SBO conditioned medium and with the control groups.

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Figure 3. Effects of conditioned medium prepared from subchondral bone osteoblasts (SBOs) obtained from normal subjects and patients with osteoarthritis (OA) on normal articular cartilage chondrocyte (ACC) matrix deposition and gene expression in indirect coculture. A, ACCs were cultured with conditioned medium from normal or OA SBOs and control ACC micromasses were cultured in nonconditioned medium. Deposition of glycosaminoglycans (GAGs) decreased and matrix mineralization increased in ACCs cultured for 7 days with OA SBO conditioned medium. B and C, The expression of COL2 and AGG was decreased (B), but the expression of CBFA1, COL10, and ALP was increased (D), in ACC micromasses cultured for 7 days in OA SBO conditioned medium, as determined by quantitative reverse transcription–polymerase chain reaction. Values are the mean ± SD of 3 combination studies of ACCs cultured with conditioned medium. = P < 0.05. Color figure can be viewed in the online issue, which is available at http://www.arthritisrheum.org.

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Phospho-p38 and phospho–ERK-1/2 kinase signaling patterns in cocultured ACCs.

The effect of normal and OA SBOs on the ACCs was assessed with respect to alterations in the MAPK signaling cascade in both direct and indirect coculture systems. The results showed that the phosphorylation of ERK-1/2 was significantly augmented when ACCs were cocultured with OA SBOs, in both the direct and the indirect coculture models, as compared with noncocultured ACCs and ACCs cocultured with normal SBOs. Phosphorylation of p38, on the other hand, was considerably down-regulated in the ACCs cocultured with normal SBOs as compared with ACCs alone. Nonetheless, the coculture of ACCs with OA SBOs led to a complete attenuation of p38 phosphorylation. These results suggest that up-regulation of ERK-1/2 phosphorylation and down-regulation of p38 phosphorylation are involved in the interaction between ACCs and SBOs, which in turn, leads to hypertrophic changes in ACCs (Figures 4A–D).

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Figure 4. MAPK signaling pattern in direct and indirect cocultures of articular cartilage chondrocytes (ACCs). A, ACC pellets were cocultured directly in monolayers with subchondral bone osteoblasts (SBOs) obtained from normal subjects and patients with osteoarthritis (OA). After 14 days, the ACC pellet protein was isolated, and phosphorylation of ERK-1/2 and p38 were determined. ACC pellets cultured alone were used as control. Tubulin was used as a loading control. Increased phospho–ERK-1/2 and decreased phospho-p38 were noted in the coculture with OA SBOs. B, ACC micromasses were cultured for 7 days in the presence or absence of conditioned medium (CM) from normal or OA SBOs. Increased phospho–ERK-1/2 and decreased phospho-p38 were detected. C and D, The densities of the phospho–ERK-1/2 (C) and phospho-p38 (D) bands identified in the Western blots shown in A and B, respectively, were quantified by densitometry using ImageJ software, and the relative densities were compared. Results are representative of protein bands from 3 separate experiments. Values are the mean ± SD of 3 combination studies of ACC pellets cocultured with SBOs. = P < 0.05.

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Reversal of phenotype changes in ACCs induced by OA SBO conditioned medium following addition of the ERK-1/2 inhibitor PD98059.

Incubation of ACCs cultured with OA SBO conditioned medium with PD98059 had the effect of decreasing the expression of phospho–ERK-1/2 and increasing the expression of phospho-p38 in a concentration-dependent manner (Figure 5A). ACCs alone did not show significant changes in response to the addition of PD98059, which indicates that the observed effects were specific to OA SBO conditioned medium. Quantitative RT-PCR analysis showed that PD98059 reversed the expression of hypertrophic gene expression of CBFA1, COL10, and ALP in ACC micromasses cultured in the presence of OA SBO conditioned medium (Figure 5C). In contrast, the expression of COL2 and AGG was up-regulated by the addition of PD98059 (Figure 5B).

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Figure 5. Reversal of the expression of hypertrophic genes in articular cartilage chondrocytes (ACCs) induced by conditioned medium prepared from subchondral bone osteoblasts (SBOs) obtained from patients with osteoarthritis (OA) following the addition of PD98059. A, ACC micromasses were cultured in OA SBO conditioned medium with or without the indicated concentrations of the ERK-1/2 inhibitor PD98059. After 7 days, total cell protein was isolated from ACC micromasses, and changes in the phosphorylation of ERK-1/2 and p38 were measured. Tubulin was used as a loading control. PD98059 decreased the phosphorylation of ERK-1/2 in a concentration-dependent manner, with a concomitant increase in the phosphorylation of p38. B and C, The expression of mRNA for COL2 and AGG was up-regulated (B), but the expression of mRNA for CBFA1, COL10, and ALP was down-regulated (C), in ACC micromasses cultured for 7 days with OA SBO conditioned medium following addition of the ERK-1/2 inhibitor PD98059. Levels of mRNA were normalized against those of GAPDH and 18S, and the relative expression is shown. Values are the mean ± SD of 3 combination studies of ACC micromasses cultured in conditioned medium from OA SBOs. = P < 0.05.

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Induction of hypertrophic gene expression in ACCs cultured in normal SBO conditioned medium following addition of the p38 inhibitor SB203580.

Inhibition of the phosphorylation of p38 by SB203580 led to a reduction of p38 phosphorylation and activated the phosphorylation of ERK-1/2 in ACCs cultured in normal SBO conditioned medium (Figure 6A). In the presence of SB203580, the hypertrophic markers CBFA1, COL10, and ALP were significantly enhanced, whereas the chondrogenic markers COL2 and AGG were down-regulated in ACCs in the presence of normal SBO conditioned medium (Figures 6C and D). These results indicate that the use of SB203580 could significantly shift ACCs toward a more hypertrophic phenotype. Similar results were obtained in normal ACCs cultured alone (data not shown).

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Figure 6. Induction of the expression of hypertrophic genes in articular cartilage chondrocytes (ACCs) cultured with conditioned medium prepared from subchondral bone osteoblasts (SBOs) obtained from normal subjects following the addition of SB203580. A, ACC micromasses were cultured in normal SBO conditioned medium with or without the indicated concentrations of the p38 inhibitor SB203580. After 7 days, total cell protein was isolated from ACC micromasses, and changes in the phosphorylation of p38 and ERK-1/2 were measured. Tubulin was used as a loading control. SB203580 had an inhibitory effect on the phosphorylation of p38, but enhanced the phosphorylation of ERK-1/2. B and C, The expression of mRNA for COL2 and AGG was decreased (B), but the expression of mRNA for CBFA1, COL10, and ALP was increased (C), in ACC micromasses cultured for 7 days with normal SBO conditioned medium following addition of the p38 inhibitor SB203580. Levels of mRNA were normalized against those of GAPDH and 18S, and the relative expression is shown. Values are the mean ± SD of 3 combination studies of ACC micromasses cultured in conditioned medium from normal SBOs. = P < 0.05.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

In this study, we demonstrated the importance of MAPK signaling pathways as the means by which OA subchondral bone osteoblasts induce altered phenotype changes in articular cartilage chondrocytes. Our experiments also provide some insight into the cross-talk taking place between the p38 and ERK-1/2 signaling pathways during this pathologic interaction process.

It was observed that ACCs isolated from OA patients produced significantly greater levels of mRNA for CBFA1, COL10, and ALP genes as compared with ACCs isolated from healthy patients. This finding indicates that the OA ACCs possessed greater potential to undergo hypertrophic differentiation; these results corroborate those of previous studies comparing normal and OA ACCs (28, 29).

Applying an in vitro indirect coculture model, Sanchez et al (12) demonstrated that sclerotic OA SBOs decreased cartilage-specific gene expression, such as SOX9 and COL2. They also showed that inhibitors of hypertrophic differentiation, such as parathyroid hormone–related protein and parathyroid hormone receptor were significantly down-regulated in ACCs cocultured with OA SBOs (12). These findings are evidence that OA SBOs can decrease the inhibitors of hypertrophic differentiation, leading to a subsequent mineralized matrix deposition in cartilage. In the present study, using both direct and indirect coculture methods, we showed that OA SBOs increased both hypertrophic gene expression and matrix mineralization.

Interestingly, hypertrophic changes are followed by a simultaneous decrease in chondrocyte-specific phenotype. A characteristic change of OA is an up-regulation of hypertrophy-related markers and mineralization-related markers (3) and a down-regulation of chondrocyte-specific markers (COL2 and AGG) in articular cartilage (30). The observations in our study suggest that the interaction of OA SBOs may lead to these typical hypertrophic changes in ACCs. It has been reported that the transition of ACCs to hypertrophic changes contributes to the activation of matrix metalloproteinases (MMPs), which precedes cartilage degeneration (31, 32), indicating that the phenotypic conversion of ACCs to hypertrophy is pathologic for the health and integrity of articular cartilage, leading to its degeneration.

The reasons why OA SBOs seem to induce the altered ACC phenotypes remain unclear; however, several potential pathways may be responsible. Both our own studies (data not shown) and those by other groups of investigators have demonstrated that OA SBOs produce abnormal levels of osteogenic markers, growth factors, and cytokines. Specifically, increased production of growth factors, such as insulin-like growth factor 1 (IGF-1) (33), and TGFβ (34), have been reported in OA SBOs. Among these factors, IGF-1 has been implicated in the induction of cartilage hypertrophic changes in growth plate chondrocytes (35, 36). In addition, it has been reported that OA SBOs produce abnormal levels of cytokines such as interleukin-1 and interleukin-6 (34), tumor necrosis factor, and MMP-13 (37, 38), all of which have the ability to activate a diverse array of signaling pathways in cartilage hypertrophy. It is therefore possible that the biomolecules secreted from OA SBOs communicate either individually or cooperatively with ACCs, thereby mediating the induction of phenotype changes of ACCs. Further studies are required to delineate the soluble factors from OA SBOs that are responsible for triggering the hypertrophic changes of ACCs in OA.

Among the signaling factors, the MAPK subtypes ERK-1/2 and p38 play a key role in the signaling process of chondrocyte cellular differentiation and homeostasis, depending on the nature of extracellular stimuli (14, 39). This knowledge prompted us to investigate MAPK signaling in the context of the influence of normal and OA SBOs on the differentiation of ACCs. This study is the first of its kind to show that OA SBOs induce ERK-1/2 phosphorylation and suppress p38 phosphorylation in ACCs, indicating that the alterations of these pathways accompany the pathologic phenotype changes in ACCs. Indeed, we have demonstrated that basal levels of ERK-1/2 phosphorylation increased and p38 decreased in OA ACCs as compared with normal ACCs, an indication of the pathologic relevance of these pathways in the pathogenesis of OA.

When the influence of ERK-1/2 phosphorylation was blocked by an inhibitor, p38 was activated in ACCs grown in the presence of OA SBO conditioned medium. The application of the ERK-1/2 inhibitor in OA SBO conditioned medium reversed ACC hypertrophy, and there was a return to the chondrogenic phenotype of ACCs. This observation implies that OA SBOs induced altered phenotype changes in ACCs via a deactivation of p38 and an activation of ERK-1/2 phosphorylation. This notion was further supported by results showing that when p38 was neutralized by an inhibitor in ACCs cocultured with normal SBOs, ERK-1/2 phosphorylation was augmented, and a weakening of chondrogenic gene expression and increase of hypertrophic gene expression was observed. Taken together, these data indicate that OA SBOs decrease p38 phosphorylation and increase ERK-1/2 activity, with a resulting reduction in chondrogenic phenotype and an increase in hypertrophic phenotype.

MAPKs are regulated at several levels, including kinase–kinase and kinase–substrate interactions and inhibition of cross-talk/output by the MAPKs themselves (40, 41). The activities of p38 are primarily governed by extensive cross-talk with ERK-1/2, a process that involves protein phosphatase, resulting in a reciprocal bidirectional equilibrium between ERK-1/2 and p38 phosphorylation, where an increase in p38 activity suppresses the activation of ERK-1/2 and vice versa (42). Such cross-talk appears to play a role in the OA SBO–regulated ACC phenotype, the existence of which has been shown in chondrocytes. For example, the opposing roles of ERK-1/2 and p38 have been demonstrated in chondrogenesis regulation (43). The finding that ERK-1/2 activation increased the hypertrophic differentiation of ACCs is consistent with study findings of a strong activation of the ERK-1/2 pathway in the hypertrophic zone of the growth plate (44). Furthermore, it has been demonstrated that the inhibition of ERK-1/2 delayed hypertrophic differentiation in growth plate chondrocytes during endochondral ossification (45).

It is possible that components of the p38 and ERK-1/2 pathways may interact directly in the transcription complex. The intermediate p38 and ERK-1/2 pathway substrates involved in these interaction are not known, but it is interesting that PD98059 (anti–ERK-1/2) significantly reduced the expression of the transcription factor CBFA1, whereas SB203580 (anti-p38) activated this transcription factor. During early skeletogenesis, chondrocyte hypertrophy is stimulated through the expression of CBFA1 in prehypertrophic chondrocytes, most likely by up-regulation of COL10 expression (46). Continuous expression of CBFA1 in chondrocytes induces hypertrophic differentiation and endochondral ossification, which is suggestive of an important role of this transcription factor in triggering hypertrophic changes (47). It is therefore possible that OA SBO–induced altered phenotype changes are triggered in ACCs via the activation of the MAPK/CBFA1 pathway.

In conclusion, this study demonstrated that OA SBOs could induce the activation of ERK-1/2 and the deactivation of p38 in articular cartilage chondrocytes, resulting hypertrophic changes of the chondrocytes. These data provide insight into the MAPK signaling pathways involved in the molecular mechanisms of the pathogenesis of OA, which may have significant clinical implications.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Xiao had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Prasadam, Crawford, Xiao.

Acquisition of data. Prasadam, van Gennip, Friis, Shi, Crawford, Xiao.

Analysis and interpretation of data. Prasadam, van Gennip, Friis, Shi, Crawford, Xiao.

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  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES
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