Microenvironment regulation of extracellular signalregulated kinase activity in chondrocytesEffects of culture configuration interleukin-1 and compressive stress.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 48, No. 3, March 2003, pp 689–699 DOI 10.1002/art.10849 © 2003, American College of Rheumatology Microenvironment Regulation of Extracellular Signal–Regulated Kinase Activity in Chondrocytes Effects of Culture Configuration, Interleukin-1, and Compressive Stress Kelvin W. Li, Aaron S. Wang, and Robert L. Sah chondrocyte monolayers exhibited a pattern that was distinct from that in other culture systems, suggesting that the extracellular matrix plays an important regulatory role in modulating the response to extracellular stimuli. Since IL-1 and dynamic loading have distinct effects on chondrocyte biosynthesis, signaling pathways other than ERK participate in the chondrocyte responses to these stimuli. Objective. To compare extracellular signal– regulated kinase (ERK) activity in response to interleukin-1 (IL-1) in chondrocytes under various culture configurations designed for the study of cartilage biology and repair, and also in response to dynamic load for chondrocytes in cartilage. Methods. Isolated bovine articular chondrocytes were maintained in serum-supplemented medium under 4 culture configurations: high-density monolayer, attached to a cut surface of cartilage, within tissueengineered constructs, or within intact cartilage explants. Samples were subjected to a change of medium with or without IL-1. Cartilage explants were also subjected to dynamic compression. Results. In chondrocyte monolayers, both basal and IL-1–stimulated ERK activities were similarly elevated at 0.5 hours after medium change, diminishing by 74% after 16 hours. In contrast, chondrocytes in other culture configurations exhibited lower basal levels of ERK activity and a moderate activation of ERK in response to IL-1 that was sustained over the 16-hour treatment time. The dynamic component of loading of cartilage explants led to a 5-fold activation of ERK, compared with free-swelling controls, that was indistinguishable from the effects of IL-1. Conclusion. ERK signaling in response to IL-1 in Articular cartilage, the load-bearing, low-friction, wear-resistant material in skeletal joints, is actively maintained by a population of chondrocytes that is sparsely distributed throughout a dense extracellular matrix (1). Under normal conditions, the chondrocytes mediate matrix remodeling through a balance between synthesis and degradation of matrix components. This balance is regulated by chemical and mechanical factors that make up the cellular microenvironment. In vitro studies have shown that chondrocyte metabolism is regulated by cytokines (2), growth factors (3), matrix adhesion (4,5), and mechanical loading (6,7). In vivo, the coordinated influences of these cues regulate the biologic behavior of chondrocytes and, thus, the state of cartilage health. In healthy joints, dynamic compression associated with joint loading is necessary for the maintenance of cartilage homeostasis (6,8). In degenerative conditions, tissue homeostasis is disrupted, perhaps partially due to the effects of interleukin-1 (IL-1), an inflammatory cytokine that is found in elevated levels in osteoarthritic joints (9). In cultures of isolated chondrocytes and cartilage explants, IL-1 induces a number of degradative processes, including inhibition of matrix (10) or DNA (11) synthesis and induction of matrix metalloproteinases (12). The signal transduction mechanisms mediating the opposing regulatory effects of IL-1 Supported by the NIH, the National Science Foundation, the National Aeronautics and Space Administration, and the Beckman Foundation. Kelvin W. Li, PhD, Aaron S. Wang, BS, Robert L. Sah, MD, ScD: Whitaker Institute of Biomedical Engineering, University of California, San Diego. Address correspondence and reprint requests to Robert L. Sah, MD, ScD, Department of Bioengineering, Mail Code 0412, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0412. E-mail: email@example.com. Submitted for publication July 1, 2002; accepted in revised form December 9, 2002. 689 690 and dynamic compression in normal cartilage and in cartilage repair situations remain to be elucidated. There is considerable overlap in the signal transduction pathways of various chemical and mechanical extracellular stimuli. Exposure of isolated chondrocytes to cytokines (13–15), growth factors (13,16), matrix adhesion (17,18), or mechanical stress (19–21) individually activates extracellular signal–regulated kinase (ERK), providing opportunities for interaction between these major regulatory cues. ERK is a member of the ubiquitous family of mitogen-activated protein kinases (MAPKs) that transmit stimuli from outside the cell to the nucleus and play important roles in a variety of cell processes by influencing transcriptional regulation (22). In chondrocytes, activation of signaling cascades involving ERK has been implicated in the regulation of proliferation (16), dedifferentiation (23), apoptosis (17), and biosynthesis of matrix molecules (19,24) and protease inhibitors (15). In most of these studies (13,14,16,19), activation of ERK was transient, reaching a peak after 15–30 minutes, but usually dissipating within a few hours. These studies on ERK signaling pathways in chondrocytes have often used isolated cells cultured in monolayer (13–17,19,20,23,24). This may be problematic, because the differentiated chondrocyte phenotype is typically unstable in monolayer culture over time (25) and can be modulated by morphology (26,27) as well as specific cell–matrix interactions (5,28). Since the responses of chondrocytes depend greatly on their culture conditions, a number of experimental models have been introduced to study various aspects of cartilage repair (Figure 1A). To simulate a clinical scenario in which chondrocytes are transplanted into a focal cartilage defect and come into contact with host cartilage, chondrocytes were seeded at a high density and allowed to attach to a cartilage surface (29,30) (Figure 1B). These transplanted chondrocytes in vitro maintained a stable cartilage phenotype during short-term culture, as indicated by deposition of macromolecules characteristic of cartilage (29). To fabricate replacement tissue for implantation, chondrocytes were formed into cartilaginous tissue after recovery from culture in alginate using the alginate-recovered chondrocyte (ARC) method (31) (Figure 1C), a procedure designed to use phenotypically stable chondrocytes (26). In addition, intact cartilage explants are in common use experimentally because, when maintained in medium supplemented with serum, they provide a stable, wellcharacterized in vitro model system for the study of chondrocytes within their native extracellular matrix (32) (Figure 1D). Relatively little information is avail- LI ET AL Figure 1. A, Schematic of cartilage repair and in vitro models. The models were used to study the regulation of chondrocytes involved in B, integrative repair following chondrocyte transplantation, C, bulk repair following implantation of a cartilaginous construct, and D, tissue homeostasis in the host cartilage. able on the regulation of ERK in chondrocytes in any of these experimental systems. This study was designed to test the hypothesis that the microenvironment of chondrocytes affects the way that ERK signaling is regulated. The first objective was to directly compare the magnitude and time course of ERK activation when chondrocytes were exposed to exogenous IL-1 in different in vitro models used in the study of cartilage repair. Specifically, the culture configurations compared were cultures of isolated chondrocytes in monolayer, isolated chondrocytes transplanted to cartilage, isolated chondrocytes precultured in alginate and then formed into constructs, and cartilage explants. The second objective was to examine the differential regulation of ERK signaling in cartilage explants by IL-1 and static and dynamic loading, stimuli that have different effects on matrix synthesis. MATERIALS AND METHODS Materials. Materials for harvest and culture of chondrocytes and cartilage explants were obtained as previously described (29–31,33). In addition, recombinant human IL-1␣ was obtained from R&D Systems (Minneapolis, MN). Leupeptin, aprotinin, and sodium orthovanadate were obtained from Sigma (St. Louis, MO). Phenylmethylsulfonyl fluoride was obtained from Calbiochem (La Jolla, CA). Rabbit polyclonal anti–ERK-2 antibody (C-14) and rabbit normal IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and rabbit polyclonal anti–phosphorylated ERK-1/2 REGULATION OF ERK IN CHONDROCYTES (anti–p-ERK-1/2) antibody (no. 9101) was purchased from Cell Signaling Technology (Beverly, MA). Activated recombinant rat ERK-2 standards were from Stratagene (La Jolla, CA). Microfiltration plates (96-well) with polyvinylidene fluoride (MultiScreen HV) and phosphocellulose (MultiScreen PH) filters were purchased from Millipore (Bedford, MA). Protein A–Sepharose 4 Fast-Flow and enhanced chemiluminescence kits were obtained from Amersham Pharmacia Biotech (Piscataway, NJ). We purchased 2-mercaptoethanol from Bio-Rad (Hercules, CA). Materials for in vitro kinase reactions were obtained as previously described (34), except that ␥32P-ATP was from ICN Biomedicals (Costa Mesa, CA) and myelin basic protein (MBP) was from Invitrogen (Carlsbad, CA). Sample preparation and culture. To generate samples for comparative studies, 4 different systems of chondrocyte cultures were prepared using cartilage harvested under aseptic conditions from the femurs of 1–3-week-old calves. Isolated chondrocytes were obtained by sequential enzymatic digestion from full-thickness cartilage slices with pronase and collagenase (30). Some of these chondrocytes were plated at high density (200,000 cells/cm2) onto tissue-culture plastic and incubated at 37°C in an atmosphere of 5% CO2 with medium (Dulbecco’s modified Eagle’s medium [DMEM], 10 mM HEPES buffer, 0.1 mM nonessential amino acids, 0.4 mM L-proline, 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 0.25 g/ml amphotericin B) supplemented with 10% fetal bovine serum (FBS). For this and all other culture systems, the volume of the medium used was at least 1 ml per 106 cells per day in culture. Chondrocyte monolayers were maintained in culture for either 1 or 4 days. Isolated chondrocytes were also used in combination with cartilage substrates to form an in vitro transplantation model, as described previously (29,30). Briefly, calf articular cartilage discs (6.4 mm diameter ⫻ 0.25 mm thick) were harvested from patellofemoral groove cartilage (after the removal of the articular surface), lyophilized overnight to lyse endogenous chondrocytes, and then stored at ⫺20°C until they were rehydrated for use as nonviable cartilage substrates. Cells (80,000) were seeded onto each cartilage substrate (250,000 cells/cm2) and allowed to attach undisturbed for 2 hours. The cartilage substrates, laden with transplanted cells, were incubated in medium (described above) supplemented with 10% FBS and 25 g/ml ascorbate for either 1 or 4 days. Other chondrocytes, following isolation from cartilage, were formed into cartilaginous constructs using the ARC method (31). Briefly, chondrocytes were resuspended at 4 ⫻ 106 cells/ml in 1.2% alginate and polymerized in 102 mM CaCl2 to form spherical beads. Alginate bead cultures were maintained at 37°C in an atmosphere of 5% CO2 for 8 days in a 1:1 ratio of DMEM:Ham’s F-12 medium, supplemented with 0.1 mM nonessential amino acids, 0.4 mM L-proline, 2 mM L-glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, 0.25 g/ml amphotericin B, 10% FBS, and 25 g/ml ascorbate. The alginate beads were dissolved using 55 mM sodium citrate in 0.15M NaCl, and the released cells, along with their associated matrix coat, were seeded (5 ⫻ 106 cells/cm2) in culture well inserts (6.5 mm diameter) with a base of porous (0.4 m) polyester. These cultures were maintained in DMEM-based medium (described above) including 10% FBS and 25 g/ml ascorbate for 12 days and then released as 691 cohesive cartilaginous discs (⬃1.3 mm thick) and maintained in free-swelling culture for an additional day. Cartilage explant discs (3 mm diameter ⫻ 1 mm thick) were harvested from the middle and deep zones of patellofemoral calf cartilage as previously described (33). Explants were maintained in free-swelling culture in medium (250 l medium per explant) including 10% FBS and 25 g/ml ascorbate for 6 days. Biochemical and biomechanical treatments. Effect of culture configuration on ERK activation by IL-1. To compare the effects of exogenous IL-1 on chondrocytes in the various culture systems, samples were maintained in culture for the durations specified and then subjected to changes in medium, some of which included IL-1. Samples were analyzed for basal ERK activity by using standard culture medium (including FBS) for the change, whereas the additional effect of IL-1 was determined by switching to medium supplemented with 5 ng/ml IL-1, a concentration shown to induce a number of degradative processes in bovine cartilage explants (35). IL-1␣ was chosen for these experiments since bovine chondrocytes are more responsive to the ␣-isoform than to the ␤-isoform (36). Fresh batches of medium were prepared in advance and preincubated at 37°C in an atmosphere of 5% CO2 for at least 1 hour prior to changes in medium. Stimulation durations were 0.5 and 16 hours to allow assessment of transient and sustained ERK activation, respectively, since chondrocyte monolayers were previously shown to respond to IL-1 stimulation with rapid MAPK activation that dissipated within an hour (13,14). As a control study to determine the effects of changing medium on chondrocytes in monolayer, some cultures were switched to fresh medium containing either 10% FBS or 0.01% bovine serum albumin, while others were subjected to a simulated change of medium in which conditioned culture medium was removed for a few seconds and then pipetted back into the original sample well. These chondrocyte monolayers were analyzed for ERK activity either immediately before changing medium or after 0.5 or 16 hours. Effect of static and dynamic compression on cartilage explants. While some cartilage explant discs underwent IL-1 stimulation, others were transferred from free-swelling culture into independent loading chambers within a standard incubator and subjected to static or dynamic unconfined compression between nonporous platens, as described previously (33). The culture medium included 10% FBS and 25 g/ml ascorbate. For statically loaded explants, a compressive stress of 84 kPa was applied and held, reaching mechanical equilibrium at a strain of ⬃15% (relative to the initial cut thickness) after 5 hours. For dynamically loaded explants, a time-varying stress waveform peaking at 280 kPa was applied to induce small oscillatory strains (⫾2.4%) of 0.01 Hz around the static compression level (33). The static loading protocol inhibits both cell proliferation (33) and glycosaminoglycan biosynthesis in calf cartilage (33,37), whereas the dynamic loading protocol stimulates glycosaminoglycan biosynthesis (33,37) while inhibiting cell proliferation (33). As with the IL-1 treatments, treatment durations for loaded samples were either 0.5 or 16 hours. Quantification of activated ERK. Following various treatments, samples in the different culture configurations were prepared for the extraction of cellular proteins by 1 of 2 methods. Chondrocytes either in monolayer culture or trans- 692 planted onto cartilage were rinsed briefly with ice-cold phosphate buffered saline (PBS) containing 100 M sodium orthovanadate and then incubated with lysis buffer (20 mM Tris, pH 7.6, 3 mM EDTA, 3 mM EGTA, 100 M sodium orthovanadate, 10 g/ml leupeptin, 2 mM dithiothreitol, 10 g/ml aprotinin, 1 mM p-nitrophenyl phosphate, 1 mM phenylmethylsulfonyl fluoride, 250 mM NaCl, 20 mM ␤-glycerophosphate, and 0.5% Nonidet P40 [NP40]) (38) for 15 minutes at 4°C on an Adams Nutator (Becton Dickinson, Franklin Lakes, NJ). Lysate solutions were collected into microfuge tubes either by vigorous pipetting (for chondrocytes in monolayer) or after scraping the cartilage surfaces with a flat-ended spatula (for transplanted cells). In the case of transplanted chondrocytes, 10 disc substrates, each seeded with 80,000 transplanted cells initially, were subjected to similar treatments, and their lysed cells were pooled to generate a single sample for later analysis. For either explant cultures or ARC constructs, treatments were terminated by replacing culture medium with ice-cold PBS containing sodium orthovanadate, followed by rapid transfer of the samples into liquid nitrogen. For explants under mechanical compression, the medium was replaced with the PBS rinse by infusion while maintaining the applied load, and the explants were flash-frozen within 5 minutes. Frozen samples were stored at ⫺70°C until they were powdered for extraction, as described previously (38). Briefly, frozen samples were powdered using a liquid nitrogen–immersible mortarand-pestle (Fisher Scientific, Fair Lawn, NJ), resuspended in 20 mM Tris (pH 7.6), 3 mM EDTA, 3 mM EGTA, and protease inhibitors (500 l/explant), and ground at 4°C for 30 seconds into particulates using a drill-mounted tissue grinder (Fisher Scientific). Solutions were lysed by the addition of NaCl, ␤-glycerophosphate, and NP40 for the final concentrations described above. To extract cellular proteins, crude lysates from all terminated cultures were incubated for an additional 30–60 minutes at 4°C on the Nutator and then centrifuged for 20 minutes at 16,000g. Pellets were routinely analyzed for DNA to provide a normalization factor for sample analysis. Pellets were solubilized overnight with proteinase K and then analyzed for DNA (using Hoechst 33258) and for cell content (assuming 7.7 pg DNA/cell) (30). The clarified supernatants, after digestion with proteinase K, contained a relatively small amount of the total recovered DNA (e.g., 5 ⫾ 2%, n ⫽ 12, and 1 ⫾ 2%, n ⫽ 12 for monolayer and explant samples, respectively). Clarified supernatants (“extracts”) were stored at ⫺70°C until use. Extracts were analyzed by a multiwell ERK activity assay. ERK-2 was first purified from cell extracts by immunoprecipitation. Extracts were transferred into 96-well filter plates (MultiScreen HV) in volumes corresponding to 200,000 cells (50–90 l) and incubated with a 20 l slurry containing 2.5 l fully swollen protein A–Sepharose and 2 g anti–ERK-2 rabbit polyclonal antibody (pAb). After overnight incubation at 4°C on an orbital shaker (500 revolutions per minute), unbound material was removed by vacuum filtration through the filter bottoms, leaving immunocomplexes bound within the Sepharose resin. After 4 rinses (200 l each) with kinase reaction buffer (13 mM Tris HCl, 0.7 mM EGTA, 10 mM MgCl2, 3.3 mM ␤-glycerophosphate, 130 M Na3VO4, 130 M dithiothreitol, 0.13 M protein kinase A inhibitor peptide, 1.3 M protein kinase C inhibitor peptide, 1.3 M calmidazolium LI ET AL chloride, and 8.3 M ATP, pH 7.5), a multiwell in vitro reaction was initiated (34). Briefly, kinase reaction buffer, supplemented with ␥32P-ATP (17 Ci/ml, 4,000 Ci/mmole) and 667 g/ml MBP, was added for a total volume of 60 l per well. After 30 minutes, reaction solutions were quenched with the addition of 60 l of 75 mM phosphoric acid, and a portion (40 l) of the quenched solution was filtered through phosphocellulose microfiltration plates (MultiScreen PH) and rinsed 5 times with phosphoric acid and 3 times with 70% ethanol to bind proteins and remove free radiolabel. The extent of substrate phosphorylation was assessed by liquid scintillation counting of membranes for 32P radioactivity, providing an index of functional kinase activity. To generate calibration curves for standards, the functional activity of activated recombinant rat ERK-2 was routinely analyzed by adding known amounts to representative extracts prior to immunoprecipitation. To measure the background signal of the assay, sham immunoprecipitations were performed by substituting 2 g normal rabbit IgG for anti–ERK-2 pAb. Samples were analyzed in duplicate, and the average counts per minute reading for each sample was related to the activity of activated ERK standards by first subtracting the corresponding background readings and then dividing by the slope of the appropriate calibration curve. To visualize the amount of total ERK-2 and p-ERK1/2, some extracts were additionally analyzed by immunoblotting. Portions of extracts, corresponding to 100,000 cells, were separated on 10% polyacrylamide minigels by electrophoresis and then transferred to nitrocellulose. Membranes were probed with a pAb against the activated form of ERK-1/2 for 3 hours at a dilution of 1:1,000. Secondary antibody reactivity was visualized by enhanced chemiluminescence and scanned with a flatbed scanner (Epson 1240U; Epson, Long Beach, CA). After storage at 4°C, membranes were stripped by incubation in 2% sodium dodecyl sulfate, 62.5 mM Tris HCl (pH 6.7), and 100 mM 2-mercaptoethanol at 50°C for 30 minutes and reprobed for total ERK-2 using anti–ERK-2 pAb. Statistical analysis. To determine the effects of IL-1 or compression in each culture configuration, data were analyzed by analysis of variance (ANOVA), where the main factors were stimulation type and duration. In the case of chondrocytes plated either on plastic or on cartilage, the incubation time (either 1 or 4 days in culture) represented a third main ANOVA factor. When a significant difference was detected, Tukey’s test was used to compare groups. Data are expressed as the mean ⫾ SEM. RESULTS Validation of multiwell ERK activity assay. While previous multiwell assay methods made use of antibodies immobilized onto a well bottom for the purpose of kinase purification (34), antigen capture was found to improve when the antibodies resided within a Sepharose resin. Since this Sepharose-based immunoprecipitation had not been previously described, it was important to verify that the results from the multiwell ERK activity assay used in this study were both sensitive REGULATION OF ERK IN CHONDROCYTES Figure 2. Typical calibration curves for multiwell extracellular signal– regulated kinase (ERK) activity assay. Activated ERK standards were immunoprecipitated in either lysis buffer (䊐) (LB), extracts of monolayer chondrocytes (F) (mono), or extracts of cartilage explants (‚) (exp). Immunoprecipitates, sham immunoprecipitations () (sham IP, prepared using nonspecific IgG), and activated ERK standards added directly into reaction wells (ƒ) (direct reactions) were all analyzed for functional ERK activity. to and specific for the activated ERK in the analyzed extracts. When recombinant rat p-ERK standards were added to various lysis buffers and immunoprecipitated with antibodies for ERK-2 prior to in vitro kinase reactions, regression analysis indicated that the amount of 32P radioactivity associated with MBP was linearly proportional to the amount of p-ERK added, up to the maximum 16 ng studied (Figure 2). Here, the sensitivity of the assay, given by the slope of the linear fit, varied somewhat with the buffer in which the immunoprecipitation was performed, ranging from 444 to 607 cpm/ng p-ERK (R2 ⫽ 0.93–0.99, P ⬍ 0.001; n ⫽ 12) when the immunoprecipitation buffer consisted of extracts of cartilage explants, extracts of monolayer chondrocytes, or fresh lysis buffer. Measured radioactivities were ⬃5-fold higher when p-ERK standards were added directly into reactions (forgoing the immunoprecipitation step), with a slope of 2,608 cpm/ng p-ERK (R2 ⫽ 0.96, P ⬍ 0.001; n ⫽ 12), suggesting that the capture efficiency of immunoprecipitation (determined as the ratio of sensitivities) ranged from 17% to 23%. In contrast, results with sham immunoprecipitations were insensitive to the addition of p-ERK standards, with a slope of 2.5 cpm/ng p-ERK (R2 ⫽ 0.74, P ⫽ 0.14; n ⫽ 4), indicating that observed 693 radioactivities were the result of specific antigen capture by anti–ERK-2 pAb. Effect of culture configuration on ERK activation by IL-1. Some chondrocyte monolayers were maintained in serum-supplemented medium until they were stimulated by switching to medium with or without IL-1. In this culture system, the effects on ERK activity were indistinguishable whether the chondrocyte monolayers were stimulated on day 1 (data not shown) or on day 4 (Figure 3) following cell plating (P ⫽ 0.7). In both cases, the inclusion of IL-1 had no additional effects (P ⫽ 0.15). Thirty minutes after monolayers underwent changes of medium including serum, ERK activity was high, with the measured activity equivalent to that of 4.4 ⫾ 0.3 ng (mean ⫾ SEM) p-ERK per 200,000 cells. The response in the initial half-hour appeared to be a transient activation of ERK due to the medium change, since 16 hours of continuous exposure led to a 74% reduction in ERK activity to 1.2 ⫾ 0.2 ng p-ERK per Figure 3. Activation of extracellular signal–regulated kinase (ERK) in chondrocyte monolayers. Isolated chondrocytes were plated at high density on tissue-culture plastic and maintained in culture in Dulbecco’s modified Eagle’s medium (DMEM) ⫹ 10% fetal bovine serum (FBS). On day 4, the culture medium was replaced with DMEM including the supplements indicated. Cultures were terminated following either a 0.5- (■) or a 16-hour (䊐) treatment period. Extracted cellular proteins were analyzed by A, Western blot for both activated and total ERK or by B, multiwell in vitro assay for functional ERK activity (n ⫽ 7 or 8 samples from 4 animals per culture condition). Values are the mean and SEM. ⴱⴱⴱ ⫽ P ⬍ 0.001, 0.5 hours versus 16 hours. IL-1 ⫽ interleukin-1; p-ERK ⫽ phosphorylated ERK. 694 LI ET AL Figure 4. Activation of ERK in chondrocytes transplanted to cartilage. Chondrocytes were passaged at high density onto a cut cartilage surface and maintained in culture in DMEM ⫹ ascorbate (asc) ⫹ 10% FBS for 1 day (A and B) or 4 days (C and D) before the culture medium was replaced with DMEM including the supplements indicated. Cultures were terminated following either a 0.5- (■) or a 16-hour (䊐) treatment period. Extracted cellular proteins were analyzed by Western blot for both activated and total ERK (A and C) or by multiwell in vitro assay for functional ERK activity (B and D) (n ⫽ 2 samples from 2 animals per culture condition). Values are the mean and SEM. ⴱ ⫽ P ⬍ 0.05. See Figure 3 for other definitions. 200,000 cells. This was comparable with the ERK activity level detected immediately before medium change (1.7 ⫾ 0.1 ng per 200,000 cells [n ⫽ 3] for time 0 controls; not shown). On the other hand, both changing to basal medium (without serum) and simulated medium changes using conditioned medium resulted in relatively low ERK activity after 0.5 hours (1.1 ⫾ 0.4 [n ⫽ 5] and 1.7 ⫾ 0.5 [n ⫽ 3] ng p-ERK per 200,000 cells, respectively), suggesting that the initially high levels of p-ERK were not nonspecific responses to fluid turbulence generated by changing medium. The attachment of chondrocytes to a cartilage substrate altered their ERK activation response to serum and IL-1 supplementation, and this was apparent after only 1 day in culture. In chondrocytes transplanted to cartilage, regulation of ERK activity by different medium formulations depended on the culture duration (P ⬍ 0.01) prior to medium exposure. For transplanted cells, overall, the inclusion of IL-1 in the medium had a significant effect (P ⬍ 0.05 by ANOVA) when it was introduced into culture on day 4 (P ⬍ 0.05 for interaction), and the effects did not vary between 0.5 and 16 hours of exposure (P ⫽ 0.4 by ANOVA). When transplanted chondrocytes were incubated on cartilage for only 1 day, a subsequent change of medium led to ERK activity equivalent to 3.4 ⫾ 0.3 ng p-ERK per 200,000 cells (n ⫽ 8) (Figures 4A and B) at both 0.5- and 16-hour time points, regardless of the presence of IL-1 (P ⫽ 1.0). On day 4, the basal response to the replacement of normal (serum-supplemented) culture medium was lower, with 0.74 ⫾ 0.14 ng p-ERK per 200,000 cells (n ⫽ 4), making the activation of ERK by IL-1 evident in comparison, with a 3.6-fold increase (P ⬍ 0.05) (Figures 4C and D) that was sustained for at least 16 hours. In cartilaginous constructs fabricated from ARCs, changing medium did not seem to elicit an increase in ERK activity unless the medium was supplemented with IL-1 (P ⬍ 0.001) (Figure 5). Exposure of constructs to fresh medium maintained low basal levels of ERK activity (0.58 ⫾ 0.19 ng p-ERK per 200,000 cells), whereas inclusion of IL-1 in the medium resulted in a 3-fold activation of ERK (to 1.71 ⫾ 0.17 ng p-ERK per 200,000 cells), and the results did not change detectably between 0.5 and 16 hours (P ⫽ 0.4). REGULATION OF ERK IN CHONDROCYTES 695 Effect of static and dynamic compression on cartilage explants. Static and dynamic compression of cartilage explants also had marked and differential effects on ERK activation, compared with free-swelling control explants (P ⬍ 0.001) (Figure 6A–D). Although treatment time (overall P ⫽ 0.5) had a slight interaction with treatment (P ⫽ 0.03), post hoc testing indicated that the ERK activities resulting from each treatment were indistinguishable between 0.5 and 16 hours (P ⫽ 0.9, 0.3, 0.9, and 0.9 for FBS, IL-1, static loading, and dynamic loading treatment groups, respectively). The low level of static compression applied here did not affect ERK signaling in explants compared with freeswelling basal controls (P ⫽ 0.3), but the application of dynamic load led to a sustained activation of 0.76 ⫾ 0.09 ng p-ERK per 200,000 cells (n ⫽ 14), which was 5.4 times greater than that in basal controls (P ⬍ 0.001) and 2.5 times greater than that in statically loaded samples (P ⬍ 0.001). Furthermore, the ERK responses to IL-1 and dynamic loading in explants were indistinguishable from one another (P ⫽ 0.9). DISCUSSION Figure 5. Activation of ERK in alginate-recovered chondrocyte (ARC) constructs. ARCs were incubated in pellet culture to form a cohesive cartilaginous construct over a 13-day period. Constructs were exposed to a medium change with the medium formulations indicated, and cultures were terminated following either a 0.5- (■) or a 16-hour (䊐) treatment period. Extracted cellular proteins were analyzed by A, Western blot for both activated and total ERK or by B, multiwell in vitro assay for functional ERK activity (n ⫽ 3 or 4 samples from 2 animals per culture condition). Values are the mean and SEM. ⴱⴱⴱ ⫽ P ⬍ 0.001. asc ⫽ ascorbate (see Figure 3 for other definitions). Cartilage explants also had a low basal level of ERK activity that was sensitive to IL-1 exposure (P ⬍ 0.001) (Figures 6A and B) but not affected by stimulation time (P ⫽ 0.5). In free-swelling culture with serumsupplemented medium, cartilage explants exhibited basal ERK activity equivalent to 0.14 ⫾ 0.03 ng p-ERK per 200,000 cells (n ⫽ 14) at both 0.5- and 16-hour time points (P ⫽ 0.9). Alternatively, the introduction of medium including IL-1 increased the level of ERK activity by 5-fold, reaching a peak at 0.85 ⫾ 0.12 ng p-ERK per 200,000 cells by 16 hours, although treatment time did not markedly affect the ERK response (P ⫽ 0.3). In this study, the effects of the biochemical and mechanical microenvironment of chondrocytes on ERK regulation were analyzed by comparing responses in various culture conditions. ERK signaling in cartilage explants (Figure 6) was distinguished from that in monolayer cultures of chondrocytes (Figure 3) by 4 general characteristics: 1) lower activity under basal conditions (after a medium change), 2) differential responsiveness to serum and IL-1, 3) lower peak activity when stimulated, and 4) sustained activity when stimulated. Among the 4 culture systems examined, monolayer cultures were unique in having a transient phase in ERK activation (Figure 3), and the other ex vivo models, which expose chondrocytes to an increasingly extensive matrix structure, either exhibited (Figure 5) or acquired over time (Figure 4) many of the ERK activation characteristics of cartilage explants. One day following chondrocyte transplantation to cartilage, cells exhibited persistently high levels of ERK activity (Figures 4A and B), but over time, ERK activity decreased and became susceptible to regulation by IL-1 (Figures 4C and D). Likewise, the trends in ERK regulation were similar in well-developed cartilaginous constructs and cartilage explants, with overall levels of ERK activity being 2 times greater in the constructs (Figures 5A and B). These results suggest that the biochemical composition of the immediate microenvironment surrounding chon- 696 LI ET AL Figure 6. Activation of ERK in cartilage explants. Calf cartilage explant disks were stimulated by free-swelling culture in different culture media (A and B) or subjected to static (stat) or dynamic (dyn) mechanical compression (C and D). Cultures were terminated following either a 0.5- (■) or a 16-hour (䊐) treatment period. Extracted cellular proteins were analyzed by Western blot for both activated and total ERK (A and C) or by multiwell in vitro assay for functional ERK activity (B and D) (n ⫽ 7 or 8 samples from 4 animals per culture condition). Values are the mean and SEM. ⴱⴱⴱ ⫽ P ⬍ 0.001. asc ⫽ ascorbate (see Figure 3 for other definitions). drocytes, as dictated by culture conditions, influences how soluble stimuli initiate signaling pathways. Furthermore, sustained activation of ERK in cartilage explants was a motif that also occurred in response to dynamic, but not static, mechanical loading (Figures 6C and D), with an effect that was identical to the activation by IL-1. Direct comparison of ERK activation between different culture systems required a number of considerations, including normalization to provide a basis for comparison between sample types. We normalized ERK activity by the amount of DNA (cell number) that was recovered during extraction, since extracted total protein, used commonly as a normalization factor (13,14,34), was likely to vary markedly among the culture systems. Qualitative inspection indicated that equivalent amounts of total ERK were made available for analysis, because immunoblots for total ERK-2 showed similar levels of total ERK-2 when extracts from different culture systems were analyzed in volumes corresponding to 100,000 cells (Figures 3A, 4A, 4C, 5A, 6A, and 6C). To allow comparison with existing studies on the response of cartilage to IL-1 (39,40) and compression (30,37), cartilage from calves was used as the cell source for the various culture systems investigated here. Also for comparison’s sake, serum was included in the culture media, and it exhibited interactive effects with IL-1 in these experiments. Chondrocytes in the monolayer culture system were characterized by a transient phase of ERK activation in response to a medium change that included either FBS or IL-1 (Figures 3A and B), but the effects were not additive when FBS and IL-1 were combined, even though the chondrocytes had been cultured with FBS prior to stimulation (Figures 3A and B). Temporary ERK activation by IL-1 has been observed both in human chondrocytes that had been serum starved (13) and in rabbit chondrocytes in serumsupplemented culture (14). Fluid-induced shear has also been reported to transiently increase ERK activation in chondrocyte monolayers either with (19) or without serum (19,20) included in the flow medium. However, the transient ERK activation associated with changing to medium that included serum in this study (Figures 3A REGULATION OF ERK IN CHONDROCYTES and B) could not be attributed to direct mechanical stimulation alone or to the abolishment of nutrient boundary layers, since changing to either serum-free or conditioned medium did not elicit the same response. Furthermore, neither significant depletion of serum components during culture nor accumulation of metabolic waste was likely, since transient ERK activation was evident even when increased medium volumes were used for culture either before or during IL-1 stimulation (data not shown). Further studies may clarify the mechanisms underlying this transient activation of ERK in serum-supplemented monolayer cultures. In culture configurations that maintained the presence of a significant matrix structure, hindered transport may have been partially responsible for the relatively low peak levels of ERK activity in the chondrocyte response to serum and IL-1. The lower degree of ERK activation in such cultures may reflect an ability of the dense cartilage matrix to buffer chondrocytes from sudden changes in their chemical and mechanical environment. The extracellular matrix presents a hindrance to mass transport, with slowing of exchange kinetics and chemical partitioning (41,42). Thus, with more matrix, both IL-1 and fresh serum components would require more time to diffuse into the tissue and reach receptor targets on cell surfaces, and would also equilibrate at lower local steady-state concentrations. For molecules similar in size to IL-1, the time constant for diffusion of IL-1 through the cartilage tissue is ⬃7 hours and the partition coefficient is 0.3–0.7 (41). Experimental observations confirm that IL-1 is able to effectively penetrate and regulate cartilage within that time frame (43). Such a partition coefficient would still result in a local, steady-state concentration of IL-1 in the explants above the threshold concentration (1 ng/ml), leading to maximal ERK activation in isolated rabbit chondrocytes (14). Thus, while the matrix does not completely protect endogenous chondrocytes from exposure to IL-1, it may physically dampen its effects. Furthermore, direct chondrocyte–matrix interactions appeared to markedly modulate ERK activity. Attachment to cartilage for 1 day eliminated the transient phase of ERK activation from the chondrocyte response to serum and IL-1 exposure (Figure 4A), suggesting that the initial adhesion to cartilage exerts an immediate influence prior to the pericellular accumulation of matrix de novo. Chondrocytes may interact with cartilage matrix through a number of receptors. The initial firm adhesion of chondrocytes to a cut surface of cartilage is mediated by engagement of ␤1 and av␤5 integrin cell surface receptors (44). In intact articular 697 cartilage, chondrocytes are embedded in matrix and use integrins to attach to matrix components (45). Integrinmediated signaling events include the activation of ERK in many cell types (46), including chondrocytes (17,18). Although ligation of some integrins alone leads to an activation of ERK that is typically transient, synergistic responses are mediated by growth factors (47,48), mechanical loading (49–51), and cytokines (52) to activate signaling cascades, including sustained ERK activation in some cases (47). In the culture systems studied here, a similar type of cooperativity may occur between attachment to extracellular matrix molecules and chemical (or mechanical) stimuli. Differences in the ERK activity and responses observed between the in vitro culture configurations examined here suggest that the adhesion of chondrocytes to components in the extracellular matrix modulates the subsequent cellular response to cytokines, affecting both the time course and the magnitude of ERK activation. The regulation of ERK activation within cartilage found here (Figure 6) is generally consistent with, and extends, previous observations. Transient activation of ERK by IL-1 was observed in human cartilage explants, but the effect required incubation in an atmosphere of low oxygen tension (53). In bovine cartilage explants, the application of static compression was previously found to induce activation of ERK in a compressionamplitude–dependent manner (21). Application of ⬎25% static compression activated ERK, while 15% compression, the degree of static compression resulting from the loading applied here, did not lead to a significant ERK response. The superimposed effect of dynamic compression on ERK activation was not investigated previously. These findings indicate that ERK signaling is operative within cartilage explants in response to a range of chemical and mechanical perturbations. The extent and time course of ERK activation in chondrocytes has certain implications. ERK signaling pathways have typically been associated with the regulation of cell proliferation (22). Despite the initially confluent seeding density, chondrocytes transplanted to cartilage initially undergo significant proliferation in this model system (30). This proliferation pattern coincides with the basal ERK activity in transplanted chondrocytes, where proliferation and ERK activity are both relatively high during culture on days 1 and 2 (Figures 4A and B). On the other hand, confluent monolayer chondrocytes in this study did not proliferate detectably when cultured on plastic (data not shown), despite the high peak level of ERK activity associated 698 LI ET AL with each medium change. This is consistent with the hypothesis that only sustained ERK activation induces cell division (54). The ERK signaling pathway does not appear to be the sole determinant of aggrecan synthesis within intact cartilage for the chemical and mechanical stimuli examined here. In articular chondrocytes, activation of ERK may have functional implications regarding aggrecan biosynthesis (19,24). However, in this study, similar patterns of ERK activation were observed whether chondrocytes in explants were exposed to IL-1 (10,39,40) or to the selected dynamic loading protocol (33,37), which have opposing effects on biosynthesis. Also, lowlevel static loading, such as IL-1, inhibits biosynthesis (33,37), but had little marked effect on ERK activity. 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