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Mechanical Regulation of Mitogen-activated Protein Kinase Signaling in Articular Cartilage

2003; Elsevier BV; Volume: 278; Issue: 51 Linguagem: Inglês

10.1074/jbc.m305107200

1083-351X

Paul Fanning, Gregory R. Emkey, Robert J. Smith, Alan J. Grodzinsky, Nóra Szász, Stephen B. Trippel,

NF-κB Signaling Pathways

Articular chondrocytes respond to mechanical forces by alterations in gene expression, proliferative status, and metabolic functions. Little is known concerning the cell signaling systems that receive, transduce, and convey mechanical information to the chondrocyte interior. Here, we show that ex vivo cartilage compression stimulates the phosphorylation of ERK1/2, p38 MAPK, and SAPK/ERK kinase-1 (SEK1) of the JNK pathway. Mechanical compression induced a phased phosphorylation of ERK consisting of a rapid induction of ERK1/2 phosphorylation at 10 min, a rapid decay, and a sustained level of ERK2 phosphorylation that persisted for at least 24 h. Mechanical compression also induced the phosphorylation of p38 MAPK in strictly a transient fashion, with maximal phosphorylation occurring at 10 min. Mechanical compression stimulated SEK1 phosphorylation, with a maximum at the relatively delayed time point of 1 h and with a higher amplitude than ERK1/2 and p38 MAPK phosphorylation. These data demonstrate that mechanical compression alone activates MAPK signaling in intact cartilage. In addition, these data demonstrate distinct temporal patterns of MAPK signaling in response to mechanical loading and to the anabolic insulin-like growth factor-I. Finally, the data indicate that compression coactivates distinct signaling pathways that may help define the nature of mechanotransduction in cartilage. Articular chondrocytes respond to mechanical forces by alterations in gene expression, proliferative status, and metabolic functions. Little is known concerning the cell signaling systems that receive, transduce, and convey mechanical information to the chondrocyte interior. Here, we show that ex vivo cartilage compression stimulates the phosphorylation of ERK1/2, p38 MAPK, and SAPK/ERK kinase-1 (SEK1) of the JNK pathway. Mechanical compression induced a phased phosphorylation of ERK consisting of a rapid induction of ERK1/2 phosphorylation at 10 min, a rapid decay, and a sustained level of ERK2 phosphorylation that persisted for at least 24 h. Mechanical compression also induced the phosphorylation of p38 MAPK in strictly a transient fashion, with maximal phosphorylation occurring at 10 min. Mechanical compression stimulated SEK1 phosphorylation, with a maximum at the relatively delayed time point of 1 h and with a higher amplitude than ERK1/2 and p38 MAPK phosphorylation. These data demonstrate that mechanical compression alone activates MAPK signaling in intact cartilage. In addition, these data demonstrate distinct temporal patterns of MAPK signaling in response to mechanical loading and to the anabolic insulin-like growth factor-I. Finally, the data indicate that compression coactivates distinct signaling pathways that may help define the nature of mechanotransduction in cartilage. Articular cartilage serves as a bearing surface for joints (1Muir H. BioEssays. 1995; 17: 1039-1048Crossref PubMed Scopus (347) Google Scholar, 2Huber M. Trattnig S. Lintner F. Investig. Radiol. 2000; 35: 573-580Crossref PubMed Scopus (210) Google Scholar). In this capacity, it is routinely exposed to mechanical loading. Articular cartilage is nearly unique among mammalian tissues in that it lacks a blood supply. This removes it from the minute-to-minute hormonal regulation available to other more vascularized tissues. In this environment, mechanical forces may play an important role in regulating cellular functions within tissues (3Urban J.P. Biorheology. 2000; 37: 185-190PubMed Google Scholar). Under normal physiological conditions, in vivo joint loading can result in peak dynamic mechanical stresses on cartilage as high as 15-20 megapascals (150-200 atmospheres) (4Hodge W.A. Fijan R.S. Carlson K.L. Burgess R.G. Harris W.H. Mann R.W. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2879-2883Crossref PubMed Scopus (510) Google Scholar). These peak stresses occur over very short durations (<1 s) and therefore lead to cartilage compressive strains of only 1-3%. In contrast, sustained (static) physiological stresses of ∼3.5 megapascals applied to cadaveric knee joints for 5-30-min durations resulted in compressive strains of various knee cartilages as high as 40-45% (5Herberhold C. Faber S. Stammberger T. Steinlechner M. Putz R. Englmeier K.H. Reiser M. Eckstein F. J. Biomech. 1999; 32: 1287-1295Crossref PubMed Scopus (168) Google Scholar). Several lines of evidence indicate that cartilage is responsive to these wide ranges of mechanical strain and stress (6Grodzinsky A.J. Levenston M.E. Jin M. Frank E.H. Annu. Rev. Biomed. Eng. 2000; 2: 691-713Crossref PubMed Scopus (521) Google Scholar, 7Talts J.F. Pfeifer A. Hofmann F. Hunziker E.B. Zhou X.H. Aszodi A. Fassler R. Ann. N. Y. Acad. Sci. 1998; 857: 74-85Crossref PubMed Scopus (18) Google Scholar, 8Quinn T.M. Maung A.A. Grodzinsky A.J. Hunziker E.B. Sandy J.D. Ann. N. Y. Acad. Sci. 1999; 878: 420-441Crossref PubMed Scopus (43) Google Scholar, 9Quinn T.M. Grodzinsky A.J. Buschmann M.D. Kim Y.J. Hunziker E.B. J. Cell Sci. 1998; 111: 573-583Crossref PubMed Google Scholar, 10Loening A.M. James I.E. Levenston M.E. Badger A.M. Frank E.H. Kurz B. Nuttall M.E. Hung H.H. Blake S.M. Grodzinsky A.J. Lark M.W. Arch. Biochem. Biophys. 2000; 381: 205-212Crossref PubMed Scopus (295) Google Scholar, 11Kim Y.J. Grodzinsky A.J. Plaas A.H. Arch. Biochem. Biophys. 1996; 328: 331-340Crossref PubMed Scopus (68) Google Scholar, 12Guilak F. Meyer B.C. Ratcliffe A. Mow V.C. Osteoarthritis Cartilage. 1994; 2: 91-101Abstract Full Text PDF PubMed Scopus (198) Google Scholar, 13Sah R.L. Kim Y.J. Doong J.Y. Grodzinsky A.J. Plaas A.H. Sandy J.D. J. Orthop. Res. 1989; 7: 619-636Crossref PubMed Scopus (741) Google Scholar). In vivo, acute (14Repo R.U. Finlay J.B. J. Bone Jt. Surg. Am. 1977; 59: 1068-1076Crossref PubMed Scopus (283) Google Scholar, 15Thompson Jr., R.C. Oegema Jr., T.R. Lewis J.L. Wallace L. J. Bone Jt. Surg. Am. 1991; 73: 990-1001Crossref PubMed Scopus (241) Google Scholar) and chronic (16Gritzka T.L. Fry L.R. Cheesman R.L. LaVigne A. J. Bone Jt. Surg. Am. 1973; 55: 1698-1720Crossref PubMed Scopus (37) Google Scholar) injurious compressive overloads can lead to cartilage degeneration. In vitro, static compression within the physiological range can reversibly inhibit the synthesis of critical components of the cartilage matrix. Such static compressive forces can down-regulate the gene expression and production of type II collagen, aggrecan core protein, and link protein (11Kim Y.J. Grodzinsky A.J. Plaas A.H. Arch. Biochem. Biophys. 1996; 328: 331-340Crossref PubMed Scopus (68) Google Scholar, 13Sah R.L. Kim Y.J. Doong J.Y. Grodzinsky A.J. Plaas A.H. Sandy J.D. J. Orthop. Res. 1989; 7: 619-636Crossref PubMed Scopus (741) Google Scholar, 17Ragan P.M. Badger A.M. Cook M. Chin V.I. Gowen M. Grodzinsky A.J. Lark M.W. J. Orthop. Res. 1999; 17: 836-842Crossref PubMed Scopus (108) Google Scholar). In contrast, cyclically applied hydrostatic pressure (18Hall A.C. Urban J.P. Gehl K.A. J. Orthop. Res. 1991; 9: 1-10Crossref PubMed Scopus (309) Google Scholar) and compressive strain (19Buschmann M.D. Kim Y.J. Wong M. Frank E. Hunziker E.B. Grodzinsky A.J. Arch. Biochem. Biophys. 1999; 366: 1-7Crossref PubMed Scopus (239) Google Scholar) can stimulate aggrecan core protein and protein synthesis. Articular cartilage biosynthetic activity is also regulated by cell signaling molecules. Insulin-like growth factor-I (IGF-I) 1The abbreviations used are: IGF-Iinsulin-like growth factor-IMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseJNKc-Jun N-terminal kinaseSEK1stress-activated protein kinase/extracellular signal-regulated kinase kinase-1FBSfetal bovine serumANOVAanalysis of varianceSAPKstress-activated protein kinaseMKK4mitogen-activated protein kinase kinase-4. is the predominant anabolic growth factor in synovial fluid (20Schalkwijk J. Joosten L.A. van den Berg W.B. van Wyk J.J. van de Putte L.B. Arthritis Rheum. 1989; 32: 66-71Crossref PubMed Scopus (119) Google Scholar) and stimulates the synthesis of both proteoglycans and collagen (21Trippel S.B. Adolphe M. Biological Regulation of the Chondrocyte. CRC Press LLC, Boca Raton, FL1992: 161-190Google Scholar). Although static compression reduces cartilage IGF-I content (22Bonassar L.J. Grodzinsky A.J. Srinivasan A. Davila S.G. Trippel S.B. Arch. Biochem. Biophys. 2000; 379: 57-63Crossref PubMed Scopus (97) Google Scholar), the short time course of inhibition of cartilage biosynthesis by static compression is not consistent with an effect that is mediated entirely by IGF-I or other soluble growth factors. Rather, it is thought that specific mechanotransduction mechanisms mediate the chondrocyte biosynthetic machinery under load (11Kim Y.J. Grodzinsky A.J. Plaas A.H. Arch. Biochem. Biophys. 1996; 328: 331-340Crossref PubMed Scopus (68) Google Scholar, 22Bonassar L.J. Grodzinsky A.J. Srinivasan A. Davila S.G. Trippel S.B. Arch. Biochem. Biophys. 2000; 379: 57-63Crossref PubMed Scopus (97) Google Scholar, 23Kim Y.J. Sah R.L. Grodzinsky A.J. Plaas A.H. Sandy J.D. Arch. Biochem. Biophys. 1994; 311: 1-12Crossref PubMed Scopus (283) Google Scholar, 24Valhmu W.B. Stazzone E.J. Bachrach N.M. Saed-Nejad F. Fischer S.G. Mow V.C. Ratcliffe A. Arch. Biochem. Biophys. 1998; 353: 29-36Crossref PubMed Scopus (131) Google Scholar). This study seeks to characterize such signaling mechanisms, including identification of pathways, time courses of pathway coactivations, and possible differences between mechanical and biochemical signal transduction. insulin-like growth factor-I mitogen-activated protein kinase extracellular signal-regulated kinase c-Jun N-terminal kinase stress-activated protein kinase/extracellular signal-regulated kinase kinase-1 fetal bovine serum analysis of variance stress-activated protein kinase mitogen-activated protein kinase kinase-4. Mechanical forces are complex multicomponent stimuli. The relatively simple case of a ramp-and-hold static compression of cartilage can result in transient interstitial fluid expression, cell deformation (25Freeman P.M. Natarajan R.N. Kimura J.H. Andriacchi T.P. J. Orthop. Res. 1994; 12: 311-320Crossref PubMed Scopus (171) Google Scholar), increased osmolarity (26Urban J.P. Hall A.C. Gehl K.A. J. Cell. Physiol. 1993; 154: 262-270Crossref PubMed Scopus (268) Google Scholar), decreased extracellular pH (27Boustany N.N. Gray M.L. Black A.C. Hunziker E.B. J. Orthop. Res. 1995; 13: 740-750Crossref PubMed Scopus (10) Google Scholar), changes in fixed charge density (28Mow V.C. Wang C.C. Hung C.T. Osteoarthritis Cartilage. 1999; 7: 41-58Abstract Full Text PDF PubMed Scopus (250) Google Scholar), and altered transport of soluble factors within the tissue (22Bonassar L.J. Grodzinsky A.J. Srinivasan A. Davila S.G. Trippel S.B. Arch. Biochem. Biophys. 2000; 379: 57-63Crossref PubMed Scopus (97) Google Scholar). Each of these physical phenomena may potentially act as a "mechano-ligand" activating or inhibiting one or more signaling pathways. Prior studies have often focused on the effects of one or a few of these components of compressive loading such as negative fixed charge density (26Urban J.P. Hall A.C. Gehl K.A. J. Cell. Physiol. 1993; 154: 262-270Crossref PubMed Scopus (268) Google Scholar) or interstitial pH (22Bonassar L.J. Grodzinsky A.J. Srinivasan A. Davila S.G. Trippel S.B. Arch. Biochem. Biophys. 2000; 379: 57-63Crossref PubMed Scopus (97) Google Scholar, 29Wilkins R.J. Hall A.C. J. Cell. Physiol. 1995; 164: 474-481Crossref PubMed Scopus (87) Google Scholar, 30Boustany N.N. Gray M.L. Black A.C. Hunziker E.B. J. Orthop. Res. 1995; 13: 733-739Crossref PubMed Scopus (20) Google Scholar). Recent studies have also demonstrated that mechanical stretching of chondrocytes can increase nitric oxide production (31Lee D.A. Frean S.P. Lees P. Bader D.L. Biochem. Biophys. Res. Commun. 1998; 251: 580-585Crossref PubMed Scopus (86) Google Scholar) and alter membrane transport phenomena, resulting in proliferative changes (32Wu Q.Q. Chen Q. Exp. Cell Res. 2000; 256: 383-391Crossref PubMed Scopus (179) Google Scholar). In many cell types, protein kinases are critical mediators of the cellular responses involved in the minute-to-minute regulation of tissue function. Little is known of the role of signaling by protein kinases in cartilage in response to load. The existing evidence for MAPK involvement in chondrocyte mechanotransduction is particularly scant. Interestingly, however, previous kinetic studies found that the chondrocytic response to static load is unlikely to be due to the intermediary action of cytokines or growth factors (22Bonassar L.J. Grodzinsky A.J. Srinivasan A. Davila S.G. Trippel S.B. Arch. Biochem. Biophys. 2000; 379: 57-63Crossref PubMed Scopus (97) Google Scholar). These findings suggest that a mechanism of direct mechanical regulation of cartilage exists. At present, published data linking any MAPK pathway activation to mechanotransduction events in cartilage are limited to the response to fluid shear flow applied over plated chondrocyte monolayers using fluid velocities that are much higher than those known to occur during cartilage loading in vivo (33Jin G. Sah R.L. Li Y.S. Lotz M. Shyy J.Y. Chien S. J. Orthop. Res. 2000; 18: 899-908Crossref PubMed Scopus (67) Google Scholar, 34Hung C.T. Henshaw D.R. Wang C.C. Mauck R.L. Raia F. Palmer G. Chao P.H. Mow V.C. Ratcliffe A. Valhmu W.B. J. Biomech. 2000; 33: 73-80Crossref PubMed Scopus (103) Google Scholar). Moreover, the temporal organization of simultaneously activated pathways has, to our knowledge, not been investigated. This is particularly true for the analysis of chondrocyte signaling under physiologically relevant loads applied to cells in situ within their native matrix. Such information would have potential relevance to the pre-pathological states leading up to the formation of degenerative joint disease. To gain further insight into the mechanisms of mechanotransduction in chondrocytes, we have studied the role of MAPK pathways in response to mechanical compression of chondrocytes within their native cartilage. We have demonstrated that activation of the ERK1/2, p38, and JNK pathways is mechano-dependent and that these pathways are differentially activated with respect to each other and with respect to IGF-I. Materials—Articular cartilage was obtained from freshly harvested intact juvenile bovine femorotibial joints (Arena, Inc., Hopkinton, MA). The protease inhibitors leupeptin, pepstatin A, aprotinin, and phenylmethylsulfonyl fluoride were from Sigma. Phosphorylation state-specific ERK1/2, anti-p38 and anti-SEK1 polyclonal antibodies; phosphorylation state-independent anti-ERK1/2 and anti-p38 antibodies; and horseradish peroxidase-conjugated goat anti-rabbit antibody were from Cell Signaling Technology, Inc. (Beverly, MA). Protran™ nitrocellulose was from Schleicher & Schüll. The enhanced chemiluminescence detection substrate (ECL) was from PerkinElmer Life Sciences. All other chemicals were from Sigma and were the purest grade available. Cartilage Explant Preparation and Compression for Dose-response Studies—Disks of articular cartilage (3 × 1 mm, diameter × thickness) were harvested from the femoropatellar groove of newborn calves and were incubated in Dulbecco's modified Eagle's medium with 10 mm HEPES, 0.1 mm nonessential amino acids, 100 units/ml penicillin/streptomycin, an additional 0.4 mm proline, and 20 μg/ml ascorbate (basal medium) containing 10% fetal bovine serum (FBS) for 2 days. Disks were subjected to graded levels of unconfined uniaxial static mechanical compression in incubator-housed compression chambers containing fresh basal medium plus designated amounts of FBS. Compression was expressed as a percentage of the original cut thickness of the disks (1 mm). Loading conditions included no compression and 0 (held at 1 mm), 12, 25, 35, and 50% compression. In each experiment, 12 cartilage disks were simultaneously compressed within a loading chamber designated for each loading condition, unless noted otherwise. Following compression for the indicated time periods, the disks from within an individual compression chamber were pooled, rinsed with serum-free medium, blotted dry, and flash-frozen in liquid nitrogen. This termination procedure was repeated for each compression chamber. To evaluate the effect of loading in the presence of low serum concentrations (2%) and in the absence of serum, experiments were performed as described above with the exception that cartilage disks were incubated in basal medium containing 2% FBS for 2 days following harvest and then segregated into two groups of 48 disks each. One group was cultured for an additional 24 h in fresh basal medium containing 2% FBS. The other was cultured in fresh basal medium containing 0.01% bovine serum albumin. All cartilage disks were then subjected to graded compressive loads (no compression and 0, 12, 25, 35, and 50% compression; eight disks per load condition) for 4 h in basal medium. Following compression, disks from within an individual compression chamber were pooled, rinsed with basal medium, and flash-frozen in liquid nitrogen. This termination procedure was repeated for each compression chamber. Cartilage Explant Preparation and Compression for Time Course Studies—In short-term time course experiments, cartilage disks were harvested and incubated in basal medium containing 2% FBS as described above. Cartilage disks were maintained at no compression and at 0 or 50% of the original cut thickness for 10, 20, 40, or 60 min. In long-term time course experiments, cartilage disks were maintained at either 0 or 50% of the original cut thickness and compressed for 4, 8, 12, or 24 h (eight disks per time condition). Following compression, the disks from within each compression chamber were pooled, rinsed with serum-free medium, briefly blotted dry, and flash-frozen in liquid nitrogen. This termination procedure was repeated for each compression chamber. IGF-I Treatment of Cartilage Explants—Cartilage was harvested as described above with the exception that explants were cut into disks of 3 × 0.5 mm (diameter × thickness) to facilitate IGF-I diffusive penetration into the full thickness of the disks. After incubation for 2 days in basal medium containing 2% FBS, the medium was removed, and all disks were serum-starved in basal medium for 24 h. The cartilage disks were then incubated in basal medium in the presence or absence of 300 ng/ml human recombinant IGF-I (PeproTech, Inc., Rocky Hill, NJ) for graded time periods (10, 20, and 40 min and 4, 12, and 24 h) under no compression. At the completion of incubation, disks comprising each individual IGF-I/time combination were pooled, rinsed with serum-free medium, blotted dry, and flash-frozen in liquid nitrogen. Tissue Preparation and Immunoblotting—Cartilage disks were pulverized under liquid nitrogen using a Bessman tissue pulverizer (Fisher) and then homogenized using a Polytron device (Brinkmann Instruments) for 45 s in buffer (20 mm Tris (pH 7.6), 120 mm NaCl, 10 mm EDTA, 10% glycerol, 1% Nonidet P-40, 100 mm NaF, 10 mm Na4P2O7, 1 mm phenylmethylsulfonyl fluoride, 2 mm Na3VO4, 40 μg/ml leupeptin, 1 μm pepstatin A, and 10 μg/ml aprotinin) at a ratio of 100 μl/10 mg of tissue. Homogenates were extracted by end-over-end rotation for 1 h at 4 °C and clarified by centrifugation at 13,000 × g for 60 min. Supernatants were quantified for protein concentration using the BCA assay (Pierce). Aliquots containing 40 μg of protein suspended in Laemmli buffer were resolved by SDS-PAGE (10% resolving gel); transferred to Protran nitrocellulose membranes; and blocked with 5% bovine serum albumin in 10 mm Tris (pH 7.6), 150 mm NaCl, and 0.1% Tween 20 (Tris-buffered saline/Tween) for 2 h at 37 °C. Membranes were incubated with phosphorylation state-specific antibody (1:1000) or phosphorylation state-independent antibody (1:1000) overnight at 4 °C, washed with Tris-buffered saline/Tween (3 × 5 min), incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:2000) for 1 h at room temperature, and again washed with Tris-buffered saline/Tween (5 × 10 min). For the ECL reaction, immunoblots were developed for 1 min in Western Lightning Chemiluminescence Reagent Plus (PerkinElmer Life Sciences). The respective phosphoproteins or total proteins were visualized using enhanced chemiluminescence on x-ray film (Blue Sensitive autoradiographic film, Marsh Biomedical Products, Inc., Rochester, NY) and then quantified using a computing densitometer (Amersham Biosciences). Band intensities were determined by calculating the sum of all pixel optical density values within selected bands minus the background (volume integration). Band intensity profiles are denoted on individual figures by use of the arbitrary term "Volume" and are meant to identify total optical density units of band intensity minus the appropriate background values. Note that specific phosphorylation state-independent antibodies were utilized in conjunction with the corresponding phosphorylation state-specific antibodies on immunoblots to demonstrate equivalency of protein loading between lanes for each phosphoprotein analyzed. In each case, protein loading was found to be equivalent, or minor differences were not correlative with phosphorylation trends between lanes. Statistical Analysis—For strain dose dependence and time course experiments, the density of bands within given blots was normalized to that of the most common test condition between the various experiments of that type. Two-way ANOVAs of strain and time were then used to determine whether significant changes were present. In case of significance, two-tailed Student's t tests and one-way ANOVAs were performed to determine where the significance occurred. Dependent values created by normalization were removed. Statistical analyses were performed using the SYSTAT Version 9.0 statistical software package (SYSTAT Software Inc., Richmond, CA). Note that each figure depicts a representative immunoblot and its accompanying band intensity plot for each type of experiment performed. However, all available data from individual experiments were utilized in the generation of statistical analyses. Mechanical Compression Activates the ERK1/2 MAPK Pathway in Bovine Cartilage Explants—Cartilage explants were subjected to graded amounts of compression ranging from 0% (cut thickness) to 50%, and the activation state of the ERK1/2 signaling pathway was assessed by immunoblot analysis using phosphorylation state-specific anti-ERK1/2 antibodies. Both the ERK1 (p44) and ERK2 (p42) kinases were phosphorylated in a dose-dependent manner within the 0-50% compression range (Fig. 1). This dose-dependent response was observed in n = 10 separate experiments. The pattern and degree of load-induced ERK1/2 activation were similar under the two serum conditions except at the maximal compression tested (50%). These data suggest that mechanical compression activates the ERK1/2 pathway and that this activation does not depend on the presence of exogenous serum growth factors. The maximal magnitudes of the increases in ERK phosphorylation were 2.2- and 3.7-fold for ERK1 and ERK2, respectively, at 50% compression for 4 h in the absence of serum. The effect of compression was consistently greater for ERK2 than for ERK1. This difference was observed at the various time points and compression levels tested in all experiments. The total ERK1/2 protein levels were assessed as described under "Experimental Procedures" and were found to be equivalent among lanes (Fig. 1, gels, lower panels). Effect of Duration of Compression on Compression-induced ERK1/2 Phosphorylation—To determine whether the dose-response pattern of compression-induced ERK1/2 activation (Fig. 1) is dependent on the duration of the compressive stimulus, further dose-response experiments were carried out for shorter (10 min and 1 h) and longer (24 h) time periods. Dose-response analyses were performed using phosphorylation state-specific ERK1/2 immunoblot assays as described above. We found a nearly linear dose-response relationship between ERK1/2 phosphorylation and the magnitude of static compression after 10 min of compression (Fig. 2A). After a 1-h compression period, ERK1/2 phosphorylation showed a dose-response curve with a stronger plateau at the higher compressions (Fig. 2B). At 24 h, the dose-response pattern changed dramatically, with a significantly lower sensitivity to compression at the lower compressive strains (Fig. 2C). At all three time points, the maximal increase in ERK1 and ERK2 phosphorylation was observed at the highest magnitude of compression. Replicate experiments (n = 10) showed similar ERK1/2 phosphorylation patterns. Time Course of ERK1/2 Phosphorylation in Response to Mechanical Compression—Cartilage explants were subjected to no compression (NC) or 0 or 50% compression for 10, 20, 40, and 60 min and then subjected to phosphorylation state-specific ERK1/2 immunoblot analysis. In shorter time course experiments, the phospho-ERK1/2 levels in the 50% compressed samples were highest at the earliest time point tested (10 min) and then decreased over the next 50 min (Fig. 3A). Similar results were obtained in n = three experiments. In longer time course experiments (n = two experiments), cartilage explants were analyzed by phosphorylation state-specific ERK1/2 immunoblotting following compression times of 4, 8, 12, and 24 h (Fig. 3B). The phospho-ERK2 levels were highest at the earliest time point (4 h) and decreased with time in a bimodal fashion (Fig. 3B). In the 8-h interval between the first three time points of the experiment (4-12 h), the phospho-ERK2 levels decreased by 82% of the total phospho-ERK2 decrease over the entire time interval of the experiment (4-24 h). This period of rapid decay was followed by an ∼12-h period of slower loss of phospho-ERK2 levels. Interestingly, at 50% compression, even after 24 h, the phospho-ERK2 levels remained elevated in comparison with samples held at 0% compression. These data indicate that one effect of static compression on cartilage explants is to produce a sustained ERK2 activation. As in the earlier dose-response experiments (Figs. 1 and 2), ERK1 phosphorylation was increased to a lesser degree than ERK2 phosphorylation by prolonged static compression. Time Course of ERK1/2 Phosphorylation in Response to IGF-I—To compare mechanically induced ERK1/2 activation with activation by a known biochemical inducer of the ERK pathway, we investigated the time course of ERK1/2 phosphorylation in response to addition of IGF-I. Cartilage explants were harvested as described above and incubated in the absence or presence of 300 ng/ml IGF-I for the same time periods used for ERK1/2 activation in mechanically loaded explants. This concentration of IGF-I was chosen on the basis of previously reported dose-response studies demonstrating that, under the conditions of these experiments, 300 ng/ml is reproducibly on the upper plateau of the dose-response curve for IGF-I stimulation of [3H]proline and [35S]sulfate incorporation, indices of protein and glycosaminoglycan synthesis, respectively (22Bonassar L.J. Grodzinsky A.J. Srinivasan A. Davila S.G. Trippel S.B. Arch. Biochem. Biophys. 2000; 379: 57-63Crossref PubMed Scopus (97) Google Scholar, 35Bonassar L.J. Grodzinsky A.J. Frank E.H. Davila S.G. Bhaktav N.R. Trippel S.B. J. Orthop. Res. 2001; 19: 11-17Crossref PubMed Scopus (193) Google Scholar). IGF-I produced an initial rapid increase in ERK1/2 phosphorylation, followed by a rapid decrease to the phospho-ERK1/2 levels present in untreated explants (Fig. 3C). This pattern of transient ERK1/2 activation is in contrast to the sustained activation produced by compressive stress (Fig. 3B). Similar results were obtained in n = two experiments. Effect of Compression on p38 MAPK Phosphorylation—The p38 MAPK pathway is an alternative to the ERK1/2 MAPK pathway in the response mechanism used by many cell types to adapt to environmental changes. To determine whether the p38 pathway is involved in mechanical regulation of cartilage, explants were subjected to graded levels of compression from 0 to 50%, and tissue extracts were prepared as described under "Experimental Procedures." The activation state of p38 was assessed by immunoblot analysis using a phosphorylation state-specific anti-p38 antibody. After a 10-min loading period, compression produced a "dose-dependent" increase in phosphorylated p38, with a maximal stimulation at 50% compression, the highest magnitude of compression tested. Maximal stimulation was ∼5-fold compared with non-compressed samples. When analyzed 1 h after the onset of loading, the phosphorylated p38 MAPK levels remained close to base line, with maximal levels ∼4-fold lower than those observed at 10 min and with a marginal dose-response (Fig. 4). Similar trends were observed in n = five experiments. Time Course Responsiveness of p38 MAPK Phosphorylation—Cartilage explants were statically compressed to either 0 or 50% of the original cut thickness for graded time periods, and phospho-p38 was assessed by immunoblot analysis. At 0% compression, the phospho-p38 levels remained low over the entire duration of the short-term (10-60 min) (Fig. 5A) and long-term (20 min to 24 h) (Fig. 5B) experiments. The data of Figs. 4 and 5 (A and B) collectively indicate that, at 50% compression, maximal p38 phosphorylation occurred between 10 min and 4 h (Fig. 5, A and B), followed by a decline in phospho-p38 to levels close to those at 0% compression by 24 h (Fig. 5B). These findings suggest that mechanical compression activates the p38 MAPK pathway in a transient fashion. Effect of Compression on SEK1 Phosphorylation—JNKs, together with the p38 kinases, constitute the SAPK subfamily of MAPKs (36Tibbles L.A. Woodgett J.R. Cell Mo