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RECEPTORS AND SIGNAL TRANSDUCTION
Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland
Submitted 14 November 2007 ; accepted in final form 10 March 2008
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ABSTRACT |
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 TOP ABSTRACT EXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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or C/EBP
, binds to the C/EBP site, whereas c-Jun binds to AP-1. Individual gene knockdown experiments demonstrate the direct regulation of C/EBPβ expression by c-Jun, and the critical roles of both c-Jun and C/EBPβ in shear-induced COX-2 synthesis. Our studies also indicate that Rac and, to a lesser extent, Cdc42 transactivate MEKK1, which is, in turn, responsible for activation of mitogen-activated protein kinase kinase 7 (MKK7). MKK7 regulates c-Jun NH2-terminal kinase 2 activation, which, in turn, triggers the phosphorylation of c-Jun that controls shear-mediated COX-2 upregulation in chondrocytes. Reconstructing the signaling network regulating shear-induced COX-2 expression and inflammation may provide insights to optimize conditions for culturing artificial cartilage in bioreactors and for developing therapeutic interventions for arthritic disorders. mechanobiology; signal transduction; shear stress; cyclooxygenase; enhancer-binding protein; mitogen-activated protein kinase kinase; c-Jun NH2-terminal kinase
60% amino acid identity, with much higher conservation of active site residues (14), and a similar molecular mass of 70 kDa. Despite their similar active site structures, products, and kinetics, COX-1 and -2 isoforms display remarkable differences in their regulation and function. COX-1 is constitutively expressed in many cell types and is presumed to be responsible for the synthesis of housekeeping prostanoids that are critical for normal physiological functions such as the regulation of vascular homeostasis, protection of gastric mucosa, and maintenance of renal integrity (28). On the other hand, COX-2 is either absent or minimally expressed in most normal tissues. However, cox-2 mRNA and protein synthesis can be induced in numerous cell types, including chondrocytes, in a time- and dose-dependent manner by oncogenes, growth factors, and cytokines (15, 20, 29, 41, 55). It is thus presumed that COX-2 is primarily responsible for the elevated production of prostanoids at sites of disease and inflammation. Indeed, aberrant expression of COX-2 protein in articular tissues is an earmark of arthritis (2, 19, 42). Studies in animal models demonstrated that COX-2 expression was detected in inflamed, but not normal, paw tissue from rats with adjuvant-induced arthritis (3, 38, 42). Oral administration of a selective COX-2 inhibitor markedly suppressed COX-2 expression, PGE2 production, paw edema, and inflammatory cell infiltration in the joints (3). Similarly, an anti-PGE2 antibody prevented the development of tissue edema and hyperalgesia (38), thereby suggesting that the major COX-2-derived prostanoid that contributes to inflammation is PGE2 (28, 38). Superinduction of COX-2 activity accompanied by markedly increased levels of PGE2 release has been reported in human osteoarthritis-affected cartilage (2). Increased COX-2 expression has also been observed in human and an animal model of rheumatoid arthritis (19). Clinical studies have documented the efficacy of COX-2-specific inhibitors in osteoarthritis and rheumatoid arthritis (5). However, recent clinical findings also disclose that certain gastrointestinal, renal (10), and, most importantly, cardiovascular (32) side effects occur with COX-2-specific drugs. Thus, elucidation of the cox-2 transcriptional activation in human chondrocytes in response to either chemical or physical stimuli such as shear stress may aid in the identification of alternative therapeutic targets.
The promoter of the cox-2 gene contains a TATA box and several regulatory elements including nuclear factor-
B (NF-B) sites, a CCAAT/enhancer-binding protein (C/EBP) motif, Sp1 sites, a cyclic AMP response element (CRE) motif, activator protein-1 (AP-1), AP-2, and polyomavirus enhancer activator 3 (PEA-3) sites (43). Different regulatory elements have been demonstrated to regulate cox-2 transcription in various cell types: NF-B binding sites (18, 49), the C/EBP motif (8, 17, 35, 43, 49), activating transcription factor/CRE sequences (35, 46, 47), and PEA-3 site (47). However, data from cox-2 promoter analysis in chondrocytes are very limited. For instance, it has been reported that binding of C/EBP and C/EBPβ factors to their cognate sites on the cox-2 promoter is required for stimulation of its activity by IL-1β in rabbit chondrocytes (49). Moreover, NF-B binding is involved in the IL-1β-induced cox-2 transcriptional regulation (49). Circumstantial evidence that the CRE motif plays a role in regulation of COX-2 in okadaic acid-stimulated human chondrocytes has been provided by electrophoretic mobility shift assays (EMSA) (29). However, as has been appropriately argued in the literature, direct evidence for the involvement of particular cis-elements in the regulation of gene transcription can only be provided by experiments using reporter gene constructs containing native and mutated promoter sequences (45). Most importantly, the signaling pathways regulating COX-2 expression are species-, tissue-, cell-, and stimulus-specific.
In diarthrodial joints, articular cartilage is subjected to mechanical loads during normal daily activity. Elegant modeling studies have documented that chondrocytes of the superficial and transitional zones are exposed to high and low fluid flow, respectively (6, 7), suggesting that fluid flow is a pathophysiologically relevant mechanical signal in cartilage biology. Notably, in cartilage tissue engineering, chondrocytes/cartilage constructs are constantly exposed to fluid shear levels ranging from 1 to 23.2 dyn/cm2 (40, 53). These studies (40, 53) also disclose that hydrodynamic shear affects construct development beyond nutrient transport, presumably by altering intracellular signaling pathways in chondrocytes (40, 53).
In this regard, our studies have been directed at delineating in a rigorous manner the regulatory elements located in the 5'-flanking region of the cox-2 promoter region and their cognate trans-acting factors that contribute to the fluid shear-induced expression of COX-2 in human chondrocytic cells. Moreover, we systematically investigate the involvement of MAPK kinases and their upstream signaling molecules in the regulation of COX-2 synthesis using selective, individual gene knockdowns via dominant negative (DN) antisense oligonucleotide or RNA interference technology.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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mutant [pCMV-Flag-p38 (agf)] was obtained from Dr. Roger Davis (University of Massachusetts Medical School) (54). The 
kinase-Raf-1 was provided by Dr. Ulf Rapp (University of Wurzburg, Wurzburg, Germany) (31). The wild-type CRE-binding protein (CREB) and the nonphosphorylatable mutant CREB-M1 were donated by Dr. Marc Montminy (Salk Institute) (26). DN Rho, DN Rac, and DN Cdc42 constructs were kind gifts from Dr. Denis Wirtz (Johns Hopkins University) (25). The mitogen-activated protein kinase kinase 7 (MKK7) short hairpin RNA (shRNA) vectors pKD-MKK7-v2 and pKD-MKK7-v5 and the negative control vector pKD-NegCon-v1 were purchased from Upstate. Validated small interfering RNA (siRNA) targeting C/EBPβ, MEKK1, as well as the negative control scramble were from Qiagen. Cell culture and shear stress exposure. Human T/C-28a2 chondrocytic cells were grown (37°C in 5% CO2) in 1:1 Ham's F-12/DMEM (BioWhittaker) supplemented with 10% FBS (1, 12). Twenty-four hours before shear stress exposure, T/C-28a2 cells were incubated in serum-free medium containing 1% Nutridoma-SP (Roche) (1, 12), a low-protein serum replacement that maintains chondrocyte phenotype.
T/C-28a2 cells were exposed to shear stress in medium containing 1% Nutridoma by use of a parallel-plate flow chamber with a recirculating flow loop (37°C in 5% CO2) (1, 13). In select experiments, cells were incubated with pharmacological agents for 2 h before shear exposure.
Transient transfection.
T/C-28a2 cells were transfected with 13 µg of total plasmid using Lipofectamine 2000 (Invitrogen, 30 µl) for 6 h in serum-containing medium. For promoter analysis studies alone, cells were transfected with 12 µg of COX-2-pGL3 or COX-1-pGL3 reporter vector and 0.8 µg of phRL-TK– control vector (15:1 ratio). For cotransfection experiments (e.g., with DN mutants), cells were transfected with 6 µg of the promoter/reporter construct, 6 µg of the additional construct, and 0.4 µg of phRL-TK–. Following transfection, cells were incubated for 48 h in serum-free medium containing 1% Nutridoma and were subsequently exposed to the indicated treatments. Transfection efficiency was assessed by flow cytometry using pEGFP-N2 (BD Biosciences). Cotransfection efficiency, defined as the population of cells receiving copies of both plasmids, was determined by flow cytometry with the plasmid of enhanced green fluorescence protein (pEGFP-N2) and the plasmid of enhanced yellow fluorescence protein (pYFP). Approximately 70–75% of the transfected cells expressed significant amounts of both GFP and YFP. For the use of siRNA or antisense RNA, cotransfection efficiency was determined in an analogous manner using pEYFP and a FITC-labeled negative control siRNA (Qiagen).
Preparation of nuclear extract. Following fluid shear exposure, nuclear extracts were collected from slides using the NE-PER nuclear extraction kit (Pierce). Briefly, the slides were washed two times in ice-cold PBS, scraped, and incubated for 20 min in hypotonic buffer, and detergent was added to lyse the cellular membrane. The lysates were incubated for an additional 10 min on ice and vortexed for 15 s, and the nuclei were collected by centrifugation (2 min, 10,000 g, 4°C). The nuclei were lysed for 40 min on ice in a hypertonic buffer and were vortexed for 15 s every 10 min. The specimens were centrifuged (13,000 g, 10 min, 4°C), and the supernatants containing the nuclear extract were stored at –80°C. Protein concentrations were determined by the bicinchoninic acid reagent (BCA, Pierce).
Promoter activity assays. Firefly and Renilla luciferase activities were measured by use of the Dual-Luciferase Report Assay kit (Promega). Firefly luciferase activities were normalized to the Renilla luciferase controls. Data are expressed as ratios of shear to static normalized firefly luciferase activity unless otherwise stated.
EMSA and supershift assay.
As shown in Figs. 1 and 2, three 5'-biotinylated oligonucleotide probes were synthesized containing the C/EBP, PEA-3/nuclear factor of activated T cells (NFATc)/AP-1, and CRE cis-elements present in the –159/–48 region of the cox-2 promoter. The PEA-3/NFATc/AP-1 and the CRE probes were partially mutated to destroy overlapping response elements, because these two sites are located in close proximity (Figs. 1 and 2). EMSAs were carried out with a commercially available nonradioisotopic EMSA kit (LightShift Chemiluminescence EMSA kit; Pierce). Briefly, nuclear extracts (4 µg) were incubated in binding buffer [10 mM Tris, 50 mM KCl, 1 mM DTT supplemented with 1 µg poly(dI-dC), 2.5% glycerol, 0.05% NP-40, and 5 mM MgCl2] containing 12 fmol biotinylated, double-stranded probes for C/EBP, PEA-3/NFATc/AP-1, and CRE for 30 min at 4°C. For competition binding, a 200-fold excess of unlabeled (cold) probe was incubated with nuclear extracts before the inclusion of the biotinylated one. For supershift assays, the nuclear extracts were preincubated for 30 min at 4°C with anti-CEBP, anti-C/EBPβ, anti-C/EBP (Active Motif), anti-PEA-3 (Santa Cruz), anti-NFATc, anti-CREB-1, anti-phospho-CREB-1, anti-CREB-2 (Santa Cruz), anti-c-Jun, or anti-c-Fos antibodies (Active Motif); the biotinylated oligonucleotide probe specific for C/EBP, PEA-3/NFATc/AP-1, or CRE was then added and incubated for another 30 min at 4°C. The protein-DNA complexes were resolved on a native 6% DNA retardation gel (Invitrogen) in 0.5x Tris-borate-EDTA running buffer (Invitrogen), transferred to a nylon membrane (Pierce), and visualized using the LightShift Chemiluminescence kit.
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Quantitative real-time PCR. PCR primers were designed using the Primer3 website (Whitehead Institute, Massachusetts Institute of Technology) (39), and their specificity was examined by National Center for Biotechnology Information Basic Local Alignment Search Tool of the human genome. In the case of multiple splice variants, the conserved regions of the gene of interest were used for primer design. Amplicon specificity was verified by first-derivative melting curve analysis using software provided by Perkin-Elmer/Applied Biosystems (27). The GenBank accession numbers and forward (F-) and reverse (R-) primers are as follows: c-jun (W91655), F-GCAGCCCAAACTAACCTCAC and R-TAGCCATAAGGTCCGCTCTC; jnk2 (AA157286), F-CTGGCCTCAGACACAGACAG and R- CCATCAACTCCCAAGCATTT; cox-2 (AA644211), F-TGAGCATCTACGGTTTGCTG and R-AACTGCTCATCACCCCATTC; cox-1 (AA454668), F-CTTTCCCTCAAGGGTCTCC, and R-AGGGACAGGTCTTGGTGTTG; β-actin (XM_004814), F-ATCGGCGGCTCCATCC and R-GGGGCACGAAGGCTCATC; and gapdh (AA777488), F-GGCCTCCAAGGAGTAAGAC and R-AGGGGTCTACATGGCAACT.
Single-stranded cDNAs were generated from reverse transcription of RNA samples using the TaqMan RT kit (Applied Biosystems), diluted, and then subjected to PCR with SYBR Green (Applied Biosystems) as the detected fluoroprobe. Incorporation of the SYBR Green dye into the PCR products was monitored in real time with the ABI Prism 7900HT sequence detection system. The carboxy-X-rhodamine passive reference dye was used to factor in well and pipetting variability. GAPDH and β-actin were used as "housekeeping" genes. Standard curves were determined for each specimen using the 18S ribosomal RNA (18S rRNA kit; Ambion, Austin, TX) and were used to normalize and quantify mRNA levels via 18S rRNA amplification.
Antisense oligonucleotides. Control and antisense oligonucleotides against c-jun, c-fos, and JNK2 were generously provided by ISIS Pharmaceuticals (1, 4). For analysis of mRNA transcript levels, T/C-28a2 cells were transfected with 400 nM of antisense oligonucleotides using Lipofectamine 2000 (30 µl) for 6 h in serum-containing media. For studies in which antisense oligonucleotides were cotransfected with reporter plasmids, 400 nM of oligonucleotide and 6 µg of reporter vector, and 0.4 µg of control vector were used. The antisense oligonucleotide targeting JNK2 and a mismatch control oligonucleotide were synthesized as uniform phosphorothioate, chimeric oligonucleotides with 2'-O-methoxyethyl-modified sugars on nucleotides 1–5 and 16–20 and 2'-deoxy sugars on nucleotides 6–15. The antisense oligonucleotides targeting c-jun or c-fos alone and a chemistry control oligonucleotide were synthesized as uniform phosphorothioate oligonucleotides and 2'-deoxy sugars on all nucleotides. The oligonucleotides were synthesized using an Applied Biosystems 380B automated DNA synthesizer (Applied Biosystems) and were purified as described previously (4). The sequences of the oligonucleotides used in these studies were previously reported (1).
Intracellular protein staining. T/C-28a2 cells were fixed with 1.0% formaldehyde for 10 min at 37°C, permeabilized in 90% methanol for 30 min on ice, and incubated at room temperature (RT) for 10 min in blocking buffer (0.5% BSA in Dulbecco's PBS). Cell specimens were then incubated with monoclonal antibodies specific for JNK1/2 or phospho-JNK1/2, ERK1/2 or phospho-ERK1/2, p38 or phospho-p38, or appropriate isotype controls for 30 min at RT. Next, cells were washed two times in blocking buffer, incubated with FITC-goat anti-rabbit IgG, and analyzed by flow cytometry.
Western hybridization. Total protein liberated using a cell lysis buffer (20 mM Tris·HCl, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, and protease inhibitor cocktail, pH 7.5) or nuclear extracts from sheared and matched static control specimens were separated by 10–14% SDS-PAGE and electrotransferred on polyvinylidene difluoride membrane (Millipore). The membrane was blocked for 1 h in Starting Block solution (Pierce) at RT, incubated with a primary antibody against MEKK1, MKK7, phospho-MKK7, phospho-JNK2, or C/EBPβ for 1 h at RT, washed four times in Tris-buffered saline-Tween 20, and incubated for 1 h with the appropriate horseradish peroxidase secondary antibody (1:5,000; Sigma) at RT. The membrane was washed four times in Tris-buffered saline-Tween 20, and reactive bands were detected using a Super Signal chemiluminescence substrate kit (Pierce). To ensure equal loading of samples in each lane, membranes were stripped and reprobed with a β-actin (total protein) or lamin (nuclear protein) antibody (24, 51).
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RESULTS |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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18 to 6 upon chondrocytic cell transfection with a plasmid deleted from –891 to –568 bp (–568/+9), but it remained nearly constant (6) upon further serial deletions up to –193 bp (Fig. 4A). However, subsequent deletion from –193 to –120 bp (use of construct: –120/+9) markedly suppressed the luciferase activity, which was completely abolished when the region from –96 to –53 bp was also deleted (Fig. 4A). Cumulatively, these data suggest that two regions (–193 to –120 bp and –96 to –53 bp) contain possible shear stress-response elements. The potential involvement of region –891 to –568 bp in the regulation of cox-2 promoter activity in response to shear is highly unlikely, since deletion of region –182 to –49 bp from the (–891/+9) construct was sufficient to abrogate the shear-induced luciferase activity (Fig. 4A). Of note, the absolute value of luciferase activity of sheared chondrocytes remains nearly constant (8) upon transfection with constructs (–891/+9, –568/+9, –459/+9, –362/+9, and –193/+9) (Fig. 4A). Rather, the absolute value of firefly luciferase activity of chondrocytes under static conditions increases upon deletion of the –891 to –568 bp region, suggesting that a repressor element may bind this region of the cox-2 promoter under static conditions.
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C/EBPβ and c-Jun are involved in the regulation of COX-2 induction in shear-activated human chondrocytic cells.
Having identified the putative cis-elements on the cox-2 gene promoter responsible for COX-2 induction in shear-activated chondrocytes, our next set of experiments aimed to determine their cognate transcription factors. To this end, EMSAs were carried out using nuclear extracts from static (0 dyn/cm2) and sheared (20 dyn/cm2; 3 h) chondrocytic cell specimens along with three biotinylated double-stranded oligonucleotide probes containing motifs of C/EBP/NF-IL-6, PEA-3/NFATc/AP-1, and CRE on the basis of their consensus sequence in the human cox-2 gene promoter (Fig. 2). When the C/EBP/NF-IL-6 probe was incubated with nuclear extracts from sheared, but not static, T/C-28a2 chondrocytes, a band indicating their complex was readily detected in the gel (Fig. 5A). The specificity of the shear-induced binding was demonstrated by the addition of a 200-fold excess of nonbiotinylated (cold) C/EBP/NF-IL-6 probe before the inclusion of the biotinylated probe, which prevented the detection the complex formation (Fig. 5A). When the C/EBP/NF-IL-6 probe and sheared nuclear extracts were incubated with an anti-C/EBPβ antibody, a marked supershift of the complex was denoted in the gel (Fig. 5A). In contrast, no supershift was detected with an anti-C/EBP or an anti-C/EBP antibody (Fig. 5A), suggesting that shear stress induced pronounced and selective C/EBPβ binding to the C/EBP/NF-IL-6 binding site. Likewise, fluid shear stress induced the formation of the complex with the nuclear extracts and PEA-3/NFATc/AP-1 probe, which was supershifted after incubation with an anti-c-Jun or anti-c-Fos antibody (Fig. 5B). No supershift was noted with an anti-PEA-3 or anti-NFATc antibody (Fig. 5B).
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To delineate the potential functional contribution of CREB-1 in the regulation of shear-induced COX-2 synthesis in human chondrocytes, a DN CREB-1 mutant was transfected to T/C-28a2 cells before their exposure to either static (0 dyn/cm2) or shear (20 dyn/cm2, 3 h) conditions. As shown in Fig. 6, this genetic intervention did not significantly impair the ratio of shear to static luciferase activity relative to the appropriate control, suggesting the lack of CREB-1 involvement in shear-mediated COX-2 induction.
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60% (Fig. 8A). Taken together, our data suggest that MEKK1 phosphorylates MKK7, which in turn activates JNK2, which is responsible for the phosphorylation of c-Jun and induction of COX-2 synthesis in shear-activated human chondrocytes. Activation of the JNK pathway has been reported to involve the small GTP-binding proteins of the Rho family, including RhoA, B, C, Rac1, 2 and Cdc42 in a cell type- and stimulus-dependent manner, which may in turn get activated by either the Ras protooncogene or the phosphatidylinositol 3-kinase (PI3K) pathway (9). Interestingly, previous work has revealed a role for Rho and Raf-1 in the induction of COX-2 synthesis in human mammary epithelial cells treated with either microtubule-interfering agents (46) or transformed with HER-2/neu (erbB-2) (47). In marked contrast with these data (46, 47), the stimulation of cox-2 promoter activity evoked by high fluid shear was not altered by overexpressing DN forms of Rho or Raf (Fig. 10A). To further establish the lack of Rho involvement in this signaling pathway, quantitative real-time PCR experiments were carried out to monitor the effect of the DN Rho mutant on the JNK2, c-jun, and cox-2 mRNA synthesis induced by shear. Our data reveal that this genetic intervention did not interfere with the shear-induced upregulation of JNK2, c-jun, and cox-2 expression (Fig. 10B). On the other hand, use of a DN mutant specific for Rac drastically inhibited shear-induced cox-2 promoter activity (Fig. 10A). Interestingly, knocking down Cdc42 expression also reduced, albeit to a lesser extent, cox-2 promoter induction (Fig. 10A). These genetic interventions were also effective, albeit to different extents, in repressing shear-induced JNK2, c-jun and cox-2 mRNA synthesis (Fig. 10B), thereby providing further evidence for their involvement in the regulation of shear-induced COX-2 expression. Taken altogether, our data provide direct evidence for the critical involvement of Rac and Cdc42 in the regulation of JNK2/c-Jun-dependent COX-2 induction in sheared activated chondrocytes (Fig. 11). Moreover, MEKK1 and MKK7 appear to lie downstream of Rac and Cdc42 and directly upstream of the JNK2/c-Jun pathway (Fig. 11).
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DISCUSSION |
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TOPABSTRACTEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONGRANTSREFERENCES |
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Our studies using a series of human cox-2 promoter-deletion constructs as well as single mutation analysis of the cox-2 promoter reveal that the C/EBP and AP-1 motifs, but neither the proximal nor the distal NF-B sites, are critically involved in the COX-2 induction in human chondrocytic cells in response to high fluid shear, whereas a minor role for CRE cannot be ruled out. These data are in clear contrast to previously published reports showing that NF-B binding to the proximal NF-B site (–222/–213) is required for the maximal stimulation of cox-2 gene transcription in IL-1β-treated rabbit chondrocytes (16, 49), and provide further support to the concept that discrete signaling pathways mediate the genesis of COX-2 synthesis by distinct activating stimuli.
On the other hand, significant similarities and disparities exist in the regulation of shear-induced COX-2 induction in MC3T3-E1 murine osteoblastic (35) and human chondrocytic cells. Most importantly, the C/EBP and AP-1 binding sites contribute to the shear stress-induced cox-2 promoter activity in both cell types (35). Although fluid shear selectively enhanced the binding of phospho-CREB to the CRE motif of the cox-2 promoter in murine osteoblastic cells (35), no such interaction was detected in shear-activated chondrocytic cells. Along these lines, introduction of a mutation into the CRE (–59/–53) site did not cause any significant reduction in the cox-2 promoter activity (i.e., ratio of shear to static luciferase activity) relative to the wild-type control in chondrocytic cells, although it had a clear inhibitory effect on murine osteoblastic cells (35). In light of previous reports showing increased binding of c-Jun to the CRE site of the human cox-2 promoter in HER-2/neu-transformed mammary epithelial cells (47) and Rous sarcoma virus-transformed fibroblasts, we examined whether fluid shear induces c-Jun binding to CRE in chondrocytic cells. However, our EMSA experiments did not disclose such an interaction. It is also noteworthy that although the kinetics of COX-2 induction in murine osteoblastic and human chondrocytic cells is similar, the former respond to fluid shear even at a wall shear stress level of 0.18 dyn/cm2 (35). COX-2 is also rapidly induced in human umbilical vein endothelial cells subjected to a stress level of 1 dyn/cm2 (17), whereas COX-2 induction is detected in human chondrocytic cells only above the threshold stress level of 5 dyn/cm2. The enhanced mechanosensitivity of endothelial cells compared with chondrocytes may be related in part to the physiological shear environment (1–4 dyn/cm2) that these cells encounter in vivo. Moreover, the role of COX-2 and its products (e.g., prostaglandins) may be distinct in different tissues. For instance, prostaglandins have anabolic effects on proliferation and differentiation of bone-forming cells, and selective inhibition of COX-2 activity has been reported to inhibit mechanical loading-induced bone formation in rats (11). In contrast, overexpression COX-2 in articular tissues is an earmark of arthritis (2, 19, 42) associated with increased numbers of apoptotic chondrocytes (13, 33, 34, 37). Thus, expression of COX-2 in response to low levels of fluid shear would offer an unfavorable phenotype for chondrocytic cells. Hence, our data demonstrating the requirement of an elevated shear stress threshold for the induction of COX-2 expression suggest that abnormally high mechanical loading is necessary to potentially elicit COX-2-mediated inflammation and cartilage degradation within articular joints.
Numerous studies in diverse cell types (46, 47, 55) including chondrocytic cells (21, 22, 34, 49, 50) have shown that the expression of COX-2 can be altered by changes in MAPK activity. To keep this discussion focused, only pathways that mediate COX-2 induction in chondrocytes will be outlined here. Evidence for the involvement of p38 in COX-2 expression in chondrocytes stimulated with either IL-1β or a nitric oxide (NO) donor was provided by experiments using a DN p38 kinase (50), overexpression of p38 active kinase (50), or specific pharmacological inhibitors (22, 34, 49, 50). The dependence of COX-2 expression in NO-treated chondrocytes on ERK1/2 signaling was documented through the use of ERK1/2 specific inhibitors (22, 34). It is noteworthy that this pharmacological intervention partially suppressed COX-2 expression (22) but abolished PGE2 production (22, 34), probably by blocking COX-2 activity. Although application of high fluid shear induces the phosphorylation of p38 and ERK1/2, neither of these kinases mediates the genesis of COX-2 synthesis in shear-activated human chondrocytic cells. In fact, we previously demonstrated and confirmed herein the pivotal role of a JNK2-dependent pathway in the regulation of COX-2 expression in chondrocytes subjected to high shear by using an antisense JNK2-specific oligonucleotide (1, 13). Whereas shear-induced COX-2 synthesis proceeds via an NF-B-independent mechanism, NF-B has been implicated in the regulation of COX-2 in NO donor-treated rabbit chondrocytes (21). NO-induced NF-B activation is negatively regulated by PKC and PKC
(21), the latter of which is in turn regulated by p38 kinase (23).
Activation of the JNK pathway has been reported to involve the small GTP-binding proteins of the Rho family, including RhoA, B, C, Rac1, 2 and Cdc42 in a cell-type- and stimulus- dependent manner, which may in turn get activated by either the Ras protooncogene or the PI3K pathway (9). Although a role for Rho has been suggested in the regulation of COX-2 expression in sheared murine osteoblastic cells (36), and in mammary epithelial cells treated with microtubule-interfering agents (46), use of a DN mutant specific for Rho failed to impair the shear-induced upregulation of JNK2, c-Jun, and COX-2. Similarly, although Raf regulates COX-2 expression in HER-2/neu-positive breast cancer cells, it does not appear to contribute to shear-induced COX-2 synthesis. On the other hand, serial, selective gene knockdown experiments via the use of DN mutants or RNA interference technology illustrate that Rac and, to a lesser extent, Cdc42 regulate MEKK1, which is responsible for the transactivation of MKK7 that lies directly upstream of JNK2, which in turn phosphorylates c-Jun. c-Jun in coordination with C/EBPβ, which is directly regulated by c-Jun, mediates the genesis of COX-2 synthesis.
Taken altogether, our data suggest that the synthesis of COX-2 expression in chondrocytes subjected to high shear stress is regulated by a Rac/MEKK1/MKK7/JNK2/c-Jun-C/EBPβ-dependent pathway, which is not shared by cytokine- or NO donor-induced stimulation. Reconstructing the biochemical pathway regulating cartilage inflammation in response to high shear may identify potential therapeutic targets for controlling arthritic pathogenesis and/or progression and may be useful in the design of bioreactors for cartilage culture.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* Z. R. Healy and F. Zhu contributed equally to this work. 
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