Postsurgery, discarded human OA articular cartilage was obtained from OA patients (mean ± standard deviation age, 67 ± 9 years) who underwent total knee arthroplasty. Informed consent had been obtained from patients with OA for the use of their tissues for research purposes. All patients were evaluated by rheumatologists who followed American College of Rheumatology criteria 22. The Clinical Research Ethics Committee of the Hôpital du Sacré-Cœur de Montréal, including clinicians, researchers and jurists, approved the study protocol and the use of human articular tissues.

OA cartilage (femoral condyles and tibial plateaus) was obtained under aseptic conditions and carefully dissected from the underlying bone in each specimen 23. OA chondrocytes were extracted by sequential enzymatic digestion with 1 mg/ml pronase (Sigma, Oakville, ON, Canada) for 1 hour at 37°C, and then with 2 mg/ml type IV collagenase (Sigma) for 6 hours in DMEM (Invitrogen, Burlington, ON, Canada) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen). The cells were seeded at high density in culture flasks at 37°C in a humidified atmosphere of 5% CO₂/95% air until they were confluent and ready for the experiments. First-passage cells were employed to ensure their phenotype and were seeded at 10⁵ cells/cm² in culture tissue plates. The DMEM containing 2% fetal bovine serum and antibiotics was replaced 24 hours before the experiments were performed in this medium with the factors under study for different incubation time periods.

For COX-2 and mPGES-1 studies (up to 24 hours), chondrocytes were treated with a single addition of 10 μM HNE or with repeated treatments by adding 10 μM HNE to the cultures at 0, 2, 4, 6, 8, 10, 12 and 14 hours. For 5-LOX and FLAP studies (up to 72 hours), cells were treated with a single addition of 10 μM HNE in the presence or absence of 50 μM naproxen or 100 μg/ml anti-transforming growth factor-beta 1 (TGFβ1).

Twenty micrograms of total proteins from chondrocyte lysates treated with HNE under the indicated conditions were loaded for discontinuous 4 to 12% SDS-PAGE. Protein transfer, immunodetection and semiquantitative measurements were performed as described previously 18. The primary antibodies were rabbit anti-COX-2 (Cayman Chemical, Hornby, ON, Canada), anti-mPGES-1 (Cayman Chemical), anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-Egr-1 (Santa Cruz Biotechnology). After serial washes, primary antibodies were detected by goat anti-rabbit IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). Immunoreactive proteins were detected with SuperSignal blotting substrate (Pierce Biotechnology, Inc., Rockford, IL, USA) and were exposed to clear-blue X-ray film (Pierce).

Total RNA was isolated with TRIzol reagent according to the manufacturer's instructions. RNA was quantitated with the RiboGreen RNA quantitation kit (Molecular Probes, Eugene, OR, USA), dissolved in diethylpyrocarbonate-treated H₂O, and stored at -80°C until use. One microgram of total RNA was reverse-transcribed with Moloney murine leukemia virus reverse transcriptase (Fermentas, Burlington, ON, Canada), as detailed in the manufacturer's guidelines. One-fiftieth of the reverse transcriptase reaction product was analyzed by traditional PCR or by real-time quantitative PCR. The following sense and antisense specific primers (Bio-Corp, Inc., Montreal, QC, Canada), were tested: human mPGES-1, 5'-GAA GAA GGC CTT TGC CAA C-3' (sense) and 5'-GGA AGA CCA GGA AGT GCA TC-3' (antisense); human 5-LOX, 5'-CTG TTC CTG GGC ATG TAC CC-3' (sense) and 5'-GAC ATC TAT CAG TGG TCG TG-3' (antisense); human FLAP, 5'-AAT GGG AGG AGC TTC CAG AG-3' (sense) and 5'-ACC AAC CCC ATA TTC AGC AG-3' (antisense); and human GAPDH, 5'-CAG AAC ATC ATC CCT GCC TCT-3' (sense) and 5'-GCT TGA CAA AGT GGT CGT TGA G-3' (antisense).

Quantitative PCR analysis was performed in a total volume of 50 μl containing template DNA, 200 nM sense and antisense primers, 25 μl SYBR Green Master Mix (Qiagen, Mississauga, ON, Canada), and 0.5 units uracil-N-glycosylase (Epicentre Technologies, Madison, WI, USA). After incubation at 50°C for 2 minutes (uracil-N-glycosylase reaction) and at 95°C for 10 minutes (uracil-N-glycosylase inactivation and activation of AmpliTaq Gold enzyme), the mixtures were subjected to 40 amplification cycles (15 seconds at 95°C for denaturation and 1 minute for annealing and extension at 60°C). Incorporation of SYBR Green dye into the PCR products was monitored in real time with a Mx3000 real-time PCR system (Stratagene, La Jolla, CA, USA), to determine the threshold cycle (C_t) at which exponential amplification of PCR products begins. After PCR, dissociation curves were generated with one peak, indicating amplification specificity. A C_t value was obtained from each amplification curve with the software provided by the manufacturer (Stratagene).

Relative mRNA expression in chondrocytes was quantified according to the ΔΔC_t method, as detailed in the manufacturer's guidelines (Stratagene). A ΔC_t value was first calculated by subtracting the C_t value for the housekeeping gene GAPDH from the C_t value for each sample. A ΔΔC_t value was then calculated by subtracting the ΔC_t value for the controls (unstimulated cells) from the ΔC_t value for each treatment. Fold changes compared with the controls were then determined by 2^-ΔΔC_t. Each PCR generated only the expected specific amplicon, as shown by melting temperature profiles of the final product and gel electrophoresis of the test PCRs. Each PCR was performed in triplicate on two separate occasions for each independent experiment.

After incubation, the culture medium from cultured OA chondrocytes was collected and the PGE₂, LTB₄ and TGF-β1 levels were measured with specific commercial kits from Cayman Chemical and R&D Systems (Minneapolis, MN, USA), according to the manufacturer's instructions. Detection sensitivity was 9, 13.7 and 4.6 pg/ml, respectively. All assays were performed in duplicate.

The human mPGES-1 promoter construct (-538/-28) was a gift from Dr Terry J. Smith (University of California, Los Angeles, CA, USA) 24. The pEgr-1Mutx3-TK-Luc reporter construct was generously provided by Dr Yuqing E. Chen (Morehouse School of Medicine, Atlanta, GA, USA) 25. The 5-LOX promoter construct was kindly donated by Dr Dieter Steinhilber (University of Frankfurt, Frankfurt, Germany).

Subconfluent human OA chondrocytes were transiently transfected in 12-well cluster plates with lipofectamine 2000™ reagents (Invitrogen Life Technology, Inc.), according to the manufacturer's protocol. Briefly, transfections were conducted for 6 hours with DNA lipofectamine complexes containing 10 μl lipofectamine reagent, 2 μg DNA plasmid and 0.5 μg pCMV-β-galactosidase (as a transfection efficiency control). In cotransfection experiments the amount of transfected DNA was kept constant using a corresponding empty vector. Using the method of Howcroft and colleagues 26, the transfection efficiency was measured and the average rate of transfected cells was 48%. After washing, the cells were incubated for different incubation periods in the presence or absence of 10 μM HNE (single addition) in fresh DMEM culture medium containing 2% fetal bovine serum in a humidified atmosphere of 95% air/5% CO₂ at 37°C. Luciferase activity was determined in cell lysate with commercially available kits (Luciferase assay system; Promega Corporation, Madison, WI, USA). The data were normalized to the β-galactosidase level, which was quantified in cell lysate by a specific ELISA (Roche Applied Science, Laval, QC, Canada).

All quantitative results were calculated as the mean ± standard error of the mean. The data were assessed by Student's unpaired t test. P < 0.05 was considered statistically significant.

As described in our previous report 20, COX-2 protein and mRNA expression decreased gradually after 8 hours of chondrocyte incubation with single addition of 10 μM HNE. To examine whether this reduction is attributed to HNE depletion, cells were stimulated with either single or repeated 10 μM HNE treatments for different incubation periods (0 to 24 hours, n = 4 independent experiments) as described in Materials and methods.

Single treatment of chondrocytes with 10 μM HNE showed an increase in COX-2 protein expression and PGE₂ release within 2 hours and plateaued at 8 hours of incubation, before declining at 16 hours and 24 hours (Figure 1a, b). PGE₂ and COX-2 reached maximum levels of 157.5 ± 33 ng/10⁵ cells and 340% of control values, respectively (P < 0.001). In experiments where the cells were stimulated with repetitive addition of 10 μM HNE, however, PGE₂ release and COX-2 protein expression were enhanced after 4 hours of incubation, with maximum values obtained at 24 hours (Figure 1c, d). Maximum PGE₂ and COX-2 values were 210 ± 29 ng/10⁵ cells and 400% of control values, respectively (P < 0.001). Single or repeated treatments with 10 μM HNE did not induce either toxic or apoptotic phenomena in chondrocytes (data not included).

These data suggest for the first time that repeated treatments at 2-hour intervals with 10 μM HNE are essential to maintain the protein expression of COX-2 up to 24 hours.

To gain insights into the role of HNE in the mechanism underlying the production of PGE₂ we evaluated its effect on the protein and mRNA expression of mPGES-1, an inducible form of PGES involved in the terminal step of PGE₂ synthesis. Human OA chondrocytes were treated once or several times for different incubation periods as described in Materials and methods (n = 4 independent experiments).

Treatment of chondrocytes with single addition of 10 μM HNE to cultures significantly increased mPGES-1 protein and mRNA levels, which plateaued at 16 hours but then declined at 24 hours (Figure 2a, c). mPGES-1 protein and mRNA reached maximum levels of 230 and 220% of control values, respectively (P < 0.01). Repeated treatments at 2-hour intervals with 10 μM HNE, however, enhanced mPGES-1 protein and mRNA levels and plateaued at 24 hours (Figure 2b, c). The maximum levels of mPGES-1 protein and mRNA were 400 and 300% of the controls, respectively (P < 0.001).

Similar to COX-2, our data indicate that the maintained mPGES-1 protein and mRNA expression might be linked to repetitive treatments with HNE.

In our previous report, we reported that HNE induced COX-2 transcription through ATF-2/CREB-1 transactivation 20. In the present study, we further examined whether HNE induced mPGES-1 at a transcriptional level through Egr-1 transactivation.

Firstly, to determine whether changes in mPGES-1 mRNA levels can be ascribed to the regulation in promoter activity, chondrocytes were transiently transfected with human mPGES-1 promoter-luciferase reporter genes (n = 6 independent experiments) and then treated with single addition of 10 μM HNE to all wells except the controls. As shown in Figure 3a, HNE induced mPGES-1 promoter activity in a time-dependent increment. Maximum promoter activity was 331% at 16 hours compared with the control (P < 0.001).

Secondly, we analyzed the effect of HNE on the transcriptional activation of a synthetic luciferase reporter construct containing three tandem repeats of the putative Egr-1 binding sequence, pEgr-1x3-TK-Luc. The transcription factor Egr-1 is believed to mediate the induction of a host of target genes such as mPGES-1, as suggested by Cheng and colleagues 8. As seen in Figure 3b, HNE increased the luciferase activity of the above construct, and this activation was time dependent. Maximum promoter activity reached 450% at 16 hours compared with the controls (P < 0.001). Western blot analysis revealed that single treatment with 10 μM HNE induced Egr-1 protein expression by 280% compared with untreated cells after 16 hours of incubation (Figure 3c).

Collectively, these data suggest that Egr-1 may be one of the transcription factors involved in HNE-induced mPGES-1 promoter activity.

In the next series of experiments, we further investigated whether the decrease of COX-2 expression in treated chondrocytes with single addition of 10 μM HNE to cultures lead to a switch to 5-LOX and FLAP mRNA expression, and consequently to LTB₄ production. LTB₄ is a potent chemoattractant and stimulator of inflammation 27 and probably contributes, along with other factors, to the upregulation of the synthesis of other catabolic factors involved in the pathophysiology of OA 28. Human OA chondrocytes (n = 5 independent experiments) were treated for different incubation periods with single addition of 10 μM HNE to all wells except the controls. HNE-induced changes in the LTB₄ level and in 5-LOX and FLAP mRNA expression and were evaluated respectively in picograms per milliliter or as a percentage of control. LTB₄ release (Figure 4a) was induced only after a long incubation period. The maximum level rose significantly after 72 hours of treatment (66.6 ± 3.7 pg/10⁵ cells, P < 0.001).

5-LOX and FLAP are essential proteins responsible for LTB₄ synthesis. To determine whether augmented LTB₄ production induced by HNE is related to 5-LOX and FLAP synthesis, we further examined the effect of HNE on their mRNA expression. As shown in Figure 4b, c, our data disclosed that both FLAP and 5-LOX mRNA expression rose significantly but differentially after incubation in human OA chondrocytes. FLAP mRNA expression occurred earlier, after 8 hours of incubation, compared with 5-LOX (48 hours). Maximum FLAP and 5-LOX levels were 630% and 560% of control values, respectively (P < 0.001).

Taken together, these results indicate that single treatment with HNE in OA chondrocytes induced the shunt from the production of PGE₂ to LTB₄ through COX-2 downregulation and 5-LOX and FLAP upregulation.

To more accurately identify whether HNE induced 5-LOX at the transcription level, the 5-LOX promoter construct with luciferase gene reporter was transiently transfected into human OA chondrocytes, followed by stimulation with single addition of 10 μM HNE to all wells excepted control for different time periods (n = 4 independent experiments). Compared with unstimulated cells, HNE induced 5-LOX gene promoter activity in transfected chondrocytes. 5-LOX promoter activity increased significantly by 310% and 580% after 48 and 72 hours of incubation, respectively (P < 0.05) (Figure 5).

These data indicate that HNE regulates 5-LOX mRNA expression at the transcriptional level.

Further experiments were aimed at elucidating the role of PGE₂ and TGF-β1 production in HNE-induced 5-LOX and FLAP expression in OA chondrocytes. Firstly, cells were treated with 50 μM naproxen (a nonselective COX inhibitor) for 1 hour followed by another treatment for 24 or 72 hours with single addition of 10 μM HNE to all wells except the controls (n = 4 independent experiments). HNE-induced changes in 5-LOX and FLAP mRNA expression were evaluated as a percentage of control. Our real-time RT-PCR results demonstrated that HNE induced FLAP expression after 24 hours of treatment (210% of control, P < 0.001) and the level was even higher after 72 hours (315% of control, P < 0.001) (Figure 6a, b). FLAP activation by HNE was abolished by naproxen, which prevents PGE₂ production. In contrast, HNE induced 5-LOX expression by 600% above control values (P < 0.001) (Figure 6d) only after 72 hours of incubation. Incubation of cells with naproxen, however, caused a consistent increase of 5-LOX mRNA level by 330% and 570% of control values (P < 0.001) after 24 and 72 hours of incubation, respectively (Figure 6c, d). The addition of naproxen to HNE had no additive effect on the 5-LOX mRNA level. All these data suggest that PGE₂ may play a critical role in 5-LOX and FLAP regulation by HNE.

Secondly, to determine whether TGF-β1 production could be involved in HNE-induced 5-LOX and FLAP expression, chondrocytes were treated or not with a single addition of 10 μM HNE for 72 hours in the presence or absence of 100 μg/ml TGF-β1 antibody. HNE-induced changes in 5-LOX and FLAP mRNA expression were evaluated as a percentage of control (untreated cells). As illustrated in Figure 6e, the addition of anti-TGF-β1 antibody to cultured cells attenuated HNE-induced 5-LOX and FLAP by 40% (both P < 0.05). In another experiment, the determination of TGF-β1 by ELISA in the culture media from HNE-treated chondrocytes showed a significant increase in TGF-β1 levels in comparison with untreated cells (control), and reached 210, 288 and 201 pg/ml after 24, 48, and 72 hours of incubation, respectively (Figure 6f) (n = 5 independent experiments, P < 0.05, P < 0.01 and P < 0.05).

Collectively, our results indicate that HNE-induced 5-LOX and FLAP requires PGE₂ and TGF-β1 production.