Prostaglandin E2 synthesis in cartilage explants under compression: mPGES-1 is a mechanosensitive gene

Abstract

Knee osteoarthritis (OA) results, at least in part, from overloading and inflammation leading to cartilage degradation. Prostaglandin E2 (PGE2) is one of the main catabolic factors involved in OA. Its synthesis is the result of cyclooxygenase (COX) and prostaglandin E synthase (PGES) activities whereas NAD+-dependent 15 hydroxy prostaglandin dehydrogenase (15-PGDH) is the key enzyme implicated in the catabolism of PGE2. For both COX and PGES, three isoforms have been described: in cartilage, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. COX-3 (a variant of COX-1) and mPGES-2 have been recently cloned but little is known about their expression and regulation in cartilage, as is also the case for 15-PGDH. We investigated the regulation of the genes encoding COX and PGES isoforms during mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) for 2 to 24 hours. After determination of the amount of PGE2 released in the media (enzyme immunoassay), mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blotting respectively. Mechanical compression of cartilage explants significantly increased PGE2 production in a time-dependent manner. This was not due to the synthesis of IL-1, since pretreatment with interleukin 1 receptor antagonist (IL1-Ra) did not alter the PGE2 synthesis. Interestingly, COX-2 and mPGES-1 mRNA expression significantly increased after 2 hours, in parallel with protein expression, whereas COX-3 and mPGES-2 mRNA expression was not modified. Moreover, we observed a delayed overexpression of 15-PGDH just before the decline of PGE2 synthesis after 18 hours, suggesting that PGE2 synthesis could be altered by the induction of 15-PGDH expression. We conclude that, along with COX-2, dynamic compression induces mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.

Figure 1

Mouse cartilage explants and Flexercell apparatus employed for mechanical stimulation. (a,b) Rib cages were harvested from one litter of 6-day-old Swiss mice. (c) Costal cartilage was cleaned and cut into little segments. 50 mg of the costal cartilage pool were put into a Biopress culture plate and 1.5 ml of media was added. (d) Each well was hermetically sealed with a specific cap. (e) The physiological compressive stress was applied by the Flexercell Compression Plus system described by Fermor and colleagues [26] on mouse costal cartilage explants. Intermittent compression was applied using a sinusoidal waveform at 0.5 Hz and 1.0 MPa of magnitude.

Figure 2

Compression stimulates nitric oxide (NO) and prostaglandin E2 (PGE₂) release in mouse costal cartilage explants in the media. Mouse costal cartilage explants were compressed (C) or not (NC) for 2 h, 4 h, 18 h and 24 h. At each time interval, our results are expressed in fold-induction in comparison to the appropriate control. (a) The amount of NO released into the media (μmol/mg of costal cartilage) was measured by Griess reagent. Values are the mean and SEM of 3 (C 2 h and 4 h) and 2 (C 18 h and 24 h) independent experiments with n = 2/group/experiments. ***p < 0.001 versus control (NC). (b) The amount of PGE₂ released into the media (pg/mg/ml of costal cartilage) was measured by enzyme immunoassay. Values are the mean and SEM of 3 (C 2 h), 2 (C 4 h and 18 h) and 4 independent experiments (C 24 h) with n = 2/group/experiments, analyzed in duplicate. ***p < 0.001 versus control (NC).

Figure 3

Compression stimulates prostaglandin E2 (PGE₂) release in mouse articular cartilage explants. Mouse articular cartilage explants were compressed (C) or not (NC) for 2 h, 4 h, 18 h and 24 h. The amount of PGE₂ released into the media (pg/ml) was measured by enzyme immunoassay. Values are the mean ± SEM of 2 independent experiment with n = 2/group/experiments, analyzed in duplicate. *p < 0.05, **p < 0.01, ***p < 0.001 versus control (NC).

Figure 4

Over-release of prostaglandin E2 (PGE₂) in compressed costal cartilage explants is the result of mechanical stress. (a) Implication of the mechanoreceptor integrin α5β1 in PGE₂ over-release in compressed cartilage explants. Mouse costal cartilage explants treated with either the β1 non-blocking antibody VMA1997 or the α5β1 blocking antibody AB1950 at 2.5 μg/ml were compressed (C) or not compressed (NC) for 4 h. Results are normalized to the mean not-compressed control (cont) value. Data are the mean ± SEM of 2 independent experiments with n = 2/group/experiments, analyzed in duplicate. ***p < 0.001 versus control NC, *p < 0.05 versus control C. (b) Increased PGE₂ release in compressed costal cartilage explants is not due to the cytokine IL-1. Mouse costal cartilage explants treated with the IL-1 receptor antagonist (IL1-Ra) at 100 ng/ml were compressed (C) or not compressed (NC) for 4 h. Results are normalized to the mean not compressed control value. Data are the mean ± SEM of 2 independent experiment with n = 2/group/experiments, analyzed in duplicate. *p < 0.05 versus control NC.

Figure 5

Compression increases cyclooxygenase type 2 (COX-2) and microsomal prostaglandin E synthase type 1 (mPGES-1) but not cytosolic PGES (cPGES) and mPGES-2 protein expression in costal cartilage. Costal cartilage explants were (a-d) not compressed or (e-h) compressed for 18 h and immunostained with anti-COX-2, anti-mPGES-1, anti-cPGES and anti-mPGES-2 antibodies and then counterstained with toluidine blue. Increased expression of (e) COX-2 and (f) mPGES-1 protein was seen in compressed explants compared to uncompressed ((a) COX-2 and (b) mPGES-1). In contrast, (g) cPGES and (h) mPGES-2 were not overexpressed after application of a compressive stress compared to the uncompressed condition ((c) cPGES and (d) mPGES-2). Representative findings from two compressed and two uncompressed samples were tested. Scale bar = 100 μM.

Figure 6

Compression increases cyclooxygenase type 2 (COX-2) gene expression but not COX-1 nor COX-3 in mouse costal cartilage explants. (a-c) Real-time RT-PCR assays demonstrating increased COX-2 gene expression after 2 h and 4 h in compressed explants versus control and no increase for COX-1 and COX-3. Standard curves for COX-1, COX-2, COX-3 and hypoxanthine-guanine phosphoribosyltransferase (HPRT) were generated by serial dilution of a cDNA mixture. The amount of COX-1, COX-2 and COX-3 mRNA was normalized against the amount of HPRT mRNA measured in the same cDNA. Values are the mean ± SEM of 4 independent experiments with n = 1/group/experiment for COX-1 and COX-2 and of 2 independent experiments with n = 1/group/experiment for COX-3. *p < 0.05, **p < 0.01 versus control (NC). (d) Explant lysates were analyzed by SDS-PAGE using 8% gradient gels. Proteins were transferred to a nylon membrane and successively blotted with anti-COX-1, anti-COX-2, anti-COX-3 and anti-β-actin antibodies. An increased expression of COX-2 protein in compressed cartilage compared to uncompressed, but not COX-1 and COX-3, was observed after 4 hours of compression up to 24 hours. Each blot is representative of three independent experiments.

Figure 7

Compression increases microsomal prostaglandin E synthase type 1 (mPGES-1) gene expression but not mPGES-2 nor cytosolic PGES (cPGES) in mouse costal cartilage explants. The rates of mPGES-1, cPGES and mPGES-2 expression in response to compressive stress at 2 h and 4 h were analyzed by (a-c) real-time quantitative RT-PCR and (d) immunoblotting. (a-c) Up-regulation of mPGES-1 mRNA expression but not cPGES and mPGES-2 mRNA expression by mechanical stress appeared at 2 hours until 4 hours. Values are the mean ± SEM of 3 independent experiment with n = 1/group/experiment. *p < 0.05, **p < 0.01 versus control (NC). (d) Increased translational expression of mPGES-1 but not cPGES and mPGES-2 was observed on the immunoblot (15% gradient gels), from 4 hours until 24 hours. Each blot is representative of three independent experiments.

Figure 8

Compression increases NAD+-dependent 15 hydroxy prostaglandin dehydrogenase (15-PGDH) transcriptional expression in mouse costal cartilage explants. The rate of 15-PGDH mRNA expression in response to a compressive stress (C) at 2 hours and 4 hours was analyzed by real-time quantitative RT-PCR. Increased 15-PGDH mRNA expression was observed after 4 hours of a compressive stress. Values are the mean ± SEM of 3 independent experiment with n = 1/group/experiment. *p < 0.05 versus control (NC).