Our findings demonstrate that dynamic mechanical loading of costal cartilage can significantly increase PGE2
release. Moreover, we describe here for the first time that COX-2 and mPGES-1 expression is increased in mouse costal cartilage explants under compression but not their constitutive isoforms COX-1 and cPGES. Therefore, it appears that COX-2 and mPGES-1 are encoded by mechanosensitive genes implicated in the compression-induced PGE2
is the pivotal eicosanoid involved in the initiation and the development of inflammatory disease, such as rheumatoid arthritis [29
]. Notably, it is thought to be a key regulator of cartilage degradation during OA [30
]. An increase in PGE2
release induced by mechanical stress has already been described in various tissues [31
] and particularly in articular cartilage subjected to dynamic compression representative of the physiological range [6
Regulation of COX-2 mRNA expression in cartilage by mechanical stress has already been reported in the literature [6
]. Notably, elements including AP-1 sites, cyclic AMP response elements (CREs) and shear stress response elements (SSRE) are found in the promoter region of mechanical stress-response genes, such as those encoding COX-2 and inducible NO synthase. Shear stress response elements contain a TPA response element to which NFκB, which is part of a main mechanical pathway, binds [33
]. Ogasawara and colleagues [34
] have described the role of C/EBP beta, AP-1 sites and CREB in shear stress-induced COX-2 expression in osteoblasts. Moreover, post-transcriptional regulation by mRNA stabilization seems to be involved in COX-2 gene expression in vascular endothelial cells subjected to fluid shear stress [35
]. In addition to these studies at the mRNA level, we show here, for the first time in cartilage, that COX-2 is also increased at the protein level.
Interestingly, our data indicate that COX-3, also named COX-1 V1, is expressed in mouse costal cartilage. COX-3, which was cloned in 2002, was derived from COX-1 through retention of intron 1 in its mRNA. This probably resulted in the modification of the active site conformation of the enzyme. COX-3 expression has actually been found in several canine, human and rodent tissues, but never in cartilage, whatever the species [18
]. In this present study, we report for the first time the expression of COX-3 (mRNA and protein) in mouse cartilage. Moreover, we show that mechanical loading did not modify its expression. As COX-1 and COX-3 are derived from the same gene, these enzymes share the same promoter. However, no sites that are regulated through mechanical stress or pro-inflammatory cytokines have been found so far in the COX-1 promoter, which is consistent with the fact that COX-1 is constitutively and ubiquitously expressed. Thus, this might explain the lack of COX-3 regulation by compression.
The regulation of mPGES-2 expression has never been described in cartilage. mPGES-2 is ubiquitously expressed under basal conditions in many tissues and is activated by reducing agents, but its role in PGE2
release in both basal and inflammatory contexts remains unclear. In human rheumatoid synoviocytes, expression of mPGES-1 increased with severity of the disease, whereas that of mPGES-2 did not [23
]. In COX-2-deficient mouse brains, a decreased release of PGE2
and a decreased expression of mPGES-2, but not of mPGES-1 or cPGES, was observed, suggesting that mPGES-2 could be functionally coupled to COX-2 [36
]. In the present study, mPGES-2 expression was similar in compressed and uncompressed cartilage explants, suggesting that mPGES-2 is not encoded by a mechanosensitive gene.
The striking point of our study is the evidence that mPGES-1 is encoded by a mechanosensitive gene. In recent years, several studies have demonstrated that inflammation induces mPGES-1. In rat paws of the acute and chronic arthritis model, up-regulation of mPGES-1 mRNA and protein expression was observed. Moreover, levels of mPGES-1 mRNA and protein were markedly elevated in OA versus normal cartilage [37
]. Additionally, we and others have previously reported an overexpression of mPGES-1 in OA chondrocytes in primary cultures stimulated by IL-1 [21
]. Interestingly, our results identify an earlier significant transcriptional expression (as soon as 2 hours) after mechanical stress compared to the effect of IL-1 (after 12 hours). Moreover, its induction was higher with compression (five-fold) compared to IL-1 stimulation (three-fold). A structural comparison of COX-2 and mPGES-1 promoters revealed that the gene encoding mPGES-1 does not contain transcriptional elements that are classically involved in response to mechanical stress (AP-1, CRE, SSRE), as described for the COX-2 promoter [38
]. It suggests that divergent transcriptional mechanisms are responsible for inducible mPGES-1 regulation. Yokota and colleagues [39
] have described the role of the nuclear regulator CITED2 (CBP/p300-interacting transactivator with ED-rich tail 2) in shear-induced down-regulation of matrix metalloproteinase 1 and matrix metalloproteinase 13. This mechanism could also occur in compression-induced mPGES-1 expression. But post-transcriptional regulation could also be present since a stabilization of mPGES-1 mRNA has been recently described in cardiomyocytes, leading to delayed protein expression via cJNK [40
Our data suggest that loading triggers mPGES-1 and COX-2 colocalization in the perinuclear region of the chondrocytes. This result is in line with the study from Kojima and colleagues [22
] that recently described COX-2 and mPGES-1 colocalization around the nuclei of chondrocytes after IL-1 stimulation.
Selective inhibitors of mPGES-1 have been developed recently [41
]. Effectively, targeting the mPGES-1 enzyme in preference to COX-2 should represent a new therapeutical approach to treating joint diseases such as OA. Interestingly, Cheng and colleagues [42
] reported that mPGES-1-null mice exhibit no alteration in thrombogenesis or blood pressure, whereas selective COX-2 inhibitors (Coxib) did. These results suggest that inhibitors of mPGES-1 may retain their anti-inflammatory efficacy by depressing PGE2
, while avoiding the adverse cardiovascular consequences associated with COX-2 deletion [42
To understand the weak PGE2
release at 18 and 24 hours after compression, we focused on 15-PGDH mRNA expression because 15-PGDH is one of the major PGE2
-inactivating enzymes [43
]. 15-PGDH expression decreases in response to IL-1β treatment in trophoblast cells in primary culture [44
] as well as in response to lipopolysaccharide (LPS) treatment in liver and lungs of rats [45
]. Moreover, 15-PGDH expression is reduced in inflammatory bowel disease [46
]. To our knowledge, 15-PGDH expression in cartilage has never been reported and its expression under mechanical stress was unknown. The findings presented in this study first indicate that 15-PGDH is expressed in mouse cartilage and is induced after 4 hours of compression (three-fold induction). Genomic structural analysis revealed that the human 15-PGDH promoter contains binding sites for AP-1 and CREB, two transcriptional factors implicated in the regulation of gene expression subjected to mechanical stress [47
]. We suggest that the 15-PGDH enzyme could be implicated in the catabolism of PGE2
after 18 hours. So, proinflammatory stimuli (LPS or cytokines) induce monophasic PGE2
synthesis whereas mechanical stress triggers a biphasic response in cartilage, PGE2
synthesis followed by PGE2
degradation. We hypothesize that this response corresponds to an adaptation of the tissue to this stress.
The choice of an experimental model to study the physiological regulation of cartilage homeostasis by mechanical stress is challenging. Three types of mechanical stress have been described: shear stress, strain and compressive stress. Overloading of the joints, a major risk factor for OA, mainly induces compressive stress. However, shear stress and strain have also been described when overloading is experimentally applied on cartilage. The compression of chondrocytes embedded in agarose or collagen gels as well as the compression of ex vivo
porcine cartilage explants represent the main strategies developed [6
]. Among the literature, it appears obvious that the consequences to metabolism of chondrocytes vary according to the type of stress applied. Takahashi and colleagues [49
] reported that static compression promotes type II collagen, whereas cyclic loading could denature type II collagen in articular chondrocytes [50
]. Here, we used mouse cartilage explants subjected to cyclical uniaxial mechanical stress. Mouse cartilage was chosen because of the availability of a large choice of biomolecular tools suitable to study it, such as genetically modified mice. However, many labs work on discs of porcine or bovine cartilage, permitting a more homogeneous compressive stress. In our model, the compressive stress is less uniform because of the size of the explants. Enhancements to our system, in order to better define the stress applied, is ongoing.
We decided to work on costal cartilage rather than articular cartilage because of the extremely small quantity of the latter available. However, we performed comparative experiments in order to validate our model with costal cartilage explants. We observed an induction of PGE2 release in both types of cartilage, but with slight differences in kinetics.
The mechanoreceptor integrin α5β1 is considered by many authors to be the critical mechanoreceptor for transducing signal from the extracellular matrix to the chondrocyte [8
]. Our data confirm the major role of this receptor in compression-induced PGE2
Among the mechanotransduction pathways recently described, Mitogen-Activated Protein Kinase (MAPK) plays a major role. In smooth muscle cells and fibroblasts, mechanical strain increases the activity of all three MAPKs, namely p38 MAPK, ERK 1/2 and Junk kinase [51
]. In particular, ex vivo
cartilage compression has been reported to activate the three MAPKs, but with different kinetics [9
]. Preliminary results suggest that this is also the case in our model (personal communication).
Surprisingly, overexpression of COX-2 and mPGES-1 proteins was observed at 18 hours in uncompressed samples. Several authors have described the release of mediators, such as basic fibroblast growth factor (bFGF) and IL-1α, in cartilage after explantation and cutting [53
]. Moreover, Fermor and colleagues [6
] reported that uncompressed explants of articular cartilage exhibited a significant production of PGE2
(from 14 pg/mg/ml at 24 hours after explantation to 1 pg/mg/ml at 72 hours). We have considered that dissection alone induces COX-2 and mPGES-1 protein expression in the uncompressed samples.
As mature articular cartilage is an avascular tissue, the oxygen supply to resident chondrocytes could rather limit and influence chondrocyte activation under a mechanical signal. Fermor and colleagues have shown that oxygen tension influences the endogenous production of NO and PGE2
in porcine cartilage explants in response to mechanical stimulation. Under mechanical compression, PGE2
production in cartilage at 20% O2
increased 50-fold, but in cartilage at 5% O2
it increased only 4-fold and in cartilage at 1% O2
it did not increase at all [48
]. Since previous studies have suggested that the oxygen tension in the superficial layer of articular cartilage is higher than in the deeper layer [55
], our results could have occurred, at least in part, in the superficial zone of articular cartilage.