High hydrostatic pressure induces pro

The ATDC5 cell line is now a well established cell line to study chondrogenesis and other aspects of cartilage biology [20]. Using a mouse cell line also made it easier to compare our results with previous in vivo studies on mouse models of OA, the main model involving destabilization of the medial meniscus (DMM), a procedure that leads to severe osteoarthritis within 8 weeks [21]. In addition, it has been shown that stresses generated in joint cartilage is within one order of magnitude between quadruped species (i.e. mouse or cow) [22]; consequently, it is reasonable to assume that the MPa pressures that are generated in human joints may also be generated in mouse cartilage, despite the different weights.

One limitation to our study was the use of constant pressures, which, at such a high intensity, are unlikely to be found in vivo. Indeed, when studying the effects of physiological pressures, cyclical pressures are usually applied; however, high pressures (25 MPa) are just as unlikely to be generated in vivo in a cyclical manner. A more physiological approach would have been to apply a single 25 MPa impact pressure to simulate a traumatic injury, which is known to promote the onset of osteoarthritis [23]. However, traumatic injuries in vivo trigger a complex response involving several cell types such as immune cells; as our goal was to identify new gene targets of high HP, we decided to use constant pressure and limit the stimulation to 24 h.

Despite the importance of HP in cartilage homeostasis, no whole genome microarray study of the effects of HP has been carried out on chondrocytes or chondrocyte precursors. Sironen et al. used a cDNA array with under 600 probes to identify genes in a human chondrocyte cell line responsive to 30 MPa for 3, 6 or 12 h [12]. Among the genes present in their array, they found 51 modulated genes, while 151 were modulated in our array. Though the overlap was not statistically significant (p = 0.064), 18 genes were found to be modulated in common and in the same direction (representation factor of 1.4), including Fosl1, Hbegf, Ddit3 and Gadd45a, the latter suggesting that growth arrest/apoptosis may have also been observed in their cells [24].

By looking at the effects of high HP on the whole mouse transcriptome after 1, 4 and 24 h of pressurization, a broad picture of the early response of the cell to high HP could be drawn: an initial stress response followed by arrested growth, increased cell death and a decrease in extra-cellular matrix synthesis, eventually starting the chondrocyte de-differentiation process.

Therefore, among the modulated genes, several genes involved in the physiopathology of OA were identified: Adamts5, which was transiently but significantly induced under HP, produces an aggrecanase found responsible for aggrecan degradation in a mouse model of OA [25]. Arntl is implicated in chondrocytes’ circadian rhythm, and loss of this gene leads to progressive cartilage degradation in mice [26]. CD14, an antigen usually expressed by immune cells, has also been found in human chondrocytes [27] and may play a role in lymphocyte-mediated cartilage degradation [28]. CITED2 is a transcriptional co-activator known to suppress the expression of the collagen-degrading enzymes MMP-1 and MMP-13 [29][6] and its strong down-regulation further indicates anti-chondrogenic effects of high HP in ATDC5 cells. Ctgf was markedly up-regulated after 24 h of pressure and is strongly expressed in osteoarthritic cartilage [30]; although CTGF may be important for cartilage differentiation and maintenance [31], its over-expression has also been shown to cause cartilage damage [32]. Ctsk, which produces the collagen-degrading protease cathepsin K, was also transiently but strongly up-regulated by high HP, and knockdown of this enzyme in mice delays the progression of the disease in surgically induced OA [33]. Cytl1, strongly down-regulated by 24 h of HP in ATDC5 cells, is a cytokine-like protein involved in chondrogenesis, whose knockdown leads to more severe cartilage destruction in a mouse model of OA [34]. Ddit3 produces a transcription factor found to participate in cartilage degradation in a mouse model of OA [35]. Errfi1 deletion in mouse cartilage leads to an OA-like phenotype in the knees, though rarely in other joints [36]. Fosl1, markedly up-regulated in our study, produces a FOS-related transcription factor that has been found to be potentially involved in the regulation of gene expression in human osteoarthritic cartilage [37]. Hbegf, also transiently up-regulated in our study, produces an EGF-related growth factor, and has been found to be over-expressed in damaged cartilage from patients suffering from OA [38]. Ptgs2, which produces cyclooxygenase 2, was also up-regulated after 24 h; its role in OA, however, is still controversial, some studies showing that cyclooxygenase 2 inhibition protects against OA [39] while others show no effect [40].

Surprisingly, Adamts4, which is known to be expressed by chondrocytes in osteoarthritic cartilage [41] and also participates in aggrecan degradation in a model of human OA [42], was significantly down-regulated for up to 24 h. However, the respective importance of Adamts4 and Adamts5 in the onset and progression of OA is still under investigation. The expression of Nfil3, a transcription factor essential to the development of certain immune cells [43], was transiently up-regulated in our study but found down-regulated in osteoarthritic cartilage [44]; the role of NFIL3 in cartilage is still poorly understood and warrants further investigation.

Overall, the microarray results seem to point to osteoarthritic-like effects of high HP on chondrogenic cells. In agreement with the PCR results, several hundred genes modulated by HP were found to be similarly modulated in cartilage from rodent OA models. ATF3, for instance, is a transcription factor, whose expression is increased in OA cartilage and which is involved in the progression of the disease in a mouse OA model [45]. Nfe2l2 produces a transcription factor, which is known to protect cartilage from degradation in mouse OA models [46]. The overlap between the present results and the previously published microarray studies used for comparison, however, was not statistically significant. This may be due to the different time points used; 24 h of pressurization may only trigger events seen at the very onset of the disease, and longer pressurization periods may be necessary to lead to a more OA-like gene expression profile. Nevertheless, among the newly identified HP-modulated genes, may lie some yet poorly known genes essential to the pathogenesis of OA, which further studies will help to uncover.

In this study, no growth factors or hormones were added to the medium to promote ATDC5 differentiation into mature chondrocytes before HP was applied. However, ATDC5 cells were seeded at a relatively high density and were fully confluent for one day before the start of the pressurization. Real-time PCR confirmed that they thus clearly expressed Sox9, Acan and Col2a1. Moreover, we found that 25 MPa strongly inhibits Acan and Col2a1 expression; this is similar to the effects of high HP (20 to 50 MPa) reviewed by Elder et al. [4], which include a decrease in collagen and aggrecan gene expression in bovine chondrocytes. In addition, one technical difficulty with using differentiated ATDC5 cells is that the cell population obtained after differentiation may not be homogeneous, making the results difficult to interpret. Though investigating the effects of pressure on primary (differentiated) chondrocytes may be an interesting future study, we believe the present study using a homogeneous population of ATDC5 cells yielded meaningful data on the mechanobiology of hydrostatic pressure.

As cells do not contain any gas phase and are considered almost incompressible, physiological HP does not induce any visible deformation of cell compartments or the cell as a whole, making the mechanotransduction mechanisms difficult to identify. However, within 1 h, several hundreds of genes responded to pressurization, and it is quite possible that several independent mechanisms could be implicated in the sensing of HP: for instance, HP may affect the conformation of a pressure-sensitive protein, though studies showing visible changes in protein conformation usually deal with pressures above 100 MPa [47]; HP may alter membrane fluidity and indirectly affect the conformation or the binding of signaling molecules [48]; HP could also alter forces within the membrane by increasing its bending rigidity [49], generating forces sufficient to trigger a mechano-chemical response.

As physiological pressures are known to be beneficial to cartilage, and high HP induces changes in cells reminiscent of those observed in OA, identifying the pressure mechanotransducer(s) in chondrocytes and their downstream pathways may be a key to better understand and prevent the progression of OA.