5. Discussion
5.4. COX-2 and prostaglandin production in human chondrocytes
COX-2 also represents a key inflammatory enzyme and the role of PGE2 is well documented in the inflammatory process. Hyperalgesic effects of PGE2 or involvement in the generation of fever are well studied (Ferreira et al., 1978; Stock et al., 2001;
Aronoff and Neilson, 2001), however little is known about functions PGE2 plays in cartilage homeostasis and development of OA.
According to literature synovial fluid of OA patients contains high concentrations of PGE2 and cartilage as well as isolated chondrocytes of these patients spontaneously release more PGE2 than material obtained from healthy subjects (Miwa et al., 2000;
Notoya et al., 2000; Amin et al., 1997; Jacques et al., 1999). Although it was also shown that levels of PGE2 in synovial fluid of OA patients can also vary widely (38-6380 pg/ml) (Brenner et al., 2004).
The issue of COX-2 induction in chondrocytes is even more complicated because PGE2
is far from being the only prostanoid produced by chondrocytes. Different eicosanoids which are induced parallely with PGE2 act via different receptors and there are many signaling pathways involved, which can exert multiple and divergent effects on chondrocyte metabolism. Although there are many reports dealing with PGE2 in cartilage pathophysiology production of other eicosanoids were even not characterized in detail.
In this work we provide the first complete characterization of prostanoid production in human chondrocytes and hMSC undergoing chondrogenic differentiation.
In our experimental settings we used IL-1β as an OA-relevant stimulus to induce prostaglandin production. As revealed by presented Affymetrix analysis IL-1β is a very potent inducer of prostanoid pathway. We observed overexpression of PLA-2, COX-2 and PGES. This analysis revealed as well that the expression of prostanoid receptors EP2 and EP4 was up-regulated after IL-1 treatment.
Indeed, IL-1β treatment of human chondrocytes in alginate beads resulted in an average 14-fold induction of PGE2 synthesis in comparison to control. The production of 6-keto-PGF1α, PGD2, PGF2α and 8-epiPGF2α was also up-regulated (1,6-4 fold) whereas synthesis of TxB2 and isoprostanes remained unchanged. The total amount of other prostanoids was several-fold smaller than the amount of PGE2. IL-1β-induced production of prostanoids in chondrocytes was inhibited by Dexamethasone, which implicates involvement of glucocorticoid-sensitive COX-2 (Masferrer et al., 1992). In our study the failure of Dexamethasone to reduce spontaneous PGE2 and TxBB2 release in chondrocytes suggests involvement of constitutively expressed COX-1 (Masferrer and Seibert, 1994).
PGE2 was by far the predominant COX-product in IL-1β stimulated chondrocytes.
Predominance of PGE2 over other prostanoids could be related to the up regulation of microsomal prostaglandin E synthase1 (mPGES-1), the final enzyme in the PGE2
biosynthesis pathway which was recently reported to be overexpressed in OA cartilage and in human chondrocytes stimulated with IL-1β (Masuko-Hongo et al., 2004). We could also show an increase in mRNA levels of this enzyme after IL-1 stimulation.
hMSCs after 6 days of chondrogenic differentiation released PGE2 as the predominant product but PGI2, TxBB2, PGD2, and PGF2α were also generated although in much lower quantities. We did not detect F-isoprostanes except 8-epi PGF2α. Interestingly, spontaneous release of PGE2 was very high in pellets at the beginning of chondrogenic differentiation and exceeded even PGE2 production in chondrocytes after IL-1 stimulation. These results were confirmed by COX-2 PCR which revealed high COX-2 mRNA levels in unstimulated hMSCs at the beginning of chondrogenic differentiation.
Affymetrix analysis showed also up- regulation of the constitutive COX isoform after
induction of chondrogenesis. However inhibition by dexamethasone indicates that high production of prostaglandins was due to COX-2 activity. Spontaneous PGE2 production declined at later time points of differentiation (data not shown). Production of very high levels of PGE2 at the beginning of chondrogenic differentiation suggest importance of this prostaglandin in cell differentiation. COX-2 and PGE2 have been shown to be involved in the differentiation of multiple cell types, however the role of PGE2 in chondrogenic differentiation is not quite clear. There have been reports on a negative influence of PGE2 on chondrocyte differentiation (Jacob et al., 2004). On the other hand already more than twenty years ago there have been reports indicating positive effects of PGE2 and PGI2 on chondrogenic differentiation by elevating cAMP levels by these prostaglandins (Kosher and Walker, 1983; Ballard and Biddulph, 1983). Clark et al.
have recently shown that EP2 and EP4 receptor activation leading to the increase in intracellular cAMP levels may represent a central axis of events that facilitate the impact of PGE2 on the processes of hMSC commitment to chondrogenesis and ultimate chondrocyte maturation (Clark et al., 2005). Furumatsu et al. have shown that cAMP-response element binding protein (CBP/p300) acts as an important SOX-9 co-activator during chondrogenesis (Furumatsu et al., 2004). This transcription factor is necessary for chondrogenic differentiation by providing transcriptional signals for expression of the collagen 2 gene. These observations indicate that PGE2 exerts positive effects on collagen synthesis and chondrogenic differentiation (Goldring et al., 1990; Schwartz et al., 1998).
However, in addition to its modulatory role on chondrocyte differentiation and homeostasis, PGE2 is also known to play an important role in synovial inflammation indicating an additional indirect role in the pathogenesis of arthritis. High concentrations of PGE2 produced by OA tissue might have a role in the degradation of bone and cartilage associated with OA (Hardy et al., 2002). On the other hand COX inhibition is the most common form of OA therapy and leads to pain relieve but does not improve cartilage structure.
6-keto-PGF1α, TxB2, PGF2α and in very low levels of PGD2 and 8-epi-PGF2α are other eicosanoids released by human chondrocytes and hMSCs during chondrogenic
differentiation. Jacob et al. reported that PGD2 and PGF2α enhanced chondrogenic differentiation and hyaline cartilage matrix deposition (collagen 2 and glycosaminoglycans) of dedifferentiated articular chondrocytes (Jakob et al., 2004).
The role prostacyclin plays in cartilage and chondrocyte homeostasis is not clear. PGI2
could have a positive effect on chondrogenic differentiation by elevating cAMP levels as indicated before.
To our knowledge this is also the first report directly demonstrating prostacyclin synthase (PCS) staining in human cartilage and expression of the IP receptor on human chondrocytes.
IP receptor activation results in the increase of intracellular cAMP (Bley et al., 1998), which exerts anti-inflammatory effects. However activation of PGE2 receptors EP2 and EP4 leads also to elevation of cAMP levels and therefore anti-inflammatory effects. One could speculate that cAMP elevation under basal conditions is through the IP receptor and through EP2 and 4 receptors after stimulation as expression of IP receptor was down-regulated after IL-1 stimulation. In contrary the expression of EP receptors was elevated.
Isoprostanes are often used as a sensitive marker of oxidative stress and their generation has been shown to increase in several pathologies. The rate of radical formation and oxidation is increased in the lipid phase because NO and O2 are 6-20 times more soluble in lipid layers compared with aqueous fractions (Gow et al., 1996).
Isoprostanes were under detection limit in hMSCs, but human chondrocytes under basal conditions released significant amounts of isoprostanes. Interestingly 8-epi-PGF2α
and other isoprostanes were not only a product of non-enzymatic arachidonic acid oxidation, but seemed to be to large extent generated by COX-2 as their formation could be inhibited by Dexamethasone and Diclofenac. The phenomenon of COX-2 dependent formation of isoprostanes was also observed in other biological systems, which limits their use as a marker of free radical generation (Klein et al., 2001b; Pratico et al., 1995).
5.4.1. Non-enzymatic isoprostane formation in excess of AA
Increasing concentrations of exogenous AA led to a preferential formation of isoprostanes irrespective of cell stimulation, what indicates a COX-independent mechanism of their formation. Interestingly DETA NONOate and SIN-1 exerted distinct effects on the isoprostane formation. DETA NONOate in a dose-dependent manner inhibited isoprostane formation whereas SIN-1 enhanced formation of isoprostanes.
Enhancement of isoprostane formation by SIN-1 is not surprising as oxidizing agents are known for the nonenzymatic oxidation of AA to isoprostanes (Klein et al., 2001a).
A very interesting and important finding indicating that NO can be cytoprotective and reduce the generation of ROS under the conditions of oxidative stress in chondrocytes was the observation that DETA NONOate significantly inhibited the generation of isoprostanes probably due to ONOO- scavenging by NO (HOONO + 2NO → N2O3 + HONO).
Results obtained for isoprostanes indicate that their formation can be COX-dependent as shown before (inhibition by Dex of isoprostane formation), or they can result directly from interaction of AA with ROS.
However, it seems that in chondrocytes under physiological conditions formation of isoprostanes is mostly COX-dependent. This could be due to low ROS production or under these conditions the redox potential of chondrocytes is high and therefore non-enzymatic isoprostane formation does not take place.
We should not forget that the experiments with excess of AA are not relevant to the in vivo situation and they were performed only to demonstrate that COX enzymes are not inhibited by SIN-1 or DETA NONOate even in relatively high concentrations of tested agents (up to 250µM).
In long lasting experiments without exogenous AA the effect of NO donors was not prominent.
5.4.2. Redox-regulation of prostanoid synthesis
As indicated in the introduction there are contradictory opinions about the regulation of prostanoid generation by NO. Both inhibition and stimulation of COX-enzymes by NO in human chondrocytes was suggested. Therefore we performed detailed analysis of the regulation of prostanoid production in human chondrocytes by nitric oxide.
Generally the effect of NO-donors and inhibitors on prostanoid production in physiologic related conditions was not very pronounced in our experimental settings.
As shown in results NO donors had inhibitory effects on the COX-2 mRNA expression.
Therefore a slight increase in prostanoid levels after the cells were stimulated with IL-1 in the presence of iNOS inhibitors was not surprising. However experiments using NO donors did not show inhibition of prostanoid synthesis. One can speculate that this could be due to compensation of COX-2 mRNA inhibition by increased levels of peroxides and therefore higher enzymatic activity of COX (as discussed later).
In experiments with exogenously added arachidonic acid we observed a concentration-dependent increase in prostaglandin synthesis in both unstimulated and stimulated cells. These results indicate that AA availability is a limiting factor in COX-activity and further that prostaglandin synthesis is regulated by phospholipase activity.
DETA NONOate or SIN-1 in experiments with exogenous AA slightly increased formation of prostanoids in chondrocytes. This effect was more prominent in unstimulated cells and could be due to the peroxide tone.
The catalytic cycle of COX-enzymes is initiated by peroxides, which oxidize the heme prostetic group of the enzyme initiating the generation of PGG2 leading further to PGH2. Therefore the constant presence of low levels of intracellular peroxides is necessary for the activity of COX and formation of prostaglandins over the time (Capdevila et al., 1995; Margalit et al., 1998). The activation of COX by peroxides is called “peroxide tone”. The estimated peroxide tone for COX-2 was 2nM (Kulmacz and Wang, 1995) and about 10-fold higher for COX-1 (21nM). In contrast to COX-1 prostanoid generation by COX-2 is controlled rather at the transcriptional level and the regulation on the enzyme activity level due to peroxide tone is not so important.
Therefore we think that the effect of NO donors was more prominent in resting cells, where the levels of peroxides are kept low to inhibit prostanoid generation by
constitutively expressed COX-1. The addition of exogenous peroxynitrite activated COX-1 in these resting cells. Further the increased levels of prostaglandins after incubation of chondrocytes with DETA NONOate indicate that sufficient levels of ●O2
-are present in the cells to react with NO to form ONOO- and provide the peroxide tone to COX enzymes.
Prostacyclin synthase (PCS)
Nitration of PCS has been shown in a number of cell types under pathophysiological conditions like endotoxemia, ischemia-reperfusion, diabetes and atherosclerosis (Zou and Ullrich, 1996; Zou et al., 1997; Zou et al., 1999). We could not find nitration of this enzyme in OA chondrocytes. It has been shown that nanomolar levels of peroxynitrite (50-100nM) are sufficient to nitrate PCS (Zou et al., 1997(Schmidt et al., 2003) Schmidt), because of the heme nature of the enzyme.
Although we assumed that PCS in chondrocytes should be nitrated due to on the one hand, nitration susceptibility of this enzyme and on the other, production of high levels of NO and formation of ONOO- as indicated by presence of nitrotyrosine, PCS did not show any nitration in chondrocytes.
Western blots probed with anti-nitrotyrosine antibody did not reveal any nitration of this enzyme (data not shown). On the contrary stimulation of cells with IL-1 or addition of SIN-1 increased PGI2 synthesis. This could be due to a peroxide tone generated by ONOO-, increased generation of PGH2 by COX and therefore enhanced availability of substrate for PCS resulting in increased PGI2 synthesis.
Our data are in agreement with those obtained in SMC by Schildknecht et al. where 2-3-fold increase in PGI2 generation was observed after exposure to IL-1 (Schildknecht et al., 2004).
To conclude, chondrocytes produce very high levels of prostaglandins after IL-1 stimulation due to COX-2 activity, which is regulated on the transcriptional level.
The dependence of prostanoid formation on the peroxides means that prostaglandin synthesis is controlled also by the redox-state of the cell. Under conditions of oxidative
stress increased generation of prostanoids could be due to enhanced activity of constitutively expressed COX-1.
NO can interfere with prostanoid production on both transcriptional and enzymatic activity level, however its effects in chondrocytes are not very prominent.