The degradation of osteoarthritic (OA) cartilage is related to a complex interaction of mechanical and biochemical factors (1–3). Among the latter, a number of catabolic factors, including proinflammatory cytokines, nitric oxide (NO), and proteases, have been demonstrated to play major roles (1, 4).
OA cells, at both the cartilage and synovial membrane levels, are actively involved in producing an excess amount of catabolic factors (1). The increase in the metabolic activity of these cells is related to the stimulation of proinflammatory cytokines, such as interleukin-1 (IL-1), tumor necrosis factor α (TNFα), IL-6, and leukemia inhibitory factor (1, 4–6). After binding to a specific membrane-bound receptor, these cytokines initiate the activation of a number of intracellular signaling pathways that lead to the synthesis of transcription factors that will induce the expression of multiple genes, including those from catabolic factors (7).
The protein kinase pathways are believed to be of key importance among the different signaling systems activated by proinflammatory cytokines (7–12). These pathways comprise a number of signaling cascades. The three most predominant cascades culminate in the activation of the extracellular signal–regulated kinase 1/2 (ERK-1/2), c-Jun N-terminal kinase (JNK), and p38 families of mitogen-activated protein kinases (MAPKs). These cascades have been demonstrated in several studies to be essential to the synthesis of a number of catabolic factors responsible for inducing the structural changes seen in OA (7).
ERK-1/2 is activated by MAPK kinase 1/2 (MEK-1/2) as part of the MAPK pathway (7, 13). In this pathway, the Raf kinases phosphorylate and activate MEK-1/2, which in turn phosphorylates and activates ERK-1/2. Activated ERK-1/2 can then translocate in the nucleus and activate transduction factors by phosphorylation, thus altering specific gene expression. In addition, ERK-1/2 has a number of cytosolic substrates that can influence gene expression directly or indirectly (13).
The MAPK pathway has also been demonstrated to be a key factor in the induction of matrix metalloproteinases (MMPs) by cytokines (14), production of cyclooxygenase 2 (COX-2) by chondrocytes, and chondrocyte apoptosis (15). The JNK and p38 signaling cascades are implicated in the synthesis of MMP by chondrocytes (7, 14). Moreover, p38 has been shown to be involved in the expression of the inducible NO synthase (iNOS) (16) and proinflammatory cytokines (17). A recent study has demonstrated that an inhibitor of p38 very effectively reduced the synthesis in vitro of cytokines and iNOS (16). In another recent study, treatment with a specific p38 inhibitor in a rat model of inflammatory arthritis was found to reduce the progression of structural damage while simultaneously reducing the synthesis of proinflammatory cytokines (18).
The primary goal of the present study was to examine, using a rabbit experimental model of OA, the effect of a specific inhibitor of MEK-1/2, PD 198306, on the development of structural changes in articular tissues. We also examined the effect of the inhibitor on the phosphorylation level of ERK-1/2, as well as on some of the major pathophysiologic pathways of OA.
PD 198306 is a potent, selective, non-ATP competitive inhibitor of MEK-1/2. It inhibits the isolated enzyme at a concentration of 8 nM and inhibits MEK activity in synovial fibroblasts at concentrations of 30–100 nM, depending on the species. The compound is highly selective for MEK and has a 50% inhibition concentration of >1 μM for ERK, >4 μM for c-Src, >10 μM for phosphatidylinositol 3-kinase γ, and >4 μM for cyclin-dependent kinases. PD 198306 has a bioavailability of 62% when taken orally and is active in several animal models of rheumatoid arthritis, including rat streptococcal cell wall–induced arthritis (median effective dose [ED50] 11.2 mg/kg) and rat adjuvant arthritis (ED50 6.6 mg/kg) according to an internal file of Pfizer Global Research & Development (Ann Arbor, MI). Figure 1 shows the chemical structure of PD 198306.
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- MATERIALS AND METHODS
This study provides new and interesting findings about the role of MAPK in OA pathophysiology as well as the potential uses of MAPK and MEK-1/2 inhibitors for the treatment of the articular tissue structural changes in OA. This approach brings into perspective the possibility of pharmacologic intervention with regard to specific pathophysiologic targets.
The last few decades of research in the field of OA have allowed us not only to better characterize the morphologic changes taking place during the course of the disease, but also to discover a number of very important mechanisms responsible for such changes (1–4). For instance, the participation of synovial inflammation in the progression of cartilage changes at the clinical stage of the disease is becoming increasingly obvious (4). There are now a number of clinical studies that demonstrate a clear association between inflammation and disease progression (29–32). A number of factors synthesized within the inflamed synovium are responsible for stimulating the synthesis of a larger number of catabolic/anabolic factors, which in turn mediate the structural changes (1–4). Among the different synovial factors, cytokines, such as IL-1β and TNFα, are believed to play a prime role (6). The activation of cells by these cytokines is mediated by the binding to a specific cell membrane surface receptor, which triggers the activation of a number of complex intracellular signaling pathways (7, 33–36). Other inflammatory factors (e.g., NO) have also been demonstrated to activate intracellular signaling pathways (37). Among these, the protein kinase pathways are of major importance in the induction of the synthesis of transcription factors that mediate the up-regulation of a number of very important catabolic factors, such as MMP and NO (16, 38).
The findings of the present study suggest that PD 198306 could effectively reduce the phosphorylation of MEK-1/2 in situ, since this kinase is immediately upstream from ERK-1/2 and is the only kinase that has thus far been demonstrated to be able to phosphorylate ERK-1/2 (7, 13). The effect of PD 198306 was dose dependent and more pronounced in the highest-dosage treatment group, which also provides additional evidence of this inhibition. Since the MAPK pathway was involved in the synthesis of a number of catabolic factors participating in the development of the structural changes in OA, it is plausible that the action of PD 198306 was mediated through the inhibition of their synthesis. Moreover, the reduction in the severity of synovial inflammation, which was related to a reduction in the level of synovial hyperplasia, could probably have also contributed to this phenomenon. However, when considering the reduction in the histologic score of the synovial membrane, one must take into account that the synovial scoring system used in the study gave more weight to villous hyperplasia and cellular infiltration, which represented 8 of the 10 points (total score) in the system.
We have previously demonstrated in the OA experimental dog model that agents that could effectively reduce synovial inflammation (such as a selective iNOS inhibitor) could also simultaneously reduce the appearance/progression of structural changes in OA, including cartilage lesions and osteophyte formation (39). This phenomenon was associated with a concomitant reduction in the level of synthesis of IL-1β and inflammatory mediators such as prostaglandin E2 (PGE2) and NO (39, 40). This finding is most interesting, since the synthesis of PGE2, a well-known factor also capable of inducing bone remodeling (41), can be blocked by MEK-1/2 inhibition, which reduces COX-2 expression.
Treatment with therapeutic concentrations of PD 198306 was also found to be capable of reducing the progression of cartilage damage. This effect was noted on the surface of the tibial plateaus and the depth of lesions in the total joint, both of which were significantly reduced. This protective effect was also associated with a better preservation of the cartilage histologic structure. The lower score on the Mankin scale observed in this study was in great part related to this particular finding. However, it should be noted that this scoring system gives a much greater emphasis to matrix changes, since 10 of the 14 points (total score) were for structural changes and loss of Safranin O staining. Moreover, these OA lesions were not always in the same topographic location; therefore, the sampling of cartilage specimens could also explain some of the variation observed in the study.
Concerning the role of cartilage catabolic factors, a number of biochemical factors that participate significantly in the degradation of cartilage macromolecules have been identified (1–4). Among these factors, the proteases, and more specifically MMP, have been demonstrated to be major factors in the catabolism of cartilage macromolecules (42). In the MMP family, collagenases are believed to be key players in the proteolysis of cartilage type II collagen (43).
Collagenase 1 (MMP-1) has been identified in OA cartilage (both in human and in experimental models) and shown to be synthesized in an increased amount by OA chondrocytes (25, 26, 43). Previous studies have demonstrated that there is a preferential synthesis of MMP-1 by chondrocytes located in the superficial layers in OA cartilage (25, 26). These reports are consistent with the findings of our study. The involvement of the excess synthesis and activity of MMP-1 and other collagenases in the structural changes in OA cartilage has been well established (6, 43). In the present study, we demonstrated that treatment with the inhibitor of MEK-1/2 was capable of reducing the number of chondrocytes that stained positive for MMP-1. However, it should be pointed out that the differences among the OA PD 198306–treated groups and the OA placebo-treated group did not reach statistical significance. It is of interest to note that the in situ level of synthesis of MMP-1 in OA cartilage chondrocytes, as assayed by immunohistochemistry, has been recently shown by Altman and Cheung to reflect the level of MMP activity in rabbit OA (44). The differences in the extent to which PD 198306 inhibits ERK-1/2 and MMP-1 raise a number of questions about the relationship between the effect of this drug on structural changes and its effect on MMP-1 synthesis.
A possible explanation for this finding could be that, since previous studies have demonstrated that MMP synthesis depends on the simultaneous activation of many of the protein kinase pathways, including the JNK, MEK-1/2, and p38 pathways (7, 11, 14), it is therefore understandable that the selective inhibition of a single protein kinase pathway could have induced only a partial inhibition of MMP synthesis. However, since the MEK-1/2/ERK-1/2 pathway is also involved in the synthesis of other catabolic factors by chondrocytes (including NO [27,38], which is important in inducing cartilage lesions), it is likely that the effect of PD 198306 was mediated through the inhibition of synthesis of several other catabolic factors. Further studies are currently under way to explore additional mechanisms of action of this drug.
Preliminary findings from this study provide information about potential mechanisms for mediating the structure-modifying effects of this MEK-1/2 inhibitor. Treatment with the inhibitor was found to reduce the severity of synovial inflammation, which was essentially related to a significant reduction in the villous hyperplasia. Therefore, the total number of cells (including synovial cells and infiltrating mononuclear cells) that could have potentially produced catabolic factors, such as cytokines and MMP, was reduced by treatment with PD 198306. However, caution must be taken not to overinterpret these results, and the synovial scoring system used in the present study must be taken into account. Moreover, a very important study by Firestein and Manning (7) recently showed that synovial fibroblasts from OA patients expressed the 3 main MAPK families. That study also showed that MEK-1/2 was readily phosphorylated by IL-1 and was responsible for increasing MMP-1 gene expression in these cells. Therefore, although this is very speculative, it is possible that the inhibition of MEK-1/2 in OA synovium could, by reducing cartilage catabolism, also contribute to a reduction in the level of synovial inflammation (1–4).
In summary, the present study has demonstrated that the activation of the MAPK pathway could probably contribute to the pathogenesis of OA. Therapeutic intervention with the goal of MEK-1/2 inhibition may have interesting potential for the development of agents for the treatment of OA.