Osteoarthritis (OA) is a progressive, debilitating disease of the joints characterized by the erosion of articular cartilage. Although much is known about the expression and regulation of genes associated with OA (e.g., metalloprotease, tissue inhibitor of metalloproteases, inflammatory cytokines, and extracellular matrix proteins such as collagens and proteoglycans), it is likely that the expression of many other genes is also affected during this pathologic process, and more is yet to be learned in order to gain a comprehensive understanding of this disease.
Microarray technology was developed to analyze the expression of thousands of genes in a very short time. Complemented by techniques such as real-time reverse transcription–polymerase chain reaction (RT-PCR) or Northern blotting, it is a powerful tool for analyzing genetic expression on a large scale to obtain novel information about a given pathophysiology. This technology has already been applied to study a number of systems ranging from chondrogenesis to cancer and, more recently, to the field of arthritis (1–4).
Using microarray technology, we identified follistatin, a bone morphogenetic protein (BMP) and activin-binding protein, as being significantly up-regulated in OA chondrocytes. Activin and BMPs belong to the transforming growth factor β (TGFβ) superfamily of secreted signaling molecules (5). BMPs were first identified for their ability to induce bone formation. They have a wide range of biologic activities in embryogenesis and in the maintenance and repair of bone, cartilage, and other tissues in adults (5–10).
To date, very little is known about the role of the BMPs and their antagonists in OA pathophysiology. Nakase et al have shown that BMP-2/4 is present in chondrocytes in adult OA cartilage and osteophytes, as well as in neonatal growing articular cartilage, but is scarce in normal adult articular cartilage (11). It has been reported that the expression of BMP-2 is stimulated by interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα) in normal and OA human chondrocytes (12), and that BMP-2 and BMP-6 are expressed in arthritic synovium and up-regulated by inflammatory cytokines (13). However, in another recent study, Bobacz et al (14) reported that BMP-6 is expressed in both normal and adult OA human cartilage but with no significant difference of expression. BMP-2 was found to stimulate in vivo proteoglycan synthesis in normal murine joints (15) but could not counteract the catabolic effect of IL-1β (16), something that BMP-7 was capable of doing in human cultured articular chondrocytes (17). Contradictory results were reported for BMP-7. Chubinskaya et al (18) observed that BMP-7 could be up-regulated in OA cartilage, while Bobacz et al (19) did not demonstrate a differential expression between normal and OA chondrocytes. In addition, it was recently shown that each BMP has a different pattern of distribution in joint articular tissue (20).
BMP antagonists comprise a family of structurally unrelated proteins. They regulate the activities and functions of the different BMPs by forming a complex with them and preventing their proper binding to the receptors (7, 8). Representatives of the BMP antagonist family include follistatin, gremlin, chordin, and noggin. Because each antagonist differs in its specificity and affinity for a specific BMP, each plays a different role, depending on cell and tissue type, in the spatial and temporal regulation of BMP activity.
Follistatin binds BMP-2, BMP-4, and BMP-7, with a higher affinity for BMP-7, although the affinity for BMPs is lower than that for activin (21, 22). Follistatin can inhibit BMP signaling in a manner different from that of the other antagonists, by binding to BMP receptors and BMPs, forming a trimeric complex (22). Gremlin binds BMP-2, BMP-4, and BMP-7 (23, 24). It is highly expressed in nondividing and terminally differentiated cells; it has been shown to regulate limb bud development, inhibit chondrogenesis and cell replication, and induce apoptosis in vitro (25). Chordin binds BMP-2, BMP-4, and BMP-7, with a higher affinity for BMP-2 and BMP-4; it does not bind activin or TGFβ1 (26). Overexpression of chordin in the embryonic chick limb system delays chondrocyte maturation and supports a role for chordin as a negative regulator of endochondral ossification (27). Noggin binds BMP-2, BMP-4, and BMP-7, with a higher affinity for BMP-2 and BMP-4 (28). It decreases osteoblast formation (29, 30), inhibits membranous ossification, and prevents chondrogenesis and limb development (31, 32).
This study is the first to evaluate the expression/production of 4 BMP antagonists in normal and OA human chondrocytes and synovial fibroblasts, as well as the regulation of 2 of them by different factors involved in the OA process. We show that the BMP antagonists are expressed and regulated differently in normal and OA chondrocytes. Such a change in the levels of BMP antagonists in OA tissues will likely impede the biologic action of the BMPs, suggesting a role in the progression of the disease.
- Top of page
- MATERIALS AND METHODS
Microarrays are powerful screening tools that have allowed us to identify several novel genes with potential involvement in OA pathophysiology. Among the novel genes found to be modulated in OA cartilage, we selected the BMP antagonist follistatin, as well as 3 other BMP antagonists, for further characterization and regulation in normal and OA human articular tissue cells. This study is the first to show that the expression and synthesis of the BMP antagonists follistatin and gremlin are up-regulated in cultured OA chondrocytes and cartilage, suggesting their potential involvement in human OA.
The BMPs and their antagonists constitute a complex, dynamic system regulated at different levels in order to respond to specific environmental stimuli. In the adult joint, an equilibrium between the levels of BMPs and their antagonists exists to maintain the normal turnover of the tissues. This equilibrium is affected in response to inflammation or injury, when a higher level of BMP activity is required. The levels at which the BMP antagonists are expressed in OA cartilage may disturb activin/BMP activities, leading to decreased anabolic activities, affecting tissue repair and remodeling. Because BMPs are now targeted as therapeutic molecules to stimulate healing/reengineering of cartilage, BMP antagonists also should be taken into consideration when using this system.
Our results show differential expression of the antagonists not only within a given cell type (chondrocytes) but also between different cell types (chondrocytes, synovial fibroblasts). Differential expression of the antagonists is not unexpected because the role and expression/regulation of BMPs and their antagonists are different in cartilage and synovial membrane. The fact that normal and OA synovial fibroblasts do not differ significantly in their levels of follistatin and gremlin may reflect the complex interplay between stimulatory and inhibitory factors present in the synovial membrane.
In OA pathology some cytokines and other inflammatory factors (although not the primary cause of this disease) are definitely closely related to its progression. We tested several cytokines to verify whether 1 or more of these factors influenced the expression of the antagonists and contributed to the higher levels of follistatin and gremlin observed in OA cartilage. Moreover, as a number of tissue repair agents are present in these diseased tissues, we also tested the effect of some growth factors. Our results demonstrated a complex pattern of regulation, indicating that the increased expression of follistatin and gremlin in cartilage may not necessarily result from only 1 or the same stimulus. Indeed, we did not find a single factor that simultaneously triggered the increase of both. Follistatin was increased by IFNγ and TNFα and gremlin was not; gremlin expression was increased by BMP-2 and BMP-4, but follistatin was not. The overall expression of follistatin and gremlin is increased in OA cartilage, and, because of their different binding affinities, these BMP antagonists could likely play different roles and/or appear at different stages during the OA process, and could also be responsible for sequential autocrine feedback in the control of BMP/activin activities. Very few factors stimulate expression of follistatin and gremlin compared with the number of factors that are inhibitory. This supports the hypothesis that their expression is timed with specific stages in the progression of OA.
Although the factor or factors that initiate OA are still unknown, the very early stage of the disease is characterized by a hypertrophic biochemical repair reaction and an enhanced synthesis of extracellular matrix. At this stage, chondrocytes express genes involved in the repair of cartilage, such as BMP-2. The new matrix thus formed might not be quantitatively and qualitatively adequate to regenerate new functional cartilage and could result in a gradual loss of this tissue. The cartilage fragments released may trigger inflammatory reactions from the synovial membrane, or even within the cartilage, which will promote the expression of catabolic factors, including matrix metalloproteases, and contribute to the maintenance of OA.
Our data show that the growth factors BMP-2 and BMP-4 are favorite candidates for triggering the increase in gremlin expression. In OA, gremlin may first appear at the hypertrophic stage, when the increased level of BMP-2 stimulates its expression. IL-1β, which plays a pivotal role in cartilage degradation and is involved early in the disease, may also indirectly up-regulate gremlin by inducing BMP-2 in cartilage (12). Interestingly, Fukui et al (12) reported that, in moderately damaged OA cartilage, BMP-2 was expressed mostly in the middle and deep layers; in severely damaged OA cartilage, it was not seen at the fibrillated surface, a pattern that parallels the expression of gremlin-positive chondrocytes. Thus, the absence of gremlin near the surface of OA cartilage may result from the absence of BMP-2 as well as its inhibition by inflammatory factors. The overall gremlin expression levels could be influenced by both the presence of BMP-2 and the inhibitory effects of inflammatory factors. Thus, the IL-1β catabolic effect combined with the inhibition of the BMP anabolic activity would eventually result in severe cartilage degradation and an increased level of synovitis and inflammatory factor production. The presence of the inflammatory factor TNFα produced later by the diseased articular cells could also stimulate BMP-2 (12), which could in turn induce gremlin. Gremlin would then be present in both the early and late stages of OA.
Follistatin, in contrast to gremlin, may have a stronger link to the inflammatory aspect of OA and may appear later during the OA process, possibly induced by the presence of TNFα and IFNγ, 2 cytokines involved in inflammation and in severe OA. Follistatin binds activin A, a molecule expressed in inflammatory arthropathies (38) that is capable of inducing cell proliferation of rheumatoid arthritis (RA) synovial fibroblasts (39) and is released during inflammatory episodes at about the same time as the release of TNFα (40). Furthermore, Thornton et al (41) showed that a mouse follistatin-like gene was highly expressed along the margin of contact between the inflammatory synovial pannus and eroding bone in collagen-induced arthritis.
We showed that TNFα and IFNγ significantly increased follistatin levels, and that follistatin was mainly localized at the superficial zone of OA cartilage; this is the zone where TNFα had been previously located (42). Although the involvement of IFNγ in OA could be debated, it was reported that both OA and RA synovial lymphocytes have a similar Th1 profile, and that OA lymphocytes could produce IFNγ, although not as much as lymphocytes from RA (43). During the inflammatory episodes, follistatin would be preferentially increased over gremlin, the expression of which is decreased by most cytokines. Activin A and the other growth factors are not likely to trigger the increase in follistatin, because these molecules inhibit rather than stimulate its expression.
It has been suggested that BMPs could induce apoptosis (44–46). Because the major role of the BMP antagonists is to modulate BMP activity, changes in the levels of the antagonists may indirectly affect the level of apoptosis in the tissue. Gremlin, for example, was found to regulate programmed cell death in the developing avian limb (25). Whether or not follistatin and gremlin protect OA cartilage from apoptosis remains to be evaluated.
The finding that BMP antagonists are expressed in chondrocytes and are subjected to differential regulation opens the door to a new field of investigation, and experiments are under way to understand the role of each antagonist in OA pathophysiology. Normal chondrocytes might express low levels of BMPs and their antagonists, which are responsible for the normal turnover of the tissues. In OA, there is a reexpression of genes normally involved in embryogenesis and development in an attempt to repair the cartilage: the gene coding for BMPs, matrix metalloproteinase 13, wingless, and frizzled, for example, respond to the healing signal (20, 47–49) and are found at a higher level in human OA cartilage. Therefore, the follistatin and gremlin genes, normally expressed in embryogenesis, should be added to the list of genes reactivated during OA. The balance of BMP antagonist levels, as well as inflammatory or BMP factors, during the OA process may play a critical role in influencing the progression of the disease, making these antagonists interesting targets for the treatment of this pathologic condition. The patients with OA who were selected for this study had long-established OA disease, and future experiments with OA animal models will help in establishing expression levels of the BMP antagonists in the early stages of the disease.
In summary, new players in the field of OA pathophysiology have been identified. The study of the biologic functions of follistatin and gremlin is a challenging new area of research and may open new directions for the treatment of OA.