We investigated the role of TGFβ in two osteoarthritis models reflecting aspects of human osteoarthritis. TGFβ3 and TGFβ signalling, assessed by SMAD‐2P staining, were studied. TGFβ induces SMAD‐2 phosphorylation in a time‐dependant and dose‐dependent manner, giving an insight into active TGFβ signalling.4,18,19
One of the main features of osteoarthritis is destruction of cartilage, which is preceded by loss of proteoglycans. In both osteoarthritis models proteoglycan loss is accompanied by reduced TGFβ3 and SMAD‐2P staining. Osteoarthritis lesions in the spontaneous model were predominantly observed on the medial side of the joint corresponding with previous findings of Dunham et al
describing progressive disorganisation of proteoglycans in the medial cartilage plateau of STR/ort mice younger than 30 weeks of age.3
We observed that TGFβ3 and SMAD‐2P staining also diminished fastest on the medial side in both osteoarthritis models. The STR/ort mice were studied over a longer time period, enabling us to study more progressive cartilage damage. The severely damaged articular cartilage was totally negative for TGFβ3 and SMAD‐2P, whereas intact cartilage, even in older STR/ort mice, contained TGFβ3 and SMAD‐2P immunopositive cells. Supportive of our findings, Verdier et al22
showed decreased TGFβ expression in degraded human osteoarthritis cartilage and diminished TGFβ II receptor expression in fibrillated cartilage. Furthermore, Wang et al23
found a correlation between expression of TGF‐β II receptor/TGFβ1 and intracellular levels of tissue inhibitor of metalloproteinase in human cartilage chondrocytes. Tissue inhibitors of metalloproteinase inhibit matrix metalloproteinases, thereby facilitating accumulation of ECM products, indicating a role for the TGFβ pathway in ECM homeostasis of the cartilage. Kizawa et al24
show an asporin polymorphism in which asporin D14 has a greater inhibitory effect on TGFβ‐mediated expression of cartilage matrix gene than does the common form D13. The D14 variant was over‐represented in people with arthritis, indicating the lack of TGFβ responsiveness and its association with osteoarthritis progression.24
Taken together, these data strongly suggest that loss of TGFβ signalling is associated with cartilage damage.
In the papain model, TGFβ was found to be increased in damaged cartilage.25
However, papain breaks down the ECM of the cartilage, thereby directly affecting cartilage integrity. It can be anticipated that chondrocytes respond differently to this insult when compared with instability‐induced cartilage damage, probably involving different roles for TGFβ.26,27,28,29
To confirm that our findings were not the effect of an overall drop in cell viability in osteoarthritis‐affected cartilage, but were TGFβ specific, we studied the expression of another TGFβ family member, BMP‐2. In contrast with TGFβ3, BMP‐2 was increased with osteoarthritis progression, indicating that the reduced TGF‐β3 expression is not simply the result of decreased cell viability.
Another prominent feature of osteoarthritis is osteophyte formation. The chondrocyte‐like cell clusters that are observed in early osteophyte development are all positive for TGFβ3 and SMAD‐2P. More developed, but not fully ossified, osteophytes have a core of bone‐like tissue covered with a layer of cartilage‐like tissue and an outer layer with fibroblast‐like cells. In this stage, TGFβ3 is observed in the bone marrow of the osteophyte. The cartilage‐like tissue is negative, whereas the fibroblast‐like cells are all positive for TGFβ3. In contrast, SMAD‐2P is found in every layer of the osteophyte. TGFβ3 expression is low to absent in this phase of development or obscured by matrix development. The first seems more plausible, in which case the TGFβ might diffuse from the fibrous layer to the cartilage, inducing TGFβ signalling. Either way, there is SMAD‐2P staining indicating active TGFβ signalling.
Overexpression of TGFβ in murine knee joints has been shown to induce osteophytes, whereas blocking endogenous TGFβ in an osteoarthritis model reduces osteophyte formation.3,5,6,11,25
These findings suggest an important role for TGFβ in osteophyte formation. However, in older STR/ort mice the osteophytes no longer express TGF‐β3 or SMAD‐2P. The osteophytes in the instability model have been monitored over a shorter time period, showing only the early phases of development. TGFβ seems to be particularly important in these phases. In later phases of osteophyte development, as seen in older STR/ort mice, TGFβ function is supposedly substituted by factors such as BMP. BMP is associated with osteophyte maturation30,31
and was found to be strongly expressed in late osteophytes in our experiment. Uchino et al13
described TGFβ expression in human osteophytes. They were not able to discriminate between different stages of osteophyte formation, which might explain why they found various amounts of TGFβ in osteophytes. However, the location of TGFβ expression mainly in the fibrous, superficial layer of the human osteophytes corresponds to our findings in mice. This confirms that the process of osteophyte formation observed in our murine osteoarthritis models closely resembles that in human osteoarthritis.
In the spontaneous osteoarthritis model, we observed ectopic bone formation in the collateral ligament. Collins et al32
also found these chondro‐osseous structures in synovia and ligaments in STR/ort mice. This ectopic bone formation is observed initially by red staining in the ligament in Safranin O‐stained sections, indicating proteoglycan deposition. This particular area contains cells positive for TGFβ3 and SMAD‐2P, whereas the rest of the ligament, which looks normal, is negative for both. Further changes in ligaments resemble those observed in developing osteophytes with respect to TGFβ3 and SMAD‐2P expression. In the early process of chondrogenesis TGFβ3 and SMAD‐2P are abundantly expressed, whereas fully developed pseudo joints are negative for TGFβ3 and SMAD‐2P, again suggesting a role for TGFβ in the early developmental stages of ectopic bone.
Although TGFβ expression is reduced in damaged cartilage, its expression is strongly increased in other compartments of the joint (osteophytes, but also synovial tissue). Therefore, it can be expected that TGFβ will still be released into the joint cavity and could ultimately reach other tissues. We show that TGFβ signalling in cartilage is down regulated or even absent in osteoarthritis‐affected cartilage. TGFβ that is produced outside the cartilage does not reach the chondrocytes, either because of scavenging by the extracellular matrix or because the cells have lost the ability to respond. The loss of ability to respond is in concordance with the loss in TGFβ receptor II expression, as has been shown in rabbits with osteoarthritis.34
Loss of the ability to respond to TGFβ might be related to high levels of proinflammatory cytokines such as IL1 and tumour necrosis factor present in osteoarthritis‐affected cartilage. The absence of TGFβ3 expression could also be attributed to the effects of proinflammatory cytokines. Struder et al35
have shown that proinflammatory cytokines can indirectly, via nitric oxide production, modulate TGFβ production. In contrast, IL1 and tumour necrosis factor have been shown to stimulate BMP‐2 expression, which could explain our findings of BMP‐2 up‐regulation during osteoarthritis progression.36
From our observations, we can conclude that TGFβ and SMAD‐2P expression is reduced in damaged cartilage and is completely absent in cartilage that has started eroding, suggesting a protective role for TGFβ in cartilage. TGF‐β and SMAD‐2P are up regulated during chondrogenesis, osteophyte and ectopic bone formation, but mainly in the early stages of osteophyte development. In progressive osteoarthritis, TGFβ expression in newly formed osteophytes is, although still present, reduced compared with that in the newly formed osteophytes in early osteoarthritis. At later stages of osteophyte development TGFβ and SMAD‐2P are no longer expressed, and other factors are probably involved in further progression of osteophyte formation.