Osteoarthritis (OA) is the most common degenerative joint disease, affecting >25% of the US population age >18 years. The major pathologic changes of OA include abnormal articular chondrocyte maturation, progressive loss and destruction of articular cartilage, osteophyte formation, and subchondral sclerosis. The etiology of OA is multifactorial, including joint injury, obesity, aging, and heredity ([1-3]). There are currently no interventions to restore degraded cartilage or decelerate the progression of OA, as the precise signaling pathways involved in initiation and progression of OA are still poorly understood.
Ex vivo studies with tissues obtained from OA patients and in vivo studies with mutant mouse models suggest that the factors involved in development of OA include growth factors, such as transforming growth factor β (TGFβ) and Indian hedgehog (IHH), and signaling molecules, such as Smads, β-catenin, and hypoxia-inducible factor 2α (HIF-2α) ([4-8]). TGFβ signaling strongly inhibits chondrocyte hypertrophy and maturation. In canonical TGFβ signaling, the TGFβ ligand binds to TGFβ receptor type II (TGFβRII), which then phosphorylates the type I transmembrane serine/threonine kinase receptor. The activated kinase subsequently phosphorylates Smad2 or Smad3 (receptor-activated Smad), which then forms a heteromeric complex with Smad4 (common-mediator Smad), translocates into the nucleus, and interacts with other DNA binding proteins to regulate target gene transcription ().
Recent genetic manipulations suggest that dysregulation of TGFβ signaling induces development of OA via the TGFβ/Smad3 signaling pathway in chondrocytes ([4, 10]). Transgenic mice overexpressing a dominant-negative form of TGFβRII (dn-TGFβRII) in skeletal tissue were found to exhibit cartilage disorganization and progressive cartilage degradation resembling the features of OA in humans (). Similar to the dn-TGFβRII–transgenic mice, Smad3-knockout (KO) mice were also found to display progressive articular cartilage degradation and osteophyte formation (). A recent study showed that a single-nucleotide polymorphism in the Smad3 gene was correlated with the incidence of hip and knee OA in a cohort of 527 patients (). More recently, different types of Smad3 mutations were identified in patients with a syndromic form of aortic aneurysms and early-onset OA ([12, 13]). These observations strongly support the notion that the TGFβ/Smad3 signaling pathway in chondrocytes plays an essential role in the development of OA. However, the critical downstream target genes of TGFβ signaling involved in the development of OA remain unknown.
The progressive loss of articular cartilage is a fundamental feature of OA. Articular cartilage consists of a dense meshwork of interconnected collagen fibrils within which is embedded a rich matrix of negatively charged proteoglycans. The negative charge attracts ions and provides the tissue with a high osmotic pressure that resists compressive force and provides boundary lubrication (). Human clinical and animal studies have shown that matrix metalloproteinase 13 (MMP-13) plays a pivotal role during cartilage degradation. MMP-13 is a primary collagenase that preferentially cleaves type II collagen in articular cartilage (). Clinical investigations have revealed that MMP-13 expression is elevated in articular cartilage of OA patients (). Expression of the constitutively active Mmp13 gene leads to an OA-like phenotype in mice (). In addition to MMP-13, ADAMTS-5, the principal enzyme responsible for degradation of aggrecan in articular cartilage, plays a critical role during OA development. Studies have shown that expression levels of Adamts5 are significantly increased during OA development (). Deletion of the Adamts5 gene prevented articular cartilage degradation in mouse models of surgically or chemically induced OA ([19-21]).
To determine the mechanism of inhibition of TGFβ signaling in the development of OA, we generated chondrocyte-specific Tgfbr2Col2ER mice by breeding Col2-CreER–transgenic mice ([22, 23]) with Tgfbr2flox/flox mice. Gene deletion was induced by injection of tamoxifen into 2-week-old mice, and alterations in the articular cartilage were analyzed at ages 3 and 6 months. Our studies demonstrated that deletion of the Tgfbr2 gene at the postnatal/adult stage led to a severe OA-like phenotype. Our in vitro studies demonstrated that inhibition of TGFβ signaling up-regulated Mmp13 and Adamts5 expression in articular cartilage tissue. MMP-13 is a collagenase that mainly degrades type II collagen. ADAMTS-5 is an aggrecanase that degrades aggrecan. Type II collagen and aggrecan are the principal matrix components present in articular cartilage. Because both MMP-13 and ADAMTS-5 play critical roles in the development of OA ([12, 21, 24]), we reasoned that Mmp13 and Adamts5 might be the key downstream target genes of TGFβ signaling in articular chondrocytes during OA development. In this study, we demonstrated that deletion of the Mmp13 or Adamts5 gene in mice of the Tgfbr2Col2ER background significantly prevented the OA-like phenotype observed in Tgfbr2Col2ER mice, which suggests that MMP-13 and ADAMTS-5 are critical downstream targets of TGFβ signaling during OA development.
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Although dn-TGFβRII–transgenic mice and Smad3-KO mice have already been shown to have an OA-like phenotype ([10, 29, 34]), it remains unclear whether TGFβ signaling plays a critical role in postnatal OA development, since in dn-TGFβRII–transgenic mice and Smad3-KO mice, TGFβ signaling was inhibited at an early embryonic stage. The Col2-CreER–transgenic mouse model is a valuable tool, allowing chondrocyte-specific gene targeting in an inducible manner ([22, 23, 35]). In the present study, the Tgfbr2 gene was deleted specifically in chondrocytes at the postnatal stage. TGFβRIICol2ER mice exhibit a phenotype similar to that of human OA, including increased chondrocyte hypertrophy observed in the superficial zone of articular cartilage, tears and clefts in the articular surface, severe loss of articular cartilage tissue, osteophyte formation at the margins of cartilage tissue, especially at the late stage of disease (in 6-month-old mice), and subchondral sclerosis.
It is interesting to note that inhibition of TGFβ signaling at the postnatal stage leads to articular cartilage damage and a severe OA–like phenotype, while the morphology of growth plate cartilage is relatively normal. In the present study, we induced Tgfbr2 gene deletion at age 2 weeks to compare roles of TGFβ signaling in both articular and growth plate chondrocytes. We found that deletion of the Tgfbr2 gene in chondrocytes at the postnatal stage (in 2-week-old mice) led to a severe OA–like phenotype in articular cartilage but had no significant effect on growth plate cartilage, which suggests that TGFβ signaling plays a specific role in maintaining integrity of articular cartilage at the postnatal stage. To further determine changes in growth plate cartilage development, we also performed gene expression analysis using primary sternal chondrocytes isolated from Cre-negative and TGFβRIICol2ER mice. We found that expression of chondrocyte marker genes such as Runx2, Alp, Oc, Mmp13, and Col10 was not significantly changed (data not shown). We also found that there was no significant change in type X collagen protein expression in growth plate chondrocytes from TGFβRIICol2ER mice. This finding suggests that TGFβ signaling may play a specific role in maintaining integrity of articular cartilage at the postnatal and adult stages and in preventing abnormal differentiation of articular chondrocytes. This unique feature distinguishes articular chondrocytes from growth plate chondrocytes.
In the present study, we demonstrated that Mmp13 mRNA levels were significantly up-regulated in TGFβRIICol2ER mice. Our in vitro studies demonstrated that inactivation of TGFβ signaling stimulates Mmp13 gene transcription in a RUNX-2–dependent manner. Deletion of the Mmp13 gene significantly prevented the articular cartilage destruction observed in TGFβRIICol2ER mice. Treatment of TGFβRIICol2ER mice with an MMP-13 inhibitor also significantly inhibited articular cartilage degradation. While the MMP-13 inhibitor used in this study has nonspecific inhibitory effects on other MMPs, our studies suggest that MMP-13 inhibition is a possible therapeutic strategy for the treatment of OA. Although deletion of the Adamts5 gene did not protect against articular cartilage degradation and other features of OA observed in 6-month-old TGFβRIICol2ER mice, deletion of the Adamts5 gene fully prevented the articular cartilage defects observed in 3-month-old TGFβRIICol2ER mice. These results further suggest that up-regulation of Adamts5 may play an important role in the development of early-stage OA in TGFβRIICol2ER mice.
While deletion of the Mmp13 gene significantly prevented the degradation of articular cartilage observed in TGFβRIICol2ER mice, the rescue was incomplete. These findings are consistent with those in a study in which MMP-13Col2ER mice were subjected to meniscal injury (). There are 2 possible reasons for these findings. First, the Cre recombination efficiency mediated by Col2-CreER is ∼85% in articular chondrocytes; thus, MMP-13 activity in the other 15% of chondrocytes remains normal in TGFβRII/MMP-13–double-KO mice. Second, it is known that deletion of Adamts4 and Adamts5 genes protects against OA development ([19-21]), and deletion of the Adamts5 gene may be compensated for by ADAMTS-4, a member of the same family. In exploring the roles of both collagenase and aggrecanase in OA development in TGFβRIICol2ER mice, further investigation is required to determine if double deletion of the Mmp13 and Adamts5 genes could fully protect against the OA-like phenotype observed in TGFβRIICol2ER mice.
Deletion of the Mmp13 gene significantly prevented several features of OA observed in TGFβRIICol2ER mice, such as articular cartilage degradation, osteophyte formation, and subchondral sclerosis. However, Mmp13 deletion failed to inhibit chondrocyte hypertrophy, since type X collagen was still highly expressed in the superficial zone of articular cartilage in the TGFβRII/MMP-13–double-KO mice. This is probably because the Mmp13 gene is downstream of RUNX-2. Most hypertrophic chondrocyte marker genes are induced by the Runx2 gene, which is independent of Mmp13 gene expression. It would be interesting to determine whether deletion of the Runx2 gene completely prevents the OA-like phenotype observed in TGFβRIICol2ER mice.
The current study shows that TGFβ signaling regulates the expression of genes that are critical in the maintenance of the articular cartilage matrix. In the absence of TGFβ signaling, Mmp13 and Adamts5 are induced and lead to articular cartilage tissue degeneration and the development of OA. However, it is still not known how TGFβ signaling is reduced or inactivated during the development of OA in patients. Potential causes of TGFβ signaling inactivation in articular chondrocytes include a loss-of-function mutation in the Tgfbr2 gene or mutation of other genes responsible for mediating TGFβ signaling, such as Smad3. There are several lines of evidence that mechanical loading leads to inactivation of TGFβ signaling in bone cells (). Using immunohistochemistry, we recently demonstrated that levels of TGFβRII and phosphorylated Smad3 were significantly down-regulated in a mouse model of OA induced by meniscus injury (results not shown), which suggests that 1 potential mechanism of meniscus injury–induced OA may be mediated by down-regulation of TGFβ signaling in articular chondrocytes. Finally, tumor necrosis factor α and interleukin-1β are 2 important proinflammatory cytokines involved in the development of OA. Recent studies demonstrate that these cytokines inhibit TGFβ/Smad signaling in other cell types ([37-39]). Since many other signaling pathways and transcription factors are involved in OA development, it would be interesting to investigate the interaction of TGFβ signaling with other signaling pathways during OA development, such as the Wnt/β-catenin, IHH, and HIF-2α pathways.
Previous studies demonstrate that TGFβ signaling has different effects on cells at various stages of differentiation. In mesenchymal progenitor cells, TGFβ promotes chondrogenesis ([40, 41]). However, in chondrocytes, TGFβ inhibits differentiation and hypertrophy ([42, 43]). It would be interesting to know whether inhibition of TGFβ signaling in mesenchymal progenitor cells leads to a phenotype similar to the one we observed in the current study. This question could be addressed by generating and analyzing Prx1-CreER/TGFβRIIflox/flox mice or Nestin-CreER/TGFβRIIflox/flox mice. It has been reported that exogenous application of TGFβ may cause fibrosis and osteophyte formation ([36, 44]). In the present study, we found that deletion of the Tgfbr2 gene does not cause synovial fibrosis and does lead to slight osteophyte formation. A possible explanation for this discrepancy is that we have selectively deleted the Tgfbr2 gene in articular chondrocytes. However, exogenous application of TGFβ could nonspecifically target other tissues in the joint.
In the present study, we induced Tgfbr2 gene deletion in 2-week-old mice. Since mice are still growing at this stage, which may affect articular cartilage structure, further investigation is still required using deletion of the Tgfbr2 gene in mice 2 months of age or even older. Using Col2-CreER– and Aggrecan-CreER–transgenic mice, we are currently testing whether deletion of the Tgfbr2 gene at different ages of adult mice (2, 4, and 6 months) will also produce an OA-like phenotype. These studies will help us determine the effect of Tgfbr2 gene deletion on OA development and progression in adult mice.
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All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Chen had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Study conception and design. Im, O'Keefe, Chen.
Acquisition of data. Shen, Li, B. Wang, Jin, M. Wang, Im, Chen.
Analysis and interpretation of data. Shen, Zhang, Yang, Im, O'Keefe, Chen.