Abnormal hypertrophy with subsequent ossification of articular chondrocytes plays a pivotal role in OA pathogenesis. Therefore, prevention of such changes is a rational strategy for the development of novel OA therapeutics. Studies using different genetic mouse models demonstrate that intact TGF-β signaling is essential for the maintenance of healthy articular cartilage [17
Treatment of OA by intra-articular injection of TGF-β has produced contradictory results, suggesting the involvement of several different downstream effectors [3
]. In addition to the classical TGF-β-Smad pathway, the TAK1-MKK3/6-p38-ATF-2 signaling pathway also mediates the effect of TGF-β on chondrocytes. Notably, constitutive activation of MKK6 in cartilage inhibits chondrocyte hypertrophy [28
]. Consistent with this finding, the genetic inhibition of p38 activity in cartilage leads to the development of severe OA [29
]. In addition, ATF-2 deficient mice show a dysregulated differentiation process in the epiphyseal growth plate leading to a phenotype similar to human chondrodysplasia [32
]. Previous studies suggest that the target genes of ATF-2 include chondrocyte/osteoblast-related genes such as fibronectin, osteopontin, osteocalcin, and alkaline phosphatase; as well as cell cycle related genes such as cyclin D1, cyclinA, cFos and pRb [32
]. Our present study shows that retroviral delivery of ATF-2 to mouse primary chondrocytes inhibits their terminal differentiation and blocks BMP-2-mediated effects. In addition, ATF-2 over-expression represses abnormal hypertrophic changes in Smad3−/−
chondrocytes, while dn-ATF-2 has the opposite effect. Collectively, TAK1-ATF-2 signaling inhibits chondrocyte maturation and disturbance of this pathway may, therefore, play a role in the pathogenesis of OA.
We have previously reported that ATF-2 works synergistically with Smad3 to inhibit hypertrophic changes in chicken chondrocytes [30
]. The present study provides further insight into the molecular basis for the crosstalk between the Smad3 and ATF-2 pathways. Taking advantage of the Smad3 mutant mouse model, we demonstrate here that TAK1-ATF-2 signaling is disrupted in Smad3−/−
chondrocytes at the level of p38 activation. Consequently, the DNA-binding ability of ATF-2 is decreased in Smad3−/−
cells. Reintroducing Smad3 into Smad3−/−
chondrocytes restores their response to TGF-β with regard to p38 phosphorylation. The results indicate that Smad3 is indispensable for TGF-β signal transduction along the TAK1-ATF-2 pathway.
In the classical TGF-β signaling pathway phosphorylated Smad3 forms a complex with Smad4 and this complex then translocates to the nucleus to regulate gene expression. The present study shows that Smad4-deficient cells can still activate p38 in response to TGF-β. These experiments imply that the disruption of p38 activation in Smad3-deficient cells involves a cytoplasmic mechanism. These findings are somewhat surprising because the classic role of Smad3 is that of transcriptional regulation in the nucleus. However, based on our observations, we hyposthesize that Smad3 maintains p38 in its phosphorylated state. Indeed, our immunoprecipitation experiments reveal that Smad3 forms a complex with phospho-p38. Whether TGF-β-inducible phosphorylation of Smad3 is also involved in this interaction remains to be determined. Transfection with either full-length Smad3 or different Smad3 domains indicates that the phospho-p38 binding site in the Smad3 molecule is located between the MH1 and linker domains. Thus, Smad3 has genomic and non-genomic functions that are necessary for the maintenance of healthy articular cartilage.
The magnitude and duration of MAP kinase activity is regulated by MKPs. MKPs dephosphorylate and inactivate MAP kinases through a complex negative regulatory network [41
]. TGF-β-induced p38 activation is associated with a concomitant induction of MKP-1 [42
]. Our results show that in the presence of TGF-β, MKP-1 transfection significantly reduces the level of phosphorylated p38. Co-transfection of Smad3 with MKP-1 partially blocks MKP-1 mediated dephosphorylation of phospho-p38. The p38 kinase assay further confirms that upon over-expression, Smad3 prevents MKP-1-induced p38 dephosphorylation and thus sustains the activation of p38. We, therefore, conclude that the physical interaction of Smad3 with phospho-p38 prevents its dephosphorylation by MKP-1 and that this mechanism accounts for the disruption of the TAK1-ATF-2 signaling pathway in Smad3-deficient chondrocytes.
While Smad3 and ATF-2 are likely important in the pathogenesis of OA, transcription factors are poor drug targets. Modulation of the upstream regulatory components, however, may be a more practical approach for the development of novel OA therapies. Downregulation of Col10a1 expression and upregulation of Col2a1 expression in chondrocytes was achieved by treating cells with anisomycin, an activator of p38 kinase activity. Additionally, we showed that anisomycin abrogates the enhanced Col10a1 expression observed in Smad3−/− chondrocytes. These effects closely mimic the effect of ATF-2 in chondrocyte differentiation, suggesting a possible application in OA. Our immunohistochemical analysis also revealed that p38 is expressed in articular cartilage and perichondrium (data not shown), strengthening the rationale for use of p38 activators to treat OA.
Matrix metalloproteinases (MMPs) may be involved in the pathogenesis of OA and their regulation by p38 kinase in chondrocytes should be considered. MMP-13, for example, metabolizes type II collagen and is upregulated in osteoarthritic articular chondrocytes [43
]. Mice deficient in MMP-13 exhibit decreased cartilage erosion in a surgically induced model of OA while mice over-expressing MMP-13 in chondrocytes develop an osteoarthritic phenotype [44
]. Several signaling pathways, including TGF-β, are shown to induce MMP13
expression dependent upon p38 activation. In human gingival fibroblasts, however, activation of p38 alone was not sufficient to induce MMP-13 expression [46
]. Our previous studies show no change in MMP13
mRNA levels in chondrocytes from Smad3−/−
mice when compared to WT mice even though Smad3-deficiency leads to an osteoarthritic phenotype and, as shown here, a decrease in p38-ATF-2 signaling [19
]. Additionally, treatment of mouse limb bud chondrocytes with a p38 inhibitor produced only a modest decrease in levels of MMP13
]. Collectively, these data suggest that p38 signaling alone may not largely affect MMP13
expression in chondrocytes. Nevertheless, this will need to be confirmed in future experiments should an activator of p38 be considered for use in treatment of OA.
It is known that p38 activation can incite an inflammatory response such that p38 inhibitors are currently studied for their potential use in the treatment of RA [48
]. Compared to RA, OA shows more severe articular cartilage destruction but much milder synovial inflammation. With regard to the clinical application of a p38 activator for OA treatment, the potential complication of local inflammation must be overcome. In this respect, the avascular and aneural hyaline cartilage structure may respond differently than the cells residing in the well-vascularized and well-innervated synovial membrane. However, to completely eliminate the potential adverse effects of synovial inflammation, therapeutics must target a specific p38 isoform that is able to prevent abnormal maturation/ossification of articular chondrocytes without inciting joint inflammation if delivered intra-articularly. Therefore, four currently known p38 isoforms, namely, α(MAPK14), β(MAPK11), γ(MAPK12), and δ(MAPK13), were studied in chondrocytes. We examined the expression of p38 isoforms in mouse sternal chondrocytes by immunoblotting and detected isoforms α, β, and γ, but not δ, in these cells. Immunofluorescence labeling revealed a similar expression pattern in mouse articular chondrocytes. Future work will focus on the viral delivery of different p38 isoforms intra-articularly and observation of changes in articular cartilage and the synovial membrane to test their chondroprotective vs. inflammatory effects on cartilage and synovium, respectively. Once the most suitable isoform is identified, isoform-specific activators can be developed as potential new OA therapeutics.
In summary, the present findings establish that Smad3 is a critical molecule in the TGF-β pathway that is involved in both the initiation and progression of OA. Loss of Smad3 not only results in reduced TGF-β-Smad3 signaling but also leads to the disruption of TAK1-ATF-2 signaling. Our results demonstrate that Smad3 forms a complex with phosho-p38 preventing its dephosphorylation by MKP-1. Both loss of Smad3 and meniscal injury result in a significant repression of phospho-ATF-2 expression in articular cartilage. ATF-2 and its upstream activating kinase, p38 maintain cartilage homeostasis by inhibiting abnormal maturation of articular chondrocytes. Cartilage-specific activators of p38 isoforms may in the future provide a new avenue for the development of chondroprotective drugs for OA.