Attachment of SFs to LM‐111 in the presence of TGFβ induced a significant expression of MMP‐3, a matrix protease that has been known for its involvement in tissue destruction in RA for a long time.30,31
MMP‐3 has a rather broad substrate specificity and degrades a variety of extracellular matrix proteins, including proteoglycans, collagens, fibronectin and LMs.32
Hence, MMP‐3 plays an important role in the pathomechanisms of RA. The induction of MMP‐3 is associated with inflammatory cytokines, especially IL1β or TNFα.
The additive effects of attachment of SFs to LMs together with TGFβ have not been investigated in detail so far, but some hints may have been derived from studies using Matrigel for investigation of the invasive properties of SF.33
In the present study, we show that attachment of SFs to LM‐111 sensitises these cells to TGFβ‐mediated signalling and significantly increases the expression of MMP‐3, a relevant protease in RA.
In recent studies, RA‐SF showed a higher invasive score on Matrigel when compared with control cells, and this correlated significantly with a higher expression of MMP‐1, MMP‐3 and MMP‐10.33
Matrigel contains LM‐111 as a major component. It therefore may activate SFs in a way comparable to our experiments. Using a ribozyme approach to limit the expression of MMP‐1 in RA‐SF confirmed that this increased invasiveness of SF on Matrigel was associated with the expression of MMP‐1.34
Our data, presented here, provide a molecular explanation for these findings, as we see an increased expression of MMP‐1 in SF on attachment to LM‐111. Addition of anti‐TGFβ antibodies to SF attached to LM‐111 did not change the LM‐induced effects in our experiments (data not shown). This finding confirms that our LM‐111 preparations used did not contain TGFβ. For elevated production of MMP‐1, cosignalling of LM‐111 and TGFβ is not sufficient, but LM‐111 together with TGFβ significantly elevated the expression of both MMP‐3 mRNA and protein.
As shown recently, most extracellular proteins derived from articular cartilage or connective tissue enhanced the attachment of SF mediated by integrins on the SF.8
Stronger adherence of RA‐SF to cartilage oligomeric protein was associated with their expression of αvβ1‐integrin.35
In other studies, elevated expression of α6‐ and β1‐integrins on RA‐SF correlated with their enhanced attachment to matrix proteins.9
In mice lacking β1‐integrin expression in chondrocytes, cell motility and proliferation were impaired,36
and the expression of MMP‐3, MMP‐9 and MMP‐13 was significantly reduced in these cells.37
We corroborated these results as treatment of SF with mAb 4B4 blocked the attachment of SF to LM‐111 and significantly reduced the induction of MMP‐3. Further, the induction of MMP‐3 described here was induced by TGFβ and LM‐111, without addition of IL1β or TNFα to the cells.
TGFβ is a multifunctional factor regulating cell division and differentiation. Depending on the individual experimental context, TGFβ may activate or reduce proliferation and expression of target genes in a dose‐ and time‐dependent manner.38
This factor has also been shown to influence the homeostasis of the extracellular matrix.39
TGFβ may repress the PMA‐induced expression of MMP‐1 by SMAD signalling.40
Without prestimulation by PMA, TGFβ activates the expression of MMPs including MMP‐3 using a p38MAPK signalling pathway.41
In our experiments, addition of TGFβ activated the ERK and SMAD2 but not the p38MAPK pathways. The importance of SMAD2 for regulation of MMPs is supported by the fact that SMAD2 transgenic mice show elevated expression of MMP‐1 and MMP‐2.42
Activation of ERK1/2 by expression of a recombinant constitutively active MEK1 was sufficient to induce MMP‐1 and MMP‐3, but strong expression of MMP‐1 and MMP‐3 was observed when ERK1/2 were activated in combination with either SAPK/JNK or p38MAP kinases. Further, ERK was associated with the induction of transcription factors c‐jun, junB
, whereas p38α regulated MMP mRNA stability.24
In our experiments, SF from one patient failed to induce the expression of MMP‐3 when attached to LM‐111 in the presence of TGFβ. Investigation of these cells showed that phosphorylation of ERK had not occurred (data not shown). However, attachment to LM‐111 seems to address the ERK pathway in different mesenchymal cells.43
In our experiments, phosphorylation of p38MAPK was not observed by TGFβ alone but on coactivation by LM‐111 and TGFβ. We also quantified less MMP‐3 mRNA after blocking p38MAPK activity in activated SF by the kinase blocker SB203580, corroborating that p38MAPK is involved in LM‐111 and TGFβ induced expression of MMP‐3 expression. A very weak signal of phospho‐SAPK/JNK was noted in LM‐111 and TGFβ activated SF, but not in SF activated by TGFβ alone (see supplementary fig S2 and files at http://ard.bmj.com/supplemental
), indicating that additional MAP kinases are addressed by these stimuli.44
However, a detailed investigation of the contribution of other MAP kinases to the TGFβ and LM‐111 induced expression of MMPs in SF must await additional experiments.45
In summary, we show that attachment of RA‐SF and OA‐SF to LM‐111 in the presence of TGFβ is sufficient to yield a significant MMP‐3 expression. Our data corroborate the importance of ERK, p38MAPK and SMAD2 signalling for efficient MMP‐3 induction by TGFβ and attachment to LM‐111 in SF. Other cytokines such as IL1β or TNFα were not necessary for the expression of MMP‐3 under these conditions. We conclude that pathological expression of LMs in the synovium contributes to the elevated expression of MMP‐3 in RA. This pathway may also explain in part the progression of cartilage destruction when patients with RA are treated with anti‐TNF (or anti‐IL1) therapies, as levels of TGFβ did not change during etarnecept treatment.46