The contraction processes in wound and fibrotic tissue mainly depend on a specialised form of fibroblasts known as myofibroblasts, which express the procontractile protein α-SMA. It is also well know that a number of integrins are responsible for cell contraction within different types of cells [39, 40]. Our previous data had found that dermal fibroblasts from SSc lesions are characterised by enhanced contractile ability of SSc fibroblasts and expression of a cohort of overexpress profibrotic genes, including α-SMA and integrins [7, 10]. TGFβ1 is a key factor in mediating both in fibroblasts' participation in wound repair and in a promoting pathological fibrosis, including SSc. Treatment of fibroblasts with TGFβ results in their differentiation into myofibroblasts and also stimulates their production of extracellular matrix, and adhesive proteins such as integrins [7, 41]. In monolayer culture, TGFβ is partially responsible for the phenotype of lesional SSc fibroblasts [7, 10]. However, it remains unclear whether activation of TGFβ signalling plays a role in ECM contraction in three-dimensional models of contraction. The data presented in this investigation shown that TSP1 is tightly linked with the enhanced contractility of SSc fibroblasts in the context of a three-dimensional culture system, as knockdown of the TSP1 gene or a blocking anti-TSP1 peptide, which prevents activation of latent TGFβ, reduced the cell contractility of fibrotic SSc fibroblasts. In parallel, antagonising TSP1 impaired expression of α-SMA, integrin α3, and integrin β5. Blocking TSP1 expression and activity also reduced the basal contractility of normal fibroblasts. We have found that endogenous TGFβ signalling contributes to the basal contractility of normal and SSc fibroblasts in three-dimensional FPCL. The results from our current report indicate that increased activation of latent TGFβ by TSP1 contributes to the overall activity of exogenous TGFβ during the process of ECM contraction in a three-dimensional culture. After mechanical loading of fibroblasts within the FPCL system, TGFβ activity and TSP1 expression were increased. All these results indicate that TSP1 contributes to the contractile ability of fibroblasts by promoting myofibroblast differentiation by TGFβ. Our data are also consistent with the notion that TSP1 is a key mediator contributing to the enhanced contractile ability displayed by lesional SSc dermal fibroblasts. In summary, blocking TSP1 may be a viable antifibrotic strategy.
The ability of TGFβ1 to induce TSP1 in fibroblasts is ERK dependent . TSP1 can also induce ERK phosphorylation via β1 integrin . Prior data from our laboratory have shown heparan sulfate-dependent ERK activation contributes to the enhanced contractile ability demonstrated by lesional dermal scleroderma fibroblasts . Consistent with these results, in the current study we have shown that anti-TSP1 strategies not only reduced fibroblast contractility but also decreased ERK activation in fibroblasts subjected to ECM contraction and mechanical loading. We have also shown that TGFβ and PDGF-induced contractility in normal and SSc fibroblasts corresponded with elevated expression of TSP1 and ERK activation. It has been shown that TSP1 can bind and stabilise PDGF, enhancing the biological effect of PDGF in proliferative tissue repair . It is interesting to note that the overexpression of TSP1, whether induced by TGFβ and PDGF in normal fibroblasts or basally in SSc lesional dermal fibroblasts, was inhibited by the MEK/ERK inhibitor (U0126). All these results indicate that, as an endogenous activator of TGFβ, TSP1 contributes to the pathological contractile activity of SSc fibroblasts. Moreover, TSP1 may also potentially mediate responses to PDGF in the pathogenesis of SSc. Our results are consistent with a previous suggestion that constitutive overexpression of TSP1 in SSc fibroblasts depends on autocrine TGFβ signalling .
Lesional SSc dermal fibroblasts overexpress syndecan 4, CCN2 and TSP1 [7, 9, 10]. CCN2 is expressed by mesenchymal cells undergoing active tissue remodelling, and is characteristically overexpressed in connective tissue pathologies such as fibrosis and cancer [6, 44]. Heparan sulfate chains of syndecan 4 mediate response to growth and differentiation factors such as TGFβ . Syndecan 4 also binds CCN and acts as a coreceptor for CCN2 . Although the precise nature of the interactions among syndecan 4, CCN2 and TSP1 is still unclear, our previous investigations found low expression of TSP1 in fibroblasts isolated from syndecan 4 -/- or CCN2 -/- mice [11, 47]. In our current study, TSP1 knockdown with siRNA did not alter expression of syndecan 4 and CCN2. Collectively, these results suggest expression of TSP1 in fibroblast culture is downstream of both syndecan 4 and CCN2. It has been reported that, in a mouse model of arthritis, injection of TSP1 blocking peptides for 16 days reduced joint infiltration and inflammation and CCN2 message and protein levels . However, this reduced CCN2 could result indirectly due to the ability of TSP1 to activate latent TGFβ. Alternatively, a mechanism involving activation of cell types other than fibroblasts might be involved. Therefore, whether TSP1 directly affects CCN2 expression in vivo in SSc still needs to be investigated.
We have previously shown that the ras/MEK/ERK 'classical' MAP kinase cascade is important for several features of fibrogenesis. For example, MEK/ERK mediates the induction of CCN2 expression in normal mesenchymal cells [48, 49]. In addition, MEK/ERK is required for α-SMA stress fibre assembly, via a syndecan 4-dependent mechanism [7, 10]. Moreover, the enhanced constitutive ERK activation in lesional SSc fibroblasts is due to an increase in syndecan 4 expression [7, 10]. The MEK-ERK pathway and HSPGs contribute to the overexpression of profibrotic proteins and enhanced contractile forces in SSc dermal fibroblasts, and the procontractile signals from TGFβ are integrated through syndecan 4 and MEK/ERK . TGFβ has long been hypothesised to be a major contributor to pathological fibrotic diseases . In this investigation we showed that TSP1-mediated TGFβ activation contributes to the pathological contractile activity of SSc fibroblasts via an ERK-dependent mechanism. In contrast, as a multifunctional cytokine, TGFβ is not only a key regulator of extracellular matrix assembly and remodelling but also affects a wide variety of cellular processes. Therefore, therapeutic strategies focusing on non-specific, systemic blockade of TGFβ ligand-receptor interactions may have a problematic side effect profile considering the complex function of TGFβ in vivo. Conversely, TSP1 is a multicellular protein that modulates cell functions and cell-matrix interactions . Abnormalities observed in TSP1-null animals resemble those observed in TGFβ1 deficient animals, but are much less severe . Collectively, our results suggest that, as compared to broad targeting of TGFβ, TSP1 may be an ideal therapeutic target for fibrotic diseases such as SSc.