Western blot assays were also per formed with fibroblasts treated with TSP1 siRNA. After TSP1 knockdown in fibroblasts from normal and SSc patients, p ERK activation was reduced, concomitant with decreased expression of integrin a3. Consistent with prior data sellekchem using an ALK5 inhibitor, extremely modest reduction of a SMA and integrin b5 were observed. Expression of CCN2 and syndecan 4 was not altered in normal and SSc fibroblasts confirming previous evidence that basal expression of these proteins is independent of the TGFb pathway. TSP1 expression and p ERK activation were enhanced by the external mechanical force loading stimulation It has been suggested that TSP1 plays a significant role in wound healing.

Fibroblasts loaded by biomechanical forces within the three dimensional FPCL system remodel their matrix resulting in potent differentiation into myofibroblasts similar to that observed in wound tis sue and pathological scarring. As our previous data suggested that TSP1 mediated activation of TGFb played a key role in matrix contraction by normal and fibrotic fibroblasts, we wondered if fibroblast induced ECM con traction itself was sufficient to induce TSP1 expression. Thus, fibroblasts from normal and SSc patients were mechanically loaded to a magnitude similar to that seen in skin wounds. During mechanical loading, cells within the FPCL system went through normal gel contrac tion for 12 h, after which cyclical mechanical forces were exerted on cells controlled by a computer. Each cycle con sisted of force loading for 9 min followed by a 15 min rest ing phase prior to unloading for an additional 9 min followed by a further 15 min resting phase.

Cycles were repeated 15 times for an additional 12 h. ERK activation contributes to the overexpression of fibrotic proteins and the enhanced contraction by lesional dermal scleroderma fibroblasts. Therefore, after force loaded gel contraction, TSP1 expression and p ERK activation were assessed by western blotting. We found that TSP1 expression and p ERK activation were significantly increased in force loaded fibroblasts isolated from both normal individuals and SSc patients. TSP1 expression is therefore regulated by contractile activity of fibroblasts within the three dimensional FPCL model. TSP1 is induced by PDGF and TGFb during fibroblast mediated matrix contraction Our previous GSK-3 research had demonstrated that TGFb enhanced contractile ability of fibroblasts partly depends on ERK activation.

Inhibiting the TGFb type I receptor reduced the contractility especially of fibroblasts, but not a SMA expression and stress fibre formation. Conversely, the PDGF/c abl inhibitor Gleevec reduced ECM contraction and a SMA expression. Previously, it was shown that the antifibrotic effect of interferon b in lung fibrosis occurred via inhibiting TGFb activation and decreasing TSP1/2 expression.