This was suggested by the known interaction with the KRTK motif of thrombospondin, the major activator of TGF-β in vivo,40 leading us Pirfenidone ic50 to propose that the KTFR sequence could play a similar

role in ADAMTS1 (Fig. 3). The inhibition of ADAMTS1-mediated activation of TGF-β by KTFR peptides indicates that the mechanism of interaction is similar—if not identical—to that reported for thrombospondin-mediated activation.24 In this case, a “conformational” mechanism is in full agreement with the results of our experiments using proteolysis-deficient ADAMTS1 mutants and protease inhibitors, which show that TGF-β activation by ADAMTS1 is independent of the proteolytic activity of the latter. Although it might occur via similar interactions, activation of TGF-β by ADAMTS1 and thrombospondin are unlikely to overlap in vivo. Thrombospondin is expressed in freshly isolated human HSCs,22 but we show that their activation induces a decrease in thrombospondin expression MG-132 research buy counterbalanced by a dramatic increase in ADAMTS1 expression. The physiological relevance of this activation process is fully supported by our finding that depleting ADAMTS1 in HSCs strongly diminishes the release of TGF-β-dependent transcriptional activity. A conformational model of TGF-β

activation by ADAMTS1 predicts that interfering with KTFR/SLKL interactions should lead to a decrease Terminal deoxynucleotidyl transferase in available active TGF-β. We demonstrate that such a mechanism is, indeed, at play in vivo, using a murine model of induced liver fibrosis. In full agreement with the activation pathway described above, injection of the KTFR peptide in CCl4-treated mice that develop liver fibrosis reduces the levels of biological markers associated with hepatic damage (Fig. 7). This conclusion is further borne out by the demonstration, using highly sensitive SHG analysis,41 that concomitant injection of KTFR dramatically reduces collagen deposition

associated with the early onset of fibrosis (Fig. 8). The full conservation of KTFR and LSKL motifs between humans and mice suggests that this important proof of concept can be extrapolated to humans, identifying ADAMTS1 as a new therapeutic target during chronic liver injury. Many factors have been implicated in TGF-β activation, including thrombospondin, proteases (e.g., plasmin, thrombin, and MMP), and integrins, but also heat, acid, reactive oxygen species, and mechanical force. All these components build up an environmental network, in which the role of each one is obviously part of the sum and depends on dynamic tissue changes, especially as the liver proceeds from a healthy to a fibrotic state.