MMP-19 Deficiency Is Hepatoprotective In Vivo Figure 6. Isolated hepatocytes from MMP19KO livers show impaired responsiveness to treatment with TGF-ß1. Primary hepatocytes were isolated from WT and MMP19KO livers, seeded on collagen I and stimulated with TGF-ß1. (A, B) mRNA levels of vimentin and Snail1 were measured in control, untreated hepatocytes, and hepatocytes stimulated with TGF-ß1 for 6, 24, or 48 h. (C) Phosphorylation of Akt was slightly increased in MMP19KO compared to WT hepatocytes. Noncontiguous lanes from one Western blot are shown. n = 5 different hepatocyte isolations from both WTs and MMP19KOs. (D) mRNA levels of Snail1 in livers from animals treated with CCl4 for 4 weeks showed slightly lower expression of Snail1 in MMP19KOs than in WTs. 4–7 mice from each strain were analyzed for each time point. *p,0.05 MMP19KOs vs. WTs. doi:10.1371/journal.pone.0046271.g006 During the earlier phase of injury, the expression of MMP-2 and MMP-9 and processing of MMP-2 was weaker in MMP19KOs. This lower expression and processing of gelatinase, in turn, likely contributes also to lower processing of collagen IV in MMP19KO livers as MMP-2 efficiently cleaves this type of collagen [37]. After 6 weeks of injury, the expression of both gelatinases was comparable between MMP19KOs and WTs; however, the proportion of activated form of MMP-2 was significantly higher in MMP19KOs. This finding might be pointing to the activity of an additional protease, which converts pro-MMP-2 into the active form. Such a protease might be MMP-14 (MT1-MMP, a membrane type-1 MMP) that together with TIMP-2 activates MMP-2, as all these components are expressed in injured liver [38,39]. Moreover, MT1-MMP can be upregulated by IGF-1 [40] whose signaling we also observed to be higher in MMP19KOs. The role of MT1-MMP in liver pathology has not been established yet, however, its direct proteolytic activity towards ECM and the activation of MMP2 in the initial phase of liver damage could accelerate the destruction of normal ECM-structure in the liver. Proteolytic activity of MT1-MMP towards collagen I could be beneficial for the resolution of fibrosis [41]. Moreover, MMP-2 appears to also have a protective role in mouse models of liver fibrosis by inhibiting collagen I synthesis and suppressing TIMP-1 upregulation [11] which could thus contribute to lower fibrosis in MMP19KOs. Interestingly, the MMP13 mRNA induction was impaired in MMP19KOs during the initial phase of injury, returning to WT levels during recovery. As MMP-13 was reported to affect acceleration of fibrogenesis in cholestatic livers by mediating the initial inflammation [12], it is likely that downreg- ulation of this interstitial collagenase might be involved in the decreased development of injury in MMP19KOs. This is also in agreement with our observation of increased levels of IGF-1- mediated signaling as IGF-1 was reported to downregulate MMP13 expression [42]. These data suggest that MMP-19 contributes to liver injury, not only by direct processing of components of normal liver ECM, but also by affecting expression and activity of other MMPs probably via the IGF-1 signaling pathway. Liver injury leads to massive hepatocyte necrosis and hepatic stellate cell activation and is accompanied by decrease in IGF-1 serum levels and induction of TGF-ß. TGF-ß-mediated signaling is pro-apoptotic [43], inhibits hepatocyte proliferation [44], and it was shown that interruption of TGF-ß signaling results in diminished liver fibrosis [45,46]. IGF-1 reduces formation of fibrosis and enhances liver regeneration [47]. Our data clearly show tendency toward lower TGF-ß-mediated signaling in MMP19KO animals, as we observed less SMAD3 phosphoryla- tion at the end of chronic injury and during the recovery period. As MMP-2 and MMP-9 are both able to activate latent TGF-ß1 [48], lower amounts of active gelatinases in MMP19KOs in the PLOS ONE | www.plosone.org 7 October 2012 | Volume 7 | Issue 10 | e46271