MMP-19 Deficiency Is Hepatoprotective In Vivo Figure 5. Analysis of TGF-ß- and IGF-1-mediated signaling pathway mediators during hepatic fibrosis and its resolution. (A–C) SMAD3 (A), Akt kinase (B) and IRS1 (C) phosphorylation was analyzed by immunoblotting liver lysates of treated and control animals. (D) IGFBP3 protein expression was significantly lower in untreated MMP19KO than in WT livers. (E) qRT-PCR analysis showed slightly higher IGF-1 and slightly lower TGF-ß1 mRNA levels in MMP19KO livers than in WT livers during fibrosis resolution. *p,0.05, MMP19s vs. WTs. 4–7 mice from each strain were analyzed at each time point. In some instances, noncontiguous lanes from one Western blot are shown. doi:10.1371/journal.pone.0046271.g005 treatment resulted in an increase in their mRNA levels, although this induction was always lower in MMP19KO compared to WT cells. Similar to our observations in vivo, we also observed a trend toward slightly increased phosphorylation of Akt in MMP19KO than in WT hepatocytes, both untreated and treated with TGF-ß1 (Figure 6C). Based on these observations, we also analyzed mRNA expression of Snail1 in liver from treated animals. This analysis also showed slightly lower expression of Snail1 in MMP19KOs than in WTs after 4 weeks of CCl4 treatment (Figure 6D). Altogether, these results indicate a reduced susceptibility of MMP19KO hepatocytes to TGF-ß1 stimulation and are consis- tent with our observation of lower TGF-ß-mediated changes in MMP19KO livers in vivo. Discussion In the present study, MMP-19-deficient mice showed resistance to chronic liver injury caused by CCl4 intoxication and recovered faster than WT animals upon removal of the toxin. The protective effect of MMP19 deficiency was primarily a result of milder hepatocellular damage, as plasma ALT and AST levels were significantly lower in the knockout animals throughout the CCl4 treatment. We also observed decreased liver hydroxyproline content and fibrillar collagen deposition in MMP19KOs through- out disease progression and recovery together with lower phosphorylation of SMAD3, and slightly increased levels of Akt and IRS1 phosphorylation during injury development, suggesting the involvement of TGF-ß- and IGF-1-mediated signaling. Degradation of normal liver ECM contributes to the patho- genesis of liver fibrosis, particularly in the early stages of injury [36].The ameliorated disease development in MMP19KOs likely originates from slower kinetics of basement membrane destruction and ECM remodeling, as MMP-19 is known to process a number of basement membrane proteins such laminin 2c chain, tenascin C, collagen IV, and nidogen-1, which all are abundant compo- nents of normal liver ECM [20,22,23]. After 4 weeks of intoxication, we observed higher collagen IV protein levels in MMP19KO livers after 4 weeks of intoxication, suggesting decreased degradation of this protein. This observation also correlated with the expression and activity of both gelatinases. PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e46271