MMP-19 Deficiency Is Hepatoprotective In Vivo Figure 4. MMPs expression and processing during liver damage progression. (A, B) MMP-2 and MMP-9 expression and activity in liver lysates was analyzed using gelatin zymography. (A) At 4 weeks, WT livers showed higher MMP2 processing and pro-MMP-2 and pro-MMP-9 expression. n = 4 for each strain. (B) At 6 weeks, levels of activated MMP-2 were significantly increased in MMP19KO liver and pro-MMP2 and -MMP9 expression was comparable in WTs and MMP19KOs. n = 6 for each strain. (C) Western blot analysis showed increased collagen IV expression in MMP19KO liver at 4 weeks of CCl4. As loading control, the membrane was stained with Ponceau S. n = 10 for each strain. (D) mRNA analysis showed slightly higher collagen IV expression in MMP19 KO than in WT mice. n = 5 animals of each strain analyzed at each time point. (E) qRT-PCR analysis showed slightly reduced expression of MMP13 mRNA levels in MMP19KO mice; 4–9 mice of each strain were analyzed at each time point. *p,0.05, **p,0.01 MMP19KOs vs. WTs. doi:10.1371/journal.pone.0046271.g004 ting analysis revealed slightly increased phosphorylation of Akt in MMP19KOs in the recovery period (Figure 5B), showing a higher activity of this anti-apoptotic signal in the MMP19KOs. Phosphorylation of IRS1 was slightly higher in MMP19KO livers at 6 weeks of CCl4-treatment although this reversed and after 10 days of recovery, pIRS1 was significantly lower in MMP19KO than in WT livers (Figure 5C). Analysis of IGFBP3, an MMP19 substrate involved in IGF- mediated signaling, showed significantly decreased protein levels in untreated MMP19KO livers compared to untreated WT livers (Figure 5D). This difference was smaller after 4 weeks of intoxication (not shown) and the levels of IGFBP3 at 6 weeks were almost identical between WTs and MMP19KOs (Figure 5D). As previously described [34], IGFBP3 processing was significantly diminished in MMP19KO livers compared to WTs (Figure S5). IGFBP3 protein expression was similar between MMP19KO and WT livers during the recovery phase, likely reflecting the increase in IGFBP3 expression as IGFBP3 mRNA levels increased during the recovery phase by 4 fold in both MMP19KO and WT livers compared to the mRNA expression during the intoxication phase (Figure S5). IGF-1 mRNA levels also showed a trend toward being higher in MMP19KOs compared to WTs during the later recovery period (Figure 5E). Thus, these data suggest that MMP-19-deficiency results in lower TGF-ß-mediated hepatocyte damaging signaling and higher IGF-1-mediated, anti-apoptotic and regenerative activity. Primary MMP-19-deficient hepatocytes show decreased reaction to TGFß-1 treatment ex vivo As CCl4-intoxication targets primarily hepatocytes, we used primary hepatocytes to further understand the protective mech- anisms in MMP19KO livers. We first studied the effect of CCl4 treatment on hepatocytes seeded on collagen I and observed no differences in ALT and AST levels between WT and MMP19KO cells (not shown). Further, the hepatocytes susceptibility to TGF- ß1 stimulation was tested. We measured mRNA expression levels of vimentin and Snail1 in both WT and MMP19KO hepatocytes after TGF-ß1 treatment and studied phosphorylation of the Akt signaling molecule. Vimentin and Snail 1 mRNAs were expressed at low levels in untreated hepatocytes (Figure 6A, B). TGF-ß1 PLOS ONE | www.plosone.org 5 October 2012 | Volume 7 | Issue 10 | e46271