earlier phases of liver injury might also contribute to inhibition of TGF-ß-signaling. Along the same line, phosphorylation of Akt showed an increasing trend in MMP19KO livers during the recovery period, suggesting higher anti-apoptotic signaling either via IGF-1 or another pathway. The Akt pathway also controls a specific cell division program that leads to generation of binucleated tetraploid liver cells and inhibition of Akt was shown to reduce numbers of binucleated cells [49]. Indeed, higher frequencies of binucleated cells in MMP19KO mice in the absence of cellular proliferation markers correlated with increased levels of pAkt. Moreover, there was also a clear tendency towards higher activation of IRS1 during the progression of liver injury, further supporting the possibility that IGF-1 signaling may impact the better outcome in the injury in MMP19KOs. Also, as the hepatocytes are the main source of IGF-1 in liver [50], it is obvious that lower hepatocellular damage in the case of MMP19KO animals by itself may contribute to better regeneration of the disease in these animals. Interestingly, we observed reduced phosphorylation of IRS1 in MMP19KO animals during the recovery period indicating possible deregulation of IGF-1 signaling pathway or other defect in the MMP19KO animals during the recovery. This situation might be reflecting the increased MMP-19 mRNA level in WT animals during recovery suggesting thus an additional role for MMP19 within the recovery period when it is necessary to degrade fibrotic matrix and/or increase the IGF-1 mediated anti-apoptotic signaling. It is plausible, that the increased levels of IGF-1 mRNA levels during the recovery period in MMP19KO animals are part of compensatory mechanism for the impaired IGFBP3 processing and expression, allowing thus for higher levels of bioavailable IGF-1, as there has to be a very stringent regulation of both of these proteins in the liver [50,51]. There is an increasing evidence that hepatocytes could play a crucial role in orchestrating the TGF-ß1-mediated pro-fibrotic response to chronic exposure to hepatotoxic agents [52]. Our observation that primary hepatocytes isolated from MMP19KO livers express lower levels of vimentin and Snail1 (i.e. genes directly linked to fibrosis progression) mRNA in response to TGF- ß1 stimulation suggests that the MMP19KO hepatocytes are less susceptible to TGF-ß1. This is in agreement with the observation of tendency toward higher phosphorylation of Akt in the MMP19KO hepatocytes as Akt causes resistance to TGF-ß1- induced apoptosis [53]. Consequently, activation of the profibrotic programs in MMP19KO hepatocytes, including those controlled by Snail1 [52], is delayed in comparison to WT cells. Provided that down-regulation of Snail1 in hepatocytes leads to slower fibroproliferative disease progression [52], it seems plausible that the lower activation of Snail1 in MMP-19-deficient hepatocytes not only reflects the observed situation in vivo but might also represent a plausible mechanism mediating hepatoprotective effect of MMP-19-deficiency. Thus we showed that MMP-19 plays an essential role in the development of hepatic damage by toxins and subsequent fibrosis as it affect the destruction and remodeling of hepatic basement membrane components, which in turn influence TGF-ß and IGF- 1 signaling pathways and leads to differential expression and activation of other MMPs. As MMP-19 negatively impacts the initial phase of liver injury and may play an additional role during the fibrosis recovery, it may serve as a valuable target for pharmacologic treatment of liver pathologies. MMP-19 Deficiency Is Hepatoprotective In Vivo Supporting Information Materials and Methods. (DOC) Methods S1 Detailed Materials and Figure S1 No differences in ALT, AST, ALP, and bilirubin serum levels in untreated and olive oil-treated control WT or MMP19KO mice were observed. Control mice were treated with olive oil twice a week for 6 weeks. n = 4 for both strains. (JPG) Figure S2 CCl4 intoxication leads to ameliorated liver damage in MMP19KOs. (A) After 4 weeks of CCl4 intoxica- tion, H&E staining showed centrolobular hepatocyte damage more serious in WTs than in MMP19KOs. Sirius red staining revealed larger fibrotic areas in WTs than in MMP19KOs. Images were originally obtained at 1006 (H&E) and 506 (Sirius red) magnification, scale bars = 200 mm. (B) Fibrosis, as measured by image analysis of Sirius red staining, was slightly larger in WT than in MMP19KO mice, 5–9 animals of each strain were analyzed at each time point. (JPG) Figure S3 MMP19 mRNA expression during fibrosis development and resolution in WT animals, n = 5. (JPG) Figure S4 MMP19KO and WT livers show no difference in pro-MMP2 and -MMP9 expression in olive oil-treated control animals. (A,B) Gelatinase zymography showed no difference in pro-MMP2 and -MMP9 expression in animals treated with olive oil and no activated MMP2 form expression. (C) No differences were observed in MMP2 mRNA expression after 4 weeks of CCl4 treatment between WT and MMP19KO livers; n = 5 olive oil controls, 8 WT and 7 MMP19KO mice. (JPG) Figure S5 As previously described, IGFBP3 degradation is lower in MMP19KO livers. (A, B) IGFBP3 degradation products were lower in MMP19KO livers after 4 and 6 weeks of CCl4 treatment. (C) During the recovery period, the IGFBP3 mRNA increased 4-fold in both WT and MMP19KO mice. 4–7 mice from each strain were analyzed at each time point. (JPG) Table S1 MCP1 and KC levels in MMP19KO and WT mice after 4 or 6-week treatment with CCl4 were measured in serum using an Elisa kit. (DOC) Acknowledgments We thank Eliska Machalova, Zuzana Dvorakova, and Ivana Marsalkova for their outstanding technical assistance, Dr Eva Honsova, Department of Pathology, Institute for Clinical and Experimental Medicine, Prague, for supportive discussions, and Dr Petr Stavek, Laboratory of Atherosclerosis Research, Institute for Clinical and Experimental Medicine, for help with serum liver enzyme analysis. The authors are grateful to Dr Epp for critical reading of the manuscript. Author Contributions Conceived and designed the experiments: M. Jirouskova OZ MG KC LS RS. Performed the experiments: M. Jirouskova OZ MG KC LS. Analyzed the data: M. Jirouskova OZ MG KC LS MH JE M. Jirkovska. Wrote the paper: M. Jirouskova OZ MG KC RS. PLOS ONE | www.plosone.org 8 October 2012 | Volume 7 | Issue 10 | e46271