Transforming Growth Factor-β1 Induces an Epithelial-to-Mesenchymal Transition State in Mouse Hepatocytes in Vitro

Abstract

Liver fibrosis is a progressive pathologic process that involves deposition of excess extracellular matrix leading to distorted architecture and culminating in cirrhosis. The role of transforming growth factor-β (TGF-β) as a key molecule in the development and progression of hepatic fibrosis via the activation of hepatic stellate cells, among other fibroblast populations, is without controversy. We hereby show that TGF-β1 induces an epithelial-to-mesenchymal transition (EMT) state in mature hepatocytes in vitro. EMT state was marked by significant upregulation of α1(I) collagen mRNA expression and type I collagen deposition. Similar changes were found in a “normal” mouse hepatocyte cell line (AML12), thus confirming that hepatocytes are capable of EMT changes and type I collagen synthesis. We also show that in hepatocytes in the EMT state, TGF-β1 induces the snail-1 transcription factor and activates the Smad2/3 pathway. Evidence for a central role of the TGF-β1/Smad pathway is further supported by the inhibition of EMT by Smad4 silencing using small interference RNA technology. In conclusion, TGF-β1, a known pro-apoptotic cytokine in mature hepatocytes, is capable of mediating phenotypic changes and plasticity in the form of EMT, resulting in collagen deposition. Our findings support a potentially crucial role for EMT in the development and progression of hepatic fibrogenesis. Liver fibrosis is a progressive pathologic process that involves deposition of excess extracellular matrix leading to distorted architecture and culminating in cirrhosis. The role of transforming growth factor-β (TGF-β) as a key molecule in the development and progression of hepatic fibrosis via the activation of hepatic stellate cells, among other fibroblast populations, is without controversy. We hereby show that TGF-β1 induces an epithelial-to-mesenchymal transition (EMT) state in mature hepatocytes in vitro. EMT state was marked by significant upregulation of α1(I) collagen mRNA expression and type I collagen deposition. Similar changes were found in a “normal” mouse hepatocyte cell line (AML12), thus confirming that hepatocytes are capable of EMT changes and type I collagen synthesis. We also show that in hepatocytes in the EMT state, TGF-β1 induces the snail-1 transcription factor and activates the Smad2/3 pathway. Evidence for a central role of the TGF-β1/Smad pathway is further supported by the inhibition of EMT by Smad4 silencing using small interference RNA technology. In conclusion, TGF-β1, a known pro-apoptotic cytokine in mature hepatocytes, is capable of mediating phenotypic changes and plasticity in the form of EMT, resulting in collagen deposition. Our findings support a potentially crucial role for EMT in the development and progression of hepatic fibrogenesis. Liver fibrosis results from increased deposition of type I collagen within the hepatic extracellular space and constitutes a common cardinal signature to all forms of liver injury, regardless of etiology (1Friedman S.L. J. Biol. Chem. 2000; 275: 2247-2250Abstract Full Text Full Text PDF PubMed Scopus (1867) Google Scholar). End-stage liver fibrosis is recognized clinically as cirrhosis. Since their initial description, hepatic stellate cells (HSC) 3The abbreviations used are: HSC, hepatic stellate cells; TGF-β, transforming growth factor-β; EMT, epithelial-to-mesenchymal transition; siRNA, small interference RNA; ANOVA, analysis of variance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's media; PBS, phosphate-buffered saline; qRT, quantitative reverse transcription; α-SMA, α-smooth muscle actin; IP, immunoprecipitation; IF, immunofluorescence; DAPI, 4′,6-diamidino-2-phenylindole; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; IB, immunoblotting.3The abbreviations used are: HSC, hepatic stellate cells; TGF-β, transforming growth factor-β; EMT, epithelial-to-mesenchymal transition; siRNA, small interference RNA; ANOVA, analysis of variance; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DMEM, Dulbecco's modified Eagle's media; PBS, phosphate-buffered saline; qRT, quantitative reverse transcription; α-SMA, α-smooth muscle actin; IP, immunoprecipitation; IF, immunofluorescence; DAPI, 4′,6-diamidino-2-phenylindole; ERK, extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; IB, immunoblotting. have dominated the field of liver fibrogenesis (2Friedman S.L. Roll F.J. Boyles J. Bissell D.M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 8681-8685Crossref PubMed Scopus (737) Google Scholar, 3McGee J.O. Patrick R.S. J. Pathol. 1972; 106: viGoogle Scholar, 4Sato M. Suzuki S. Senoo H. Cell Struct. Funct. 2003; 28: 105-112Crossref PubMed Scopus (261) Google Scholar). Indeed, their role is central in hepatic fibrosis (5Reeves H.L. Friedman S.L. Front. Biosci. 2002; 7: D808-D826Crossref PubMed Scopus (420) Google Scholar). Unfortunately, despite several discoveries pertaining to HSC activation and mechanisms of collagen deposition, no substantial anti-fibrotic therapies have been developed in order to halt the progression to cirrhosis and or reverse established fibrosis. Although resident tissue fibroblasts are traditionally considered as the principal source of fibrosis, there has been increasing interest in the ability of epithelial cells to assume not only a mesenchymal phenotype (known as epithelial-to-mesenchymal transition (EMT)) but also to undertake mesenchymal function(s), i.e. contribute to fibrosis formation. Indeed, EMT has been established as a major mechanism for the deposition of extracellular matrix in renal and pulmonary fibrosis injury models (6Iwano M. Plieth D. Danoff T.M. Xue C. Okada H. Neilson E.G. J. Clin. Investig. 2002; 110: 341-350Crossref PubMed Scopus (1681) Google Scholar, 7Willis B.C. Liebler J.M. Luby-Phelps K. Nicholson A.G. Crandall E.D. du Bois R.M. Borok Z. Am. J. Pathol. 2005; 166: 1321-1332Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar, 8Xie L. Law B.K. Aakre M.E. Edgerton M. Shyr Y. Bhowmick N.A. Moses H.L. Breast Cancer Res. 2003; 5: R187-R198Crossref PubMed Scopus (105) Google Scholar). Several lines of evidence support an important role for TGF-β1 signaling in the initiation and progression of liver fibrosis (9Dooley S. Streckert M. Delvoux B. Gressner A.M. Biochem. Biophys. Res. Commun. 2001; 283: 554-562Crossref PubMed Scopus (67) Google Scholar). In mature (i.e. adult) hepatocytes, TGF-β1 is responsible for inhibition of cell proliferation and induction of apoptosis (10Bissell D.M. Roulot D. George J. Hepatology. 2001; 34: 859-867Crossref PubMed Scopus (335) Google Scholar, 11Gressner A.M. Weiskirchen R. Breitkopf K. Dooley S. Front. Biosci. 2002; 7: D793-D807Crossref PubMed Google Scholar, 12Oberhammer F.A. Pavelka M. Sharma S. Tiefenbacher R. Purchio A.F. Bursch W. Schulte-Hermann R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5408-5412Crossref PubMed Scopus (679) Google Scholar). Interestingly, TGF-β1 is the most established mediator and regulator of EMT (13Zavadil J. Bottinger E.P. Oncogene. 2005; 24: 5764-5774Crossref PubMed Scopus (1358) Google Scholar). It has been shown that TGF-β1 mediates EMT by inducing snail-1 transcription factor and tyrosine phosphorylation of Smad2/3 with subsequent recruitment of Smad4 (14Vega S. Morales A.V. Ocana O.H. Valdes F. Fabregat I. Nieto M.A. Genes Dev. 2004; 18: 1131-1143Crossref PubMed Scopus (659) Google Scholar, 15Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (891) Google Scholar, 16Zeisberg M. Kalluri R. J. Mol. Med. 2004; 82: 175-181Crossref PubMed Scopus (419) Google Scholar). Hepatocyte Isolation and Cell Culture—12-Week-old C57BL-6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were anesthetized with ketamine (50 mg/kg intraperitoneally) and xylazine (10 mg/kg intraperitoneally). Livers were perfused in situ through the portal vein, initially with calcium- and magnesium-free Hanks' balanced salt solution (Invitrogen) containing 0.5 mm EDTA (Invitrogen) followed by Dulbecco's modified Eagle's media (DMEM; Invitrogen) containing collagenase (Sigma) until the liver lost its firm texture. The soft liver was removed and gently shaken in DMEM containing collagenase at 37 °C for 10-15 min. The homogenate was filtered and centrifuged for 2 min. The pellet was washed three times with DMEM and then resuspended in DMEM supplemented with 10% fetal bovine serum (Invitrogen), 100 units/ml penicillin G, 100 μg/ml streptomycin, 250 ng/ml amphotericin B (Invitrogen), and 5 μg/ml insulin (Sigma) (Culture Medium). Cell number and viability were assessed by counting an aliquot in the presence of 0.1% trypan blue. Cells were plated on 60-mm culture plates previously coated with collagen (Cohesion, Palo Alto, CA). Incubation was carried out at 37 °C under 5% CO2. After 18 h, the culture medium was replaced with serum-free medium (same composition as Culture Medium except that fetal bovine serum was replaced with 9.6 ng/ml dexamethasone (Fujisawa, Deerfield, IL), 10 μg/liter epidermal growth factor, 0.5 5 μg/liter 0.5 and 0.5 Culture Medium). transforming growth was i.e. at of to serum-free at a of 2 The cells without TGF-β1 and with TGF-β1 were at and Hepatocyte Cell Culture and mouse hepatocyte cell i.e. was purchased from The cell line was in medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), 5 μg/ml 5 μg/ml 5 ng/ml ng/ml dexamethasone and 100 ng/ml amphotericin B (Invitrogen) Medium). cells were at in The cells were to serum-free 18 with calcium- and magnesium-free phosphate-buffered Similar to hepatocytes, TGF-β1 was at a of 2 The cells without TGF-β1 and with TGF-β1 were at and TGF-β1 was by using CA). and reverse were purchased from analysis was using was used for expression of Cell cells and mouse hepatocytes were with cell mm mm mm mm mm mm mm μg/ml and on Cells were and to and for at followed by at for 10 at The resulting were in at °C until in the cell solution was using cell was with mm 10% supplemented with 5% were at 100 °C for 10 and on 10% or were to a in containing mm mm and at °C for to the was for at with 5% in mm mm and 0.1% were then for at °C with in containing 5% at the by the The were mouse I collagen mouse mouse (Sigma) and mouse After in the were with for at in 5% in were then washed with and the were using the were with of in was to that of or of was to of cell and the was with for at After the of of the was with at was centrifuged for 5 at at and the were then washed three times with cell mm mm mm mm mm mm mm μg/ml The pellet was resuspended in (50 mm 10% 0.1% 5% and for 5 min. were on and by hepatocytes for of and were plated on in culture and in the as hepatocytes for of I collagen were plated on in culture of type I Incubation was carried out at 37 °C under 5% CO2. After 18 h, culture medium was replaced with serum-free TGF-β1 was at the of the (i.e. of to serum-free at a of 2 cells for were plated on by in culture and using the as and to serum-free cells without TGF-β1 and with TGF-β1 were washed with three times and with for 10 on Cells were with in for 10 at and in for 5 at After with for 5 cells were with for at then with 5% serum as in for and then with the The were used for and I collagen CA). or were used Cells were with to the cells were with medium and by and are among RNA medium was the of hepatocytes, and the cells were in the presence of or Smad4 for of was to the 10 was to the most for Smad4 10 with in serum-free culture medium was for 10 at and to the The hepatocyte cells were plated with and for The hepatocytes and the cells with the were to serum-free medium with TGF-β1 was at a of 2 The cells without TGF-β1 and with TGF-β1 were and are as were from of at three for were with analysis of and was used for the of and TGF-β1 at the was considered to and were using CA). TGF-β1 in in mouse hepatocytes were in serum-free medium with or without TGF-β1 After in hepatocytes were for and and by In hepatocytes, was in the with a and cells a with of the the cell phenotypic that mature hepatocytes a mesenchymal of in and cells, that the cells are hepatocytes the of expression of α-smooth muscle a of hepatic stellate cells in and hepatocytes using analysis to that results are not of HSC or fibroblast of the were using a “normal” mouse hepatocyte cell line cells, but not a of to hepatocytes TGF-β1 TGF-β1 an EMT in is a epithelial that a key role in the of and of its expression EMT M. Kalluri R. J. Mol. Med. 2004; 82: 175-181Crossref PubMed Scopus (419) Google Scholar, Y. J. C. C. Y. J. Clin. Investig. 2003; PubMed Scopus Google Scholar). We the of expression in to TGF-β1 mRNA were in the hepatocytes as with analysis that were in the hepatocytes, the phenotypic changes and of epithelial were by of mesenchymal the expression of a used to mesenchymal cells, as as cells in EMT (10Bissell D.M. Roulot D. George J. Hepatology. 2001; 34: 859-867Crossref PubMed Scopus (335) Google Scholar). of hepatocytes (i.e. and increased the expression of mRNA was increased in the hepatocytes as with for and by of cell by of the in with of in the hepatocytes and TGF-β1 changes were in the an EMT in the further the EMT changes in hepatocytes in the mouse hepatocyte cell mRNA were by TGF-β1 analysis that were also in the cells TGF-β1 Similar to hepatocytes, mRNA were in cells at and h, by using was also on cells in order to further the of epithelial and mesenchymal The findings are with in hepatocytes, and further that results are to EMT and not to fibroblast and TGF-β1 I in the of a mesenchymal phenotype by hepatocytes by changes in and is by mesenchymal function(s), hepatocytes contribute to the of extracellular matrix H. R.M. T. Z. Res. 2005; PubMed Scopus Google Scholar). the expression of α1(I) collagen mRNA by TGF-β1 α1(I) collagen mRNA the expression of a of the of that is to fibroblasts M. Kalluri R. J. Mol. Med. 2004; 82: 175-181Crossref PubMed Scopus (419) Google Scholar). of TGF-β1 further that increased α1(I) collagen mRNA expression by TGF-β1 is not of fibroblast the type I collagen expression in cell and as shown in type I collagen was by of type I collagen were in the hepatocytes in their the hepatocytes were with I collagen shown in collagen were in the extracellular space and TGF-β1 the of in cells collagen by TGF-β1 I by further that the source of type I collagen was the hepatocytes, fibroblasts or other cell that have potentially hepatocyte the cell line was for type I collagen α1(I) collagen mRNA and type I collagen were TGF-β1 that hepatocytes I collagen in to TGF-β1 for type I collagen in hepatocytes TGF-β1 for and I collagen in cells in the or presence of 2 ng/ml TGF-β1 at and in and in serum-free by Cell are by of type I collagen is in cells at the in is with the hepatocyte of the TGF-β1 snail-1 and Smad4 in a of is by TGF-β1 H. M. A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The as a key regulator of EMT in and in (14Vega S. Morales A.V. Ocana O.H. Valdes F. Fabregat I. Nieto M.A. Genes Dev. 2004; 18: 1131-1143Crossref PubMed Scopus (659) Google Scholar, J. A. A. P. 2003; PubMed Scopus Google Scholar). the expression of in and hepatocytes is with In mRNA was at 0.5 and TGF-β1 in and hepatocytes, and is with other and with of R. Y. T. Mol. Biol. 2001; PubMed Scopus Google Scholar). the TGF-β1 that are with EMT and (i.e. and have been in EMT in epithelial cell L. Law B.K. Aakre M.E. Edgerton M. Shyr Y. Bhowmick N.A. Moses H.L. Breast Cancer Res. 2003; 5: R187-R198Crossref PubMed Scopus (105) Google Scholar, H. R.M. T. Z. Res. 2005; PubMed Scopus Google Scholar, J. Pathol. 2004; PubMed Scopus Google Scholar, L. Law B.K. A.M. Aakre M.E. Moses H.L. 2004; PubMed Scopus Google Scholar). the pathway by and The and expression of and the forms of and were i.e. not and hepatocytes and and of expression in and cells that signaling is not in mediating the signaling i.e. Smad2/3 and Smad4 of Smad2/3 to their activation increased TGF-β1 and the (i.e. of the Smad2/3 to the of hepatocytes 0.5 TGF-β1 cells were for evidence of Smad2/3 activation and were with Smad2/3 and Smad4 and the signaling pathway was in and hepatocytes and not in their that EMT changes in hepatocytes were via signaling and not via the pathway. EMT in in the central role of the TGF-β1/Smad on EMT was used for silencing in and hepatocytes A. K. J. PubMed Scopus Google Scholar, L. H. L. H. H. J. Google Scholar). The of and silencing in and hepatocytes was by IP, and using (i.e. was to all to out that to In hepatocytes, the activation of the was with the of Smad4 to a of the TGF-β1/Smad was as by silencing of Smad4 was by The Smad4 mRNA were to in with Smad4 TGF-β1 to as by Smad4 mRNA to the was not in the reverse the EMT was then In the of mRNA expression by TGF-β1 was but a of was shown to TGF-β1 shown in snail-1 mRNA was by in mRNA expression was but not by in hepatocytes the other the of mRNA expression by TGF-β1 was the upregulation of α1(I) collagen mRNA by TGF-β1 was by The of on the expression of and was then by with was Smad4 RNA in was with the in mRNA a and a to the of type I collagen by in hepatocytes, were with I collagen shown in hepatocytes that were with a significant in type I was not in the of hepatocytes from a to a was not in and hepatocytes that a type I collagen in hepatocytes, under in were for and I collagen and by blue. type I collagen and deposition in TGF-β1 with the results with hepatocytes, the of Smad4 to Smad2/3 in cells, as by of was of Smad4 as by a significant in Smad4 mRNA expression in the was not cells that were with to the Smad4 The of mRNA was snail-1 mRNA expression was by in cells expression of mRNA TGF-β1 was but silencing the in mRNA by TGF-β1 was but by the induction of α1(I) collagen mRNA by TGF-β1 was by Smad4 silencing the was by Smad4 silencing in cells Similar to hepatocytes, the of from to in cells as shown in Smad4 silencing in hepatocytes the type I collagen results support a central role of the pathway in EMT type I collagen in mouse hepatocyte cell for and I collagen in cells (same as in was by blue. type I collagen deposition by In that hepatocytes type I collagen in to TGF-β1 of the most and of fibrosis Our results the by M. J. Clin. Investig. PubMed Scopus Google that hepatocytes are to findings were by that the ability of hepatocytes to contribute to hepatic fibrosis I. S. T. K. Hepatology. PubMed Scopus Google Scholar). is that only HSC, portal and mesenchymal cells of have a in the liver P. M. N.A. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, B. Investig. 2004; PubMed Scopus Google Scholar). Although other have also shown that hepatocytes the that hepatocytes have an role in the of liver fibrosis, and progression to cirrhosis. We that hepatocytes a potentially important role in the of liver fibrosis and progression to cirrhosis. the of epithelial cells to is not a In EMT to that in its that epithelial cells are at the of of EMT that epithelial cells their epithelial phenotype and in of mesenchymal has been and to in epithelial cells as and L. Law B.K. Aakre M.E. Edgerton M. Shyr Y. Bhowmick N.A. Moses H.L. Breast Cancer Res. 2003; 5: R187-R198Crossref PubMed Scopus (105) Google Scholar, Y. J. C. C. Y. J. Clin. Investig. 2003; PubMed Scopus Google Scholar, L. 2005; PubMed Scopus Google Scholar, Sci. 2004; PubMed Scopus Google Scholar). in EMT and in from the that mesenchymal the and the phenotypic changes that hepatocytes in in that hepatocytes epithelial in of mesenchymal and inducing type I collagen a known fibroblast not that hepatocytes fibroblasts but that assume a phenotype and but to a the epithelial In the EMT was to the i.e. cells, in the fetal liver and was recognized in fetal and hepatocytes with serum J. A. A. P. 2003; PubMed Scopus Google Scholar, F. A.M. A. S. B. M. M. Nieto M.A. Fabregat I. Mol. Cancer Res. 2002; Google Scholar). Although has been that EMT is not a major in mature hepatocytes of no have been to the role of EMT in hepatic fibrogenesis. Since its in other and has been to EMT in mature hepatocytes and its in liver fibrogenesis L. Law B.K. Aakre M.E. Edgerton M. Shyr Y. Bhowmick N.A. Moses H.L. Breast Cancer Res. 2003; 5: R187-R198Crossref PubMed Scopus (105) Google Scholar, H. R.M. T. Z. Res. 2005; PubMed Scopus Google Scholar, L. Law B.K. A.M. Aakre M.E. Moses H.L. 2004; PubMed Scopus Google Scholar, L. 2005; PubMed Scopus Google Scholar, Sci. 2004; PubMed Scopus Google Scholar, J. C. Breast Cancer Res. 2004; PubMed Scopus Google Scholar). a have to EMT in liver M. J. M. A.M. Am. J. PubMed Scopus Google Scholar, W. A. J. D. L. R. Bottinger E.P. Mol. Biol. PubMed Scopus Google Scholar). Several to EMT in a hepatocyte the most with resident fibroblasts stellate TGF-β1, a known of apoptosis in hepatocytes, in the of major to as with and has shown of and as as collagen and I collagen was and in cells, further that hepatocytes are the source of is collagen in the form of then in the form of to extracellular The is as for and a stellate out with HSC and for and with to of cells, was not out HSC for not show TGF-β1 thus that results were of EMT in hepatocytes and not to or We then to a “normal” hepatocyte cell i.e. EMT changes were also in the mouse hepatocyte cell thus confirming that the changes in hepatocytes were of EMT changes to HSC, or other mesenchymal cell to EMT of hepatocytes in is that hepatocytes in hepatocytes a and Although cells have to a in is for in the TGF-β1 are from by not for collagen Our results further support the and have important EMT a an all or that further in with a is to the hepatocytes are to as of hepatic as of known of the EMT mechanism in other epithelial and fetal is that the of TGF-β1 on hepatocytes is to apoptosis (10Bissell D.M. Roulot D. George J. Hepatology. 2001; 34: 859-867Crossref PubMed Scopus (335) Google Scholar, 12Oberhammer F.A. Pavelka M. Sharma S. Tiefenbacher R. Purchio A.F. Bursch W. Schulte-Hermann R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5408-5412Crossref PubMed Scopus (679) Google Scholar, T. Y. R. K. K. A. Biochem. Biophys. Res. Commun. 1985; PubMed Scopus Google Scholar). It is that hepatocytes by EMT F. A.M. A. S. B. M. M. Nieto M.A. Fabregat I. Mol. Cancer Res. 2002; Google Scholar). transcription factor is central to EMT (14Vega S. Morales A.V. Ocana O.H. Valdes F. Fabregat I. Nieto M.A. Genes Dev. 2004; 18: 1131-1143Crossref PubMed Scopus (659) Google Scholar, J. A. A. P. 2003; PubMed Scopus Google Scholar, R. Y. T. Mol. Biol. 2001; PubMed Scopus Google Scholar). snail-1 is by and is in the of Indeed, is known to EMT development and mice at of a EMT R. Y. T. Mol. Biol. 2001; PubMed Scopus Google Scholar). is of mesenchymal as K. R. S. M. M. H. M. J. 2003; Google Scholar). TGF-β1 its by to the type and the type I J. Mol. Cell Biol. 2000; PubMed Scopus Google Scholar). Smad2/3 and Smad4 are known of by the TGF-β1 with Smad4 and to the TGF-β1 A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (891) Google Scholar, J. Mol. Cell Biol. 2000; PubMed Scopus Google Scholar, F. M. Cell Res. 2003; PubMed Scopus Google Scholar). It has been shown that Smad2/3 activation is a central role for as of EMT and their important role in fibrosis H. R.M. T. Z. Res. 2005; PubMed Scopus Google Scholar, J. L. Bottinger E.P. EMBO J. 2004; PubMed Scopus Google Scholar, U. M. H. Heldin C.H. A. Mol. Biol. 2005; 16: PubMed Scopus Google Scholar, S. J. Breitkopf K. E. J. Dijke P. Gressner A.M. 2003; Full Text Full Text PDF PubMed Scopus (335) Google Scholar, H. J. E. Gressner A.M. J. Breitkopf K. Dooley S. J. Cell Mol. Med. PubMed Scopus Google Scholar). have also been in the induction of EMT in cell L. Law B.K. A.M. Aakre M.E. Moses H.L. 2004; PubMed Scopus Google Scholar, A. J. F. W. M. L. Cancer Res. 2003; Google Scholar). In cells, TGF-β1 EMT via signal-regulated and that expression H. M. A. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, R. 2003; PubMed Scopus Google Scholar, D. E.D. Mol. Biol. PubMed Scopus Google Scholar). In cells, pathway was found to for EMT A.V. Bhowmick N.A. Moses H.L. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Valdes F. A.M. B. A. M. M. Fabregat I. Cell Res. 2004; PubMed Scopus Google activation of the pathway in fetal hepatocytes that We have also shown that TGF-β1 mediates EMT in hepatocytes by the induction of snail-1 and activation of is further supported by the of EMT Smad4 RNA silencing D. Cancer Res. PubMed Scopus Google Scholar). In of and hepatocytes with EMT as the epithelial phenotype and and most type I collagen was not in hepatocytes, there was a of its of to a with EMT The that Smad4 silencing EMT in hepatocytes that Smad4 is central to EMT in hepatocytes and hepatic fibrogenesis. Although therapies to the progression of liver fibrosis are not that findings to the of the development and progression of hepatic fibrogenesis. to for hepatic fibrosis. We and of the of The for their and