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-␤ (TGF-␤) 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-␤1 induces an epithelial-to-mesenchymal transition (EMT) state in mature hepatocytes in vitro. EMT state was marked by significant upregulation of ␣ 1 (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-␤1 induces the snail-1 transcription factor and activates the Smad2/3 pathway. Evidence for a central role of the TGF-␤1/Smad pathway is further supported by the inhibition of EMT by Smad4 silencing using small interference RNA technology. In conclusion, TGF-␤1, 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 (1). End-stage liver fibrosis is recognized clinically as cirrhosis. Since their initial description, hepatic stellate cells (HSC) 3 have dominated the field of liver fibrogenesis (2-4). Indeed, their role is central in hepatic fibrosis (5). 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 (6 -8).Several lines of evidence support an important role for TGF-␤1 signaling in the initiation and progression of liver fibrosis (9). In mature (i.e. adult) hepatocytes, TGF-␤1 is responsible for inhibition of cell proliferation and induction of apoptosis (10 -12). Interestingly, TGF-␤1 is the most established mediator and regulator of EMT (13). It has been shown that TGF-␤1 mediates EMT by inducing snail-1 transcription factor and tyrosine phosphorylation o...

Weinberg and coworkers have used serial transduction of a human, primary fibroblast cell line with the catalytic domain of human telomerase, large T antigen, small T antigen, and an oncogenic allele of H-ras to study stages leading toward a fully transformed cancerous state. We performed a three-dimensional screening experiment using 4 cell lines, 5 small-molecule perturbagens (2-deoxyglucose, oxamate, oligomycin, rapamycin, and wortmannin), and a large number of metabolic measurements. Hierarchical clustering was performed to obtain signatures of the 4 cell lines, 24 cell states, 5 perturbagens, and a number of metabolic parameters. Analysis of these signatures and sensitivities of the cell lines to the perturbagens provided insights into the bioenergetic states of progressively transformed cell lines, the effect of oncogenes on small-molecule sensitivity, and global physiological responses to modulators of aerobic and anaerobic metabolism. We have gained insight into the relationship between two models of carcinogenesis, one (the Warburg hypothesis) based on increased energy production by glycolysis in cancer cells in response to aberrant respiration, and one based on cancer-causing genes. Rather than being opposing models, the approach described here suggests that these two models are interlinked. The cancercausing genes used in this study appear to increase progressively the cell's dependence on glycolytic energy production and to decrease its dependence on mitochondrial energy production. However, mitochondrial biogenesis appears to have a more complex dependence, increasing to its greatest extent at an intermediate degree of transduction rather than at the fully transformed state.cancer ͉ metabolic profiling ͉ metabolism ͉ small molecules ͉ warburg effect C ancer cells override mechanisms for controlling cellular proliferation, differentiation, and death during malignant transformation. Weinberg and coworkers (1) have shown that ectopic expression of the telomerase catalytic subunit (hTERT) in combination with simian virus 40 large T antigen (LT), small T antigen (ST), and an oncogenic allele of H-ras results in the tumorigenic conversion of normal human epithelial and fibroblast cells (1, 2). In this study, we used the four BJ fibroblast cell, and 4-[hTERT ϩ LT ϩ ST ϩ H-ras], which we abbreviate here as CL1, CL2, CL3, and CL4, respectively. To study physiological changes on the path toward tumorigenic conversion, we performed a three-dimensional screening experiment that yielded a matrix of data derived from variations in cell states, cell measurements, and small molecules (Fig. 1). To assess the bioenergetic status of each cell line CL1-CL4, we selected small-molecule inhibitors of metabolic and nutrient-sensing pathways. Each cell line was incubated individually with (i) DMSO; (ii) oxamic acid, an inhibitor of lactate dehydrogenase, which is an enzyme involved in anaerobic glycolysis; (iii) 2-deoxyglucose, an inhibitor of the glycolysis enzyme hexokinase; (iv) oligomycin, an inhibitor of mitochondrial A...