In hepatocytes TGF-β induces Epithelial-to-Mesenchymal Transition (EMT)

Introduction: Chronic intoxication of the liver with alcohol or hepatotoxic viruses is known to induce liver fibrosis which progresses to cirrhosis or hepatocellular carcinoma. During fibrogenesis liver cells, especially activated hepatic stellate cells (HSCs) release the pro-fibrogenic cytokine TGF-β and we have previously shown that general hepatic overexpression of the TGF-β antagonist Smad7 abrogates development of experimentally induced fibrosis and inhibits activation of HSCs in vitro.

Aims: To investigate TGF-β signal transduction especially in hepatocytes.

Methods: We studied primary mouse hepatocytes and two cell lines (Hep1–6 and AML12) after stimulation with TGF-β1 using RT-PCR, Western blot, immunocytochemistry and reporter gene assays.

Results: During only 4 days of culture, cellular morphology of primary cells changes from the typical honeycomb shape to a more fibroblastic appearance. As during EMT, E-cadherin is down-regulated and fibronectin and vimentin are upregulated, while typical hepatocyte markers, e.g., albumin and transferrin are unaffected. We further found that during this process only E-Cadherin is delocalized from the cell membrane spontaneously while other typical cell-cell contact proteins, like ZO-1 and β-Catenin become internalized only in the presence of TGF-β. The cell lines tested did not undergo such changes in culture but with constant TGF-β stimulation we also found clear phenotypic changes with disintegration of cell-cell contacts. Using adenoviral reporter constructs containing either Smad3/4 or Smad1/5/8 response elements, we found at culture day 1, a dose dependent response to TGF-β1 for both pathways while at day 3 the Smad3/4 response was completely inverted, showing the strongest activation with the lowest dose. The TGF-β type III receptor betaglycane was not expressed but endoglin was upregulated during culture.

Conclusion: Our data show that hepatocytes stimulated with TGF-β1 undergo EMT like phenotypic changes that are accompanied by a modulation of TGF-β responsiveness and since EMT is often the first step towards cirrhosis and HCC, these findings suggest that hepatocytes are much more involved in the fibrotic response of the liver than previously assumed.