Abstract
BackgroundDedifferentiation and loss of hepatocyte polarity during primary culture of hepatocytes are major drawbacks for metabolic analyses. As a prominent profibrotic cytokine and potent inducer of epithelial mesenchymal transition (EMT), TGF-β contributes to these processes in liver epithelial cells. Yet, a distinction between culture dependent and TGF-β driven hepatocyte dedifferentiation has not been shown to date.ResultsHere, we show that in both settings, mesenchymal markers are induced. However, upregulation of Snai1 and downregulation of E-Cadherin are restricted to TGF-β effects, neglecting a full EMT of culture dependent hepatocyte dedifferentiation. Mechanistically, the latter is mediated via FAK/Src/ERK/AKT pathways leading to the induction of the oncogene caveolin-1 (Cav1). Cav1 was recently proposed as a new EMT marker, but our results demonstrate Cav1 is not up-regulated in TGF-β mediated hepatocyte EMT, thus limiting validity of its use for this purpose. Importantly, marking differences on Cav1 expression exist in HCC cell lines. Whereas well differentiated HCC cell lines exhibit low and inducible Cav1 protein levels - by TGF-β in a FAK/Src dependent manner, poorly differentiated cell lines display high Cav1 expression levels which are not further modulated by TGF-β.ConclusionsThis study draws a detailed distinction between intrinsic and TGF-β mediated hepatocyte dedifferentiation and elucidates cellular pathways involved. Additionally, by evaluating the regulation of the oncogene Cav1, we provide evidence to argue against Cav1 as a reliable EMT marker.
Highlights
Dedifferentiation and loss of hepatocyte polarity during primary culture of hepatocytes are major drawbacks for metabolic analyses
Culture induced hepatocyte dedifferentiation leads to upregulation of mesenchymal markers and caveolin-1 Hepatocytes cultured on collagen monolayer rapidly dedifferentiate
At day 1, primary hepatocytes still exhibited a polar organization with actin fibers forming a belt on the inner side of the cell membrane (Figure 1A)
Summary
Dedifferentiation and loss of hepatocyte polarity during primary culture of hepatocytes are major drawbacks for metabolic analyses. Basic functional hepatocyte features such as bile canaliculi formation, bile secretion, polarity and metabolic activities (including detoxification by Cyp and other drug-metabolizing enzymes) are rapidly lost during culture on a collagen layer (monolayer or 2D culture). To overcome these limitations, alternative hepatocyte culture systems have been developed, including co-culture systems, bioreactors and 3D systems, where hepatocytes are embedded in a soft collagen matrix [1,2]. Transforming growth factor beta (TGF-β), a pleiotropic cytokine, is considered as the most potent profibrotic mediator in diverse organ diseases [5] It is recognized as a strong EMT inducer in various epithelial cell types including hepatocytes cultured on a stiff collagen matrix [3,6,7]. These observations raise the question whether TGF-β acts as an accelerator of the intrinsic dedifferentiation process or whether culture and TGF-β mediated EMT are distinct processes
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