Extracellular matrix configuration modulates TGF-β effects in primary hepatocytes

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In vitro models to study TGF-β effects on the liver include cell lines, primary fetal cells and primary hepatocytes, the last resembling closer most of the phenotipic features of an intact liver. We compared the behaviour of primary mosue hepatocytes cultured on collagen type I as a dry monolyer or as gel sandwich and their response to TGF-β. Hepatocytes cultured on dry matrix displayed a typical cuboidal shape for the initial 24h, but started to spread and dedifferentiate constantly thereafter. Bile canaliculi was absent in this culture condition. Cells cultured in collagen sandwich started to form bile canaliculi from day 1 which persisted even after 5 days. Bile canaliculi function was evaluated by CMFDA secretion. Cell morphology was consistently better preserved as compared to dry matrix, as judged by nuclear/cytoplasmic ratio, cytoplasm granulosity and cell refringence. Upon TGF-β stimulation, cells in dry matrix displayed slight enhanced dedifferentiation. Very weak apoptosis induction was observed. Contrary to the dry collagen system, TGF-β treated cells showed strong morphologycal changes in time, progressing through a clear dedifferentiation resembling epithelial to mesenchymal transition. This changes were acompanied with expression of profibrogenic markers like CTGF. After 72h of stimulation, strong apoptosis was observed. This was confirmed by PARP degradation and Caspase–3 activation. Antiapoptotic proteins Bcl–2 and Bcl-xL were downregulated by TGF-β treatment. Interestingly, the p38 inhibitor SB203580 strongly atenuated apoptosis induction, but had no effect on dedifferentiation and CTGF expression, while ALK5 inhibitor SB431542 inhibited every feature induced by TGF-β. Our findings indicate that hepatocytes cultured on gel matrix preserve their features better and for a longer time than on a dry matrix, which allows a more sensitive response to TGF-β.