Effects of growth and differentiation factors on the epithelial-mesenchymal transition in cultured neonatal rat hepatocytes

Background/Aims: Loss of specific differentiation markers, adoption of a mi grating morphology and progressive replacement of the cytokeratin network b y vimentin intermediate filaments characterize the epithelial-mesenchymal t ransition of cultured neonatal rat hepatocytes. In a previous study (Hepato logy 1997; 25: 598-606), we reported that this process can be differentiall y regulated by EGF and DMSO, two agents that affect hepatocyte growth and d ifferentiation. The aim of the present study was to determine if growth act ivation or differential gene expression could explain the differences in EG T observed between these two factors. Methods: We compared the effects of EGF, HGF, TGF-beta(1) and DMSO on growt h, proto-oncogene expression, epithelial-mesenchymal transition markers and expression of liver transcription factors in cultured neonatal rat hepatoc ytes using thymidine incorporation, Northern blotting and Western blotting analysis. Results: When TGF-beta(1) or DMSO was added to the cultures supplemented wi th EGF and HGF, the mitogenic activity induced by these factors was inhibit ed. DMSO down-regulated c-myc and c-fos expression. mRNA levels of some liv er-specific genes such as albumin, or liver-enriched transcription factors such as C/EBP delta, HNF-4 and HNF-1 beta were slightly different in cultur es supplemented with DMSO or TGF-beta(1) However, no differences were found when DMSO or TGF-beta(1) was added to the cultures supplemented with EGF W estern blotting analysis showed that TGF-beta(1) decreased cytokeratin and increased vimentin levels, while DMSO decreased both cytokeratin and viment in. When DMSO or TGF-beta(1) was added in combination with EGF or HGF, both factors maintained the increase in albumin and cytokeratin induced by the growth factors although DMSO, but not TGF-beta(1), inhibited vimentin expre ssion. Conclusions: Activation of vimentin expression produced in cultures supplem ented with the mitogenic factors (EGF and HGF) is independent of the activa tion of cell growth, because DMSO but not TGF-beta(1) can abolish vimentin synthesis, although both inhibited growth. Moreover, the vimentin expressio n in these cultures seems to be independent of the mRNA levels of transcrip tion factors associated with the differentiated liver phenotype.