Effects of growth and differentiation factors on the epithelial-mesenchymal transition in cultured neonatal rat hepatocytes.
OBJECTIVE
Loss of specific differentiation markers, adoption of a migrating morphology and progressive replacement of the cytokeratin network by vimentin intermediate filaments characterize the epithelial-mesenchymal transition of cultured neonatal rat hepatocytes. In a previous study (Hepatology 1997; 25: 598-606), we reported that this process can be differentially regulated by EGF and DMSO, two agents that affect hepatocyte growth and differentiation. The aim of the present study was to determine if growth activation or differential gene expression could explain the differences in EMT observed between these two factors.


METHODS
We compared the effects of EGF, HGF, TGF-beta1 and DMSO on growth, proto-oncogene expression, epithelial-mesenchymal transition markers and expression of liver transcription factors in cultured neonatal rat hepatocytes using thymidine incorporation, Northern blotting and Western blotting analysis.


RESULTS
When TGF-beta1 or DMSO was added to the cultures supplemented with EGF and HGF, the mitogenic activity induced by these factors was inhibited. DMSO down-regulated c-myc and c-fos expression. mRNA levels of some liver-specific genes such as albumin, or liver-enriched transcription factors such as C/EBPdelta, HNF-4 and HNF-1beta were slightly different in cultures supplemented with DMSO or TGF-beta1. However, no differences were found when DMSO or TGF-beta1 was added to the cultures supplemented with EGF. Western blotting analysis showed that TGF-beta1 decreased cytokeratin and increased vimentin levels, while DMSO decreased both cytokeratin and vimentin. When DMSO or TGF-beta1 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-beta1, inhibited vimentin expression.


CONCLUSIONS
Activation of vimentin expression produced in cultures supplemented with the mitogenic factors (EGF and HGF) is independent of the activation of cell growth, because DMSO but not TGF-beta1 can abolish vimentin synthesis, although both inhibited growth. Moreover, the vimentin expression in these cultures seems to be independent of the mRNA levels of transcription factors associated with the differentiated liver phenotype.
Pagan
R
R
Department de Biologia Cel.lular, Universitat de Barcelona, Catalonia, Spain.
Sánchez
A
A
Martin
I
I
Llobera
M
M
Fabregat
I
I
Vilaró
S
S
D000818:Animals; D000831:Animals, Newborn; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects; D016762:Genes, fos / Q000187:drug effects; D016259:Genes, myc / Q000187:drug effects; D006133:Growth Substances / Q000494:pharmacology*; D017228:Hepatocyte Growth Factor / Q000494:pharmacology; D007633:Keratins / Q000235:genetics; D008099:Liver / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D008648:Mesoderm / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014746:Vimentin / Q000235:genetics
D006133:Growth Substances; D016760:Proto-Oncogene Proteins c-fos; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D014746:Vimentin; D004815:Epidermal Growth Factor; D017228:Hepatocyte Growth Factor; D007633:Keratins; D004121:Dimethyl Sulfoxide