Matrix induced re-differentiation of cultured rat hepatocytes and changes of CCAAT/enhancer binding proteins.
Rat hepatocytes de-differentiate and proliferate when cultured on collagen-coated dishes in a chemically defined Hepatocyte Growth Medium in the presence of hepatocyte growth factor and epidermal growth factor. The addition of biomatrix derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma stops this process and leads to re-differentiation of the cells. We monitored DNA binding activity and protein levels of CCAAT/Enhancer Binding Proteins (C/EBPs) during these events by electrophoretic mobility shift assays and western blot analysis. We used plasma protein gene expression as a marker for the proliferation and differentiation phases. During the initial proliferation phase the DNA binding activity of C/EBPs decreased about 5-10 fold, mainly due to reduction of C/EBP alpha protein to nearly undetectable levels. Addition of EHS-gel prevented the further loss of C/EBP alpha protein and established a new steady state level. Since C/EBP beta proteins were affected to a much lesser extent, the C/EBP alpha:C/EBP beta ratio was greater in the presence of EHS-gel. Transferrin, alpha 1-antitrypsin, and albumin mRNA expression increased substantially. Thus stabilized C/EBP alpha expression, an increased C/EBP alpha:C/EBP beta ratio, and increased expression of liver specific mRNAs all correlated with the transition of proliferative to differentiated cells.
Runge
D
D
Department of Pathology, University of Pittsburgh, PA 15261, USA.
Runge
D M
DM
Bowen
W C
WC
Locker
J
J
Michalopoulos
G K
GK
D000418:Albumins / Q000378:metabolism; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003094:Collagen / Q000494:pharmacology; D003470:Culture Media; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000378:metabolism; D005782:Gels; D008099:Liver / Q000166:cytology*; D008297:Male; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344; D000515:alpha 1-Antitrypsin / Q000378:metabolism
D000418:Albumins; D022762:CCAAT-Enhancer-Binding Proteins; D003470:Culture Media; D004268:DNA-Binding Proteins; D005782:Gels; D009687:Nuclear Proteins; D000515:alpha 1-Antitrypsin; D004815:Epidermal Growth Factor; D003094:Collagen