MATRIX INDUCED RE-DIFFERENTIATION OF CULTURED RAT HEPATOCYTES AND CHANGES OF CCAAT ENHANCER BINDING-PROTEINS/

Rat hepatocytes de-differentiate and proliferate when cultured on coll agen-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) mo use sarcoma stops this process and leads to re-differentiation of the cells. We monitored DNA binding activity and protein levels of CCAAT/E nhancer Binding Proteins (C/EBPs) during these events by electrophoret ic mobility shift assays and western blot analysis. We used plasma pro tein gene expression as a marker for the proliferation and differentia tion phases. During the initial proliferation phase the DNA binding ac tivity of C/EBPs decreased about 5-10 fold, mainly due to reduction of C/EBP alpha protein to nearly undetectable levels. Addition of EHS-ge l 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 m uch lesser extent, the C/EBP alpha:C/EBP beta ratio was greater in the presence of EHS-gel. Transferrin, alpha(1)-antitrypsin, and albumin m RNA expression increased substantially. Thus stabilized C/EBP alpha ex pression, an increased C/EBP alpha: C/EBP beta ratio, and increased ex pression of liver specific mRNAs all correlated with the transition of proliferative to differentiated cells.