Matrix-mediated changes in the expression of HNF-4alpha isoforms and in DNA-binding activity of ARP-1 in primary cultures of rat hepatocytes.
Recently, we have developed a culture system in which rat hepatocytes dedifferentiate and proliferate and after the addition of EHS-gel redifferentiate. During both developmental stages HNF-4alpha2 mRNA was more abundant than HNF-4alpha1 mRNA. However, Western blot analysis using COS-7 cell-expressed HNF-4alpha1 and HNF-4alpha2 proteins as standards revealed that (i) HNF-4alpha2 protein was not expressed in dedifferentiated hepatocytes and (ii) either HNF-4alpha2 protein or a highly phosphorylated HNF-4alpha1 protein was the dominating isoform in redifferentiated hepatocytes. The changes in HNF4-isoform expression could not be mimicked by DMSO, suggesting them to be matrix specific. Furthermore, DMSO was less efficient than EHS-gel in reinducing liver-specific gene expression. EHS-gel overlay also led to reduction of ARP-1 DNA binding activity, while overall ARP-1 protein levels did not change. These results suggest that EHS-matrix overlay regulates the expression of different HNF-4alpha isoforms on a posttranscriptional level while ARP-1 DNA binding activity is regulated by posttranslational mechanisms.
Runge
D
D
Department of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, 15261, USA.
Runge
D M
DM
Daskalakis
N
N
Lubecki
K A
KA
Bowen
W C
WC
Michalopoulos
G K
GK
D000818:Animals; D051837:COUP Transcription Factor II; D051836:COUP Transcription Factors; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004338:Drug Combinations; D005109:Extracellular Matrix / Q000737:chemistry*; D051557:Hepatocyte Nuclear Factor 4; D007797:Laminin / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008297:Male; D010750:Phosphoproteins / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011509:Proteoglycans / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology; D051381:Rats; D011916:Rats, Inbred F344; D011987:Receptors, Steroid; D014157:Transcription Factors / Q000378:metabolism*
D051837:COUP Transcription Factor II; D051836:COUP Transcription Factors; D004268:DNA-Binding Proteins; D004338:Drug Combinations; D051557:Hepatocyte Nuclear Factor 4; D007797:Laminin; C497588:Nr2f2 protein, rat; D010750:Phosphoproteins; D011509:Proteoglycans; D011987:Receptors, Steroid; D014157:Transcription Factors; C059485:matrigel; D003094:Collagen