Expression of laminin gamma 1 cultured hepatocytes involves repeated CTC and GC elements in the LAMC1 promoter.
Laminin gamma 1 chain is present in all basement membranes and is expressed at high levels in various diseases, such as hepatic fibrosis. We have identified cis- and trans-acting elements involved in the regulation of this gene in normal rat liver, as well as in hepatocyte primary cultures and hepatoma cell lines. Northern-blot analyses showed that laminin gamma 1 mRNA was barely detectable in freshly isolated hepatocytes and expressed at high levels in hepatocyte primary cultures, as early as 4 h after liver dissociation. Actinomycin D and cycloheximide treatment in vivo and in vitro indicated that laminin gamma 1 overexpression in cultured hepatocytes was under the control of transcriptional mechanisms. Transfection of deletion mutants of the 5' flanking region of murine LAMC1 gene in hepatoma cells that constitutively express laminin gamma 1 indicated that regulatory elements were located between -594 bp and -94 bp. This segment included GC- and CTC-containing motifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of the GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obtained with total liver and similar to that with hepatoma cells. Southwestern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that A M(r) 60,000 polypeptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various proto-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures.
Levavasseur
F
F
Institut National de la Santé et de la Recherche Médicale, Unité de Recherches Hépatologiques U-49, CHRU Pontchaillou, Rennes, France.
Liétard
J
J
Ogawa
K
K
Théret
N
N
Burbelo
P D
PD
Yamada
Y
Y
Guillouzo
A
A
Clément
B
B
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D016384:Consensus Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D018900:Dinucleotide Repeats; D005786:Gene Expression Regulation; D007797:Laminin / Q000235:genetics*; D008099:Liver / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D017207:Rats, Sprague-Dawley; D017384:Sequence Deletion; D018911:Trinucleotide Repeats; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D007797:Laminin; C089141:laminin gamma 1; D004247:DNA