EXPRESSION OF LAMININ GAMMA-1 IN CULTURED-HEPATOCYTES INVOLVES REPEATED CTC AND GC ELEMENTS IN THE LAMC1 PROMOTER

Laminin gamma 1 chain is present in all basement membranes and is expr essed at high levels in various diseases, such as hepatic fibrosis. We have identified cis- and trans-acting elements involved in the regula tion of this gene in normal rat liver, as well as in hepatocyte primar y cultures and hepatoma cell lines. Northern-blot analyses showed that laminin gamma 1 mRNA was barely detectable in freshly isolated hepato cytes 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 overexp ression in cultured hepatocytes was under the control of transcription al mechanisms. Transfection of deletion mutants of the 5' flanking reg ion of murine LAMC1 gene in hepatoma cells that constitutively express laminin gamma 1 indicated that regulatory elements were located betwe en -594 bp and -94 bp. This segment included GC- and CTC-containing mo tifs. Gel-shift analyses showed that two complexes were resolved with different affinity for the CTC sequence depending on the location of t he GC box. The pattern of complex formation with nuclear factors from freshly isolated and cultured hepatocytes was different from that obta ined with total liver and similar to that with hepatoma cells. Southwe stern analysis indicated that several polypeptides bound the CTC-rich sequence. Affinity chromatography demonstrated that a M(r) 60 000 poly peptide was a major protein binding to the CTC motif. This polypeptide is probably involved in the transcriptional activation of various pro to-oncogenes and extracellular matrix genes that are expressed at high levels in both hepatoma cells and early hepatocyte cultures.