The LAMC1 promoter contains two sequences, interacting with the large subunit of replication factor C and SP1, which are involved in laminin γ1 expression in Faza 567 rat hepatoma cell line.
摘要
Laminins, the major non-collagenous glycoproteins of all basement membranes are scarce in the normal adult liver. However, several laminin chains, particularly laminin γl, can be expressed at high levels in various liver diseases, such as fibrosis and carcinogenesis, as well as in oth hepatocytes and hepatic stellate cells after a few days in culture and in hepatoma cell lines. We have investigated the molecular mechanisms involved in the expression of laminin γl in rat hepatoma cells. cis and trans acting elements were identified in the promoter region of LAMC1 gene coding for laminin γl. Transfection of deletion mutants of the 5′ flanking region of murine LAMC1 gene in the Faza 567 hepatoma cell line indicated that two enhancers and a silencer segment which contains GC- and CTC-containing sequences, were located between −594 bp and −94 bp. Two complexes were formed when nuclear extracts from hepatoma cells were used in gel-shift analyses with double-stranded synthetic oligonucleotides corresponding to the CTC- and GC-containing regions. Transactivation of LAMC1 promoter was achieved when cotransfected in Drosophila cells with a Sp1 expression vector. Immunocytochemistry, western-blotting and northern-blot analyses indicated that Faza 567 hepatoma cells expressed both Sp1 and Alp145, the large subunit of replication factor C (RFC), which interacted respectively with the GC- and CTC-motives in the silencer regulatory element of LAMC1 promoter. A combination of immunoprecipitation and western-blotting with Faza 567 cell extracts revealed that Alp145 was in complex with PCNA. These data show that LAMC1 gene expression in Faza 567 hepatoma cells is governed by nuclear factors interacting with CTC- and GC-rich regions within the promoter, including RFC and Sp1, thus suggesting that there is a relationship between laminin γl expression and the cell cycle.