Gene expression and synthesis of fibronectin isoforms in rat hepatic stellate cells. Comparison with liver parenchymal cells and skin fibroblasts.

Fibronectins are multifunctional glycoproteins that are important components of the extracellular matrix in normal and fibrotic liver. Multiple fibronectin isoforms are generated from a single gene by alternative splicing of the primary transcript at the domains EIIIA, EIIIB, and V. The aim of this study was to investigate the fibronectin isoforms expressed by activated hepatic stellate cells, the most important connective tissue-producing cells in injured liver. Hepatocytes and skin fibroblasts were also studied for comparison. Activation of hepatic stellate cells in vivo was induced by injecting CCl4 twice weekly for 3 weeks. Activation in vitro was achieved by culturing cells on plastic. The level of activation was evaluated by alpha-smooth muscle actin immunocytochemistry. Steady-state levels of fibronectin isoform messenger RNA were examined by Northern hybridization analysis using specific cDNA probes for the EIIIA, EIIIB, and V domains. The de novo synthesis of fibronectin isoforms was examined by metabolic labelling and immunoprecipitation using domain-specific monoclonal antibodies. Fibronectin transcripts were not detectable in freshly isolated hepatic stellate cells from normal liver. Cultured hepatic stellate cells, as well as skin fibroblasts, expressed EIIIA+, EIIIB+, and V95+ transcripts. They were detectable as early as day 3 and increased with time in culture. At 3 days in culture, more than 90 per cent of stellate cells were alpha-smooth muscle actin-positive. In vivo activated hepatic stellate cells expressed EIIIA+ and V95+ transcripts; EIIIB+ fibronectin mRNA was absent. Less than 20 per cent of in vivo activated stellate cells expressed alpha-smooth muscle actin. Freshly isolated parenchymal cells from normal liver as well as from CCl4-treated liver expressed V95+ transcripts, but were negative for EIIIA or EIIIB fibronectin mRNA. Immunoprecipitation results were in accordance with Northern hybridization analysis. Hepatic stellate cells in culture synthesized and secreted fibronectin molecules that contained EIIIA, EIIIB, and V fragments. Our results indicate that hepatic stellate cells synthesize and secrete fibronectin isoforms that are distinct from those of parenchymal cells.