Rat hepatocytes in primary culture synthesize and secrete cellular fibronectin

Abstract

Fibronectins, involved in cell-matrix interactions and cell attachment, are glycoproteins which show a remarkable heterogeneity, due to alternative splicing. The type III-related domains, ED-A and ED-B, are present in cellular fibronectin in a variety of ratios whereas they are absent in circulating plasma fibronectin. Fibronectin synthesis by hepatocytes which are accepted as suppliers of plasma fibronectin was studied in primary cultures during a 6-day culture period. Using site-specific antibodies we demonstrate that rat hepatocytes are also able to synthesize and secrete fibronectin bearing the ED-A domain from Day 3 on after inoculation. By immunocytological characterization of the hepatocyte monolayer with antibodies directed against desmin, laminin, collagen IV, alpha-SM-actin, or ED-1 or factor VIII-related antigen, contaminating mesenchymal hepatic cell-types as a source for cellular fibronectin production could be ruled out. Dexamethasone treatment caused enhanced fibronectin synthesis and cellular fibronectin was already detectable at Day 1 after plating. Elevation of cellular fibronectin synthesis after prolonged culture-terms and by dexamethasone could also be demonstrated on mRNA steady-state level, using ED-A cDNA as a probe in hybridization analysis. Dot blot hybridisation proved a prominent response of cellular fibronectin mRNA level to dexamethasone at Day 1 when dexamethasone treatment resulted in an increased contribution of ED-A-positive fibronectin transcripts to total fibronectin mRNA level.