Dexamethasone can modulate the synthesis and organization of cytokeratins in cultured differentiating rat hepatocytes

Abstract

Dexamethasone, added to suckling rat hepatocytes cultured in serum-free medium supplemented with insulin and epidermal growth factor (EGF), caused a selective dose-dependent increase in cytokeratin synthesis. The response was dependent on the initial hepatocyte density. At 5 × 104 hepatocytes/cm2 a concentration of 1 or 10 μM dexamethasone, in the presence of insulin, enhanced the synthesis of a 55 000 relative mass (Mr) cytokeratin and to a lesser degree a 49 000 Mr cytokeratin, whereas at 1 × 105 hepatocytes/cm2 10 μM dexamethasone preferentially stimulated a 51 000 Mr component. Preferential synthesis of the 51 000 Mr component also occurred when either 1 or 10 μM dexamethasone was added to cultures seeded at 2 × 105 hepatocytes/cm2. The inclusion of EGF along with dexamethasone and insulin in cultures at 2 × 105 cells/cm2 yielded a differential effect of dexamethasone concentration equivalent to that observed at the lower cell density in absence of EGF. Under conditions where increased cytokeratin synthesis was observed, the hepatocytes maintained a high production of albumin and lost their capacity to produce α-fetoprotein, a change in gene expression associated with the normal differentiation of suckling rat hepatocytes. In contrast, no enhancement of cytokeratin synthesis was observed in hepatocytes following addition of EGF and insulin only, a condition that promoted hepatocyte growth and the maintenance of α-fetoprotein production. The dexamethasone-induced enhancement of cytokeratin synthesis was still present at 3 days postseeding. At this time, morphological observations by phase-contrast and immunofluorescence microscopy using monoclonal antibodies against the 55 000 and 49 000 Mr components revealed that under growth-promoting conditions the hepatocytes were spread and the cytokeratin filaments were stretched, whereas under differentiation-promoting conditions the cultures constitute a compact monolayer of cells exhibiting highly ordered filaments. These data suggest a close relationship between synthesis and organization of cytokeratins and promotion of differentiation of suckling rat hepatocytes by glucocorticoids.