Modulation of α6/β1 Integrin Expression during Differentiation of F9 Murine Embryonal Carcinoma Cells to Parietal Endoderm

Abstract

In vitrodifferentiation of the murine embryonal carcinoma (EC) cell line F9 parallels that of the early blastocyst, where visceral (VE) and parietal endoderm (PE) diverge from a common precursor, the primitive endoderm. This differentiation pathway is induced by retinoic acid (RA) and dibutyryl cyclicAMP (dcAMP) and is accompanied by progressive and dramatic changes in cell morphology and functions. Within 7 days of treatment the cells organize their cytoskeleton and synthesize large amounts of extracellular matrix proteins, becoming fully differentiated migratory cells; all these changes are likely to involve integrins expression and organization. We have investigated the changes in β1 integrin expression, its maturation, and organization on the cell surface in association with α6, during the transition from undifferentiated F9 stem cells to migrating PE cells. By Western blotting and immunoprecipitation we showed a gradual decrease in the amount of the β1 subunit on the cell surface and a parallel progressive accumulation of immature protein, indicating that the control of β1 expression during F9 cells differentiation occurs first at posttranslational level and then at the level of transcription. Moreover, the induction of differentiation produces a marked decrease of α6B and its association to a high molecular weight protein, while α6A level increases. By immunofluorescence we found that upon differentiation there is a relocation of the β1 and α6B integrin subunits from cell–cell contacts to focal contacts where they colocalize with vinculin. On the contrary α6A, weakly present in F9 stem cells, is present in the focal contacts of PE cells and along the stress fibers. We suggest different roles for the two α6 isoforms.