Adhesion to Fibronectin Induces Megakaryocytic Differentiation of JAS-REN Cells

Abstract

Background: Binding of integrins to the extracellular matrix elicits various responses. We have previously reported a megakaryocytic-erythroid cell line (JAS-R) that showed phenotypic changes after adhesion to plastic dishes. However, the matrix protein and the mechanism responsible for megakaryocytic differentiation still remain unknown. Materials and Methods: JAS-REN (erythroid) cells were cultured on dishes coated with various proteins. The cells were treated with RGDS, a tetrapeptide ligand to integrins, or phorbol ester (12-o-tetradecanoylphorbol-13-acetate, TPA) for 48 hours and then were harvested. Subsequently, the cell surface markers were analyzed using flow cytometry and gene expression was studied by RT-PCR. Results: The JAS-REN cells adhered to fibronectin-coated dishes, but showed poor adhesion to dishes coated with collagen, laminin or poly-D-lysine. The TPA-stimulated JAS-REN cells showed an increase in the expression of integrin α_IIb, β₃ complex (CD41a) and integrin, β₃ (CD61), while glycophorin A (CD235a) expression was decreased. JAS-REN cells that were adherent to fibronectin-coated dishes also showed a similar pattern of phenotype to TPA-treated cells, but the changes were not so prominent. RT-PCR revealed that TPA treatment altered the gene expression profile of JAS-REN cells, making it similar to that of JAS-RAD (megakaryocytic) cells. The RGDS-treated and fibronectin adherent JAS-REN cells also showed a mostly similar expression profile to JAS-RAD cells, but these two stimuli did not alter the gene expression profile as TPA stimulation did. Transcription factors, FLI1 and GFI1, were induced by all stimuli. Conclusion: Signals triggered by adhesion to fibronectin result in the induction of FLI1 that may play a pivotal role in the lineage shift of JAS-REN cells from erythroid to megakaryocytic.