Effects of Spred2 on megakaryocytic differentiation of K562 cells

To investigate the regulatory role of Sprouty-related protein with enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domain 2 (Spred2) in megakaryocytic differentiation of K562 cells.

K562 cells with downregulated Spred2 were established using short hairpin RNA (shRNA) transfection, while those with overexpression of Spred2 were established using Spred2 overexpression plasmid vector transfection. Morphological changes during differentiation were analyzed via Wright-Giemsa staining. mRNA expression levels of differentiation-specific markers, including early growth response factor 1 (EGR-1), GATA binding protein 2 (GATA-2), glycophorin A (GPA), and GATA binding protein1 (GATA-1), were quantified via quantitative real-time PCR (qPCR). Expressions of platelet glycoprotein Ⅱb (CD41a) and GPA were determined by flow cytometry, while Spred2 protein levels were analyzed by Western blotting.

Transient Spred2 silencing induced megakaryocytic differentiation in K562 cells, characterized by cellular enlargement, enhanced intercellular adhesion, upregulation of CD41a, and downregulation of GPA. Stable Spred2 silencing promoted megakaryocytic differentiation induced by phorbol 12-myristate 13-acetate (PMA), as evidenced by increased cell attachment/spreading, elevated expressions of CD41a, EGR-1, and GATA-2, and reduced levels of GPA and GATA-1. Conversely, Spred2 overexpression upregulated GPA expression.

Spred2 may modulate the balance between megakaryocytic and erythroid differentiation of K562 cells.