The Cytoskeleton Proteins VASP and Zyxin Participate in Hematopoiesis and in the BCR-ABL Signaling Pathway.
Authors: Bernusso Vanessa Aline , Machado-Neto Joao , Saad Sara T. Olalla , Barcellos Karin Spat Albino 
Published: October 2019

Abstract
Abstract 2780

Background:
VASP and Zyxin are cytoskeleton regulatory proteins. They act as a protein complex involved in the signal transduction for actin polymerization, in the control of cell adhesion, cell division and cellular motility. VASP and Zyxin are abnormally expressed in epithelial tumors and are related with tumor progress. VASP is a substrate of the BCR-ABL oncoprotein and is tyrosine-phosphorylated in BCR-ABL leukemic cells. However, the function of VASP and Zyxin in hematopoietic cells and in the BCR-ABL pathway is not yet known; in addition their possible participation in chronic myeloid leukemia (CML) remains an interesting issue to be clarified.

Aims:
To evaluate the effects of VASP and Zyxin silencing in cell proliferation, apoptosis and differentiation of BCR-ABL K562 cells.

Methods:
shRNA-lentiviral delivery was used to silence VASP and Zyxin expression in K562 cell line. The shRNA-lentiviral control, VASP and Zyxin cells were treated with different Imatinib concentrations (0, 0.1, 0.5 and 1μM) during 48 hours. Cellular proliferation was measured by MTT assay and apoptosis by flow cytometry with annexin-V. To differentiate cells into megakaryocytes, K562 cells were treated with 20nM of PMA during 4 days and cells were evaluated by the presence of CD61 and CD41 cell markers by flow cytometry. The expression of VASP and Zyxin in cells submitted to megakaryocyte differentiation was evaluated by quantitative PCR and western blotting; protein phosphorylation was also analyzed by western blotting. The interaction of BCR-ABL and VASP after imatinib treatment was evaluated by co-immunoprecipiation.

Results:
Zyxin silenced cells treated with 0.5μM and 1μM of Imatinib showed a decrease of 17% (P<0.05) and of 22% (P<0.01) in cell proliferation, respectively, compared to the control treated cells. In K562 cells treated with 1μM of Imatinib, VASP and Zyxin silencing increased apoptosis in 21% (P<0.05) and 40% (P<0.05), respectively. VASP and Zyxin gene expressions were upregulated during megakaryocyte differentiation of K562 cells (8.7-fold increase, P=0.0115, and 3.6-fold increase, P=0.015, respectively). In HEL cells (BCR-ABL negative cell line) VASP and Zyxin protein expressions were increased during megakaryocyte differentiation, including the active form of these proteins (phosphorylated VASP serine 157/239 and phosphorylated Zyxin serine 142). VASP silencing in K562 cells resulted in a 40% decrease of CD61 expression at the end of the megakaryocyte differentiation (P<0.05), whereas Zyxin silencing resulted in a 15% decrease of CD41 expression (P<0.01). VASP expression was reduced during Imatinib treatment of K562 cells, as was also its interaction with BCR-ABL protein. In addition, VASP silencing resulted in a decrease of FAK phosphorylation, an effector of the BCR-ABL pathway involved in cellular adhesion of K562 cells.

Conclusions:
VASP and Zyxin proteins have a role in hematopoiesis, including megakaryocyte differentiation. Alterations in VASP and Zyxin expression affect differentiation and apoptosis of hematopoietic cells. VASP may participate in the BCR-ABL signaling pathway of leukemic cells, affecting leukemic cell adhesion through FAK activity. The elucidation of VASP and Zyxin functions will help elucidate the mechanisms of hematopoietic disorders, such as CML and others.

Disclosures:
No relevant conflicts of interest to declare.