ARHGAP21 Is Upregulated and Triggers the Modulation of Rho Gtpase Signaling Pathways during Megakaryocytic Differentiation
Authors: Bernusso Vanessa Aline , Lazarini Mariana , Machado-Neto João Agostinho , Barcellos Karin Spat Albino , Saad Sara Teresinha Olalla 
Published: October 2019

Abstract
Introduction: During differentiation, the megakaryocyte goes through profound changes in the cytoskeleton of actin and tubulin through Rho GTPase activity. Microtubules provide the elongation of proplatelets, whereas actin microfilaments mediate force to increase branching and release of platelets. ARHGAP21 is a RhoGAP for RhoA, RhoC and Cdc42, which has been shown to interact with α-tubulin in cancer cells. Moreover, arhgap21+/- mice exhibit significant reduction in platelet number and increased platelet volume.
Aim: To evaluate ARHGAP21 function in the activity of Rho GTPase and their effectors during megakaryocytic differentiation.
Materials and Methods: Megakaryocyte differentiation was stimulated in HEL cells through treatment with 20 nM of phorbol myristate acetate -13 -12 (PMA) for 4 days and was confirmed by the expression of CD41a, CD42b and CD61 and polyploidy using flow cytometry. Morphological changes were observed by optical microscopy. The localization of ARHGAP21, F-Actin and α-Tubulin cytoskeletal proteins was assessed by confocal microscopy. The expression of ARHGAP21, and the Rho GTPases RhoA, RhoC, Cdc42 and downstream proteins Rock1 e2, phospho-MLC2, MLC2, phospho-cofilin, cofilin and mDia1 were analyzed by western blotting. Rho GTPases activity was determined through pull down assays using Rhotekin-GST (RhoA, RhoB and RhoC) and WASP-GST (Cdc42) constructions. Tubulin polymerization was evaluated by soluble and insoluble tubulin precipitation assay. ARHGAP21 silencing was performed by siRNA, after PMA treatment for 2 and 3 days and was followed by the analysis of the expression and activity of Rho GTPases and their effectors, ploidy and differentiation markers.
Results: Megakaryocytic differentiation of HEL cells was accompanied by intense rearrangement of the cytoskeleton, increased cell size, polyploidy and increased expression of the membrane receptors CD61, CD41a and CD42b. Interestingly, a gradual upregulation of ARHGAP21 was observed during differentiation, especially on days 2 and 3 of treatment (both 9.33-fold increase) and mainly in extracts containing polymerized tubulin. ARHGAP21 upregulation was concomitant with the reduction of RhoA and Cdc42 activities (92% decreased and 52% decreased, respectively), but not in RhoC. Silencing of ARHGAP21 by siRNA was confirmed by western blot. Downregulation of ARHGAP21 in HEL cells trigged increased phosphorylation on serine 19 of myosin light chain2 (MLC2) on the day 2. Moreover, mDia1, a common effector of RhoA and Cdc42, was also increased at the same point. ARHGAP21 silencing induced an increase in CD42b on day 3 (5% increased, P<0.015). No difference was observed in the expression of CD61 and CD41a and in the ploidy of ARHGAP21 silenced cells compared to control.
Conclusion: Our results suggest that the upregulation of ARHGAP21 during megakaryocytic differentiation is important to control the dynamics of the cytoskeleton through the regulation of RhoA and Cdc42. Silencing of ARHGAP21 induces increased phosphorylation of MLC2 and the expression of mDia1, which may impair megakaryocytic differentiation. Furthermore, ARHGAP21 appears to regulate the acquisition of CD42b receptor, participating in the final stages of megakaryopoiesis.

Disclosures
No relevant conflicts of interest to declare.