RhoJ/TCL Regulates Endothelial Motility and Tube Formation and Modulates Actomyosin Contractility and Focal Adhesion Numbers

Abstract

Objective-RhoJ/TCL was identified by our group as an endothelial-expressed Rho GTPase. The aim of this study was to determine its tissue distribution, subcellular localization, and function in endothelial migration and tube formation. Methods and Results-Using in situ hybridization, RhoJ was localized to endothelial cells in a set of normal and cancerous tissues and in the vasculature of mouse embryos; endogenous RhoJ was localized to focal adhesions by immunofluorescence. The proangiogenic factor vascular endothelial growth factor activated RhoJ in endothelial cells. Using either small interfering (si)RNA-mediated knockdown of RhoJ expression or overexpression of constitutively active RhoJ (daRhoJ), RhoJ was found to positively regulate endothelial motility and tubule formation. Downregulating RhoJ expression increased focal adhesions and stress fibers in migrating cells, whereas daRhoJ overexpression resulted in the converse. RhoJ downregulation resulted in increased contraction of a collagen gel and increased phospho-myosin light chain, indicative of increased actomyosin contractility. Pharmacological inhibition of Rho-kinase (which phosphorylates myosin light chain) or nonmuscle myosin II reversed the defective tube formation and migration of RhoJ knockdown cells. Conclusion-RhoJ is endothelial-expressed in vivo, activated by vascular endothelial growth factor, localizes to focal adhesions, regulates endothelial cell migration and tube formation, and modulates actomyosin contractility and focal adhesion numbers. (Arterioscler Thromb Vasc Biol. 2011;31:657-664.)

The Formin FMNL3 Controls Early Apical Specification in Endothelial Cells by Regulating the Polarized Trafficking of Podocalyxin

“…Like the related formins FMNL1 and FMNL2 [4,5], FMNL3 interacted with activated Cdc42 ( Figure 1A) but also with RhoJ, RhoD, RhoH, and Rnd1. We focused on Cdc42 and RhoJ, as both signaling proteins are required for angiogenesis [6][7][8]. We confirmed the interactions with FMNL3 by co-immunoprecipitation in HEK293 cells.…”

“…Next, we sought to link FMNL3 to known endothelial functions of Cdc42 and RhoJ. RhoJ controls focal adhesion disassembly in endothelial cells (ECs) through regulation of the GIT-PIX complex [7,9]. We silenced FMNL3 in primary ECs (Figure S1B); however, this had no effect on focal adhesion number (data not shown), suggesting that FMNL3 is not involved in this aspect of RhoJ signaling.…”

Modulation of VEGF-induced migration and network formation by lymphatic endothelial cells: Roles of platelets and podoplanin

“…This model was based on one previously used with vascular endothelial cells (VEC) in the study of angiogenesis [36][37][38], and we report the first description of formation of comparable stable networks for LEC. Addition of platelets to these networks or crosslinking of podoplanin caused their disruption.…”

“…Having shown that platelets and podoplanin crosslinking modulate VEGF-mediated LEC migration, we also examined their effects on networks of LEC, adapting a method described for vascular endothelial cells [36][37][38] but not yet reported for LECs. We found for the first time that stable networks of LEC could be established on monolayers of fibroblasts.…”

The role of RhoJ in endothelial cell biology and angiogenesis

“…We explored the role of RhoJ in endothelial migration and tubulogenesis, and its regulation of the actin cytoskeleton, and mapped its subcellular localization and tissue distribution [14]. There have subsequently been two further reports noting an endothelial expression pattern and characterizing a role for RhoJ in endothelial cell biology [15,16].…”